Inflammation and Cancer: Methods and Protocols (Methods in Molecular Biology, 2691) [2 ed.] 1071633309, 9781071633304

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Methods in Molecular Biology 2691

Brendan J. Jenkins  Editor

Inflammation and Cancer Methods and Protocols Second Edition

METHODS

IN

MOLECULAR BIOLOGY

Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, UK

For further volumes: http://www.springer.com/series/7651

For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in PubMed.

Inflammation and Cancer Methods and Protocols Second Edition

Edited by

Brendan J. Jenkins Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, VIC, Australia

Editor Brendan J. Jenkins Centre for Innate Immunity and Infectious Diseases Hudson Institute of Medical Research Clayton, VIC, Australia

ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-0716-3330-4 ISBN 978-1-0716-3331-1 (eBook) https://doi.org/10.1007/978-1-0716-3331-1 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2018, 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature. The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.

Preface Dysregulated activation of both the innate and adaptive arms of the host immune system contributes to the pathogenesis of a wide range of chronic diseases, in particular autoimmune and inflammatory disorders, infectious diseases, and cancer. With respect to cancer, at least one third of all cancers are associated with chronic inflammatory responses, with prime examples being cancers of the lung, stomach, pancreas, and colon, among a range of others. Over the last two decades, laboratory-based investigations using in vivo disease models and clinical samples, coupled to state-of-the-art molecular and cellular biological methodologies, along with next-generation sequencing, proteomics, and functional genomics technologies, have driven research efforts worldwide to understand the pathogenesis of these disease states. Such knowledge is critical to our collective efforts to discover new immunebased biomarkers and therapeutic strategies against disease, especially as we enter the phase of precision medicine. This book, entitled Inflammation and Cancer: Methods and Protocols, Second Edition, is the latest instalment of the highly successful Methods in Molecular Biology laboratory protocol-based book series, and is a follow-up to the initial 2018 edition of Inflammation and Cancer: Methods and Protocols. Written by leading experts in the fields of inflammation and cancer, this book comprises 25 individual chapters and provides a timely update on a broad spectrum of research models, techniques, and protocols, which are employed by basic and clinical research laboratories. Each chapter is divided into sections providing detailed information on the background and context for the chosen topic of interest, a list of the materials and reagents needed for each topic, the step-by-step methodology for the successful and reproducible execution of each topic, as well as notes to provide tips, troubleshooting advice, and elaborate further on specific materials, reagents, or methods. Considering the enormity of the rapidly evolving and large number of research models (in vitro, ex vivo, and in vivo) and techniques which are collectively used by researchers, it is impractical to provide their sufficient coverage in detail in a single book. Therefore, this edition is divided into two parts: Part I, “Experimental Model Systems,” which provides an up-to-date snapshot of the development and characterization of representative research models for chronic immune-based (i.e. infectious, autoimmune, inflammatory) diseases and inflammation-associated cancers, and Part II, “Experimental Techniques,” which covers a range of biochemical, molecular, and cellular biological techniques that are commonly utilized to dissect the molecular mechanisms and cellular processes (including cell type(s)) which drive the pathogenesis of certain disease states. With a strong emphasis on practicality, Inflammation and Cancer: Methods and Protocols, Second Edition will appeal to a readership with a very diverse range of laboratory-based experience, ranging from undergraduate students with limited research exposure to established basic and/or clinical researchers wishing to diversify their existing portfolio of practical knowledge. In addition, this book aims to supplement researchers with the necessary practical expertise and know-how to assist their efforts to publish their research findings, dissemination of which in the literature will enhance our collective knowledge of the processes which drive inflammation and cancer. Clayton, VIC, Australia

Brendan J. Jenkins

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Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

PART I

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EXPERIMENTAL MODEL SYSTEMS

1 Identifying Adult Stomach Tissue Stem/Progenitor Cells Using the Iqgap3-2A-CreERT2 Mouse. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Junichi Matsuo, Linda Shyue Huey Chuang, Jasmine Jie Lin Tong, Daisuke Douchi, and Yoshiaki Ito 2 In Vitro and In Vivo Models for Metastatic Intestinal Tumors Using Genotype-Defined Organoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Atsuya Morita, Mizuho Nakayama, Hiroko Oshima, and Masanobu Oshima 3 On Target: An Intrapulmonary Transplantation Method for Modelling Lung Tumor Development in its Native Microenvironment . . . . . . . . . . . . . . . . . . 31 Jackson A. McDonald, Leanne Scott, Jessica Van Zuylekom, Steven Holloway, Benjamin J. Blyth, and Kate D. Sutherland 4 Endoscopic Ultrasound-Guided Fine-Needle Biopsies to Generate Preclinical Disease Models to Study Inflammation in Pancreatic Ductal Adenocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 Joanne Lundy and Daniel Croagh 5 Modeling Intestinal Carcinogenesis Using In Vitro Organoid Cultures . . . . . . . . 55 Wing Hei Chan, Diana Micati, Rebekah M. Engel, Genevieve Kerr, Reyhan Akhtar, Thierry Jarde´, and Helen E. Abud 6 An In Vitro Model for Assessing Acute Lung Injury During Pancreatitis Development Using Primary Mouse Cell Co-cultures . . . . . . . . . . . . . . . . . . . . . . . 71 Mohamed I. Saad and Brendan J. Jenkins 7 Tracking the Host Response to Infection in Peritoneal Models of Acute Resolving Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 David Millrine, Christopher M. Rice, Javier U. Fernandez, and Simon A. Jones 8 Assessing Lung Inflammation and Pathology in Preclinical Models of Chronic Obstructive Pulmonary Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Ross Vlahos, Hao Wang, and Steven Bozinovski 9 Preclinical Mouse Model of Silicosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 Maggie Lam, Ashley Mansell, and Michelle D. Tate

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Contents

PART II 10

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EXPERIMENTAL TECHNIQUES

Tracking Cardiovascular Comorbidity in Models of Chronic Inflammatory Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Aisling S. Morrin, Simon Eastham, Anwen S. Williams, and Gareth W. Jones Live Imaging of Pyroptosis in Primary Murine Macrophages . . . . . . . . . . . . . . . . . Caroline L. Holley and Kate Schroder A Streamlined Method for Detecting Inflammasome-Induced ASC Oligomerization Using Chemical Crosslinking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sebastian A. Hughes and James E. Vince DNA Methylation Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Naoko Hattori, Yu-Yu Liu, and Toshikazu Ushijima Flow Cytometry Identification of Cell Compartments in the Murine Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Joel J. D. Moffet, Zachery Moore, Shannon J. Oliver, Tahnee Towers, Misty R. Jenkins, Saskia Freytag, James R. Whittle, and Sarah A. Best Expression and Purification of Inflammasome Sensor NOD-Like Receptor Protein-1 Using the Baculovirus-Insect Cell Expression System . . . . . . . . . . . . . . Hariharan Sivaraman and Wilson Wong Dissecting Interleukin-6 Classic and Trans-signaling in Inflammation and Cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Christoph Garbers and Stefan Rose-John Selecting Therapeutic Antisense Oligonucleotides with Gene Targeting and TLR8 Potentiating Bifunctionality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sunil Sapkota and Michael P. Gantier Exploring Allosteric Inhibitors of Protein Tyrosine Phosphatases Through High-Throughput Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Takeru Hayashi and Masanori Hatakeyama Intravital Imaging of Regulatory T Cells in Inflamed Skin . . . . . . . . . . . . . . . . . . . Michael J. Hickey and M. Ursula Norman Confocal Endomicroscopy Monitoring of Tumor Formation. . . . . . . . . . . . . . . . . Adele Preaudet, Ka Yee Fung, and Tracy L. Putoczki Detection of Free Bioactive IL-18 and IL-18BP in Inflammatory Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Se´bastien Fauteux-Daniel, Charlotte Girard-Guyonvarc’h, Assunta Caruso, Emiliana Rodriguez, and Cem Gabay MAC-Seq: Coupling Low-Cost, High-Throughput RNA-Seq with Image-Based Phenotypic Screening in 2D and 3D Cell Models . . . . . . . . . . Xiang Mark Li, David Yoannidis, Susanne Ramm, Jennii Luu, Gisela Mir Arnau, Timothy Semple, and Kaylene J. Simpson Primary Intestinal Fibroblasts: Isolation, Cultivation, and Maintenance. . . . . . . . Abhimanu Pandey, Melan Kurera, and Si Ming Man

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Lipid Nanoparticle-Mediated Delivery of miRNA Mimics to Myeloid Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337 Elaine Kang and Marcin Kortylewski 25 Engineering Cell Lines for Specific Human Leukocyte Antigen Presentation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351 Dongbin Jin, Khai Lee Loh, Tima Shamekhi, Yi Tian Ting, Terry C. C. Lim Kam Sian, James Roest, Joshua D. Ooi, Julian P. Vivian, and Pouya Faridi Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors HELEN E. ABUD • Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC, Australia; Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Clayton, VIC, Australia REYHAN AKHTAR • Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC, Australia GISELA MIR ARNAU • Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, VIC, Australia; Molecular Genomics Core, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia SARAH A. BEST • Personalised Oncology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia; Department of Medical Biology, The University of Melbourne, Parkville, VIC, Australia BENJAMIN J. BLYTH • Models of Cancer Translational Research Centre, Peter MacCallum Cancer Centre, Parkville, VIC, Australia; Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, VIC, Australia STEVEN BOZINOVSKI • Centre for Respiratory Science and Health, School of Health and Biomedical Sciences, RMIT University, Bundoora, VIC, Australia ASSUNTA CARUSO • Department of Pathology and Immunology, University of Geneva, Faculty of Medicine, Geneva, Switzerland; Division of Rheumatology, Department of Medicine, Geneva University Hospitals, Geneva, Switzerland WING HEI CHAN • Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC, Australia; Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Clayton, VIC, Australia LINDA SHYUE HUEY CHUANG • Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore DANIEL CROAGH • Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, VIC, Australia; Department of Molecular Translational Science, School of Clinical Sciences, Monash University, Clayton, VIC, Australia; Department of Surgery, School of Clinical Sciences, Monash University, Clayton, VIC, Australia DAISUKE DOUCHI • Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore; Department of Surgery, Tohoku University Graduate School of Medicine, Sendai City, Japan SIMON EASTHAM • School of Cellular and Molecular Medicine, Biomedical Sciences Building, University of Bristol, Bristol, UK REBEKAH M. ENGEL • Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC, Australia; Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Clayton, VIC, Australia; Cabrini Monash University Department of Surgery, Cabrini Hospital, Melbourne, VIC, Australia POUYA FARIDI • Department of Medicine, School of Clinical Sciences, Monash Univesity, Clayton, VIC, Australia; Monash Proteomics & Metabolomics Facility, Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia

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Contributors

SE´BASTIEN FAUTEUX-DANIEL • Department of Pathology and Immunology, University of Geneva, Faculty of Medicine, Geneva, Switzerland; Division of Rheumatology, Department of Medicine, Geneva University Hospitals, Geneva, Switzerland JAVIER U. FERNANDEZ • Division of Infection & Immunity, School of Medicine, Cardiff University, Cardiff, Wales, UK; Systems Immunity University Research Institute, Cardiff University, Cardiff, Wales, UK SASKIA FREYTAG • Personalised Oncology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia; Department of Medical Biology, The University of Melbourne, Parkville, VIC, Australia KA YEE FUNG • Personalised Oncology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia; Department of Medical Biology, The University of Melbourne, Parkville, VIC, Australia CEM GABAY • Department of Pathology and Immunology, University of Geneva, Faculty of Medicine, Geneva, Switzerland; Division of Rheumatology, Department of Medicine, Geneva University Hospitals, Geneva, Switzerland MICHAEL P. GANTIER • Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, VIC, Australia; Department of Molecular and Translational Science, Monash University, Clayton, VIC, Australia CHRISTOPH GARBERS • Medical Faculty, Department of Pathology, Otto-von-GuerickeUniversity Magdeburg, Magdeburg, Germany; Health Campus Immunology, Infectiology and Inflammation (GC:I3), Otto-von-Guericke-University, Magdeburg, Germany; Center for Health and Medical Prevention (CHaMP), Otto-von-Guericke-University, Magdeburg, Germany CHARLOTTE GIRARD-GUYONVARC’H • Department of Pathology and Immunology, University of Geneva, Faculty of Medicine, Geneva, Switzerland; Division of Rheumatology, Department of Medicine, Geneva University Hospitals, Geneva, Switzerland MASANORI HATAKEYAMA • Laboratory of Microbial Carcinogenesis, Institute of Microbial Chemistry (BIKAKEN), Microbial Chemistry Research Foundation, Tokyo, Japan; Center of Infection-Associated Cancer, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan NAOKO HATTORI • Division of Epigenomics, Institute for Advanced Life Sciences, Hoshi University, Tokyo, Japan TAKERU HAYASHI • Laboratory of Microbial Carcinogenesis, Institute of Microbial Chemistry (BIKAKEN), Microbial Chemistry Research Foundation, Tokyo, Japan MICHAEL J. HICKEY • Centre for Inflammatory Diseases, Department of Medicine, Monash Medical Centre, Monash University, Clayton, VIC, Australia CAROLINE L. HOLLEY • Institute for Molecular Bioscience, and Centre for Inflammation and Disease Research, The University of Queensland, St. Lucia, QLD, Australia STEVEN HOLLOWAY • Bioservices Department, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia SEBASTIAN A. HUGHES • The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia; The Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia YOSHIAKI ITO • Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore; Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore THIERRY JARDE´ • Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC, Australia; Development and Stem Cells Program, Monash Biomedicine

Contributors

xiii

Discovery Institute, Clayton, VIC, Australia; Cancer Program, Monash Biomedicine Discovery Institute, Clayton, VIC, Australia BRENDAN J. JENKINS • Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, VIC, Australia; Faculty of Medicine, Nursing and Health Sciences, Department of Molecular and Translational Sciences, Monash University, Clayton, VIC, Australia; South Australian Immunogenomics Cancer Institute, University of Adelaide, Adelaide, SA, Australia MISTY R. JENKINS • Department of Medical Biology, The University of Melbourne, Parkville, VIC, Australia; Immunology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia DONGBIN JIN • Department of Medicine, School of Clinical Sciences, Monash Univesity, Clayton, VIC, Australia GARETH W. JONES • School of Cellular and Molecular Medicine, Biomedical Sciences Building, University of Bristol, Bristol, UK SIMON A. JONES • Division of Infection & Immunity, School of Medicine, Cardiff University, Cardiff, Wales, UK; Systems Immunity University Research Institute, Cardiff University, Cardiff, Wales, UK ELAINE KANG • Department of Immuno-Oncology, Beckman Research Institute at City of Hope, Duarte, CA, USA GENEVIEVE KERR • Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC, Australia; Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Clayton, VIC, Australia MARCIN KORTYLEWSKI • Department of Immuno-Oncology, Beckman Research Institute at City of Hope, Duarte, CA, USA MELAN KURERA • Division of Immunology and Infectious Disease, The John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia MAGGIE LAM • Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, VIC, Australia; Department of Molecular and Translational Sciences, Monash University, Clayton, VIC, Australia XIANG MARK LI • Victorian Centre for Functional Genomics, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia; Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, VIC, Australia TERRY C. C. LIM KAM SIAN • Department of Medicine, School of Clinical Sciences, Monash Univesity, Clayton, VIC, Australia; Monash Proteomics & Metabolomics Facility, Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia YU-YU LIU • Division of Epigenomics, Institute for Advanced Life Sciences, Hoshi University, Tokyo, Japan KHAI LEE LOH • Centre for Inflammatory Diseases, Department of Medicine, Monash University, Monash Medical Centre, Clayton, VIC, Australia JOANNE LUNDY • Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, VIC, Australia; Department of Molecular Translational Science, School of Clinical Sciences, Monash University, Clayton, VIC, Australia; Department of Surgery, School of Clinical Sciences, Monash University, Clayton, VIC, Australia; Peninsula Clinical School, Central Clinical School, Faculty of Medicine Nursing and Health Sciences, Monash University, Clayton, VIC, Australia JENNII LUU • Victorian Centre for Functional Genomics, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia

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Contributors

SI MING MAN • Division of Immunology and Infectious Disease, The John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia ASHLEY MANSELL • Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, VIC, Australia; Department of Molecular and Translational Sciences, Monash University, Clayton, VIC, Australia JUNICHI MATSUO • Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore JACKSON A. MCDONALD • ACRF Cancer Biology and Stem Cells Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia; Department of Medical Biology, The University of Melbourne, Parkville, VIC, Australia DIANA MICATI • Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC, Australia; Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Clayton, VIC, Australia DAVID MILLRINE • Medical Research Council Protein Phosphorylation & Ubiquitylation Unit (MRC-PPU), School of Life Sciences, University of Dundee, Dundee, UK JOEL J. D. MOFFET • Personalised Oncology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia ZACHERY MOORE • Personalised Oncology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia; Department of Medical Biology, The University of Melbourne, Parkville, VIC, Australia ATSUYA MORITA • Division of Genetics, Cancer Research Institute, Kanazawa University, Kanazawa, Japan AISLING S. MORRIN • Division of Infection and Immunity, and Systems Immunity University Research Institute, School of Medicine, Cardiff University, Cardiff, Wales, UK MIZUHO NAKAYAMA • Division of Genetics, Cancer Research Institute, Kanazawa University, Kanazawa, Japan; Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Kanazawa, Japan M. URSULA NORMAN • Centre for Inflammatory Diseases, Department of Medicine, Monash Medical Centre, Monash University, Clayton, VIC, Australia SHANNON J. OLIVER • Personalised Oncology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia JOSHUA D. OOI • Centre for Inflammatory Diseases, Department of Medicine, Monash University, Monash Medical Centre, Clayton, VIC, Australia HIROKO OSHIMA • Division of Genetics, Cancer Research Institute, Kanazawa University, Kanazawa, Japan; Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Kanazawa, Japan MASANOBU OSHIMA • Division of Genetics, Cancer Research Institute, Kanazawa University, Kanazawa, Japan; Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Kanazawa, Japan ABHIMANU PANDEY • Division of Immunology and Infectious Disease, The John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia ADELE PREAUDET • Personalised Oncology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia TRACY L. PUTOCZKI • Personalised Oncology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia; Department of Medical Biology, The University of Melbourne, Parkville, VIC, Australia; Department of Surgery, The University of Melbourne, Parkville, VIC, Australia

Contributors

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SUSANNE RAMM • Victorian Centre for Functional Genomics, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia; Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, VIC, Australia CHRISTOPHER M. RICE • School of Cellular & Molecular Medicine, University of Bristol, Bristol, UK EMILIANA RODRIGUEZ • Department of Pathology and Immunology, University of Geneva, Faculty of Medicine, Geneva, Switzerland; Division of Rheumatology, Department of Medicine, Geneva University Hospitals, Geneva, Switzerland JAMES ROEST • St Vincent’s Institute of Medical Research, Fitzroy, VIC, Australia STEFAN ROSE-JOHN • Institute of Biochemistry, Kiel University, Kiel, Germany MOHAMED I. SAAD • Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, VIC, Australia; Faculty of Medicine, Nursing and Health Sciences, Department of Molecular and Translational Sciences, Monash University, Clayton, VIC, Australia SUNIL SAPKOTA • Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, VIC, Australia; Department of Molecular and Translational Science, Monash University, Clayton, VIC, Australia KATE SCHRODER • Institute for Molecular Bioscience, and Centre for Inflammation and Disease Research, The University of Queensland, St. Lucia, VIC, Australia LEANNE SCOTT • ACRF Cancer Biology and Stem Cells Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia TIMOTHY SEMPLE • Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, VIC, Australia; Molecular Genomics Core, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia TIMA SHAMEKHI • Department of Medicine, School of Clinical Sciences, Monash Univesity, Clayton, VIC, Australia KAYLENE J. SIMPSON • Victorian Centre for Functional Genomics, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia; Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, VIC, Australia; Department of Biochemistry and Pharmacology, University of Melbourne, Parkville, VIC, Australia HARIHARAN SIVARAMAN • Centre for Innate Immunity and Infectious Diseases, Centre for Cancer Research, Hudson Institute of Medical Research, Melbourne, Clayton, VIC, Australia; Department of Molecular and Translational Science, School of Clinical Sciences, Monash University, Clayton, VIC, Australia KATE D. SUTHERLAND • ACRF Cancer Biology and Stem Cells Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia; Department of Medical Biology, The University of Melbourne, Parkville, VIC, Australia MICHELLE D. TATE • Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, VIC, Australia; Department of Molecular and Translational Sciences, Monash University, Clayton, VIC, Australia YI TIAN TING • Centre for Inflammatory Diseases, Department of Medicine, Monash University, Monash Medical Centre, Clayton, VIC, Australia JASMINE JIE LIN TONG • Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore TAHNEE TOWERS • Personalised Oncology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia TOSHIKAZU USHIJIMA • Division of Epigenomics, Institute for Advanced Life Sciences, Hoshi University, Tokyo, Japan

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Contributors

JESSICA VAN ZUYLEKOM • Models of Cancer Translational Research Centre, Peter MacCallum Cancer Centre, Parkville, VIC, Australia JAMES E. VINCE • The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia; The Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia JULIAN P. VIVIAN • St Vincent’s Institute of Medical Research, Fitzroy, VIC, Australia; Department of Medicine, The University of Melbourne, Melbourne, VIC, Australia ROSS VLAHOS • Centre for Respiratory Science and Health, School of Health and Biomedical Sciences, RMIT University, Bundoora, VIC, Australia HAO WANG • Centre for Respiratory Science and Health, School of Health and Biomedical Sciences, RMIT University, Bundoora, VIC, Australia JAMES R. WHITTLE • Personalised Oncology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia; Department of Medical Biology, The University of Melbourne, Parkville, VIC, Australia; Department of Medical Oncology, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia; Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, VIC, Australia ANWEN S. WILLIAMS • Division of Infection and Immunity, and Systems Immunity University Research Institute, School of Medicine, Cardiff University, Cardiff, Wales, UK WILSON WONG • Centre for Innate Immunity and Infectious Diseases, Centre for Cancer Research, Hudson Institute of Medical Research, Melbourne, Clayton, VIC, Australia; Department of Molecular and Translational Science, School of Clinical Sciences, Monash University, Clayton, VIC, Australia DAVID YOANNIDIS • Molecular Genomics Core, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia

Part I Experimental Model Systems

Chapter 1 Identifying Adult Stomach Tissue Stem/Progenitor Cells Using the Iqgap3-2A-CreERT2 Mouse Junichi Matsuo, Linda Shyue Huey Chuang, Jasmine Jie Lin Tong, Daisuke Douchi, and Yoshiaki Ito Abstract Identification of unique gene markers of normal and cancer stem cells is an effective strategy to study cells of origin and understand tumor behavior. Lineage tracing experiments using the Cre recombinase driven by a stem cell-specific promoter in the CreERT2 reporter mouse model enables identification of adult stem cells and delineation of stem cell activities in vivo. In our recent research on the mouse stomach, Iqgap3 was identified as a homeostatic stem cell marker located in the isthmus of the stomach epithelium. Lineage tracing with the Iqgap3-2A-CreERT2;Rosa26-LSL-tdTomato mouse model demonstrated stem cell activity in Iqgap3-expressing cells. Using the Iqgap3-2A-CreERT2 mouse model to target oncogenic KrasG12D expression to Iqgap3-expressing cells, we observed the rapid development of precancerous metaplasia in the stomach and proposed that aberrant Iqgap3-expressing cells may be critical determinants of early carcinogenesis. In this chapter, we detail a lineage tracing protocol to assess stem cell activity in the murine stomach. We also describe the procedure of inducing KrasG12D expression in Iqgap3-expressing homeostatic stem cells to explore their role as cells of origin and to trace the early cellular changes that precede neoplastic transformation. Key words Mouse model, Lineage tracing, Stomach, Stem cells, Carcinogenesis, Metaplasia, Iqgap3, KrasG12D

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Introduction Adult tissue stem/progenitor cells maintain organ homeostasis and effect tissue repair. Because of their proliferative capacity and inherent plasticity, these cells are an ideal candidate for cells of origin of cancer. The understanding of the molecular underpinnings of adult tissue stem/progenitor cells advances regenerative medicine and cancer research through the discovery of novel drug targets and prognostic biomarkers. The past decade has seen extensive research on various stem cell markers and potential cells of origin in the stomach. Two types of stem cells, homeostatic stem cells and reserve stem cells, have been identified in corpus gastric units,

Brendan J. Jenkins (ed.), Inflammation and Cancer: Methods and Protocols, Methods in Molecular Biology, vol. 2691, https://doi.org/10.1007/978-1-0716-3331-1_1, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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Fig. 1 Histology of corpus of the mouse stomach. The corpus epithelium of composed of gastric units. Gastric units can be divided into four regions, pit, isthmus, neck, and base. Mucin-secreting pit cells reside in the pit. Homeostatic isthmus stem cells are located in the isthmus. The neck contains two types of cells, acid-secreting parietal cells and mucus-secreting mucus-neck cells. Chief cells reside in the base

which are the epithelial structures that line the stomach [1– 6]. Stomach gastric units are primarily divided into pit, isthmus, neck, and base; of note, homeostatic stem cells reside in the isthmus of gastric units (Fig. 1) [7–11]. The isthmus stem cells are highly proliferative—as shown by their expression of proliferation marker Ki67—and are necessary for maintenance of the stomach epithelium under homeostatic (normal) conditions [1–3]. Reserve stem cells are a subpopulation of terminally differentiated chief cells located in the base of the gastric units and are marked by the expression of Wnt-target gene and stem cell marker Lgr5, as well as chief cell markers such as Pgc and Gif [5, 6, 12]. Interestingly, while reserve stem cells are dormant under homeostatic conditions, the cells acquire stem cell activities to regenerate gastric units after tissue injury [5, 6, 13, 14]. Although the identification of several stomach stem cell markers has considerably contributed to our understanding of the cellular hierarchy in the stomach, the distinct molecular mechanisms underlying the regulation of homeostatic and reserve stem cells are not fully understood. We recently identified cytoskeletal scaffold protein IQ motif containing GTPase activating protein 3 (Iqgap3) as a stem cell marker, which is expressed specifically in the Ki67+ isthmus cells of gastric units in mice [3]. Lineage tracing experiments using the Iqgap32A-CreERT2;Rosa26-LSL-tdTomato mouse model uncovered stem cell activities of Iqgap3-expressing isthmus cells. In this mouse model, Iqgap3-expressing cells in the isthmus maintain whole gastric units for at least 12 months under homeostatic conditions [3]. Furthermore, in vitro culture of Iqgap3-expressing cells generated gastric organoids, suggesting self-renewal and

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differentiation of Iqgap3-expressing cells [3]. On the other hand, damage induction by using high-dose tamoxifen to stomach tissue demonstrated the induction of Iqgap3 expression in Lgr5+ reserve stem cells [3]. In addition, lineage tracing with damage induction showed that regenerated epithelial cells were derived from Iqgap3expressing cells, suggesting the contribution of Iqgap3+/Lgr5+ cells to tissue repair [3]. Iqgap3 is a member of the Iqgap protein family, which interacts with components of major oncogenic signaling pathways to promote signal transduction [15, 16]. Of direct relevance to proliferation and cancer is the interaction of Iqgap protein with the Ras-Erk pathway, particularly the active form of Ras (e.g., KrasG12D), Erk1/ 2, Mek1/2, Raf, Egfr, Rac, and Cdc42 [15, 16]. The lqgap protein also interacts with key Wnt pathway components β-catenin and Apc [15, 17]. These protein interactions might contribute to proliferative capacity and stem cell potential. Gene expression analysis of human clinical samples revealed elevated IQGAP3 expression in gastric cancer, compared to normal stomach [3]. Notably, the depletion of IQGAP3 expression in less differentiated cancer cell line HGC-27 resulted in the downregulation of key stem cell factors, including NANOG/OCT4/SOX2, and the upregulation of stomach differentiation markers pepsinogen C (PGC) [3]. These findings indicated that Iqgap3 might potentially regulate cancer stem cell activities. Lineage tracing is commonly used for adult tissue stem cell studies and to identify cells of origin. Lineage tracing provides stagewise in vivo visualization of stem cell behavior, such as selfrenewal and differentiation [18]. In this chapter, we describe how we use lineage tracing experiments to investigate the stem cell activity in Iqgap3-expressing cells in the mouse stomach. To conduct lineage tracing, two types of mouse models, stem cell markerspecific CreERT2 mouse model and Rosa26-LSL-tdTomato, are required. Using stem cell marker Iqgap3, we generated the Iqgap3-2A-CreERT2;Rosa26-LSL-tdTomato mouse model (Fig. 2). In this mouse model, CreERT2 is selectively expressed in Iqgap3+ cells. Low-dose tamoxifen treatment activates CreERT2 to remove the Lox-Stop-Lox (LSL) DNA sequence from the Rosa26 locus to induce tdTomato expression permanently. At one-day post tamoxifen treatment of Iqgap3-2A-CreERT2; Rosa26-LSL-tdTomato mouse model, Iqgap3-expressing cells in the isthmus were labeled by a fluorescent protein tdTomato (Figs. 3, 4, and 5) [3]. At 12 months post tamoxifen treatment, whole gastric units were labeled by tdTomato, indicating that tdTomato-/Iqgap3-expressing cells possess stem cell activities and maintained gastric units for at least 12 months (Figs. 3, 4, and 5) [3]. If Iqgap3 were terminally differentiated cells, limited numbers of tdTomato labeled cells would be observed 12 months post tamoxifen treatment (Figs. 4 and 5).

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Fig. 2 Genetic construct of mouse models. (a) In the Iqgap3-2A-CreERT2 mouse, 2A-CreERT2 was inserted in exon 38 of the Iqgap3 gene. (b) In the Rosa26-LSLtdTomato mouse, LSL-tdTomato was inserted in the Rosa26 locus. (c) In the LSL-KrasG12D mouse, G12D mutation is introduced in the Kras gene and LSL is placed upstream of the mutation

Fig. 3 Schematic showing experimental procedure of Iqgap3 lineage tracing and induction of KrasG12D in the isthmus. (a) For lineage tracing, the mice are treated with tamoxifen and analyzed at 1 day, 6 months, and 12 months postinjection (p.i.). (b) For induction of KrasG12D, the mice are treated with tamoxifen and analyzed at 1 month postinjection

KRAS is frequently amplified or mutated in human gastric cancer [19]. Previous mouse studies have shown that the expression of an active form of Kras in homeostatic or reserve stem cells induced metaplasia in stomach epithelium [1, 6, 12, 20, 21]. Mutated Kras might therefore represent a major first “hit” oncogenic lesion in stem cells and may be used to recapitulate the contribution of stem cells in early gastric carcinogenesis. In this chapter, we also describe how the expression of KrasG12D induction in Iqgap3-expressing cells by using the Iqgap3-2A-CreERT2;LSLKrasG12D mouse influences stem cell behavior in early carcinogenesis (Fig. 2). Indeed, rapid development of metaplasia was observed in the stomach at 1-month post tamoxifen treatment (Figs. 3 and 6) [3].

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Fig. 4 Determination of stem cell activity in the stomach using lineage tracing. One day after tamoxifen treatment, CreERT2-expressing cells are labeled by fluorescent protein tdTomato. If the cells possess stem cell activity, they will continuously produce daughter cells for a long term (12 months), and whole gastric units are labeled by tdTomato. If the cells are differentiated cells, the gastric units will show limited tdTomato expression in certain cells

Fig. 5 Expression of tdTomato (tdTom), E-cadherin (E-cad), and Ki67 on corpus gastric units. At 1 day post tamoxifen injection (p.i.), tdTomato expression was observed in Ki67+ isthmus cells. tdTomato-expressing cells were expanded at 6 months after tamoxifen treatment. At 12 months post tamoxifen treatment, almost whole gastric units were labeled by tdTomato. Scale bar = 100 μM

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Fig. 6 Hematoxylin-eosin staining of the stomach from a representative Iqgap32A-CreERT2;LSL-KrasG12D mouse. Metaplastic epithelial structures were observed 1 month post tamoxifen treatment (see Fig. 1 for normal stomach)

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Materials All reagents should be prepared with Milli-Q water.

2.1

Mouse Model

All mice should be handled in strict accordance with good animal practice as defined by the appropriate Institutional Animal Care Use Committee. 1. Iqgap3-2A-CreERT2 mouse strain: This strain was newly generated by the National University of Singapore and Cyagen (Santa Clara, CA) (Fig. 2) [3]. 2. Rosa26-lsl-tdTomato strain: This strain (B6.Cg-Gt(ROSA) 26Sortm14(CAG-tdTomato)Hze/J) was obtained from The Jackson Laboratory (Bar Harbor, ME) (Fig. 2). 3. LSL-KrasG12D strain: The strain (B6.129S4-Krastm4Tyj/J) was obtained from The Jackson Laboratory (Bar Harbor, ME) (Fig. 2).

2.2 Animal Treatment

1. 10 mg/mL tamoxifen: Dissolve 100 mg of tamoxifen in 1 mL of 100% ethanol at 30 °C. Add 9 mL of sunflower oil (autoclaved) into tamoxifen in ethanol solution. Make aliquots and store at -20 °C (see Note 1). 2. Syringe (1 mL) and 26-gauge needle (13 mM) (see Note 2).

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2.3 Formalin-Fixed Paraffin Embedded (FFPE) Mouse Tissue

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1. Tissue wash buffer: 10% FBS in PBS. Dissolve 10% of fetal bovine serum (FBS) in phosphate-buffered saline (PBS: 137 mM sodium chloride [NaCl], 2.7 mM potassium chloride [KCl], 8 mM sodium phosphate dibasic [Na2HPO4], 2 mM potassium phosphate monobasic [KH2PO4]). Prepare PBS by dilution of 10× PBS in Milli-Q water. 2. Fixative: 4% paraformaldehyde (PFA) in PBS. Dissolve 4 g of PFA in 80 mL of Milli-Q water with 25 μL of 10 N sodium hydroxide (NaOH) at 80 °C. Add 10 mL of 10× PBS in PFA solution. Add Milli-Q water into PFA in PBS to make volume of 100 mL (see Note 3). 3. 50%, 70%, 80%, 100% of ethanol: Prepare 50%, 70%, 80% of ethanol by using 100% of ethanol and Milli-Q water. 4. Butanol/ethanol: Add 100% butanol to 100% ethanol to obtain the butanol-ethanol ratio of 1:4, 2:3, 3:2, and 4:1. 5. Paraffin: Place paraffin in 60 °C oven (see Note 4). 6. Microtome. 7. Adhesive glass slide. 8. Forceps and scissors. 9. 50-mL tube. 10. 6-well culture plate.

2.4

Antibodies

1. Rabbit anti-RFP antibody (PM005, MBL, 1:500) (see Note 5). 2. Goat anti-RFP antibody (MBS448122, MyBioSource, 1:500) (see Note 5). 3. Rat anti-Ki67 antibody (14–5698-82, Thermo Fisher Scientific, 1:2000). 4. Mouse anti-E-cadherin Alexa647-conjugated (560,062, BD Bioscience, 1:200).

antibody

5. Goat anti-rabbit IgG Alexa546-conjugated antibody (A11035, Thermo Fisher Scientific, 1:200) (see Note 5). 6. Donkey anti-goat IgG Alexa555-conjugated antibody (A31572, Thermo Fisher Scientific, 1:200) (see Note 5). 7. Donkey anti-rat IgG Alexa488-conjugated antibody (A21208, Thermo Fisher Scientific, 1:200). 2.5 Immunofluorescence Staining

1. Antigen retrieval solution: DAKO target retrieval solution 10× concentrate (pH 6) (see Note 6). 2. Wash buffer: PBST, 0.1% of Tween 20 was added in PBS (see Note 7). 3. Blocking reagent: DAKO Protein Block Serum-Free (see Note 8).

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4. 1 mg/mL DAPI. 5. Antifade mounting media (see Note 9). 6. Coverslip: Size is 24 by 60 mM (#1,5). 7. Autoclave. 8. Coplin jar (staining jar). 9. Pap Pen. 2.6 HematoxylinEosin Staining

1. Mayer’s Hematoxylin (Lillie’s Modification). 2. Eosin. 3. 0.001% of ammonium hydroxide in Milli-Q water. 4. Eukitt quick-hardening mounting medium.

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Microscope

1. Confocal laser scanning microscope (e.g., Zeiss LSM880 Airyscan). 2. Fluorescence microscope (e.g., Zeiss Axioplan2).

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Methods

3.1 Mice and Treatment

1. Breed stem cell marker-specific CreERT2 strain Iqgap3-2ACreERT2 mice with Rosa26-LSL-tdTomato mice to generate Iqgap3-2A-CreERT2;Rosa26-LSL-tdTomato (Iqgap3CreERT2;Rosa-tdTom) strain (see Note 10). 2. To conduct lineage tracing experiment, perform intraperitoneal injection of 2 mg/20 g low dose of tamoxifen into 7-week-old Iqgap3-CreERT2;Rosa-tdTom mice. Analyze mice at 1 day, 3 months, 6 months, and 12 months posttreatment (Fig. 3) (see Note 11). 3. To investigate the contribution of Iqgap3-expressing cells during early carcinogenesis, breed Iqgap3-2A-CreERT2 with LSLKrasG12D mice to generate the Iqgap3-2A-CreERT2; LSL-KrasG12D (Iqgap3-CreERT2;KrasG12D) strain (see Note 12). 4. Perform intraperitoneal injection of 2 mg/20 g of tamoxifen into 7-week-old mice, and analyze at 1-month posttreatment (see Note 13).

3.2 Formalin-Fixed Paraffin Embedded (FFPE) Stomach at Room Temperature

1. Resect the stomach from mice and cut along the greater curvature by scissors. 2. Wash the tissues three times with ice-cold 10% FBS in PBS and paste on the silicon board (Fig. 7).

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Fig. 7 Schematic diagram showing the procedure of tissue fixation for mouse stomach. After cutting the greater curvature of the stomach, the stomach is placed and pinned on the silicon board. The stomach pasted on a silicon board is fixed with 4% PFA/PBS

3. Fix the washed tissues with 4% PFA in PBS in 50-mL tubes at 4 °C for 18–24 h (Fig. 7). 4. Incubate the fixed tissues with 50% and 70% of ethanol at 4 °C for 1 h in 50-mL tubes, and then incubate tissues with 80% of ethanol for 18–24 h (see Note 14). 5. Incubate the samples with 100% ethanol in 50-mL tubes at 4 ° C for 1 h, and then incubate with 100% ethanol at room temperature for 1 h (see Note 15). 6. Incubate the dehydrated samples with 1:4, 2:3, 3:2, and 4:1 of butanol/ethanol solutions in 50-mL tubes at room temperature for 15 min, and then incubate with 100% butanol twice at room temperature for 15 min (see Note 16). 7. Incubate the samples with 1:1 of butanol/paraffin mixture in 6-well plates for 1 h at 60 °C (see Note 17). 8. Incubate the samples with 100% paraffin in 6-well plates for 1 h, and later incubate with 100% paraffin for 2 h, at 60 °C. 9. Embed the samples in paraffin (FFPE sample). 10. Slice FFPE samples at 5-μM thickness using a microtome. 11. Add 37–40 °C warmed water onto adhesive glass slides, and then place the sliced tissues on top of the warmed water (Fig. 8) (see Note 18). 12. Remove water from the glass slides, and dry the slides completely in a 37 °C oven. 3.3 Immunofluorescence (IF) Staining and Visualization

1. Deparaffinize sliced tissues on the glass slides by incubation in xylene, ethanol, and Milli-Q water at room temperature for 3 min. 2. Perform antigen retrieval in DAKO target retrieval solution at 100 °C for 20 min in an autoclave, and cool samples at room temperature for 20 min.

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Fig. 8 Schematic showing the procedure of FFPE slide preparation. (a) Glass slides are covered by 37 °C warmed Milli-Q water. (b) Sliced FFPE samples are placed on the water. (c) Remove Milli-Q water from slides and completely dry the slide

3. Wash the slides three times with wash buffer (PBST) in a Coplin jar. 4. Draw a liquid barrier between the paraffin tissue sample and glass slide label with a Pap Pen (see Note 19). 5. Block the slides with DAKO Protein Block Serum-Free at room temperature for 30 min (see Note 20). 6. Dilute rat anti-Ki67 antibody and mouse anti-E-cadherin Alexa647-conjugated antibody in blocking reagent at 1:2000 and 1:200, respectively. Then add rabbit anti-RFP antibody or goat anti-RFP antibody at 1:500 dilution (see Note 21). 7. Incubate the slides with primary antibody mixture at 4 °C for 18–24 h (see Note 22). 8. Wash the slides three times with PBST. 9. Dilute donkey anti-rat IgG Alexa488-conjugated antibody in 5% skim milk in PBST at 1:200 dilution. Then add goat antirabbit IgG Alexa546-conjugated antibody or donkey anti-goat IgG Alexa555-conjugated antibody at 1:200 dilution (see Note 23). 10. Incubate the slides with secondary antibodies at room temperature for 1 hour (see Note 24). 11. Wash the slides three times with PBST. Add 10 μL of 1 mg/mL DAPI in the first wash (see Note 25). 12. Drop antifade mounting media on the slides, and place coverslips on the slides. 13. Visualize stained tissues on the slides by a confocal laser microscope with 405 nM, 488 nM, 561 nM, and 633 nM laser (Fig. 5) (see Note 26). 3.4 HematoxylinEosin (HE) Staining and Visualization

1. Deparaffinize sliced tissues on glass slides by incubation in xylene, ethanol, and Milli-Q water. 2. Stain the sliced tissues with hematoxylin for 1 min, and wash three times with Milli-Q water at room temperature (see Note 27).

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3. Incubate the sliced tissues with 0.001% ammonium hydroxide for 1 min, and wash three times with Milli-Q water (see Note 28). 4. Stain the sliced tissues with eosin for 1 min, and wash three times with Milli-Q water (see Note 27). 5. Dehydrate the sliced tissues by incubation in ethanol, and then incubate in xylene. 6. Drop mounting media on the slides, and place a coverslip on each slide. 7. Visualize stained tissues by fluorescence microscopy (Fig. 6).

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Notes 1. It is necessary to dissolve tamoxifen in 30 °C pre-warmed ethanol before adding sunflower oil; tamoxifen does not dissolve easily in sunflower oil. 2. Two different lengths, 25 mM and 13 mM, are available for the 26-gauge needle. A shorter needle eases the injection of tamoxifen in mice. Moreover, although a higher needle gauge reduces stress on mice, a 26-gauge needle is recommended due to the viscosity of sunflower oil. 3. Adding 10 N NaOH to Milli-Q water helps to dissolve PFA. After dissolved PFA, 10× PBS is added to make the pH neutral. Alternatively, neutralized buffered formalin can be used for fixation instead of PFA. 4. Paraffin solidifies at room temperature and liquid paraffin is required to process and embed tissue. To melt paraffin, solid paraffin is placed in a 60 °C oven overnight. 5. Two types of antibodies, rabbit anti-RFP antibody (PM005) and goat anti-RFP antibody (MBS448122), can be used for tdTomato staining. Secondary antibody for rabbit anti-RFP antibody is goat anti-rabbit IgG Alexa546-conjugated antibody (A11035). Secondary antibody for goat anti-RFP antibody (MBS448122) is donkey anti-goat IgG Alexa555conjugated antibody (A31572). 6. Alternatively, DAKO target retrieval solution pH 9 (10×) or sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) can be used. In order to prepare sodium citrate buffer, trisodium citrate (dehydrate) is dissolved in Milli-Q water. After adjusting pH to 6.0 with HCl, Tween 20 is added in the buffer. The choice of antigen retrieval solution depends on the choice of antibody. Some antibodies work with all antigen retrieval solutions, but some only work with DAKO target retrieval solution pH 9.0.

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7. Alternatively, TBST (140 mM NaCl, 2.7 mM KCl, 25 mM Tris, 0.1% Tween 20, pH 7.5) can be used as wash buffer instead of PBST. 8. DAKO Protein Block Serum-Free was used in this chapter. Five percent skim milk in PBST, 3% bovine serum albumin (BSA) in PBST, or 2% normal donkey serum in PBST can be used for blocking instead of DAKO protein block. The choice of blocking reagent is highly dependent on the choice of antibody. 9. Confocal laser microscope induces photobleaching to immunofluorescent stained samples due to the high energy level of the laser. Antifade mounting media helps to prevent photobleaching. 10. Iqgap3-2A-CreERT2 mice express CreERT2 in Iqgap3expressing cells, while leaving Iqgap3 expression intact [3]. Tamoxifen treatment activates CreERT2 specifically in Iqgap3-expressing cells. In the Rosa26-LSL-tdTomato mouse strain, the lox-stop-lox (LSL) DNA sequence inhibits tdTomato expression. Thus, tamoxifen treatment in the Iqgap3-2ACreERT2;Rosa26-LSL-tdTomato mouse induces CreERT2 activity in Iqgap3-expressing cells, and this active CreERT2 removes LSL sequence to induce tdTomato expression. 11. Previous studies demonstrated that tamoxifen treatment damages the stomach epithelium, particularly in parietal cells [22–24]. In order to minimize tissue damage, a low dosage of tamoxifen treatment is required, and 0.5–1 mg/20 g dosage is recommended. By contrast, 5 mg/20 g high-dose tamoxifen treatment has been used for tissue damage repair studies. High-dose tamoxifen treatment concurrently induces tissue damage and CreERT2 activation. Lineage tracing studies through monitoring tdTomato expression are used to determine the nature and behavior of marker-derived CreERT2expressing cells. If the CreERT2-expressing cells were fully differentiated, localized and limited numbers of tdTomatoexpressing cells are observed. In the case of the CreERT2expressing cells possessing stem cell properties, the whole gastric unit is labeled by tdTomato at 1-year post tamoxifen treatment. 12. KrasG12D is a constitutively active form of Kras, and this mutation has been frequently detected in various cancers [25]. In the LSL-KrasG12D mouse strain, the LSL sequence inhibits KrasG12D expression. Tamoxifen administration to Iqgap32A-CreERT2; LSL-KrasG12D mouse induces expression of KrasG12D in Iqgap3-expressing cells. 13. KrasG12D expression in stomach stem cells, including homeostatic stem cells and reserve stem cells, induces metaplasia which represents the early step of gastric carcinogenesis [1, 6,

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12, 20, 21]. In Iqgap3-2A-CreERT2; LSL-KrasG12D mice, a Ushape-like structure of metaplasia is expected at 1–2 months post tamoxifen treatment [3]. 14. This ethanol incubation series is required to gradually remove water from the tissues. Prolonged incubation of tissues with 80% ethanol promotes effective dehydration. The tissues can be stored in 4 °C of 80% ethanol for a maximum of 1 year. 15. This step helps to remove water in tissue completely. 16. Compared to ethanol, an organic compound, butanol, has better compatibility with paraffin. This step helps to replace ethanol in tissues with butanol. 17. This step helps to replace butanol in tissues with paraffin. To maintain paraffin in its liquid state, this procedure is required to be performed at 60 °C. We perform this step inside a 60 ° C oven. 18. Sliced paraffin tissue unfolds and spreads on 37–40 °C warm water. If the temperature exceeds 40 °C, the tissue disperses in the water. We placed none sliced tissues on a glass slide. 19. A liquid barrier separating the paraffin tissue sample and label is required to prevent the backflow and contain any reagents used in tissue staining. 20. The slides were rocked at least ten times to agitate the blocking reagent. This rocking helps uniform staining. 21. Concentration of antibodies used is determined by optimization. RFP, Ki67, and E-cadherin antibodies used in this chapter can also be used with another blocking reagent, including 5% skim-milk in PBST and 3% BSA in PBST. 22. The slides were rocked at least ten times to agitate the primary antibody solution. This rocking helps uniform staining. 23. Five percent skim milk in PBST is used for all secondary antibody diluents. The concentration of secondary antibodies is fixed at 1:200. 24. The slides were rocked at least ten times to agitate the secondary antibody solution. This rocking helps uniform staining. 25. DAPI integrates into DNA and helps to visualize the nuclei in cells. 26. Instead of the confocal laser microscope, the fluorescent microscope can be used to visualize immunofluorescence staining. However, using the confocal laser microscope is highly recommended to obtain an enhanced high-resolution image. 27. Hematoxylin stains the nucleus while Eosin stains the cytoplasm of the cell. 28. Ammonium hydroxide enhances the blue color of hematoxylin staining.

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Acknowledgements This work is supported by the National Research Foundation Singapore and the Singapore Ministry of Education under its Research Centre of Excellence initiative. The work is also supported by the Singapore Ministry of Health’s National Medical Research Council’s Open Fund Large Collaborative Grant (OFLCG18May0023), the Singapore Ministry of Health’s National Medical Research Council under its Clinician-Scientist Individual Research Grant (MOH-CIRG21jun-0003), and the National University of Singapore School of Medicine (NUSMed) Internal Grant Funding (NUHSRO/2019/086/StomachStemCell and NUHSRO/ 2022/043/NUSMed/25/LOA). References 1. Matsuo J, Kimura S, Yamamura A et al (2017). Identification of stem cells in the epithelium of the stomach corpus and antrum of mice. Gastroenterology 152:218–231. e14 2. Han S, Fink J, Jorg DJ et al (2019). Defining the identity and dynamics of adult gastric isthmus stem cells. Cell Stem Cell 25:342–356. e7 3. Matsuo J, Douchi D, Myint K et al (2021) Iqgap3-Ras axis drives stem cell proliferation in the stomach corpus during homoeostasis and repair. Gut 70:1833–1846 4. Hata M, Kinoshita H, Hayakawa Y et al (2020). GPR30-expressing gastric chief cells do not dedifferentiate but are eliminated via PDK-dependent cell competition during development of metaplasia. Gastroenterology 158:1650–1666. e15 5. Stange DE, Koo BK, Huch M et al (2013) Differentiated Troy+ chief cells act as reserve stem cells to generate all lineages of the stomach epithelium. Cell 155:357–368 6. Leushacke M, Tan SH, Wong A et al (2017) Lgr5-expressing chief cells drive epithelial regeneration and cancer in the oxyntic stomach. Nat Cell Biol 19:774–786 7. Karam SM, Leblond CP (1993) Dynamics of epithelial cells in the corpus of the mouse stomach. I. Anat Rec 236:259–279 8. Karam SM, Leblond CP (1993) Dynamics of epithelial cells in the corpus of the mouse stomach. II. Anat Rec 236:280–296 9. Karam SM, Leblond CP (1993) Dynamics of epithelial cells in the corpus of the mouse stomach. III. Ten Anat Recd 236:297–313 10. Karam SM, Leblond CP (1993) Dynamics of epithelial cells in the corpus of the mouse stomach. IV. Anat Rec 236:314–332

11. Karam SM, Leblond CP (1993) Dynamics of epithelial cells in the corpus of the mouse stomach. V. Anat Rec 236:333–340 12. Douchi D, Yamamura A, Matsuo J et al (2021) Induction of gastric cancer by successive oncogenic activation in the corpus. Gastroenterology 161:1907–1923 13. Miao ZF, Adkins-Threats M, Burclaff JR et al (2020). A metformin-responsive metabolic pathway controls distinct steps in gastric progenitor fate decisions and maturation. Cell Stem Cell 26:910–925. e6 14. Miao ZF, Lewis MA, Cho CJ et al (2020). A dedicated evolutionarily conserved molecular network licenses differentiated cells to return to the cell cycle. Dev Cell 55:178–194. e7 15. Hedman AC, Smith JM, Sacks DB (2015) The biology of IQGAP proteins: beyond the cytoskeleton. EMBO Rep 16:427–446 16. Nojima H, Adachi M, Matsui T et al (2008) IQGAP3 regulates cell proliferation through the Ras/ERK signalling cascade. Nat Cell Biol 10:971–978 17. Briggs MW, Sacks DB (2003) IQGAP proteins are integral components of cytoskeletal regulation. EMBO Rep 4:571–574 18. Hsu YC (2015) Theory and practice of lineage tracing. Stem Cells 33:3197–3204 19. Deng N, Goh LK, Wang H et al (2012) A comprehensive survey of genomic alterations in gastric cancer reveals systematic patterns of molecular exclusivity and co-occurrence among distinct therapeutic targets. Gut 61: 673–684 20. Choi E, Hendley AM, Bailey JM et al (2016). Expression of activated Ras in gastric chief cells of mice leads to the full Spectrum of

Lineage Tracing of Stomach Stem Cells metaplastic lineage transitions. Gastroenterology 150:918–930. e13 21. Min J, Vega PN, Engevik AC et al (2019) Heterogeneity and dynamics of active Krasinduced dysplastic lineages from mouse corpus stomach. Nat Commun 10:5549 22. Huh WJ, Khurana SS, Geahlen JH et al (2012). Tamoxifen induces rapid, reversible atrophy, and metaplasia in mouse stomach. Gastroenterology 142:21–24. e7 23. Khurana SS, Riehl TE, Moore BD et al (2013) The hyaluronic acid receptor CD44

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coordinates normal and metaplastic gastric epithelial progenitor cell proliferation. J Biol Chem 288:16085–16097 24. Manning EH, Lapierre LA, Mills JC et al (2020). Tamoxifen acts as a parietal cell protonophore. Cell Mol Gastroenterol Hepatol 10: 655–657. e1 25. Huang L, Guo Z, Wang F et al (2021) KRAS mutation: from undruggable to druggable in cancer. Signal Transduct Target Ther 6:386

Chapter 2 In Vitro and In Vivo Models for Metastatic Intestinal Tumors Using Genotype-Defined Organoids Atsuya Morita, Mizuho Nakayama, Hiroko Oshima, and Masanobu Oshima Abstract It has been established that the accumulation of driver gene mutations causes malignant progression of colorectal cancer (CRC) through positive selection and clonal expansion, similar to Darwin’s evolution. Following this multistep tumorigenesis concept, we previously showed the specific mutation patterns for each process of malignant progression, including submucosal invasion, epithelial mesenchymal transition (EMT), intravasation, and metastasis, using genetically engineered mouse and organoid models. However, we also found that certain populations of cancer-derived organoid cells lost malignant characteristics of metastatic ability, although driver mutations were not impaired, and such subpopulations were eliminated from the tumor tissues by negative selection. These organoid model studies have contributed to our understanding of the cancer evolution mechanism. We herein report the in vitro and in vivo experimental protocols to investigate the survival, growth, and metastatic ability of intestinal tumor-derived organoids. The model system will be useful for basic research as well as the development of clinical strategies. Key words Colon cancer, Organoids, Collagen gel, Metastasis, Imaging

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Introduction The accumulation of driver gene mutations causes malignant progression of cancer, a well-known concept of multistep tumorigenesis [1, 2]. This model is based on the positive selection of tumor cells that acquire growth advantages, similar to Darwin’s evolution theory. We previously established genetically engineered mouse models that carried driver gene mutations in various combinations and examined the genotype-phenotype relationships [3–5]. Notably, intestinal tumor-derived organoids carrying quadruple mutations in ApcΔ716, KrasG12D, Tgfbr2–/–, and Trp53R270H, hereafter “AKTP organoids,” showed a metastatic ability with a high incidence after loss of wild-type Trp53, and the loss of heterogeneity (LOH) cells achieved dominance in the liver metastatic foci through positive selection [6].

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We previously examined the cancer evolution mechanism based on the belief that cancer cells always progress toward a malignant state. However, controversy remains concerning the reverse evolution of cancer cells, namely, the loss of malignant characteristics and elimination of these cells from tumors by negative selection [7– 9]. We therefore established several subclones from metastatic AKTP organoid cells after long passage. Although cell proliferation was not altered under standard two-dimensional (2D) culture conditions, some subclones showed a significantly suppressed survival and growth in collagen gel culture conditions. Furthermore, these subclones showed complete loss of their metastatic ability, as confirmed by in vivo imaging [10]. These in vitro and in vivo analyses using genetically defined organoids can aid in the analysis of the survival, stemness, proliferation, and metastatic ability of cancer cells. We herein report in vitro and in vivo experimental methods for evaluating the stemness as well as the metastatic ability of (colorectal) cancer cells using AKTP and AKTP-derived subcloned organoids.

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2.1 Mouse Intestinal Tumor-Derived Organoids

1. AKTP organoids: Established from ApcΔ716 (A), KrasG12D (K), Tgfbr2 –/– (T), and Trp53R270H (P) quadruple mutant mouse intestinal tumors [5]. The wild-type Trp53 gene was lost by LOH [6]. AKTP organoids develop liver metastasis at a high incidence when transplanted to the spleen. 2. tdTomato- or Venus-labeled AKTP organoids: These lines can be used for in vitro growth assay as well as in vivo metastatic analyses. tdTomato and Venus can be detected directly by immunofluorescent microscopy or indirectly by immunohistochemistry using anti-green fluorescent protein (GFP) and antired fluorescent protein (RFP) antibody, respectively. tdTomato and Venus are expressed under transcriptional regulation of the CAG promoter [11]. 3. Luciferase-expressing AKTP organoids: These lines can be used for in vitro proliferation and in vivo imaging of liver metastatic foci. The luciferase gene is expressed under transcriptional regulation of the CAG promoter [10]. 4. These organoid lines can be maintained in either 2D or threedimensional (3D) culture conditions. All organoids will be provided as a collaboration effort upon request.

2.2 Standard Culture (2D)

1. CO2 incubator. 2. Cell culture treated 6-well plates and dishes. 3. 1.5-mL microtube.

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4. Phosphate-buffered saline (PBS), pH 7.4, and PBS-EDTA. 5. 0.25% trypsin-EDTA (Gibco). 6. Cell freezing media (e.g., Bambanker, Nippon Genetics) 7. 1.8 mL cryotube. 8. 35 μm cell strainer with round-bottom polystyrene tube. 2.3 Collagen Gel Culture (2D or 3D)

1. Auto cell counter (e.g., Bio-Rad). 2. 96-well black/clear bottom plates for 3D collagen gel culture. 3. Cellmatrix Type I-A (collagen, Type I, 3 mg/mL, pH 3.0; Nitta Gelatin). 4. Reconstitution buffer (Nitta Gelatin): 50 mM sodium hydroxide, 260 mM sodium bicarbonate, 200 mM HEPES. 5. Concentrated culture solution, DF culture solution (Nitta Gelatin). 6. Collagenase, type I. 7. 15 mL conical tube. 8. Fluorescence inverted microscope.

2.4

Culture Medium

1. Standard culture medium: Advanced Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) medium (Gibco), 10% fetal bovine serum (FBS), supplemented with 5 μM A83-01 (Sigma), 5 μM CHIR99201 (Tocris Bioscience), and 10 μM Y27632 (Wako) (see Note 1). 2. Collagen gel culture medium (2D or 3D): Advanced DMEM/ F-12 medium, 10 mM HEPES (Gibco), supplemented with 2 mM Glutamax, 1× B27, 1× N2 (Gibco), 100 ng/mL recombinant murine Noggin (Peprotech), and 1 μM N-acetylcysteine (Sigma) (see Note 2).

2.5 Spleen Transplantation for Liver Metastasis Model

1. 27-gauge needle syringe. 2. Immunodeficient mice (Crl:SHO-PrkdcscidHrhr (SCID hairless outbred, SHO) or immunocompetent mice (C57BL/6) (Charles River) (see Note 3). 3. Isoflurane. 4. Inhalation anesthesia device and induction chamber (e.g., NARCOBIT-E; Natsume Seisakusho, Japan). 5. Hair clippers for small animals. 6. Hair depilatory cream. 7. 70% ethanol for disinfection. 8. Surgical instruments (forceps, scissors, sutures, clips).

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2.6 Bioimaging of Tumor Growth by Luciferase Activity

1. 15 mg/mL D-luciferin potassium solution dissolved in PBS. 2. IVIS Lumina LT in vivo imaging system (Perkin Elmer) (see Note 4) 3. Living image software program (Perkin Elmer). 4. Isoflurane anesthesia device (e.g., MK-AT210; Muromachi Kikai, Japan).

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Methods

3.1 AKTP Organoid Standard Culture on 2D Culture Plates (Fig. 1)

1. Wash the AKTP 3D organoids or semi-confluent cultured organoid cells in a 6-well plate with PBS-EDTA, and treat the cells with 100 μL of 0.25% trypsin-EDTA for 5–10 min at 37 ° C. 2. Add 1 mL of Advanced DMEM/F-12 with 10% FBS to stop the trypsin reaction, and suspend the dissociated AKTP cells by pipetting. 3. Spin down the cells in a 1.5-mL tube at 300 × g for 5 min, suspend the cells in 2 mL of standard culture medium, and seed them onto a new 6-well plate. 4. Culture the AKTP cells at 37 °C in a 5% CO2 incubator. 5. Passage cells on reaching 70–80% confluence (repeat Subheading 3.1, steps 1–4). 6. To make AKTP cell freeze stocks, suspend the cell pellets in Bambanker cell freezing media (Subheading 3.1, step 3), and transfer to a cryotube (Fig. 1). Store the tubes at -80 °C in a deep freezer overnight, and then move them to liquid nitrogen for long-term storage.

Fig. 1 Schematic drawing of AKTP organoid standard culture on 6-well plates with preparation of frozen stocks and subcloning

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7. To establish AKTP-derived subclones, dissociate AKTP organoid cells to single cells by trypsinization and a cell strainer. Plate single cells onto a 96-well plate at 1 cell/well (Fig. 1; see Note 5). After confirmation of single colony formation in each 96-well plate, expand cells by passaging to establish subclones. 3.2 Organoid Growth Analyses in 3D collagen Gel (See Note 6)

1. Prepare collagen gel by mixing Cellmatrix Type I-A, concentrated culture solution DF culture solution, and reconstitution buffer at an 8:1:1 volume ratio according to the manufacturer’s instructions. 2. Prepare the dissociated parental AKTP cells or luciferaseexpressing AKTP-derived subcloned cells by trypsinization (Subheading 3.1, steps 1–3). 3. Count the number of cells with an auto cell counter, and then suspend them in ice-cold collagen gel at 2 × 102 cells/20 μL. 4. Fill 96-well black/clear bottom plates with ice-cold collagen gel (see Note 7) as the bottom gel (70 μL/well), and then pour 20 μL of cell suspension onto the bottom gel (Fig. 2a).

Fig. 2 Organoid culture experiment in 3D collagen gel. (a) Schematic drawing of AKTP organoid culture in collagen gel. (b, c) Example data showing cell proliferation of stemness-high and stemness-low subclones examined by the luciferase activity (b) and the size and number of organoids (c). Bars in the photographs, 1 mm

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5. Leave the plate at 37 °C to polymerize the collagen gel, and then add 100 μL of warm collagen gel culture medium to each well. 6. When analyzing luciferase-expressing AKTP cells, the proliferation rate can be determined by calculating the luciferase activity relative to the initial luciferase measurement (2 × 102 cells at day 0). Add D-luciferin/PBS to each well at 150 μg/mL (see Note 8), and then analyze luciferase activity using IVIS Lumina LT and the Living Image software program. An example of cell proliferation assay data for stemness-high and stemness-low cells by measuring the relative luciferase activity is provided [10] (Fig. 2b). 7. To examine the size and numbers of developed organoids in collagen gels, take bright-field microscopic photographs under a dissection microscope and measure the values in the photographs. An example of an organoid growth assay of stemnesshigh and stemness-low cells is provided [10] (Fig. 2c). 3.3 Competition Coculture Analysis on 2D Collagen Gel (See Notes 9 and 10)

1. Fill a 6-well plate with 1 mL of ice-cold collagen gel (Subheading 3.2, step 1), and leave it at 37 °C to polymerize as the bottom gel. 2. Prepare trypsin-dissociated Venus-labeled AKTP-derived subcloned cells and tdTomato-labeled parental AKTP cells from organoids or a 2D standard culture plate (Subheading 3.1, steps 1–3, 7). 3. Spin down cells in the 1.5-mL tube at 300 × g for 5 min, suspend the cells in collagen culture medium, and count the number of cells with an auto cell counter. 4. Mix Venus-labeled AKTP-derived subcloned cells and tdTomato-labeled parental AKTP cells at a 1:1 ratio (2 × 105 cells for each), and then seed the cell mixture onto the bottom gel (step 1). Add 2 mL of collagen gel culture medium to each 6-well plate, and culture the cells in an incubator at 37 °C (Fig. 3a). 5. When the cells reach semiconfluency at approximately 90–100%, examine the cell ratio by counting the Venus- and tdTomato-expressing cells using a fluorescent microscope (at least 3 fields/well) (see Note 11). Imaging examples of a competition co-culture analysis using stemness-low subclones (green) and parental cells (red) are provided [10] (Fig. 3b). 6. For passage of the cells cultured on a collagen gel, treat the cultured well with 0.1% collagenase type I/PBS at 37 °C for 30 min to digest the collagen gel. Wash the cells twice with 10 mL PBS in a 15-mL tube, and spin down the cells at 300 × g for 5 min.

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Fig. 3 Competition co-culture experiment. (a) Schematic drawing of the competition co-culture analysis of parental AKTP cells (red) and subcloned cells (green) on 2D collagen gel culture. (b) Representative photographs of co-culture of Venus-labeled stemness-low subclone cells and tdTomato-labeled parental AKTP cells. Note that the subcloned cells are gradually eliminated from culture. Bars in the photographs, 500 μm

7. Treat the cell pellets with Trypsin-EDTA at 37 °C for 5 min. 8. Add Advanced DMEM/F-12 with 10% FBS, and suspend cells by pipetting to stop the trypsin reaction. 9. Spin down dissociated cells in a 1.5-mL tube at 300 × g for 5 min, suspend the cells in collagen gel culture medium, seed the cells onto a new collagen bottom gel, and continue the competition co-culture analysis (Subheading 3.3, steps 4 and 5). 3.4 Liver Metastasis Model (See Note 12)

1. Prepare the dissociated AKTP organoid cells from 2D standard culture (Subheading 3.1, steps 1–3). 2. Count the number of cells with an automatic cell counter, and suspend cells in PBS at 5 × 105 cells/40 μL (see Note 13). 3. Leave the cell-containing tube on ice until transplantation (Fig. 4a). 4. Anesthetize immunodeficient SHO mice or isogenic C57BL/6 mice by isoflurane inhalation, and place the mice in the right lateral recumbent position (see Notes 3 and 14). 5. Scrub the surgical site of the skin (left lateral side) with 70% ethanol for disinfection, cut the skin and abdominal wall 100 organoids, mean ± SEM). Asterisks indicate pairs of means that are significantly different using unpaired t-test (*p < 0.05; **p < 0.01). Scale bars = 100 μm

6. Incubate the organoids with 100 μL of IF buffer blocking buffer overnight at 4 °C. 7. Incubate the organoids with 30 μL of primary antibodies (e.g., Fig. 4a, b; 1:200 of anti-Ki67 and 1:200 of anti-E-cadherin) diluted in 1% BSA v/v in PBS overnight at 4 °C. 8. Wash gently twice with 150 μL of IF buffer for 4 h each at room temperature and then overnight at 4 °C. 9. Incubate the organoids with 30 μL of secondary antibodies diluted in 1% BSA v/v in PBS overnight at 4 °C and protect from light. 10. Counterstain using 100 μL of 1 μg/mL DAPI (diluted in 1% BSA/PBS) for 30 min. 11. Wash gently thrice with 150 μL of PBS for 10 min.

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12. Add 200 μL of PBS to the well and keep the plate at 4 °C (protected from light) until ready for imaging. For long-term storage, store in 3 mM sodium azide in PBS. 13. Image the plate using an inverted fluorescence microscope. 3.7 Phenotypic Analysis of Organoids

In-depth characterization of organoids can be achieved by measuring organoid size and shape [17]. 1. Seed organoids as single cells or fragments in a 48-well plate (see Note 4). 2. After 5 days in culture, wash the organoids with PBS and fix in 200 μL 4% PFA per well for 30 min at room temperature, remove, and wash three times in PBS for 5 min. 3. Counterstain the organoids by adding 1 μg/mL DAPI (diluted in PBS) to each well. Wrap the plate in foil and incubate at room temperature for 1 h. Remove the DAPI and wash twice in PBS for 5 min. 4. Image the plate using the ImageXpress Pico Automated Cell Imaging System (see Fig. 4d–g) using the 4× objective. 5. Quantify organoid shape and size using the MetaXpress Analysis Software (see Fig. 4h, i). Perform automated image processing using the Custom Module Editor (CME) to define and segment organoids identified by DAPI nuclear staining (see Fig. 4f, g). Output parameters to include shape factor (circularity = 4π × area/perimeter2; see Fig. 4h) and size (average area in μm2; see Fig. 4i). Data demonstrate that colonic organoids are significantly more cystic and larger than small intestinal organoids reflecting morphological differences observed under the microscope.

4

Notes 1. Bottles of Matrigel should be stored at -20 °C to -80 °C for long-term storage. To make up smaller working stocks, warm Matrigel to 4 °C in the fridge overnight, and then aliquot. Smaller (i.e., 1 mL) stocks can be stored at -20°C. Avoid multiple freeze-thaw cycles where possible. Keep Matrigel on ice prior to use. Do not allow Matrigel to warm to room temperature as it will solidify and this process cannot be reversed. 2. Make up working stocks to the following concentrations: Noggin 100 μg/mL; EGF 50 μg/mL; Jagged-1 500 μM; R-spondin1 100 μg/mL; Wnt3a 100 μg/mL; CHIR 99021 10 mM; Y-27632 10 mM.

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3. Organoid basal medium can be prepared and stored at 4 °C for 2–3 weeks. Complete crypt or single-cell medium should be prepared on the day of use. 4. NUNC cell culture-treated plates are recommended. Matrigel does not adhere as well to some other plate brands making seeding more difficult. 5. When scraping the epithelial surface of the intestine with a glass coverslip, do not apply too much pressure. Only light pressure is required to remove mucus and villi. Scraping too firmly may compromise crypt structure. 6. All reagents and samples should be kept on ice at all times during organoid isolation. Cell viability and therefore organoid forming ability will be compromised if samples are allowed to warm to room temperature. 7. All centrifugation steps are performed at 423 × g. Do not centrifuge samples at higher speeds. This will cause more single-cell contamination and will not yield a pure crypt culture. 8. There are numerous Matrigel seeding methods described in the literature. More Matrigel can be used per well when passaging and maintaining lines (i.e., 50 μL per well of a 24-well plate). However, when seeding organoids directly from crypts or isolated single cells, a thinner layer of Matrigel facilitates organoid growth. This allows for optimal diffusion of the required growth factors to cells. 9. Do not allow cells to sit in Matrigel for extended periods of time (>30 min) without media as organoid viability may be compromised. 10. TAT-Cre recombinase protein (EMD Millipore) is sold in units where 100 units is defined as the amount of TAT-Cre (μg) required to induce 50% GFP expression in a HEK293T reporter cell line assay. This amount varies for every batch of TAT-Cre and must be considered to generate the 8 μM TAT-Cre final concentration. Activity varies between batches. A range of concentrations (from 2 to 10 μM) should be tested for each batch. In our hands, 8 μM TAT-Cre induced a high degree of recombination (72% recombined GFP+ cells) (see Fig. 3c) and higher concentrations did not significantly increase efficiency. 11. Both the resazurin dye and resorufin product in PrestoBlue are light sensitive leading to increased background fluorescence. Store the reagent in the dark where possible. If exposure to light is unavoidable (i.e., during assay setup), ensure a no cell control well is included to correct for background fluorescence.

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12. If the PrestoBlue plate cannot be read immediately after assay completion, the black plate can be stored at 4 °C protected from light for up to 48 h. 13. Matrigel dissolves after fixation in 4% PFA. Handle the plate with extra care when adding and removing liquid in the well to avoid washing the organoids off the plate.

Acknowledgements This work was supported by the National Health and Medical Research Council of Australia grants (1100531, 1188689 and 2003693) and an Australian Research Council grant (DP200103589). Authors wish to acknowledge support from the Monash Biomedicine Discovery Institute Organoid Program, Monash FlowCore, and Monash Microimaging platform facilities. References 1. Sato T, Vries RG, Snippert HJ et al (2009) Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459:262–265 2. Nefzger CM, Jarde T, Rossello FJ et al (2016) A versatile strategy for isolating a highly enriched population of intestinal stem cells. Stem Cell Rep 6:321–329 3. Lindemans CA, Calafiore M, Mertelsmann AM et al (2015) Interleukin-22 promotes intestinal-stem-cell-mediated epithelial regeneration. Nature 528:560–564 4. Jarde T, Chan WH, Rossello FJ et al (2020) Mesenchymal niche-derived neuregulin-1 drives intestinal stem cell proliferation and regeneration of damaged epithelium. Cell Stem Cell 27:646–662 5. Mileto SJ, Jarde T, Childress KO et al (2020) Clostridioides difficile infection damages colonic stem cells via TcdB, impairing epithelial repair and recovery from disease. Proc Natl Acad Sci USA 117:8064–8073 6. Holik AZ, Krzystyniak J, Young M et al (2013) Brg1 is required for stem cell maintenance in the murine intestinal epithelium in a tissuespecific manner. Stem Cells 31:2457–2466 7. Horvay K, Jarde T, Casagranda F et al (2015) Snai1 regulates cell lineage allocation and stem cell maintenance in the mouse intestinal epithelium. EMBO J 34:1319–1335 8. el Marjou F, Janssen KP, Chang BH et al (2004) Tissue-specific and inducible

Cre-mediated recombination in the gut epithelium. Genesis 39:186–193 9. Peitz M, Pfannkuche K, Rajewsky K et al (2002) Ability of the hydrophobic FGF and basic TAT peptides to promote cellular uptake of recombinant Cre recombinase: a tool for efficient genetic engineering of mammalian genomes. Proc Natl Acad Sci USA 99:4489– 4494 10. Jarde T, Evans RJ, McQuillan KL et al (2013) In vivo and in vitro models for the therapeutic targeting of Wnt signaling using a Tet-ODeltaN89beta-catenin system. Oncogene 32:883– 893 11. Morin PJ, Sparks AB, Korinek V (1997) Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science 275:1787–1790 12. Hill DR, Spence JR (2017) Gastrointestinal organoids: understanding the molecular basis of the host-microbe interface cell mol. Gastroenterol Hepatol 3:138–149 13. Soriano P (1999) Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21:70–71 14. Shibata H, Toyama K, Shioya H et al (1997) Rapid colorectal adenoma formation initiated by conditional targeting of the Apc gene. Science 278:120–123 15. Madisen L, Zwingman TA, Sunkin SM et al (2010) A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat Neurosci 13:133–140

Inducing Conditional Mutations in Organoids 16. Engel RM, Chan WH, Nickless D et al (2020) Patient-derived colorectal cancer organoids upregulate revival stem cell marker genes following chemotherapeutic treatment. J Clin Med 9:128

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17. Nefzger CM, Jarde T, Srivastava A et al (2022) Intestinal stem cell aging signature reveals a reprogramming strategy to enhance regenerative potential. NPJ Regen Med 7:31

Chapter 6 An In Vitro Model for Assessing Acute Lung Injury During Pancreatitis Development Using Primary Mouse Cell Co-cultures Mohamed I. Saad and Brendan J. Jenkins Abstract Acute pancreatitis is a serious inflammatory disease of the pancreas that can lead to lung injury. Despite extensive research, the mechanisms underlying this complication are ill-defined. In recent years, in vitro co-culture systems have emerged as powerful tools for studying complex interactions between different cell types in disease. In the context of pancreatitis, pancreatic acinar epithelial cells produce and secrete digestive enzymes, and their cellular damage, death, and/or dysfunction is a major contributing factor to the onset of pancreatitis. Here, in this chapter we describe a co-culture system of acinar cells and lung epithelial progenitor/stem cells to model for lung injury associated with pancreatitis. Key words Pancreatitis, Acute lung injury, In vitro co-cultures, Acinar cells, Lung stem cell niche, Organoids

1

Introduction Pancreatitis is a complex and multifactorial disorder that is characterized by inflammation of the pancreas. It is one of the most common gastrointestinal causes for hospital admission in the USA and is associated with substantial morbidity and mortality [1]. The etiology of pancreatitis is diverse and can be classified into two main categories: acute and chronic. Acute pancreatitis is usually caused by gallstones or alcohol consumption, while chronic pancreatitis is associated with genetic and environmental factors such as smoking, high triglycerides, and chronic alcohol consumption [2, 3]. The pathogenesis of pancreatitis is not fully understood; however, it is believed to involve the activation of enzymes within the pancreas, leading to the destruction of the glandular tissue. This activation is thought to occur due to the obstruction of the ducts

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that drain the pancreas, leading to the accumulation of enzymes within the gland. The excess enzymes then activate the inflammatory cascade, which leads to the characteristic symptoms of pancreatitis [4]. Systemic complications of acute pancreatitis are usually exacerbations of preexisting comorbidities such as chronic lung disease, chronic liver disease, or congestive heart failure, culminating into the failure of respiratory, cardiovascular, and renal systems [5]. Acute lung injury (ALI) is a well-known complication of pancreatitis, which is characterized by the leakage of fluid into the air spaces of the lungs, leading to a reduction in oxygenation and ventilation [6]. The exact mechanism by which pancreatitis leads to ALI is not fully understood, although it is thought to be related to the systemic release of inflammatory mediators and proinflammatory cytokines that lead to an acute systemic inflammatory response. Additionally, there may be direct injury to the lung tissue from the leakage of pancreatic enzymes or from the formation of blood clots. The development of ALI can lead to a significant increase in morbidity and mortality in patients with pancreatitis, highlighting the importance of early recognition and management of this complication [7–9]. Here, we describe an in vitro co-culture system to study the crosstalk between pancreatic acinar cells (the main cell of origin for pancreatitis) and lung three-dimensional (3D) organoids comprising the lung progenitor/stem cell niche and its differentiated epithelial cells during the initiation and development of pancreatitis. The co-culture system is established using a Transwell insert, which separates the acinar cells from the lung epithelial progenitor/stem cells (Fig. 1). The acinar cells are isolated from the pancreas of mice and are cultured in a medium containing pancreatic enzymes and growth factors, using well-established protocols [10, 11]. The lung epithelial progenitor/stem cells (namely, bronchioalveolar stem cells [BASCs] and alveolar type II [AT2] cells) are isolated from the lungs of mice and are cultured in Matrigel supported with lung endothelial/stromal cells and growth factors, using wellestablished protocols [12–15]. This co-culture system also allows for the exogenous addition of various inducers of pancreatitis and inhibitors of signaling pathways in a controlled and reproducible manner, which can facilitate the discovery of new therapeutic targets for the treatment of this devastating complication of acute pancreatitis. In summary, these co-cultures provide a powerful in vitro tool for studying the complex interactions between acinar cells and lung epithelial progenitor/stem cells in lung injury associated with pancreatitis.

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Fig. 1 Assessing lung injury during pancreatitis development in vitro using primary mouse cell co-cultures. The figure shows a schematic representation of the Transwell co-culture system used. Three-dimensional lung organoids are developed in Transwells from primary mouse lung epithelial progenitor/stem cells (BASCs and/or AT2 cells) supported by mouse lung endothelial cells (MECs) and growth factors. Two-dimensional acinar cells are isolated from mouse pancreatic tissues and cultured at the bottom of 24-well plates in the presence of growth factors

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Materials

2.1 Isolation and Culturing of Mouse Lung Endothelial Cells (MECs)

1. Personal protective equipment (PPE): surgical gown, sterile gloves, and face mask. 2. Autoclaved surgical equipment: forceps, scalpel blade, and scissors. 3. Mice at 4–6 weeks of age (see Note 1). 4. Hank’s Balanced Salt Solution (HBSS). 5. Dulbecco’s Modified Eagle’s Medium (DMEM). 6. Fetal bovine serum (FBS). 7. 100-μM strainer. 8. Falcon tubes. 9. Digestion medium: 100 μL of collagenase type II (100 mg/ mL), 12 μL of DNase (10 mg/mL) and HBSS (up to 1 mL). 10. Histopaque-1077. 11. Bovine serum albumin (BSA). 12. Phosphate-buffered saline (PBS). 13. MACS® buffer: 0.5% BSA, 2 mM EDTA and PBS (up to 500 mL). 14. Anti-CD45- and anti-CD3-conjugated magnetic beads and column (Miltenyi Biotec®).

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15. MEC growth media: 5 mL of 2 mM N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES), 20 μg of heparin, 20 μg of endothelial cell growth supplement (ECGS), 20% FBS, 1% glutamine, 1% penicillin/streptomycin, and advanced DMEM (up to 200 mL). 2.2 Isolation and Culturing of Mouse Lung Progenitor Cells

1. PPE (see Subheading 2.1, step 1). 2. Autoclaved surgical equipment: forceps, scalpel blade, and scissors. 3. Mice at 6 to 10 weeks of age (see Note 1). 4. Needles (20 gauge) and syringes (3 mL). 5. Dispase. 6. Collagenase/Dispase. 7. DNase I. 8. Low-melting-point agarose. 9. 100-μM and 40-μM strainers. 10. Falcon tubes. 11. DMEM. 12. PBS. 13. 10% FBS/PBS. 14. Trypsin. 15. Red blood cell (RBC) lysis buffer: 0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, in 1 L H2O, and filtered with 0.45-μ M filter and stored at room temperature. 16. Conjugated antibodies: Sca-1-FITC, CD31-APC, CD45APC, and EPCAM-PE-CY7. 17. Lung progenitor cell media: 500 μL of insulin/transferrin/ selenium, 10% FBS, 1% glutamine, 1% penicillin/streptomycin, 1 mM HEPES and DMEM/F12 (up to 50 mL). 18. 0.4-μM 24-well plate Transwell inserts. 19. Matrigel (see Note 2).

2.3 Isolation and Culturing of Mouse Pancreatic Acinar Cells

1. PPE (see Subheading 2.1, step 1). 2. Autoclaved surgical equipment: forceps, scalpel blade, and scissors. 3. Mice at 6–10 weeks of age (see Note 1). 4. Falcon tubes. 5. Serological pipettes. 6. 100-μM strainer. 7. HBSS.

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8. Digestion solution: HBSS containing 10 mM HEPES, 200 U/ mL of collagenase IA, and 0.25 mg/mL of trypsin inhibitor. 9. Washing solution: ice-cold HBSS containing 5% FBS and 10 mM HEPES. 10. Resuspension media: Waymouth’s medium containing 2.5% FBS, 1% penicillin-streptomycin, 0.25 mg/mL of trypsin inhibitor, and 25 ng/mL of recombinant human epidermal growth factor (EGF).

3

Methods

3.1 Isolation and Culturing of Mouse Lung Endothelial Cells (MECs)

1. Euthanize the mouse humanely using CO2 asphyxiation (see Note 3) and collect lung tissues in ice-cold DMEM. 2. Chop the lungs using sharp scissors into small pieces, and then digest in digestion medium (1 mL per lung) for 30 min at 37
C (preferably on a shaker). 3. Filter cell suspension through a 100-μM strainer, followed by quenching collagenase activity using an equal volume of FBS. 4. Discard supernatant carefully (as cell pellet is usually loose), and then wash in 10 mL HBSS. 5. Resuspend cells in 5 mL HBSS and carefully load them on 5 mL of Histopaque in a 15-mL falcon tube (i.e., adding 1 mL of cell suspension at a time slowly). 6. Centrifuge at 2000 rpm for 20 min while deactivating the deceleration on the centrifuge (deceleration should be 0). This step will take 45–60 min for the centrifuge to stop completely. 7. Collect the cloudy interface containing MECs carefully (i.e., small whitish layer in the middle of the tube) by removing and discarding the top layer first until the cloudy layer is exposed. Transfer cells in a new 15-mL falcon tube. 8. Wash cells with 10 mL of 0.5% BSA in HBSS, centrifuge the suspension at 1000 rpm for 10 min, and then discard the supernatant. 9. Resuspend cells in 2 mL of sterile MACS® buffer, and then perform cell counting. 10. Subject cells to negative selection with anti-CD45-conjugated magnetic beads (Miltenyi Biotec®; discard CD45-bound cells) followed by positive selection with anti-CD31-conjugated magnetic beads (Miltenyi Biotec®). 11. Collect and centrifuge CD31-bound cells at 1000 rpm for 5 min, then resuspend them in MEC growth media, and plate into a gelatin-coated flask until they reach 70–80% confluency.

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12. If necessary, reselect cultured MECs again using anti-CD31conjugated magnetic beads followed by replating in MEC growth media. MECs are usually used for co-culture experiments between passages 4 and 8 (see Note 4). 3.2 Isolation and Culturing of Mouse Lung Progenitor/Stem Cells

1. Euthanize the mouse humanely using CO2 asphyxiation (see Note 3). 2. Expose the trachea, lungs, and heart by cutting through the rib cage without piercing lung tissues. 3. Exsanguinate the mouse through the left ventricle, and then flush 10 mL of ice-cold PBS via the right ventricle. 4. Dissect the heart out. 5. Inject 1.5–2 mL ice-cold Dispase into the trachea using a 20-gauge needle and a 3-mL syringe. 6. Immediately after, inject 0.5–1 mL warmed low-melting-point agarose. 7. Dissect the lungs out and place them on ice. 8. Chop the lungs using sharp scissors into small pieces, and then add 3 mL of PBS per mouse. 9. Add 60 μL collagenase/Dispase (100 mg/mL) per mouse then mix. 10. Incubate on a shaker at 37
C for 45 min. 11. Add 7.5 μL DNase I per mouse, then mix, and incubate for 2–3 min on ice. 12. Filter cell suspension through a 100-μM strainer, followed by a 40-μM strainer. 13. Centrifuge cell suspension at 800 rpm for 6 min at 4
C. 14. Carefully aspirate and discard the supernatant while avoiding the thin layer of cells, which contains lung progenitor/stem cells. 15. Perform RBC lysis using 1 mL of RBC lysis buffer for 90 s at room temperature. 16. Quench with 6 mL serum-free ice-cold DMEM. 17. Add 0.5 mL ice-cold FBS slowly to the bottom on the tube. 18. Centrifuge cell suspension at 800 rpm for 6 min at 4
C. 19. Aspirate and discard the supernatant, leaving the FBS interface that contains cells of interest. 20. Resuspend cells in 10% FBS/PBS and proceed to cell counting and staining for flow cytometry and cell sorting (approximately 1  106 cells per 100 μL).

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21. Use the following conjugated antibodies (1:100) in 10% FBS/PBS to stain cells on ice for 20 min: Sca-1-FITC, CD31-APC, CD45-APC, and EPCAM-PE-CY7 (see Note 5). 22. Pellet the cells, remove the supernatant, and resuspend cells in 300–500 μL 10% FBS/PBS. 23. Perform flow sorting of cells to isolate lung progenitor/stem cells, BASCs (Sca-1+ CD45 CD31 EPCAM+ cell population), and/or AT2 (Sca-1 CD45 CD31 EPCAM+ cell population) in ice-cold tubes pre-coated with FBS. 24. Trypsinize MEC flask followed by quenching trypsin activity using an equal volume of MEC media, and then resuspend them in Matrigel at a concentration of 2  104 cells per 50 μL of Matrigel per Transwell. 25. Centrifuge the sorted lung progenitor/stem cells (BASCs or AT2 or both), discard supernatant, and resuspend in progenitor cell media at a concentration of 2  103 cells per 50 μL of media per Transwell. 26. For each Transwell, mix 2  104 MECs in 50 μL of Matrigel with 2  103 lung progenitor cells in 50 μL of media, and transfer directly onto a 0.4-μM 24-well plate Transwell insert. 27. Incubate at 37
C for 30 min to allow the Matrigel to solidify, and add 450 μL lung progenitor media at the bottom of the Transwell (i.e., in the 24-well plate). 28. Change media at the bottom of Transwell every 2–3 days until 3D lung organoids form, which takes 7–14 days (Fig. 1). 3.3 Isolation and Culturing of Mouse Pancreatic Acinar Cells

1. Euthanize the mouse humanely using CO2 asphyxiation (see Note 3). 2. Expose the abdominal cavity, use the stomach and the spleen to locate the pancreas, and then use the spleen to carefully cut and liberate the pancreas from the rest of the digestive tract. Once out, dissect the pancreas carefully from the spleen and collect it in ice-cold HBSS. 3. Chop the pancreas using sharp scissors into small pieces of 1–3 mM3 in 3–5 mL ice-cold HBSS. 4. Transfer the suspension in a falcon tube and centrifuge for 2 min at 450  g and 4
C. 5. Aspirate and discard the supernatant to remove cell fragments and blood cells. 6. Add 10 mL of digestion solution to the pellet and transfer the suspension to a 25 cm2 flask using a 25-mL serological pipette. 7. Incubate the flask for 15–20 min at 37
C while performing a mechanical dissociation by moving the fragments 5–10 times using serological pipettes (25 mL, then 10 mL).

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8. Stop the enzymatic reaction of the dissociated pancreas fragments by adding 10 mL of ice-cold washing solution. 9. Transfer the suspension in a falcon tube and centrifuge for 2 min at 450  g and 4
C. 10. Discard the supernatant and repeat the washing step two more times. 11. Resuspend the cell pellet in 7 mL of resuspension medium. 12. Filtrate the cell suspension through a 100-μM strainer. 13. Rinse the filter with 6 mL of resuspension medium. 14. Transfer the isolated acini to a 6-well plate (2 mL per well) or a 75 cm2 flask (10–15 mL) and incubate at 37
C overnight. 15. Coat a 24-well plate with type I collagen (5 μg/cm2) by adding 1 mL of type I collagen solution (50 μg/mL in 0.02 M acetic acid, 0.2 μM-filtered) to each well and allow it to passively adsorb on plastic for 1 h at 37
C (or preferably overnight at 4
C). 16. Aspirate the type I collagen solution and rinse the coated well twice with PBS and leave to dry for 2–4 h. 17. Transfer the acini (in suspension) into the coated 24-well plate and discard any contaminant cells and cellular remnants that have adhered overnight. 18. After 2–3 days, change the culture medium with new resuspension medium to eliminate non-viable cells. 3.4 Co-culturing 3D Lung Organoids with Acinar Cells

1. When 3D lung organoids are fully developed, transfer the Transwells containing lung organoids to 24-well plates containing the 2D acinar cell cultures. 2. Adjust the medium in the acinar cell compartment to 450 μL (225 μL of resuspension medium plus 225 μL of lung progenitor medium). Serum-free media could be used. 3. Add pancreatitis inducers (i.e., cerulein, nicotine-derived nitrosamine ketone [NNK], lipopolysaccharide [LPS], taurocholate, ethanol) at the desired concentrations to the acinar compartment and incubate for the desired time points. Inhibitors of certain signaling pathways can also be added to the medium. At the end of the experiment, collect lung cells, acinar cells, and cell culture supernatants and store them appropriately for further molecular analyses (i.e., qPCR, Western blotting, ELISA) or use them directly to assess inflammation and acute injury (i.e., viability assays, mitochondrial function, cell death assays). Lung 3D organoids can also be embedded in Histogel and subjected to further histopathological and immunohistochemical analyses (see Notes 6 and 7).

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Notes 1. Three to six mice of the same genotype can be used and tissues can be combined once collected. Alternatively, tissues from each mouse can also be processed separately. 2. Matrigel can be thawed at 4
C overnight (preferred method), or on ice for at least 30 min prior to procedure. Freeze in small aliquots to avoid wastage and repeat freeze-thaw cycles. Keep Matrigel on ice at all times, as it will solidify if left at room temperature. 3. For mouse studies, these must be approved by the appropriate institutional animal ethics committee or board and be performed in compliance with the guidelines of the country’s governmental bodies that regulate animal experimentation. 4. MECs can be maintained in culture by changing media every 2–3 days until used in experiments, or frozen down in normal freezing media (i.e., 10% DMSO in FBS) using cryovials and liquid nitrogen until needed for experiments. 5. 40 ,6-Diamidino-2-phenylindole (DAPI) or propidium iodide (PI) can be used to check for viability at this step. 6. To study the role of immune cells in pancreatitis-associated lung injury, isolated immune cells (i.e., T-cells from spleen or bone marrow-derived macrophages) could be added to the acinar compartment. 7. To study the role of specific genes/proteins in the development of pancreatitis and/or its associated lung injury, reciprocal co-culture systems could be developed in which one or both compartments are isolated and maintained from mice lacking (or overexpressing) the genes/proteins of interest.

Acknowledgements B.J.J. is supported by a National Health and Medical Research Council of Australia (NHMRC) Senior Research Fellowship (APP1154279). This work was also financially supported in part through the Victorian State Government Operational Infrastructure Support Scheme. We would like to thank Associate Professor Rebecca Lim (Hudson Institute of Medical Research, Melbourne, Australia) and her team for their technical assistance in setting up 3D lung organoids.

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References 1. Lee PJ, Papachristou GI (2019) New insights into acute pancreatitis. Nat Rev Gastroenterol Hepatol 16:479–496 2. Weiss FU, Laemmerhirt F, Lerch MM (2019) Etiology and risk factors of acute and chronic pancreatitis. Visc Med 35:73–81 3. Vidarsdottir H, Mo¨ller PH, Vidarsdottir H et al (2013) Acute pancreatitis: a prospective study on incidence, etiology, and outcome. Eur J Gastroenterol Hepatol 25:1068–1075 4. Habtezion A (2015) Inflammation in acute and chronic pancreatitis. Curr Opin Gastroenterol 31:395–399 5. Wu BU, Banks PA (2013) Clinical management of patients with acute pancreatitis. Gastroenterol 144:1272–1281 6. Akbarshahi H, Rosendahl AH, WestergrenThorsson G et al (2012) Acute lung injury in acute pancreatitis–awaiting the big leap. Respir Med 106:1199–1210 7. Elder AS, Saccone GT, Dixon D-L (2012) Lung injury in acute pancreatitis: mechanisms underlying augmented secondary injury. Pancreatol 12:49–56 8. Bhatia M, Zemans RL, Jeyaseelan S (2012) Role of chemokines in the pathogenesis of acute lung injury. Am J Respir Cell Mol Biol 46:566–572

9. Zhang H, Neuho¨fer P, Song L et al (2013) IL-6 trans-signaling promotes pancreatitisassociated lung injury and lethality. J Clin Invest 123:1019–1031 10. Gout J, Pommier RM, Vincent DF et al (2013) Isolation and culture of mouse primary pancreatic acinar cells. J Vis Exp 78:e50514 11. Saad MI, Weng T, Lundy J et al (2022) Blockade of the protease ADAM17 ameliorates experimental pancreatitis. Proc Natl Acad Sci USA 119:e2213744119 12. Leeman KT, Fillmore CM, Kim CF (2014) Lung stem and progenitor cells in tissue homeostasis and disease. Curr Top Dev Biol 107:207–233 13. Tan JL, Lau SN, Leaw B et al (2018) Amnion epithelial cell-derived exosomes restrict lung injury and enhance endogenous lung repair. Stem Cells Transl Med 7:180–196 14. Zhu D, Kusuma GD, Schwab R et al (2020) Prematurity negatively affects regenerative properties of human amniotic epithelial cells in the context of lung repair. Clin Sci 134: 2665–2679 15. Kim CFB, Jackson EL, Woolfenden AE et al (2005) Identification of bronchioalveolar stem cells in normal lung and lung cancer. Cell 121: 823–835

Chapter 7 Tracking the Host Response to Infection in Peritoneal Models of Acute Resolving Inflammation David Millrine, Christopher M. Rice, Javier U. Fernandez, and Simon A. Jones Abstract Antimicrobial host defense is dependent on the rapid recruitment of inflammatory cells to the site of infection, the elimination of invading pathogens, and the efficient resolution of inflammation that minimizes damage to the host. The peritoneal cavity provides an accessible and physiologically relevant system where the delicate balance of these processes may be studied. Here, we describe murine models of peritoneal inflammation that enable studies of competent antimicrobial immunity and inflammationassociated tissue damage as a consequence of recurrent bacterial challenge. The inflammatory hallmarks of these models reflect the clinical and molecular features of peritonitis seen in renal failure patients on peritoneal dialysis. The development of these models relies on the preparation of a cell-free supernatant derived from an isolate of Staphylococcus epidermidis (termed SES). Intraperitoneal administration of SES induces a Toll-like receptor 2-driven acute inflammatory response that is characterized by an initial transient influx of neutrophils that are replaced by a more sustained recruitment of mononuclear cells and lymphocytes. Adaptation of this model using a repeated administration of SES allows investigations into the development of adaptive immunity and the hallmarks associated with tissue remodelling and fibrosis. These models are therefore clinically relevant and provide exciting opportunities to study innate and adaptive immunity and the response of the stromal tissue compartment to bacterial infection and the ensuing inflammatory reaction. Key words Peritonitis, Inflammation, Cytokine, Fibrosis

1

Introduction Inflammation associated with bacterial infections of the peritoneal membrane (termed peritonitis) is a major cause of treatment failure in patients undergoing peritoneal dialysis in response to renal failure. Using bacterial isolates from a patient with clinical peritonitis, we have generated a cell-free supernatant of Staphylococcus epidermidis, a gram-positive bacterium typically associated with the commensal microflora. Administration of this cell-free isolate to the

Brendan J. Jenkins (ed.), Inflammation and Cancer: Methods and Protocols, Methods in Molecular Biology, vol. 2691, https://doi.org/10.1007/978-1-0716-3331-1_7, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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peritoneal cavity triggers an acute inflammatory reaction [1]. This model has proven remarkably versatile and provides insights into cytokine/chemokine signalling networks; the tracking of immune cell recruitment, activation, and survival within a site of local infection; and mechanisms of inflammatory resolution [2–6]. These investigations have enabled the generation of new hypotheses that may be applied to a range of inflammatory settings or model systems. For instance, the requirement for interleukin-6 (IL-6) signalling in the recruitment and maintenance of leukocyte populations at the site of infection, originally observed in SES-induced peritonitis [5, 7], was readily translated into murine models of inflammatory arthritis [8, 9], where blockade of this pathway was seen to prevent synovitis and the development of cartilage and bone damage [10, 11]. Insights have also been gained into the molecular events leading to fibrosis, an untreatable condition that is currently the subject of intense research interest [12]. By modifying the protocol of SES delivery, we have developed a model that allows monitoring of adaptive immune memory and the underlining mechanisms responsible for peritoneal tissue damage and fibrosis [12, 13]. Taken together, these studies support the broad relevance of SES-induced murine peritonitis as an adaptable model for understanding inflammatory activation and maintenance, investigating the molecular mechanisms of leukocyte recruitment and clearance, providing opportunities to explore the relationships between innate and acquire immunity, and how recurrent bacterial infections may contribute to pathogenesis. Studies have characterized many of the temporal events associated with acute inflammatory activation following SES administration. These include the rapid influx of neutrophils (within 24 h), their apoptotic clearance, and replacement with a population of mononuclear leukocytes (2–4 days post administration) that include inflammatory monocytes and effector CD4+ T-cell subsets [13]. The model has also been applied to the understanding of immune homeostasis and proliferative regulation of resident tissue monocytic cells [1, 12, 14]. The very nature of the peritoneal cavity allows analysis of the inflammatory infiltrate (e.g., multiparameter flow cytometry), the generation of inflammatory mediators (e.g., ELISA; mass spectrometry), and studies of the stromal compartment (e.g., immunoblotting, electrophoretic mobility shift assays, immunocytochemistry, and studies of functional genomics). Thus, the SES-induced model of peritoneal inflammation is highly versatile and amenable to manipulation to allow mechanistic insight into the working of acute inflammation. In this regard, the model shows a number of features reflecting the clinical context in end-stage renal failure patients on peritoneal dialysis and provides opportunities to investigate antimicrobial host defense and tissue damage.

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Materials Prepare all solutions using deionized water and analytical grade reagents. Store all reagents at room temperature (unless otherwise specified). Ensure all reuseable glassware is autoclaved before starting. Where appropriate use sterile disposable plasticware and good aseptic technique.

2.1 Preparation of Staphylococcus epidermidis Cell-Free Supernatant

1. Bacterial specimen. Slope of S. epidermidis dormant bacteria stored in glycerol at
80 C (a clinical strain may be used or an ATCC-derived S. epidermidis isolate; ATCC-12228). 2. Nutrient broth no. 2: Dissolve 25 g of nutrient broth no. 2 per 1 L of deionized H2O to a total volume of 2 L (2  1 L bottles). Autoclaved media might be stored at room temperature for approximately 1 week. 3. Diagnostic sensitivity test (DST) agar: Dissolve 40 g of DST agar in 1 L of deionized H2O. Autoclave and cool the bottle sufficiently to allow handling. Ensure the agar is fully liquefied. Pour the molten agar broth into petri dishes to an approximate depth of 1 cm. This should be performed in a laminar flow hood using appropriate aseptic techniques. Allow the molten agar to solidify. Wipe any moisture for the petri dish lids with an alcohol swab and seal the petri dishes with Parafilm. Plates may be stored at 4  C for approximately 1 week. 4. Tyrode’s salt (0.012 M NaHCO3): Dissolve a vial of Tyrode’s salt powder (9.6 g) in 1 L of deionized H2O. Add 1 g NaHCO3 and filter in a Stericup filter bottle. Store in the fridge at 4  C and use within 1 month. 5. Petri dishes 90 mM. 6. 500 mL centrifuge tubes. 7. 125-, 250-, and 500 mL Erlenmeyer flasks. 8. Dialysis tubing MWCO 12–14000 daltons. 9. Plastic clips for dialysis tubing. 10. Graduated 25 mL sample tubes (universals). 11. MillexGP 0.22 μM filter unit. 12. Phosphate-buffered saline (PBS), sterile.

2.2

SES Bioassay

1. 24-well plates. 2. RPMI 1640 supplemented media (RPMI-complete; RPMIc): RPMI 1640, 10% (v/v) FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin, 100 mg/mL streptomycin, 55 mM β-mercaptoethanol.

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2.3 Acute Resolving Inflammatory Challenge

1. Syringes (1 mL and 5 mL).

2.4 Tracking Innate Immune Responses

1. Fluorescent dye for cellular labelling (e.g., Trace Far Red, Life Technologies–Invitrogen).

2. Needles (21 and 25 gauge).

2. 30 -(p-aminophenyl) fluorescein. 2.5 Tracking Stromal Tissue Responses in Mice

1. Dissection kit, including scissors. 2. Liquid nitrogen. 3. 1.7-mL Eppendorf tube. 4. Extraction buffer: 50 mM HEPES (pH 7.5), 150 mM NaCl, 25 mg/mL digitonin. 5. Solubilization buffer for cytosolic fractions containing 50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% (v/v) NP40. For membrane fractions, the following buffer is recommended (50 mM Tris–HCl [pH 7.5], 150 mM NaCl, 0.2% digitonin). 6. LDS sample buffer. 7. BCA protein detection assay. 8. RNA extraction kits. 9. A handheld electric homogenizer. 10. Tris-buffered saline 150 mM NaCl).

(50

mM

Tris–HCl

pH

7.5,

11. Proteinase-K. 12. Ribosomal depletion kit.

3

Methods Perform the following procedure in a sterile hood using standard aseptic technique. A schematic flowchart summarizing the experimental protocol required for the generation of SES is presented in Fig. 1.

3.1 Isolation and Culture of S. epidermidis Single Colonies

Allow all agar petri dishes to reach room temperature before bacterial inoculation. 1. Inoculate agar plate with a loop of dormant S. epidermidis bacteria (Fig. 2). Streaking of bacteria will ensure isolation of single colonies for subsequent expansion. Incubate at 37  C for 48 h (see Note 1). 2. Using a flamed or disposable inoculation loop, capture a single bacterial colony of S. epidermidis and transfer it into a 250-mL

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Inoculate an agar plate with S. epidermidis (clinical isolate or ATTC12228)

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See Fig. 2

48 h, 37oC

Transfer a single colony into 250 ml of nutrient broth No2 Overnight, 37oC

Transfer 2ml of the liquid culture to 400 ml of nutrient broth No2

Gram staining

Overnight 37oC 1800 x g, 20 min, 20oC

Resuspend in 50 ml of Tyrode's salt.

Check the optical density (OD). Dilute in Tyrode’s salt until an OD of 0.5-0.6 at 560 nm

18-24 hours, 37oC 5000 x g, 30 min, 20oC

Collect and filter the supernatant

Microbiology check 1

Dialyze the cell free extract against deionized water 8-12 hours, 4oC

Pool and divide in 10 ml aliquots

Overnight, -20oC

Lyophilize

Microbiology check 1 Store at -80oC

SES Bioassay (See Fig. 3)

Fig. 1 Summary schematic of depicting a flowchart of the experimental protocol required for the generation of SES

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b

c a

d Fig. 2 Schematic representation of how to inoculate an agar plate to obtain single bacterial colonies. (a) Streak one line in the direction indicated by the arrow with a sterile loop of dormant S. epidermidis from the glycerol stock. (b, c, and d) Streak four lines in the direction indicated by the arrow using a new sterile loop for each (b, c, and d)

Erlenmeyer flask bottle containing 50 mL of nutrient broth no. 2. Incubate at 37  C overnight with agitation at 170 rpm (see Note 2). 3. Transfer 2 mL of the S. epidermidis liquid culture into 500-mL Erlenmeyer flask bottle containing 400 mL of nutrient broth no. 2 and incubate overnight at 37  C with agitation at 170 rpm (see Note 3). 3.2 Preparation of SES

1. Visibly check the turbidity of the bacterial culture medium. Transfer the S. epidermidis liquid culture into 500-mL centrifuge tubes. Centrifuge at 1800  g for 20 min at 20  C. 2. Decant supernatant and fully suspend the bacterial pellet in 50 mL Tyrode’s salt. This can be achieved by gentle agitation using an automatic pipette. Centrifuge at 1800  g for 20 min at 20  C. Decant the supernatant and resuspend Tyrode’s salt solution as before. Check the optical density of the suspension on a spectrophotometer at 560 λ. Dilute the bacterial suspension in Tyrode’s salt solution to an optical density of 0.5–0.6 at 560 nM (corresponding to 5  108 cfu/mL) (see Note 4). 3. Incubate the resuspended culture for 18–24 h at 37  C. Centrifuge at 5000  g for 30 min at 20  C. Collect the supernatant and filter using a 0.2-μM Stericup filter bottle system (see Note 5). 4. Microbiology check 1. SES is a cell-free supernatant that is used to induce sterile, resolving inflammation. To ensure that no living bacteria remains in the suspension after the incubation

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with Tyrode’s salt solution, streak a DST agar plate with a 50 μL sample of the filtered suspension. Incubate at 37  C for 48 h and assess bacterial growth. The culture should be negative for any microorganism. 5. Dialyze the cell-free extract using deionized water in a ratio of 100 mL/5 L in a cold room at 4  C (see Note 6). Prepare the dialysis tubing as instructed by the manufacturer. Knot one end of the dialysis tubing, then pour approximately 50 mL of the cell-free extract into the dialysis tubing, and knot the other end. Clip one end so that the tube floats and place it in the water (see Note 7). 6. Leave for 8–12 h. Change water after 8–12 h and repeat a total of three times. Pool the dialyzed extract and divide into 10 mL aliquots in sterile universals. Remove the lids and cover with Parafilm. Freeze the samples at
20  C overnight (see Note 8). 7. Lyophilize the SES. Freeze-dry the samples overnight or until the water has completely sublimated. Replace the lids of the sterile universals and store at
80  C. Vials may be stored for approximately 1 year. 8. Microbiology check 2. To ensure that the lyophilized SES is free of any viable bacteria, reconstitute one aliquot of SES in 400 μL of sterile PBS; pass through a 0.22 μM filter unit and streak on a DST agar plate. Incubate at 37  C for 48 h and assess the growth of S. epidermidis in the plate by eye. The culture should be negative for any microorganism. 3.3 Test Validation: SES Bioassay

(Optional) It is recommended that the bioactivity of each SES batch is tested prior to use in vivo. Murine peritoneal mononuclear cells will generate inflammatory cytokines (e.g., IL-1β, IL-6, CXCL8, TNFα) in response to SES stimulation. Isolation of these resident tissue cells can be used as a reliable bioassay. Stimulation of these cells with SES triggers the activation of Toll-like receptor2 (TLR2) [15, 16]. The TLR2 agonist PAM3CSK4 may be used as a positive control. 1. Isolate resident peritoneal mononuclear cells. Perform a peritoneal lavage of C57BL/6 mice with 5 mL of RPMIc using a 5-mL syringe and a 25-gauge needle. Centrifuge at 350  g for 5 min to pellet the cell isolate, remove the supernatant, and resuspend the cells to a concentration of 2  106 cells/mL (see Note 9). 2. Plate 1  106/0.5 mL cells in 24-well tissue culture microtiter plates. 3. SES titration. Reconstitute SES at the ratio of 1 mL/vial in RPMIc and pass through a 0.22 μM filter unit. Prepare serial dilutions of SES (neat, 1/2, 1/4, 1/8, 1/16, 1/32, and 1/64) in RPMIc.

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IL-6 (ng/ml)

60 40 20

C M

1/ 8 1/ 16 1/ 32 1/ 64

1/ 4

1/ 2

1/ 1

0 SES dilution

Fig. 3 Representative plot of the levels of IL-6 produced by murine peritoneal resident cells as a response of different concentrations of SES. SES was reconstituted with 1 mL of RPMIc per vial and filtered (0.22 μM). SES serial dilutions (neat 1/1, 1/2, 1/4, 1/8, 1/16, 1/32, and 1/64) were prepared with RPMIc as specified in Subheading 3.3. Blank corresponds to RPMIc alone. For positive control, murine peritoneal resident cells can be stimulated with 10–100 pg/mL of IL-1β or 1–10 ng/mL of TNFα

4. Add 0.5 mL of each dilution of SES to each well containing 1  106 of resident peritoneal mononuclear cells (see Note 10). 5. Incubate the culture at 37  C, 5% CO2 overnight. Carefully transfer the culture media to Eppendorf tubes and render cellfree by centrifugation (2000  g, 5 min). 6. Carefully transfer the supernatant to a clean tube and store at
80 C. 7. Quantify cytokine production using a commercial ELISA (Fig. 3). 3.4 In Vivo Administration of SES and Assessment of Acute Resolving Inflammation

1. Reconstitute the SES in sterile PBS at a ratio of 0.75 mL PBS/vial of SES. 2. Filter the reconstituted SES with a 0.22 μM filter unit to a new, sterile tube. Load the 1-mL syringes with the required volume of SES (see Note 11). 3. Intraperitoneally (i.p.) administer 0.5 mL of SES to each mouse (see Note 12). 4. At defined time points (0–96 h), euthanize each mouse by an intraperitoneal (i.p.) overdose of anesthetic (240 mg/kg sodium pentobarbital). 5. Dissect out slices (e.g., 1 cm  1 cm) of the peritoneal membrane, and place in an Eppendorf tube for immediate downstream processing on ice (80 mg tissue), or snap-freeze in liquid nitrogen for subsequent
80  C storage.

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6. These time points (step 4) are selected to conduct analysis of the leukocyte infiltrate (e.g., flow cytometry, differential cell count), production of inflammatory mediators (e.g., ELISA, mass spectrometry), and stromal tissue responses (see Note 13). In wild-type mice, a single episode of acute resolving inflammation is characterized by a rapid influx of neutrophils that peaks at 3–6 h, which resolve within 24 h post stimulation. Neutrophils are replaced by a more sustained population of infiltrating monocytic cells (24–72 h post stimulation), which are characterized by flow cytometry as F4/80intCD11bint and lymphocytes (including interferon-γ [IFNγ] and IL-17-secreting populations) [1, 4, 5, 7, 17] (see Note 14). 7. Peritoneal tissue can be harvested to determine gene expression or protein activation within the stromal compartment (e.g., immunohistochemistry, real-time PCR, electrophoretic mobility shift assays, immunoblotting) (see Note 15). 3.5 Repeated Administration of SES and Profiling of Recurrent Inflammatory Challenge

This acute model can be adapted to a repeat administration model to investigate the cellular and molecular events that lead to the development of memory responses and the onset of inflammation-induced tissue damage (see Note 16). 1. Deliver SES by i.p. administration at 7-day intervals (day 0, 7, 14, and 21) to mice (see Subheading 3.4, steps 1–3; Note 12). 2. At each time point (0–21 days), as well as at 21 and 28 days after the last administration of SES (i.e., days 42 and 49) (see Note 16), euthanize each mouse and dissect out peritoneal membrane for immediate use or storage (see Subheading 3.4, steps 4 and 5)

3.6 Live Model of S. epidermidis Infection

SES-driven peritonitis provides an extremely versatile model of sterile inflammation. This approach allows investigations of inflammatory regulation without the complications arising from live bacterial infections. However, this model may not be suitable for addressing other questions relevant to the control of infections— e.g., mechanisms of bacterial clearance and dissemination, or systemic responses to local infection. Thus, a modification of the SES acute resolving inflammatory challenge can be achieved using live S. epidermidis [18–21]. 1. Inoculate an agar plate as before with a loop of dormant S. epidermidis bacteria (ATTC 12228 strain). Incubate for 48 h at 37  C. 2. Using a sterile loop, capture a single colony of S. epidermidis and transfer it to an Erlenmeyer flask (125 mL) containing 10 mL of nutrient broth no. 2. Incubate for 6 h at 37  C and 170 rpm agitation. Transfer 60 μL of the liquid culture into a

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250-mL Erlenmeyer flask with 60 mL of fresh nutrient broth no. 2. Incubate for 18 h at 37  C and 170 rpm agitation. 3. Assess the desired inoculum size of bacteria (colony-forming units [cfu]) by spectrometry. Dilute the bacteria culture to an optical density of 1.1–1.15 at 600 nM (corresponding to 1  109 cfu/mL). 4. Wash three times in sterile PBS in order to remove the excess of nutrient broth no. 2 and reconstitute to the initial volume. 5. Administer mice with 5  108 cfu of S. epidermidis (delivered in a volume of 0.5 mL PBS). This bacterial dose is nonlethal and causes no advice changes in body weight. Acute infection and is resolved within 24–36 h. 6. Collect samples at different time points as before. Here, the model can be used to track dissemination of infection into the bloodstream and other organs and can be adapted to investigate the acute phase response and systemic inflammatory outcomes including changes in body temperature. 3.7 Tracking Innate Responses to Infection

Access to the peritoneal cavity provides opportunities to evaluate temporal changes in leukocyte populations (e.g., via differential cell counting or multiparameter flow cytometry) and inflammatory mediator production (e.g., chemokines, cytokines, lipid mediators, and other soluble regulators) using standard immuno-detection assays. However, methods can also be used to track bacterial infection (e.g., clearance or dissemination) using fluorescently labelled bacteria. 1. Prepare an inoculum of Staphylococcus epidermidis (see Subheading 3.6, steps 1–4). 2. Resuspend the bacteria in pre-warmed PBS (to 37  C) containing 8 μM CellTrace Far Red tracker dye. Incubate for 20 min at 37  C. 3. Wash three times in sterile PBS to remove any excess dye and resuspend in sterile PBS. Fluorescently labelled bacteria can be directly infected into the peritoneal cavity (see Subheading 3.6, step 5). 4. At a set time interval (e.g., 3–6 h following infection), mice are terminated by an approved protocol. 5. Lavage the peritoneal cavity and screen for the phagocytic uptake of fluorescent bacteria using standard flow cytometry or imaging flow cytometry (e.g., using Ly6G immunostaining to gate infiltrating neutrophils). 6. Extending this approach, the method can also be adapted to evaluate neutrophil phagocytosis in combination with a study of respiratory burst activity. Fluorescently labelled bacteria are administered (i.p.) as described above. However, the peritoneal

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cavity is now lavaged with 2 mL of RPMI-1640 (serum-free) containing 5 μM of 30 -(p-aminophenyl) fluorescein, a redoxsensitive reporter dye that responds to reactive oxygen species. 7. Cells are incubated at 37  C for 15 min and immediately transferred to an ice water bath to stop the reaction. Cells are maintained at 4  C for all further staining protocols and analyzed using standard flow cytometry or imaging flow cytometry (see Note 17). 3.8 Biochemical and Genomic Analysis of Stromal Tissue

SES-induced inflammation stimulates the activation of immune signalling pathways in peritoneal stromal tissues leading to transcription factor activation, chromatin remodelling, and changes in gene regulation. Advances in next-generation sequencing methods (e.g., RNA-seq, ATAC-seq, ChIP-seq) now provide exciting opportunities to investigate the stromal tissue response to infection and the mechanisms that support innate and adaptive immunity. Consecutive challenge with SES leads to the development of adaptive immune responses in the peritoneal cavity that include the presence of IFNγ-secreting (Th1-polarized) CD4+ T-cells. These shift signalling dynamics in the peritoneal membrane to favor pro-fibrotic gene expression programs. Hence, the SES model supports the comparative study of acute resolving and non-resolving inflammatory responses. Analysis may be performed at the biochemical level through separation of cytosolic, membrane, and nuclear fractions, or at the genomic level through the isolation of mRNA and nuclear chromatin (Fig. 4). 1. Peritoneal membrane sections are excised postmortem and snap-frozen in liquid nitrogen for downstream processing (80 mg tissue). 2. For biochemical analysis by immunoblot, membrane is pulverized using a pestle and mortar. This may be aided through the parallel administration of small volumes of liquid nitrogen to aid generation of a fine cell powder and/or small tissue fragments. These are gathered in PBS and centrifuged at full speed in a microcentrifuge at 4  C. The tissue pellet is next resuspended in the appropriate lysis buffer. 3. To obtain cytosolic and membrane protein pellets for biochemical analysis, extracts are resuspended in extraction buffer and incubated on ice for 10 min. After centrifugation, pellets are resuspended in the respective solubilization buffers to obtain solubilized cytosolic and membrane fractions. 4. After centrifugation, the pellet is resuspended in Tris-buffered saline containing 1% (w/v) SDS to obtain the nuclear fraction. 5. Aliquots may be mixed with 4 LDS sample buffer supplemented with 10% (v/v) β-mercaptoethanol for analysis by SDS-PAGE.

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a

b

Analysis of peritoneal exudates

Analysis of stromal tissue

Methods ELISA Immunodetection methods Flow cytometry Imaging Quantitative PCR Next-generation sequencing methods

Peritoneal Lavage Detection of inflammatory mediators Peritoneal infiltrate Leukocyte numbers Leukocyte phenotype Leukocyte activity

Methods Peritoneal Tissue Histopathology Transcription factor activity Cell signalling Transcriptomic analysis mRNA expression Epigenetic studies Chromatin remodelling Transcription factor activity

Immunohistochemistry Western blot Immunoprecipitation (IP) Quantitative PCR RNA-seq Chromatin-IP ChIP-seq ATAC-seq

Fig. 4 (a) Peritoneal exudates are prepared from lavage samples. These can be used to investigate changes in inflammatory mediator production or leukocyte infiltration. (b) Parietal peritoneal membranes are identified as two sections (1 and 2) and can be harvested to evaluate histopathology and changes in genetic parameters. Methods listed in red highlight the types of approaches already used to evaluate inflammatory processes in response to SES or bacterial challenge

6. For biochemical fractionation (e.g., sucrose gradient or fast protein liquid chromatography) or enzymatic analysis (e.g., kinase assay, DUB assay), we recommend lysis in the absence of detergents using mechanical stress (e.g., resuspend in water and incubate on ice for 10 min prior to ~20–30 passes through a syringe). 7. For immunoblot analysis, protein is quantified using a BCA protein assay. Load ~10 μg protein/well for analysis by SDS-PAGE (based on an 8–12-well system). 8. For epigenetic studies, genomic DNA fractions may be subjected to ChIP by diluting (1:4) in Tris-buffered saline and overnight incubation with the appropriate antibody at the dilution factor recommended by the manufacturer. DNA is eluted and prepared for sequencing via standard protocols (e.g., Illumina). 9. Total mRNA may be prepared using standard extraction protocols. Extracts of peritoneal membrane (Fig. 4) are dissected directly into 1 mL RNA extraction buffer supplemented with β-mercaptoethanol and the tissue dissociated using a handheld electric homogenizer. Lysates are diluted 1:3 in distilled water and digested in 0.2 mg/mL Proteinase-K for 10 min at 55  C. Samples are cleared and RNA precipitated in 70% (v/v) ethanol.

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10. Following total RNA extraction, cytoplasmic, mitochondrial, and ribosomal RNA are depleted, and libraries prepared following manufacturer’s instructions. 11. The quality and quantity of total RNA or genomic DNA is established using nanodrop and the integrity confirmed on an Agilent 2100 Bioanalyzer.

4

Notes 1. The colonies should have a creamy and rounded aspect. 2. The suspension should have substantial turbidity. 3. It is suggested to prepare 4  400 mL of culture for each batch in order to obtain around 200 vials of SES at the end of the procedure. At this step, it is recommended to perform a Gram staining to characterize our culture. Staphylococcus epidermidis is a Gram-positive coccus. Under the microscope they should appear as rounded cells stained in dark blue following treatment with crystal violet. 4. The spectrophotometer is calibrated against Tyrode’s salt solution. Experience shows that a 1/32 dilution gives an optical density of 0.5–0.6 at 560 λ. However, it is advisable to perform serial dilutions until the optical density required is achieved. 5. Due to the composition of the SES, the filter may become blocked. If so, replace as necessary. 6. The dialysis is performed to remove the Tyrode’s salt from the SES suspension. The semipermeable membrane (MWCO 12–14,000 daltons) allows Tyrode’s salt solute diffusion into the dialysis solution. 7. To facilitate the handling of the dialysis membranes, wet them in deionized water before pouring the SES into them. Note that that preparation of dialysis membrane sometimes requires preparation in an EDTA solution. Check manufacturer instructions before commencing. 8. Make a hole in the Parafilm to allow sublimation of the frozen water during the lyophilization process. 9. Approximately 2.5–3  106 resident peritoneal tissue macrophages are isolated from each mouse lavage. Resuspend the cells in 1 mL of RPMIc and count them prior to adjustment to 2  106 cells/mL. 10. It is advised to prepare a non-stimulated control with 1  106 cells by adding 0.5 mL of RPMIc alone to 0.5 mL of cells.

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11. It is essential to ensure that no bubbles remain in the syringe or the needle before injection. To eliminate this concern always draw more SES suspension than is required for administration. 12. The experiments need to be approved by an institutional animal experimentation ethics committee and be conducted in compliance with the guidelines of the country’s governmental bodies that regulate animal experimentation. 13. To ensure optimal cell recovery by peritoneal lavage after SES stimulation, we recommend to lavage the cavity with 5 mL of RPMI. However, in order to assess the levels of inflammatory mediators, we recommend that the lavage is no bigger than 2 mL of PBS so as to avoid extreme dilution. 14. The model is also suitable for the study of resident peritoneal monocytic cells, for example, F4/80hi/CD11bhi monocytic cells, which show a classical disappearance reaction following the initial SES challenge. This population is replenished during the course of the model through proliferative mechanisms [17]. 15. The peritoneum is an asymmetrical membrane consisting of different cell types and extracellular matrix components. Therefore, different sample processing is required depending on the downstream applications. For example, a correct orientation of the peritoneal membrane is essential for immunohistochemistry analysis. For protein quantification or gene expression analysis (e.g., quantitative PCR, RNA-seq), it is recommended to snap-freeze the sample in liquid nitrogen immediately after collection (see outlined protocol). 16. This allows sufficient time for the resolution of inflammation between each SES treatment and the repeat administration promotes the focal development of fibrosis within the peritoneal membrane (days 42–49 after the first SES injection) [12]. 17. Analysis should be conducted quickly after the initial APF labelling (ideally with 30 min of sample collection). The addition of catalase and superoxide dismutase may be added to confirm the specificity of the fluorescent changes and links to the NADPH oxidase system. References 1. Hurst SM, Wilkinson TS, McLoughlin RM et al (2001) Il-6 and its soluble receptor orchestrate a temporal switch in the pattern of leukocyte recruitment seen during acute inflammation. Immunity 14:705–714 2. Fielding CA, McLoughlin RM, McLeod L et al (2008) IL-6 regulates neutrophil trafficking

during acute inflammation via STAT3. J Immunol 181:2189–2195 3. McLoughlin RM, Hurst SM, Nowell MA et al (2004) Differential regulation of neutrophilactivating chemokines by IL-6 and its soluble receptor isoforms. J Immunol 172:5676–5683 4. McLoughlin RM, Jenkins BJ, Grail G et al (2005) IL-6 trans-signaling via STAT3 directs

In Vivo Modelling of Chronic Peritoneal Inflammation T cell infiltration in acute inflammation. Proc Natl Acad Sci USA 102:9589–9594 5. McLoughlin RM, Witowski J, Robson RL et al (2003) Interplay between IFN-gamma and IL-6 signaling governs neutrophil trafficking and apoptosis during acute inflammation. J Clin Invest 112:598–607 6. Hams E, Colmont CS, Dioszeghy V et al (2008) Oncostatin M receptor-beta signaling limits monocytic cell recruitment in acute inflammation. J Immunol 181:2174–2180 7. Jones GW, McLoughlin RM, Hammond VJ et al (2010) Loss of CD4+ T cell IL-6R expression during inflammation underlines a role for IL-6 trans signaling in the local maintenance of Th17 cells. J Immunol 184:2130–2139 8. Nowell MA, Richards PJ, Horiuchi S et al (2003) Soluble IL-6 receptor governs IL-6 activity in experimental arthritis: blockade of arthritis severity by soluble glycoprotein 130. J Immunol 171:3202–3209 9. Richards PJ, Nowell MA, Horiuchi S et al (2006) Functional characterization of a soluble gp130 isoform and its therapeutic capacity in an experimental model of inflammatory arthritis. Arthritis Rheum 54:1662–1672 10. Nowell MA, Williams AS, Carty SA et al (2009) Therapeutic targeting of IL-6 trans signaling counteracts STAT3 control of experimental inflammatory arthritis. J Immunol 182:613– 622 11. Jones GW, Greenhill CG, Williams JO et al (2013) Exacerbated inflammatory arthritis in response to hyperactive gp130 signalling is independent of IL-17A. Ann Rheum Dis 72: 1738–1742 12. Fielding CA, Jones GW, McLoughlin RM et al (2014) Interleukin-6 signaling drives fibrosis in unresolved inflammation. Immunity 40:40–50 13. Jones SA (2005) Directing transition from innate to acquired immunity: defining a role for IL-6. J Immunol 175:3463–3468

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14. Jenkins BJ, Grail D, Nheu T et al (2005) Hyperactivation of Stat3 in gp130 mutant mice promotes gastric hyperproliferation and desensitizes TGF-beta signaling. Nat Med 11: 845–852 15. Colmont CS, Raby AC, Dioszeghy V et al (2011) Human peritoneal mesothelial cells respond to bacterial ligands through a specific subset of toll-like receptors. Nephrol Dial Transplant 26:4079–4090 16. Strunk T, Power Coombs MR, Currie AJ et al (2010) TLR2 mediates recognition of live Staphylococcus epidermidis and clearance of bacteremia. PLoS One 5:e10111 17. Liao CT, Rosas M, Davies LC et al (2016) IL-10 differentially controls the infiltration of inflammatory macrophages and antigenpresenting cells during inflammation. Eur J Immunol 46:2222–2232 18. Raby AC, Le Bouder E, Colmont C et al (2009) Soluble TLR2 reduces inflammation without compromising bacterial clearance by disrupting TLR2 triggering. J Immunol 183: 506–517 19. Raby AC, Holst B, Le Bouder E et al (2013) Targeting the TLR co-receptor CD14 with TLR2-derived peptides modulates immune responses to pathogens. Sci Transl Med 5: 185ra64 20. Raby AC, Colmont CS, Kift-Morgan A et al (2017) Toll-like receptors 2 and 4 are potential therapeutic targets in peritoneal dialysisassociated fibrosis. J Am Soc Nephrol 28:461– 478 21. Morgan AH, Dioszeghy V, Maskrey BH et al (2009) Phosphatidylethanolamine-esterified eicosanoids in the mouse: tissue localization and inflammation-dependent formation in Th-2 disease. J Biol Chem 284:21185–21191

Chapter 8 Assessing Lung Inflammation and Pathology in Preclinical Models of Chronic Obstructive Pulmonary Disease Ross Vlahos, Hao Wang, and Steven Bozinovski Abstract Chronic obstructive pulmonary disease (COPD) is an incurable disease that is a major cause of mortality and morbidity worldwide. Cigarette smoking is a major cause of COPD and triggers progressive airflow limitation, chronic lung inflammation, and irreversible lung damage and decline in lung function. COPD patients often experience various extrapulmonary comorbid diseases, including cardiovascular disease, skeletal muscle wasting, lung cancer, and cognitive decline which markedly impact on disease morbidity, progression, and mortality. People with COPD are also susceptible to respiratory infections which cause exacerbations of the underlying disease (AECOPD). The mechanisms and mediators underlying COPD and its comorbidities are poorly understood and current COPD therapy is relatively ineffective. We and others have used animal modelling systems to explore the mechanisms underlying COPD, AECOPD, and comorbidities of COPD with the goal of identifying novel therapeutic targets. Here we provide a preclinical model and protocols to assess the cellular, molecular, and pathological consequences of cigarette smoke exposure and the development of comorbidities of COPD. Key words COPD, Cigarette smoke, Comorbidities, AECOPD, Emphysema, Lung inflammation

1

Introduction Chronic obstructive pulmonary disease (COPD) is a major incurable global health burden and is the third leading cause of death worldwide [1]. A hallmark of COPD is airflow limitation, usually progressive, that is not fully reversible and is associated with dysregulated pulmonary inflammatory response to noxious particles and gases [1]. Cigarette smoking is the major cause of COPD and accounts for more than 95% of cases in industrialized countries, but other environmental pollutants are important causes in developing countries [1]. COPD is heterogeneous and encompasses chronic obstructive bronchiolitis with fibrosis and obstruction of small airways, leading to closure of small airways. Emphysema caused by enlargement of airspaces due to destruction of lung parenchyma and loss of lung elasticity can subsequently develop.

Brendan J. Jenkins (ed.), Inflammation and Cancer: Methods and Protocols, Methods in Molecular Biology, vol. 2691, https://doi.org/10.1007/978-1-0716-3331-1_8, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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Most patients with COPD have multiple pathologic conditions (chronic obstructive bronchiolitis, emphysema, and mucus plugging), and as the disease worsens, patients experience progressively more frequent and severe exacerbations, which are due in greatest part to viral and bacterial chest infections. Patients are also increasingly disabled by disease comorbidities, such as cardiovascular disease, skeletal muscle wasting, metabolic syndrome, cognitive decline, and lung cancer, which further reduce their quality of life [2–4]. Moreover, respiratory infections can worsen these comorbidities and further impact on the patient’s life. Current therapies for COPD are relatively ineffective, and the development of effective treatments for COPD has been severely hampered due to a poor understanding of the mechanisms and mediators that drive the induction and progression of chronic inflammation, emphysema, altered lung function, defective lung immunity, and systemic comorbidities. As cigarette smoke is the major cause of COPD, “smoking mouse” models, using either nose only or whole-body smoke exposure systems, that accurately reflect disease pathophysiology have been developed and have made rapid progress in identifying candidate pathogenic mechanisms and new therapies [5]. Many species have been used including rodents, dogs, guinea pigs, monkeys, and sheep [5]. However, mice remain the most popular choice by many researchers given the enormous information about the mouse genome, the abundance of antibody and gene probes, the ability to produce animals with genetic modifications that shed light on specific processes within COPD, the availability of numerous mouse strains with different responses to smoke, and ultimately the low cost. It has been established that there is excellent concordance between biological pathways initiated by cigarette smoke exposure in the lungs of humans and mice [6]. Regardless of the method of cigarette smoke exposure, many of the characteristic features of human COPD can be mimicked in the smoking mouse model. Acute and chronic cigarette smoke exposure protocols have been developed where acute protocols (1–4 days) are typically used to explore the mediators and mechanisms involved in the induction of cigarette smoke-induced lung inflammation [5]. Chronic cigarette smoke exposure protocols have largely been used to explore the mechanisms that drive chronic inflammation, impaired lung function, emphysema, small airway wall thickening, systemic comorbidities, and vascular remodeling [5]. Here we describe updated methodologies and protocols using a preclinical smoking mouse model to assess the cellular, molecular, and pathological consequences of cigarette smoke exposure and the development of COPD comorbidities.

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Materials Prepare all solutions described below using ultrapure water (prepared by purifying deionized water, to attain a sensitivity of 18 MΩ-cm at 25 °C) and analytical grade reagents. Prepare and store all reagents at room temperature (unless indicated otherwise). Diligently follow all waste disposal regulations when disposing waste materials.

2.1 Cigarette Smoke Exposure

1. 18-L Perspex chamber. 2. Specific pathogen-free male mice (e.g., BALB/c, C57BL/6) (see Note 1). 3. Winfield Red cigarettes (16 mg of tar, 1.2 mg of nicotine, and 15 mg of CO) (see Note 2).

2.2 Characterization of Cigarette Smoke Exposure: Total Suspended Particulate Mass and Particle Number Concentration

1. Polytetrafluoroethylene filters (47 mm Teflon, 2 μm pore size).

2.3 Bronchoalveolar Lavage and Lung Collection

1. Anesthetic (240 mg/kg sodium pentobarbital).

2. Microbalance with a resolution of 0.0001 mg.

2. Phosphate-buffered saline (PBS): 2.85 g/L NaHPO4.2H2O (16 mM HPO4), 0.625 g/L NaH2PO4.2H2O (4 mM PO4), 8.7 g/L NaCl (149 mM NaCl). 3. Weighing scales. 4. 27-gauge × ½ inch (0.43 × 13 mm) needle. 5. 1 mL and 5 mL insulin syringe. 6. Tracheotomy needle 21 gauge × 1 inch (0.80 × 25 mm). 7. Two curved tissue forceps. 8. Scissors. 9. Liquid nitrogen. 10. 1.7 mL Eppendorf tube.

2.4 Total and Differential Cell Counts

1. Ethidium bromide and acridine orange. 2. Neubauer hemocytometer. 3. Shandon CytoSpin 3 centrifuge. 4. Merck Microscopy Hemacolor and Kwik-Diff™ solution. 5. Microscope glass slides. 6. Glass coverslips. 7. Entellan® mounting medium.

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2.5 Carboxyhemoglobin Measurements

1. Anesthetic (240 mg/kg sodium pentobarbital).

2.6 RNA Extraction, cDNA Synthesis, and qPCR

1. Liquid nitrogen.

2. 27-gauge × ½ inch (0.40 × 13 mm) needle. 3. 1 mL heparinized blood gas syringe.

2. 1.7 mL Eppendorf tube. 3. RLT lysis buffer (RNeasy® Mini Kit 250). 4. β-Mercaptoethanol. 5. 21-gauge × 1 inch (0.80 × 25 mm) needle. 6. 1 mL syringe. 7. RNeasy Mini Kit 250. 8. NanoDrop 2000 spectrophotometer. 9. TaqMan primers.

2.7

Histology

1. Anesthetic (240 mg/kg sodium pentobarbital). 2. 27-gauge × ½ inch (0.40 × 13 mm) needle. 3. 10% neutral buffered formalin. 4. Hematoxylin and eosin stain.

2.8 Acute Exacerbations of COPD

1. Influenza A virus (H3N1, Mem 71 strain) (see Note 3).

2.9

1. Hank’s Balanced Salt Solution (HBSS).

Flow Cytometry

2. Fetal bovine serum (FBS), heat inactivated at 56 °C for 1 h, filtered, and stored in 50 mL aliquots at -20 °C. 3. FACS buffer: 2% FBS and 2 mM EDTA in HBSS (storage: 4 °C). 4. Ammonium-Chloride-Potassium (ACK) Red Cell Lysis Buffer: 150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA in H2O, pH 7.4 (storage: 4 °C). 5. Antibodies for flow cytometry; store at 4 °C in the dark (see Table 1). 6. 5 mL round bottom FACS tubes with 35 μm cell strainer snap caps. 7. Falcon 70 μm Cell Strainers. 8. Eppendorf 1.5 mL tubes and Falcon tubes (15 mL and 50 mL).

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Table 1 Antibodies for flow cytometry analysis Antibody

Conjugate

Clone

Dilution Supplier

CD11b

PE dazzle 594

M1/70

1/400

Biolegend

CD11c

BV650

N418

1/250

Biolegend

CD45

AlexaFluor700

30-F11

1/100

Biolegend

CD64

PE Cy7

X54–5/71

1/150

Biolegend

CD49b

BV711

HMα2

1/100

BD

LIVE/DEAD™ Fixable viability dye eFluor™ 780

1/1000 Thermo fisher scientific

Ly6C

AlexFluor488

HK1.4

1/300

Biolegend

Ly6G

BV785

1A8

1/250

Biolegend

MerTk

APC

2B10C42

1/200

Biolegend

MHCII

PerCp Cy5.5

M5/114.15.2 1/150

Biolegend

Siglec F

PE

E50–2440

BD Pharmingen

3 3.1

1/300

Methods Mice

1. Specific pathogen-free male mice (e.g., BALB/c, C57BL/6) aged 7 weeks and weighing ~20 g are obtained from an animal resource center (e.g., Animal Resources Centre, Western Australia, Australia) (see Note 1). 2. House mice at 20 °C on a 12:12 h day-night cycle in sterile microisolators and fed a standard sterile diet of Purina mouse chow with water ad libitum. The experiments need to be approved by an institutional animal experimentation ethics committee and be conducted in compliance with the guidelines of the country’s governmental bodies that regulate animal experimentation (see Note 4).

3.2 Cigarette Smoke Exposure and Euthanasia

1. Place mice in an 18-L Perspex chamber in a chemical hood and expose to cigarette smoke generated from 1 cigarette for 15 min. For sham-exposed mice, place mice in an 18-L Perspex chamber and do not expose to cigarette smoke (see Note 5). 2. The lid of the Perspex chamber is then opened for 5 min before being closed and the mice exposed again to cigarette smoke generated from 1 cigarette for 15 min. 3. Repeat step 2 such that mice are exposed to 3 cigarettes for 1 h, three times a day for 4 days (acute protocol), 8 weeks (5 consecutive days a week; sub-chronic protocol), or 24 weeks (5 consecutive days a week; chronic protocol), delivered at 9 AM, 12 noon, and 3 PM.

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4. Generate cigarette smoke in 50 mL tidal volumes over 10 s, by use of timed drawback mimicking normal smoking inhalation volume and cigarette burn rate. 5. Euthanize mice by an intraperitoneal overdose of anesthetic (240 mg/kg sodium pentobarbital) on the day after the last cigarette smoke exposure of the protocol (see Notes 6 and 7). 6. Lavage lungs with PBS as described below. 3.3 Total Suspended Particulate Mass and Particle Number Concentration

1. Collect total suspended particulate (TSP) matter on a polytetrafluoroethylene filter with a PMP support ring (47 mm Teflon, 2 μM pore size) enclosed in a polypropylene holder (47-mM holder), using a low-flow miniature diaphragm pump. 2. Calibrate pump to a volumetric flow rate of 50 mL/min using a National Association of Testing Authorities-certified soap bubble flowmeter. 3. Equilibrate the unexposed and cigarette smoke-exposed filter at a relative humidity of 50 ± 5% and a temperature of 21 ± 1 °C and weigh with a microbalance with a resolution of 0.0001 mg. 4. Determine the TSP mass concentration (in milligrams per cubic meter) by dividing the difference in mass between the cigarette exposed and unexposed filter by the volume of air sampled through the filter. The mean TSP mass concentration in the chamber containing cigarette smoke generated from one cigarette, measured from 3 min 13 s to 15 min, is typically about 420 mg/m3 for our setup [7]. 5. Measure the particle number concentration in the chamber immediately after injection of cigarette smoke into the chamber and again at the end of the 15-min period. Number concentration for a particle size range of 0.01 to >1.0 μm is measured with a handheld condensation particle counter.

3.4 Bronchoalveolar Lavage and Lung Collection

1. Euthanize mice with an intraperitoneal anesthetic overdose (240 mg/kg sodium pentobarbital) administered via a 27-gauge needle (see Notes 6 and 7). 2. Make a midline incision in the neck to expose the trachea and perform a tracheotomy using a blunt 21-gauge needle inserted into the trachea (see Note 8). 3. Lavage the lungs in situ with 0.4 mL of chilled PBS followed by three aliquots of 0.3 mL of PBS with ~1 mL of bronchoalveolar lavage fluid (BALF) recovered from each animal. 4. Clear the lungs of blood via right ventricular perfusion of the heart with 5 mL of PBS. 5. Rapidly excise the lungs, rinse in PBS, snap-freeze in liquid nitrogen, and store at -80 °C until required (see Note 9).

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BALF Cell Counts

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1. To determine the total viable cell numbers in BALF, take a 50 μL aliquot of BALF and dilute 1:2 with the fluorophores ethidium bromide/acridine orange and count on a standard hemocytometer with a fluorescence microscope (see Note 10). 2. Take 5 × 104 cells from the BALF samples and cytospin at 400 rpm for 10 min at room temperature to produce a single layer of cells on a glass slide for differential cell counts. 3. Allow cytospins to dry for 24 h, and then stain with Merck Microscopy Hemacolor and Kwik™Diff as stated by the manufacturer’s instructions, and then mount with Entellan® medium and coverslip. 4. Count the number of inflammatory cells (at least 500 cells per slide) using standard morphological criteria on a microscope equipped with a digital camera. 5. Centrifuge the remaining BALF at 3000 rpm for 5 min at 4 °C, and then collect the supernatant and store at -80 °C until required for analyses of proteins by standard ELISAs.

3.6 Carboxyhemoglobin Measurements

1. Remove the mouse from the smoke chamber, immediately euthanize with an intraperitoneal anesthetic overdose (240 mg/kg sodium pentobarbital) administered via a 27-gauge needle and collect the blood in a heparinized blood gas syringe from the abdominal vena cava. 2. Inject 85 μL of blood into a Radiometer ABL520 blood gas analyzer, which measures COHb across a range of 0–100%.

3.7 RNA Extraction, cDNA Synthesis, and qPCR

1. Crush the whole lung from each mouse into a powder using a mortar and pestle under liquid nitrogen (see Note 9). 2. Transfer 15 mg ground tissue powder into a 1.7 mL Eppendorf tube containing 600 μL RLT lysis buffer supplemented with a 1:100 dilution of β-mercaptoethanol (see Note 11). 3. Homogenize the tissue by passing it through a 21-gauge needle and 1 mL syringe 5–10 times. 4. Centrifuge at 15,000 rpm for 3 min, collect the supernatant, and purify total RNA using the RNeasy Mini Kit 250 according to the manufacturer’s instructions. 5. Determine the concentration, quality, and purity of the extracted RNA using a NanoDrop 2000 spectrophotometer to generate absorbance 260/280 ratios. A ratio of ~2.0 is generally accepted as “pure” for RNA and should fall within the generally accepted ratios of 1.8 and 2.0. 6. Synthesize single-strand cDNA by reverse transcription using 1 μg of RNA for downstream qPCR application using TaqMan primers as previously published [7] (see Note 12).

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Histology

1. Euthanize mouse by intraperitoneal anesthetic (240 mg/kg sodium pentobarbital) overdose and then perfuse fix lungs via a tracheal cannula with 10% neutral buffered formalin (NBF) at exactly 25 cmH2O pressure (see Note 13). 2. After 15 min, ligate the trachea and remove lungs from the thorax and immerse in 10% NBF for a minimum period of 24 h. 3. Once lungs are fixed, process in paraffin wax and cut 3–4 μm thick sections longitudinally through the left and right lung to include all lobes. 4. Stain lung sections with hematoxylin and eosin for general histopathology as previously published [7]. 5. Lung sections can also be investigated for signs of emphysema using the mean linear intercept method as previously published [8, 9].

3.9 Acute Exacerbations of COPD

1. Expose mice to cigarette smoke (see Subheading 3.2, steps 1–4). 2. Infect mice with influenza A virus (IAV, H3N1, Mem71 strain) as previously published [10] (see Note 3). 3. Cull mice 3–10 days post IAV infection and assess endpoints including lung inflammation, viral titers, lung pathology, body weight, and whole lung proinflammatory gene expression as previously published [10–12] (see Subheading 3.5, 3.6, 3.7 and 3.8; Note 3).

3.10 Preparation of Lung Cells for Flow Cytometry

1. Euthanize mouse by intraperitoneal anesthetic (240 mg/kg sodium pentobarbital) overdose. Clear the lungs of blood via right ventricular perfusion of the heart with 5 mL of PBS and excise the lung lobes (see Note 14). 2. Mince the lung lobes on a petri dish and transfer the lung homogenate into 1 mL Liberase™-containing HBSS. Digest the lungs by incubating at 37°C for 45 min with constant shaking. Dissociate the lung cells by passing the digested tissue through a 21-gauge needle. Stop the digestion by adding 10 mL FACS buffer. 3. Pellet the cells by centrifugation at 400 × g for 5 min at 4 °C. Resuspend the cell pellet in 1 mL ammonium-chloride-potassium (ACK) red cell lysis buffer and incubate for 1–2 min at room temperature. Stop the cell lysis by adding 10 mL HBSS to the cell suspension (see Note 15). 4. Filter the cell suspension through 70 μm cell strainers into a 50 mL Falcon tube. Take an aliquot (e.g., 200 μL) of the single-cell suspension for cell count and split the rest of the cells into desired FACS tubes. 5. Centrifuge the cells at 400 × g for 5 min at 4°C and discard supernatant.

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6. Perform the live/dead stain by adding freshly made fixable live/dead dye (80 μL per sample, 1:1000 in HBSS) to the cells and incubating on ice for 30 min in the dark. 7. Wash the cells by adding 0.5 mL FACS buffer and centrifuging at 400 × g for 5 min at 4 °C. Discard supernatants. 8. Resuspend the cells in 80 μL of 1× antibody mixture (Table 1) in FACS block (4 μg/mL anti-CD16/32 antibody in FACS buffer) and incubate on ice for 30 min in the dark. 9. Wash the cells by adding 0.5 mL FACS buffer and centrifuging at 400 × g for 5 min at 4 °C. Discard supernatants. 10. The stained cells can be resuspended in 100 μL FACS buffer for immediate analysis on a cytometer or fixed with eBioscience™ IC Fixation Buffer for analysis later (see Note 16). 3.11 Analyzing Innate Immune Cells Using Flow Cytometry

1. Start up cytometer and software and run unstained control to optimize voltages for forward scatter (FSC) and side scatter (SSC). Run single-stained controls to optimize voltages for each fluorescence channel and generate a compensation panel. Run experiment samples and apply the predetermined compensation values to the samples. 2. Set up the gating strategy (Fig. 1), which will allow for the analysis of innate immune cell populations including neutrophils, natural killer (NK) cells, eosinophils, and macrophage/ monocyte subsets: (a) FSC vs SSC to remove debris and cell doublets/ aggregates. (b) Viability versus autofluorescence to remove dead cells (see Note 17). (c) Ly6G versus CD11b to identify neutrophils. (d) From the non-neutrophils, CD64 versus CD49b to identify NK cells. (e) From the non-NK cells, CD11c versus CD11b to exclude lymphocytes. (f) From the non-lymphocytes, CD11c versus Siglec F to identify eosinophils. (g) From the non-eosinophils, CD64 versus MerTK to identify macrophages. (h) From the macrophages, CD11c versus CD11b to differentiate alveolar macrophages (AMs) and interstitial macrophages (IMs). (i) From the non-macrophage cells, identify MHCIICD11b+ monocyte subsets that are Ly6C high and Ly6C low.

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Siglec F

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Fig. 1 FACS gating for lung immune cells, including viable gate, pan immune cell marker CD45 gate, Ly6G neutrophil gate, CD49b NK cell gate, Siglec F eosinophil gate, alveolar macrophage (AM) and interstitial macrophage (IM) gates, and Ly6C high/low monocyte gate

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Notes 1. Mice are obtained from an approved and registered animal resource facility. In this case BALB/c and C57BL/6 mice were bought from the Animal Resource Facility, Western Australia, Australia. 2. Winfield Red cigarettes are purchased from a registered tobacco supply shop (e.g., Tobacco Station Group Pty Ltd., Melbourne, Australia). 3. The intermediate-virulence H3N1 (Mem71) strain of influenza A was prepared in-house as previously published [11]. Mem71 strain of influenza A is a genetic reassortant of A/Memphis/1/71 (H3N2) × A/Bellamy/42 (H1N1). Mem71 is used because, when used in combination with cigarette smoke exposure, it does not cause overt lung inflammation and pathology in mice. Endpoints are measured at day 3 or day 10 post influenza infection as these time points represent the peak and resolution (respectively) of influenza infection. There is no further cigarette smoke exposure after Mem71 infection. Mouse body weight, food consumption, and the well-being of mice are monitored daily at approximately the same time, to ensure that mice are not going through severe distress in response to cigarette smoke and influenza A virus infection.

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4. All experiments performed with mice need to be approved by an institutional animal experimentation ethics committee and be conducted in compliance with the guidelines of the country’s governmental bodies that regulate animal experimentation. 5. All smoking procedures must be performed in a chemical fume hood. Personal protective equipment (PPE) (e.g., gloves, laboratory coat, safety glasses) must always be worn. Ensure all flammable chemicals and substances are kept away from ignited cigarettes. After completion of the smoking procedure, cigarettes should be extinguished into a commercial ashtray located within the chemical fume hood. 6. When working with the mouse anesthetic sodium pentobarbital, ensure you wear a gown, gloves (irritant to skin), protective glasses (highly irritant to eyes), and closed shoes. Also note that working with and restraining mice involves risks as a result of bites, scratches, and the development of animal allergies. It is necessary that all staff and students undergo an animal handling course prior to commencing animal experimentation. 7. Working with needles and syringes involves risks to personnel because of needlestick injury. Ensure appropriate safety techniques are used. 8. For the BAL procedure, use a 21-gauge needle that has been cut to a length of ½ an inch and overlaid with a plastic cannula. This will ensure that the tracheotomy needle fits snuggly into the trachea and will not damage the trachea (i.e., pierce the tracheal wall causing the lavage fluid to leak). Clamp the trachea and tracheotomy needle together with a bulldog clamp. 9. Liquid nitrogen is a colorless, odorless, extremely cold liquid and gas under pressure. It can cause rapid suffocation when concentrations are sufficient to reduce oxygen levels below 19.5%. Contact with liquid nitrogen can cause severe frostbite. Ensure appropriate PPE is used when handling liquid nitrogen, especially cold-resistant gloves, face shield, and lab coat. 10. Ethidium bromide (EtBr) is toxic, a potent mutagen and the powder form irritant to the skin and upper respiratory tract. Wear appropriate PPE and nitrile disposable gloves when working with EtBr. 11. Buffers used for qPCR are toxic, corrosive, and flammable. Appropriate PPE (e.g., gloves, safety glasses, lab coat) must be worn when working with the solutions. All work must be conducted in a chemical hood. Waste solutions must be placed in a chemically inert bottle for removal by contracted waste management company later. Do not work near ignition sources.

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12. The TaqMan Gene Expression Assay probes contain some mild carcinogens. Wear PPE such as gloves and lab coat while performing the procedure. Place used plates, tubes, and reagents into a biohazard container. Electrical equipment is involved and must be checked yearly and tagged. 13. Neutral buffered formalin is highly toxic and contact with the skin, eyes, or mucous membrane should be avoided. Preparation should be carried out in a chemical hood, while wearing gloves, safety glasses, and a protective. 14. When flow cytometry is performed on multiple animals, excised lungs can be temporarily stored in HBSS on ice until all samples are ready for the next procedure. 15. ACK lysis buffer is formulated to lyse nonnucleated erythrocytes without affecting leukocytes. The incubation time can be extended when an excessive amount of red blood cells is present due to insufficient perfusion and removal of the blood; however, prolonged incubation (e.g., >5 min) is generally not recommended. 16. The eBioscience™ IC Fixation Buffer contains paraformaldehyde which is toxic and needs to be handled with care. Stained cells can slightly change their profiles (e.g., cell size and antibody fluorescence intensity) after fixation and hence should not be compared directly with fresh cells. 17. Alveolar macrophages tend to have high autofluorescence as demonstrated in Fig. 1. Care needs to be taken when determining the gate of live cells that are negative for fixable viability dye.

Acknowledgements This work was supported by National Health and Medical Research Council Australia Project Grants 1138915, 1139843, and 1120522 awarded to Professors Vlahos and Bozinovski. References 1. Vogelmeier CF, Criner GJ, Martinez FJ et al (2017) Global strategy for the diagnosis, management, and prevention of chronic obstructive lung disease 2017 report. GOLD executive summary. Am J Respir Crit Care Med 195: 557–582 2. Brassington K, Selemidis S, Bozinovski S et al (2019) New frontiers in the treatment of comorbid cardiovascular disease in chronic obstructive pulmonary disease. Clin Sci 133: 885–904

3. Chan SMH, Selemidis S, Bozinovski S et al (2019) Pathobiological mechanisms underlying metabolic syndrome (MetS) in chronic obstructive pulmonary disease (COPD): clinical significance and therapeutic strategies. Pharmacol Ther 198:160–188 4. Dobric A, De Luca SN, Spencer SJ et al (2022) Novel pharmacological strategies to treat cognitive dysfunction in chronic obstructive pulmonary disease. Pharmacol Ther 233:108017

Pre-Clinical Models of COPD 5. Vlahos R, Bozinovski S (2014) Recent advances in pre-clinical mouse models of COPD. Clin Sci 126:253–265 6. Morissette MC, Lamontagne M, Berube JC et al (2014) Impact of cigarette smoke on the human and mouse lungs: a gene-expression comparison study. PLoS One 9:e92498 7. Vlahos R, Bozinovski S, Jones JE et al (2006) Differential protease, innate immunity, and NF-kappaB induction profiles during lung inflammation induced by subchronic cigarette smoke exposure in mice. Am J Physiol Lung Cell Mol Physiol 290:L931–L945 8. Ruwanpura SM, McLeod L, Dousha LF et al (2016) Therapeutic targeting of the IL-6 transsignaling/mechanistic target of rapamycin complex 1 axis in pulmonary emphysema. Am J Respir Crit Care Med 194:1494–1505

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9. Dobric A, De Luca SN, Seow HJ et al (2022) Cigarette smoke exposure induces neurocognitive impairments and neuropathological changes in the hippocampus. Front Mol Neurosci 15:893083 10. Oostwoud LC, Gunasinghe P, Seow HJ et al (2016) Apocynin and ebselen reduce influenza A virus-induced lung inflammation in cigarette smoke-exposed mice. Sci Rep 6:20983 11. Gualano RC, Hansen MJ, Vlahos R et al (2008) Cigarette smoke worsens lung inflammation and impairs resolution of influenza infection in mice. Respir Res 9:53 12. Mou K, Chan SMH, Brassington K et al (2022) Influenza A virus-driven airway inflammation may be dissociated from limb muscle atrophy in cigarette smoke-exposed mice. Front Pharmacol 13:859146

Chapter 9 Preclinical Mouse Model of Silicosis Maggie Lam, Ashley Mansell, and Michelle D. Tate Abstract Silicosis is an untreatable occupational lung disease caused by chronic inhalation of crystalline silica. Cyclical release and reuptake of silica particles by macrophages and airway epithelial cells causes repeated tissue damage, characterized by widespread inflammation and progressive diffuse fibrosis. While inhalation is the main route of entry for silica particles in humans, most preclinical studies administer silica via the intratracheal route. In vivo mouse models of lung disease are valuable tools required to bridge the translational gap between in vitro cell culture and human disease. This chapter describes a mouse model of silicosis which mimics clinical features of human silicosis, as well as methods for intranasal instillation of silica and disease analysis. Lung tissue can be collected for histological assessment of silica particle distribution, inflammation, structural damage, and fibrosis in sections stained with hematoxylin and eosin or Masson’s trichrome. This approach can be extended to other chronic fibrotic lung diseases where inhalation of small damaging particles such as pollutants causes irreversible disease. Key words Intranasal, Silicosis, Inflammation, Inflammasome, Fibrosis, Histology, Microscopy

1

Introduction Repeated occupational inhalation of silica dust leads to persistent inflammation and lung fibrosis, culminating in respiratory failure [1]. Despite global efforts to improve safety regulations in mining and manufacturing industries, silicosis remains a considerable health burden with no effective therapies available [2]. The most distinctive hallmark of silicosis is the presence of silicotic nodules which are granuloma-like structures consisting of discrete fibrotic lesions surrounded by connective tissue and inflammatory cells [3]. Silicotic nodules may coalesce to form larger regions of fibrosis, known as progressive massive fibrosis (PMF), to gradually replace healthy lung parenchyma [3]. Under polarizing light, silica can be detected in dust-laden macrophages within these mass conglomerates as weak birefringent particles [4, 5]. Although the clinical features of silicosis are well documented, the underlying mechanisms which may contribute to disease progression require further

Brendan J. Jenkins (ed.), Inflammation and Cancer: Methods and Protocols, Methods in Molecular Biology, vol. 2691, https://doi.org/10.1007/978-1-0716-3331-1_9, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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investigation. Evaluation of responses in human lung tissue from silicosis patients is often limited by sample availability, relying on the provision of donor samples. Preclinical studies using mice have replicated the major hallmarks of silicosis and permitted insights into pathways involved in promoting lung inflammation and fibrosis. Respirable silica particles can disrupt or evade normal mucociliary clearance mechanisms and may reach the distal lung where they become deposited in terminal alveoli [6, 7]. Resident alveolar macrophages can engulf the silica particles, leading to lysosomal damage and oxidative stress [8–10]. Recruitment of neutrophils, T helper 17 (Th17) cells, and fibroblasts to sites of injury may further contribute to structural damage of distal airways and lung tissue. Repeated damage to the protective epithelial layer of airways and alveoli results in elevated production of the profibrotic cytokine transforming growth factor-β (TGFβ). This in turn stimulates the proliferation of fibroblast and subsequent production of excessive amounts of extracellular matrix (ECM) components including collagen and fibronectin [11, 12]. Alveolar epithelial cells also undergo a process termed epithelial mesenchymal transition where they acquire a mesenchymal phenotype to further increase ECM production [12]. In the absence of effective treatments, the combination of persistent inflammation and irreversible pulmonary fibrosis overwhelms protective immune responses and rapidly diminishes lung function. Notably, the nucleotide-binding domain and leucine-rich repeat pyrin domain-like receptor 3 (NLRP3) inflammasome has been identified as the primary immune sensor of silica and contributes to the sequalae of pulmonary inflammation and fibrosis associated with disease [1, 8, 13, 14]. Activation of the NLRP3 inflammasome leads to the autocatalysis of caspase-1 which subsequently processes the cytokines interleukin (IL)-1β and IL-18 to their mature forms [8, 15, 16]. Caspase-1 can also cleave gasdermin D to induce lytic cell death or pyroptosis [17, 18]. Membrane rupture releases danger-associated molecular patterns (DAMPs) such as high-mobility group box 1 (HMGB1), as well as the silica particle which can be re-engulfed by surrounding macrophages. While preclinical studies have identified a crucial role for the NLRP3 inflammasome in the pathogenesis of silicosis, NLRP3mediated responses have yet to be leveraged as potential biomarkers for disease diagnosis or progression and, potentially, as therapeutic targets. Studies which have assessed silica-induced cytotoxicity and immune responses suggest that particle size is an important factor in determining disease severity. Silica 10 mg/kg pentobarbitone into peritoneal cavity (see Note 4). 3. Ensure mouse is euthanized by monitoring reflexes (i.e., no toe pinch response). Lungs can be collected for histological staining for inflammation and fibrosis.

3.3 Harvesting of Lung Tissue

1. Expose the trachea of the mouse using dissection tools. 2. Make a horizontal cut on each side of the sternum, close to the bottom of the ribcage. Be careful not to puncture lung tissue. 3. Expose trachea by bluntly removing surrounding tissue. 4. Hold the mouse upright and make a small incision in the trachea using an 18-gauge needle. 5. Fill a 3 mL syringe with 2 mL of 10% NBF and connect it to the plastic cannula. 6. While holding the mouse upright, insert the plastic cannula into the incision made in the trachea. Inject 20 cells) in a T25 tissue culture dish (see Note 10). 2. Once confluent, disrupt the cell clusters through vigorous pipetting and count the cells using a hemocytometer. Passage them into 3 × T25 dishes, each in 5 mL complete RPMI, and expand to confluency.

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Fig. 1 Synergistic activation of hTLR8 by poly(dT) oligonucleotide in conjugation with uridine. HEK-TLR8 cells expressing the NF-κB–luciferase reporter were pre-treated with 1 μM dT20 for 30 min and stimulated, or not, overnight with 20 mM uridine. The NF-κB–luciferase values were normalized to the non-treated (NT) condition. Data shown are averaged from two independent experiments in biological triplicate (± standard error of the mean), and one-way ANOVA with Dunnett’s multiple comparisons to NT condition is shown. *P < 0.05, ****P < 0.0001 3.2.1 Oligonucleotide Treatment and TLR8 Stimulation

This protocol details potentiation of TLR8 sensing in nonadherent THP-1 cells, as reported in our recent publication [14]. 1. In the morning, seed 60,000 THP-1 cells per well in 150 μL of complete RPMI into 12 wells of a 96-well plate. 2. In the afternoon, prepare an intermediate 800 nM dT20 solution and 800 nM ASO solution to be tested by adding 0.6 μL of original 100 μM stock to 75 μL complete RPMI. Treat the cells with 25 μL per well of the 800 nM oligonucleotide solution and incubate the cells overnight at 37 °C in 5% CO2 (see Note 11). 3. The next morning, prepare an 8 μg/mL R848 solution by adding 0.6 μL of R848 stock solution in 75 μL complete DMEM. Stimulate the cells for 7 h by adding 25 μL of diluted R848 solution per well, giving a final concentration of 1 μg/ mL R848 and 100 nM oligonucleotides in 200 μL final volume. 4. Seven hours after R848 stimulation, collect supernatants for analysis of IP-10 production (see Note 12).

3.2.2 IP-10 Production Analysis by ELISA

1. The day before the assay (or up to 2 weeks in advance), coat a MaxiSorp™ 96-well plate with 80 μL of capture antibody diluted 1:500 in coating buffer, and leave sealed with tape at 4 °C. The morning of the assay, rinse the plate three times with PBS + 0.05% Tween-20 (PBST) and block with 100 μL assay diluent solution for 1 h at room temperature, with gentle rocking (see Note 13).

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2. Following blocking, wash the plate three times with PBST. Prepare the IP-10 standard curve following the Analysis Certificate leaflet from the kit, to give a concentration range from 1000 pg/mL to 15.6 pg/mL (seven points) recombinant IP-10. Add 60 μL of neat supernatant or standard to each well of the ELISA plate, and incubate for 2 h at room temperature, with rocking. 3. Wash the plate three times with PBST and prepare the detection antibody. Dilute both detection antibody and streptavidin–horseradish peroxidase (SAv-HRP) to 1:500 in assay diluent. Incubate for 10 min before adding 90 μL per well, and incubate for a further 1 h at room temperature. 4. Wash the plate five times with PBST and perform the enzymatic assay. Add 90 μL of pre-warmed TMB (at 25–37 °C) per well and incubate at room temperature in the dark until a blue color develops. 5. Stop the reaction with 60 μL of 0.5 M sulfuric acid. Read the absorbance in a plate reader at 450 nm within 30 min.

4

Notes 1. Cells should be passaged at least once before use in the assay. 2. HEK-TLR8 cells are poorly adherent; perform gentle and quick DPBS washes to avoid losing the cells. 3. Blasticidin-selective antibiotic should be added only when cells have been passaged twice after being brought back from frozen stocks. 4. pNF-κB-Luc4 transfection will result in the expression of firefly luciferase upon activation of TLR8. In this setup, there is no need to co-transfect a second reporter (such as Renilla luciferase), given that all subsequent conditions will use the same parental transfection of pNF-κB-Luc4. 5. These cells grow very fast and overnight incubation may lead to over-confluence. Therefore, 4 h of incubation is enough for transfection of the reporter plasmid using Lipofectamine 2000. 6. All the wells should have a final volume of 200 μL. 7. Make sure the cells are pre-treated with the oligonucleotides before stimulation with the ligand, as the other way around may not work as well. 8. Make sure that there is no supernatant left in the wells, as this will affect the total amount of lysate. Lysates can be used fresh or stored at -80 °C for later use.

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9. The luciferase assay reagent should be dispensed into working aliquots and stored at -80 °C. Multiple freeze-thaw cycles should be avoided. 10. THP-1 are suspension cells which grow as aggregates. Do not passage them harshly and always include a proportion of conditioned media when splitting into a fresh flask. After several passages the cells are likely to stop aggregating and become more individual—at that time the cells should stopped being used. 11. THP-1 cells require overnight treatment with ASO oligonucleotides. As such, short pre-treatment of the cells is not sufficient for potentiation of 2′OMe ASOs in this system. 12. THP-1 cells also have a basal level of TLR7 expression. Consequently, prolonged R848 stimulation beyond 8 h may also lead to activation of TLR7-dependent signalling. TLR8-specific ligands such as motolimod can be used to circumvent this. 13. We found that the recommended dilutions of this ELISA kit could be halved—i.e., if recommended to be used at 1/250 by the manufacturer, we use 1/500 of the antibody. Note that the individual vials of the kit may have different recommended dilutions and that these can vary between batches.

Acknowledgements We thank M. Speir for editorial assistance. This work was supported by funding from the Australian National Health and Medical Research Council (2020565 to MG) and the Victorian Government’s Operational Infrastructure Support Program. References 1. Vickers TA, Koo S, Bennett CF et al (2003) Efficient reduction of target RNAs by small interfering RNA and RNase H-dependent antisense agents. A comparative analysis. J Biol Chem 278:7108–7118 2. Wu H, Lima WF, Crooke ST (1999) Properties of cloned and expressed human RNase H1. J Biol Chem 274:28270–28278 3. Crooke ST, Liang XH, Baker BF et al (2021) Antisense technology: a review. J Biol Chem 296:100416 4. Terada C, Kawamoto S, Yamayoshi A et al (2022) Chemistry of therapeutic oligonucleotides that drives interactions with biomolecules. Pharmaceutics 14:2647

5. Eckstein F (2014) Phosphorothioates, essential components of therapeutic oligonucleotides. Nucleic Acid Ther 24:374–387 6. Crooke ST, Wang S, Vickers TA et al (2017) Cellular uptake and trafficking of antisense oligonucleotides. Nat Biotechnol 35:230–237 7. Liang XH, Sun H, Shen W et al (2015) Identification and characterization of intracellular proteins that bind oligonucleotides with phosphorothioate linkages. Nucleic Acids Res 43: 2927–2945 8. Shen W, De Hoyos CL, Migawa MT et al (2019) Chemical modification of PS-ASO therapeutics reduces cellular protein-binding and improves the therapeutic index. Nat Biotechnol 37:640–650

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9. Migawa MT, Shen W, Wan WB et al (2019) Site-specific replacement of phosphorothioate with alkyl phosphonate linkages enhances the therapeutic profile of gapmer ASOs by modulating interactions with cellular proteins. Nucleic Acids Res 47:5465–5479 10. Kariko´ K, Buckstein M, Ni H et al (2005) Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity 23:165–175 11. Judge AD, Sood V, Shaw JR et al (2005) Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nat Biotechnol 23:457–462 12. Judge AD, Bola G, Lee AC et al (2006) Design of noninflammatory synthetic siRNA mediating potent gene silencing in vivo. Mol Ther 13: 494–505 13. Zhu Y, Zhu L, Wang X et al (2022) RNA-based therapeutics: an overview and prospectus. Cell Death Dis 13:644 14. Alharbi AS, Garcin AJ, Lennox KA et al (2020) Rational design of antisense oligonucleotides modulating the activity of TLR7/8 agonists. Nucleic Acids Res 48:7052–7065 15. Valentin R, Wong C, Alharbi AS et al (2021) Sequence-dependent inhibition of cGAS and TLR9 DNA sensing by 2′-O-methyl gapmer oligonucleotides. Nucleic Acids Res 49:6082– 6099 16. Lind NA, Rael VE, Pestal K et al (2022) Regulation of the nucleic acid-sensing Toll-like receptors. Nat Rev Immunol 22:224–235 17. Tanji H, Ohto U, Shibata T et al (2013) Structural reorganization of the Toll-like receptor 8 dimer induced by agonistic ligands. Science 339:1426–1429 18. Ostendorf T, Zillinger T, Andryka K et al (2020) Immune sensing of synthetic, bacterial, and protozoan RNA by Toll-like receptor 8 requires coordinated processing by RNase T2 and RNase 2. Immunity 52:591–605.e596 19. Greulich W, Wagner M, Gaidt MM et al (2019) TLR8 is a sensor of RNase T2 degradation products. Cell 179:1264–1275.e1213 20. Tanji H, Ohto U, Shibata T et al (2015) Tolllike receptor 8 senses degradation products of single-stranded RNA. Nat Struct Mol Biol 22: 109–115 21. Shibata T, Ohto U, Nomura S et al (2016) Guanosine and its modified derivatives are endogenous ligands for TLR7. Int Immunol 28:211–222 22. Jurk M, Kritzler A, Schulte B et al (2006) Modulating responsiveness of human TLR7

and 8 to small molecule ligands with T-rich phosphorothiate oligodeoxynucleotides. Eur J Immunol 36:1815–1826 23. Gorden KK, Qiu X, Battiste JJ et al (2006) Oligodeoxynucleotides differentially modulate activation of TLR7 and TLR8 by imidazoquinolines. J Immunol 177:8164–8170 24. He M, Soni B, Schwalie PC et al (2022) Combinations of Toll-like receptor 8 agonist TL8-506 activate human tumor-derived dendritic cells. J Immunother Cancer 10:e004268 25. McWhirter SM, Jefferies CA (2020) Nucleic acid sensors as therapeutic targets for human disease. Immunity 53:78–97 26. Sun H, Li Y, Zhang P et al (2022) Targeting Toll-like receptor 7/8 for immunotherapy: recent advances and prospectives. Biomark Res 10:89 27. Baird JR, Monjazeb AM, Shah O et al (2017) Stimulating innate immunity to enhance radiation therapy-induced tumor control. Int J Radiat Oncol Biol Phys 99:362–373 28. Hornung V, Rothenfusser S, Britsch S et al (2002) Quantitative expression of Toll-like receptor 1-10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides. J Immunol 168:4531–4537 29. Lu H, Dietsch GN, Matthews MA et al (2012) VTX-2337 is a novel TLR8 agonist that activates NK cells and augments ADCC. Clin Cancer Res 18:499–509 30. Dang Y, Rutnam ZJ, Dietsch G et al (2018) TLR8 ligation induces apoptosis of monocytic myeloid-derived suppressor cells. J Leukoc Biol 103:157–164 31. Quemener AM, Bachelot L, Forestier A et al (2020) The powerful world of antisense oligonucleotides: from bench to bedside. Wiley Interdiscip Rev RNA 11:e1594 ¨ et al (2022) 32. Lorentzen CL, Haanen JB, Met O Clinical advances and ongoing trials on mRNA vaccines for cancer treatment. Lancet Oncol 23:e450–e458 33. Burris HA, Patel MR, Cho DC et al (2019) A phase 1, open-label, multicenter study to assess the safety, tolerability, and immunogenicity of mRNA-4157 alone in subjects with resected solid tumors and in combination with pembrolizumab in subjects with unresectable solid tumors (Keynote-603). JCO Glob Oncol 5:93 34. Kranz LM, Diken M, Haas H et al (2016) Systemic RNA delivery to dendritic cells exploits antiviral defence for cancer immunotherapy. Nature 534:396–401

Chapter 18 Exploring Allosteric Inhibitors of Protein Tyrosine Phosphatases Through High-Throughput Screening Takeru Hayashi and Masanori Hatakeyama Abstract High-throughput screening (HTS) using a natural or synthetic chemical or natural product library is a powerful technique for discovering novel small-molecular-weight compounds in order to develop drugs that specifically inhibit or activate molecular targets, malfunctioning of which underlies the development of diseases, especially malignant neoplasms. In contrast to a large number of successful cases in obtaining inhibitors against protein tyrosine kinases (PTKs) using HTS, however, the development of selective inhibitors for protein tyrosine phosphatases (PTPs) has lagged since PTP family members share highly conserved catalytic domain structures. Here, in this chapter we describe a novel method for exploring seed compounds of allosteric PTP inhibitors from a chemical/natural product library through HTS. Key words Protein tyrosine phosphatase, Allosteric inhibitor, High-throughput screening, Quality control

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Introduction The development of molecular-targeted inhibitors/activators that can be clinically applied for treatment is one of the goals of diseaserelated life science research. Although much progress has been made in the in silico technique of virtual screening, experimental screening of a chemical or natural product library is still a powerful tool for identifying a novel chemical structure as a seed for drug development. In the field of translational and clinical cancer research, PTKs are major targets for the treatment of cancer patients [1, 2]. Actually, a number of clinically available smallmolecule inhibitors as well as antibodies against PTKs, which provoke oncogenic intracellular signaling, have been developed for cancer treatment [1, 2].

Brendan J. Jenkins (ed.), Inflammation and Cancer: Methods and Protocols, Methods in Molecular Biology, vol. 2691, https://doi.org/10.1007/978-1-0716-3331-1_18, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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PTPs, which mediate inverse reactions to PTKs, also play important roles in intracellular signaling pathways, and deregulation of PTPs is observed in a variety of human diseases including malignancies [3–7]. Among the PTPs, SH2 domain-containing protein tyrosine phosphatase 2 (SHP2) is the best characterized pro-oncogenic tyrosine phosphatase that mediates RAS/MAPK, PI3K/AKT, and PD-1/PD-L1 pathways [8–10]. Mechanistically, SHP2 forms an autoinhibitory intramolecular interaction between N-SH2 and PTP domains and is allosterically activated by binding of tyrosine-phosphorylated effector ligands [11, 12]. We have shown that SHP2 contributes to gastric carcinogenesis through aberrant activation by binding with the tyrosine-phosphorylated Helicobacter pylori CagA protein, which is directly injected into attached gastric epithelial cells via a bacterial microsyringe termed the type IV secretion system [12–14]. The CagA-SHP2 interaction aberrantly activates mitogenic/pro-oncogenic RAS-MEK signaling. In addition, gain-of-function mutations in the PTPN11 gene, which encodes SHP2, have been found in childhood leukemias as well as solid tumors. Overexpression of SHP2 has also been found in several types of cancer. Based on these observations, SHP2 has attracted the attention of many cancer researchers as an attractive molecular target of cancer treatment. However, PTPs are noted for their structurally conserved catalytic domains with a high polarity nature of the catalytic clefts. As a consequence, catalytic sitetargeted inhibitors for PTPs show relatively low molecular selectivity [15–18]. In 2016, the research team of Novartis Institute for Biomedical Research reported the innovative allosteric SHP2 inhibitor SHP099 that binds to the inter-domain cavity in SHP2 with little to no blocking of other tyrosine phosphatases including SHP1, the closest homolog of SHP2 [19]. Thus, targeting the allosteric mechanism of PTPs represents an attractive strategy to obtain selective PTP inhibitors. Here, we describe procedures for screening allosteric PTP inhibitors from a chemical library through performing HTS. Most of the recent studies on allosteric PTP inhibitors have utilized 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as a surrogate substrate that shows fluorescence upon dephosphorylation [19–22]. Although DiFMUP is detected upon dephosphorylation with higher sensitivity than that for p-nitrophenyl phosphate ( pNPP), as an experimentally used classical substrate of PTPs, it has a high cost per assay well. Since thousands, or even up to a million, of compounds may need to be assessed via HTS, the total assay cost should be critically evaluated. A method that may enable an allosteric inhibitor of PTPs to be obtained through HTS using low-cost pNPP with proper quality control is described in this chapter.

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Materials

2.1 Reagents and Equipment for Assays

1. Falcon tubes (50 mL). 2. 10× assay buffer: 300 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), pH 7.5, 1.2 M NaCl, 0.1% (v/v) Triton X-100, 50 mM DTT. Aliquot in 50-mL Falcon tubes (e.g., 40 mL/tube) and store at -20 °C (see Note 1). 3. 1× assay buffer: 30 mM HEPES, pH 7.5, 120 mM NaCl, 0.01% (v/v) Triton X-100, 5 mM DTT. Prepare on the day of assay and leave at room temperature (see Note 2). 4. 100 mM p-nitrophenylphosphate ( pNPP): Dissolve in 1× assay buffer. Aliquot in 50-mL Falcon tubes (e.g., 40 mL/tube) and store at -20 °C. 5. Purified allosteric PTP of interest. 6. Purified isolated catalytic domain of the PTP. 7. Purified regulatory effector molecule of the PTP. 8. 384-well microplates, clear flat bottom, nonbinding. 9. Plate stickers. 10. Microplate dispenser. 11. Microplate reader, absorbance at 405 nm. 12. Microplate mixer. 13. Microplate centrifuge with swing-bucket rotor. 14. Plate incubator, 37 °C. 15. Automated 8-channel pipette. 16. DMSO. 17. Na3VO4, dissolved in DMSO. 18. Chemical library, dissolved in DMSO.

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Methods Before performing actual HTS, determine the appropriate conditions of the enzymatic reaction including final concentrations of an arbitrary enzyme, effector, and substrate and composition of the reaction buffer by a small-scale scouting experiment. On the premise that the reaction conditions have been determined, the procedures for a preliminary pilot experiment using a blank compound (DMSO) instead of a chemical library on triplicate plates, quality control of the multi-sample assay, and an actual screening experiment are described below in this section. Since the way in which the

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chemical library is provided is highly dependent on the situation, details on how to prepare the actual library plates are not provided (see Note 3). Perform all procedures at room temperature unless otherwise indicated. 3.1 Pilot Operation Using a 384-Well Microplate Format

1. Design the place of each control/sample on a microplate. We use the plate format shown in Fig. 1 and this protocol also follows it. 2. Prepare enzyme solutions (2×) and substrate solutions (2×) by diluting with 1× assay buffer in 50-mL tubes (see Note 4). (a) 40 mL solution of the allosteric enzyme (2×) (b) 40 mL solution of the isolated catalytic domain (2×) (c) 40 mL solution of the substrate containing the effector (2×) (d) 45 mL solution of the substrate without the effector (2×). 3. Add DMSO and positive controls to six 384-well microplates using an automated 8-channel pipette (see Note 5) (Fig. 1a). (a) Add 1 μL of DMSO to all the wells except I2-P2 wells on all of the plates. (b) Add 1 μL of 400 μM Na3VO4 dissolved in DMSO to I2-P2 wells on all of the plates. In the following procedures, three plates (triplicate) are used for allosterically activated phosphatase reaction (hereafter “allosteric reaction plates”) and the remaining three are used for non-allosteric reaction by an isolated catalytic domain alone (hereafter “non-allosteric rection plates”). 4. Seal with plate stickers and centrifuge at 100×g for 10 s. 5. Add enzyme solution (2×) and 1× assay buffer as follows (Fig. 1b): (a) Add 20 μL of allosteric enzyme solution (2×) to all of the wells on the allosteric reaction plates using a microplate dispenser (see Note 6). (b) Add 20 μL of isolated PTP domain solution (2×) to A2-P23 wells on the non-allosteric plates using a microplate dispenser. (c) Add 20 μL of 1× assay buffer to A1–P1 and A24–P24 wells on the non-allosteric reaction plates using an automated 8-channel pipette (see Note 7). 6. Seal with microplate stickers. 7. Shake on a microplate mixer and centrifuge at 100×g for 10 s. 8. Incubate at room temperature for 30–60 min. 9. Add substrate solution (2×) to all of the wells as follows:

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Fig. 1 A 384-well microplate format for HTS. Typical format for HTS using a 384-well microplate. (a) Two columns at each side should be used for background and controls on all of the microplates tested. Columns 3–22 are used for examination of inhibition activity of the chemical library. (b) Simplified table for preparation of reaction mixtures

(a) Add 20 μL of the substrate solution without the effector (2×) to A1–P1 and A24–P24 wells on the allosteric reaction plates using an automated 8-channel pipette. (b) Add 20 μL of the substrate solution containing the effector (2×) to A2–P23 wells on the allosteric reaction plates using a microplate dispenser.

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(c) Add 20 μL of the substrate solution without the effector (2×) to all of the wells on the non-allosteric reaction plates using an automated 8-channel pipette. 10. Seal with microplate stickers. 11. Shake on a microplate mixer and centrifuge at 100×g for 10 s. 12. Remove the plate stickers and then measure the absorbance at 405 nm at 0 min (Abs4050 min) with a microplate reader. 13. Incubate at 37 °C for 60 min with plate stickers (see Note 8). 14. Remove the plate stickers and then measure the absorbance at 405 nm at 60 min (Abs40560 min) with a microplate reader. 3.2 Quality Control of the Multi-sample Assay on a Full-Plate Format

1. Calculate net absorbance change (Abs405Diff) for all the wells by subtracting Abs4050 min from Abs40560 min. 2. Make a heat map representation of Abs4050 min, Abs40560 min, and Abs405Diff to instantly visualize the technical accuracy of preparation of the reaction mixture on a full-plate format (see Note 9) (Fig. 2). In addition to intraplate comparison, interplate stability of the assay should also be checked. 3. Calculate the average (Av0%) and standard deviation (SD0%) of background signals (0% activity) from the Abs405Diff values at the wells of A1–P1 and A24–P24. 4. Calculate average (Av100%) and standard deviation (SD100%) of intact enzymatic signals (100% activity) from the Abs405Diff values at the wells of A2–H2 and A23–P23. 5. Calculate the inhibition rate of the positive control placed in the I2–P2 wells using the following formula:

Inhibition rateð%Þ = Av100% - Abs405Diff =ðAv100% - Av0% Þ × 100

ð1Þ

6. Calculate three statistical indexes using the following formulas. (a) Coefficient of variation (CV): CV100% = SD100%/Av100%. (CV100% < 0.1 for an acceptable assay system). (b) Signal/background (S/B) ratio: S/B = Av100%/Av0%. (S/B ratio should be ≥2 at least and ideally ≥3). (c) Z′-factor: Z′ = 1 - (3×SD100% + 3×SD0%)/(Av100% Av0%). Z′-factor is the most important index to evaluate whether the assay system is appropriate. Z′ should be >0.5 (1 at maximum). 7. Confirm that each statistical factor is within the acceptable range (see Note 10).

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Fig. 2 Heat map representation of typical pilot experimental results. (a) A well-handled good result. Zero percent background, 100% controls, and positive controls showed almost the same colors in each group. (b) An example of unsuccessful result 1, where insufficient channel priming of the microplate dispenser caused gradual and regular change in absorbance along the rows and columns, respectively. (c) An example of unsuccessful result 2. E15–E19 wells showed a gradual decrease in absorbance, indicating that the reaction solution was not adequately dispensed probably due to a clogged flow path. (d) An example of unsuccessful result 3, where the center region showed lower signals. This can be caused by slow and insufficient thermal transfer within the plate during incubation (see Note 9). Abs405Diff values are colored in green (low) to yellow (high) on a 384-well format

3.3 Primary Screening Using a Chemical Library

1. Prepare 70 working microplates (see Note 11) that are all for the allosteric PTP reaction (see Note 12). The chemical library compounds, DMSO, and positive control should be appropriately arranged according to the plate format in shown Fig. 1. 2. Prepare the allosteric enzyme solution (2×) and substrate solutions (2×) by diluting with 1× assay buffer in 1-L bottles. (a) 600 mL solution of the allosteric enzyme (2×). (b) 600 mL solution of the substrate containing the effector (2×). (c) 80 mL solution of the substrate without the effector (2×).

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3. Perform basically the same operation as that for the pilot experiment described in Subheading 3.1, steps 5–14, in which the procedures for non-allosteric plates should be skipped. 4. Visualize the result by heat map representation and evaluate statistical factors (see Subheading 3.2, steps 1–7). 5. Calculate the inhibition rate for each compound in the library and extract the hit candidates. 3.4 Secondary Screening Using Extracted Hit Candidates

1. Prepare hit candidate-containing microplates that are duplicated for the allosteric and non-allosteric PTP reactions. 2. Perform the phosphatase reaction by repeating almost the same operation as that for the pilot experiment (see Subheading 3.1, steps 5–14) and analyze the data (see Subheading 3.2, steps 1–7). 3. Investigate whether there are any hit compounds for the allosteric inhibitors that inhibit only allosteric PTP activated by the effector but not the isolated PTP domain alone.

4

Notes 1. A small amount of a surfactant reduces nonspecific adsorption of the enzyme, effector, and/or substrate molecules to the assay plates, pipette tips, and dispenser tubes. Such adsorption results in data of low quality due to altered composition of the reaction mixture. 2. Freshly prepare 1× assay buffer by diluting thawed 10× assay buffer with water on the day of the assay to avoid degrading HEPES and DTT. Since the major families in PTPome have a cysteine residue to catalyze the phosphatase reaction [4], adding a reducing agent such as DTT is important for maintaining the activity of such PTPs. 3. For example, the Drug Discovery Initiative at the University of Tokyo, Japan, provides ready-to-use library plates containing specified volumes and concentrations of chemical compounds on a 384-well format. Without such an institution or department, automated microplate pipetting equipment is required for performing HTS. 4. The solution volumes to be prepared are indicated in this method upon consideration of the dead volume, which is highly dependent on the experimental environment, particularly, the microplate dispenser used. Reserve a larger amount if needed.

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5. If technically possible, the volume of DMSO can be reduced. The final concentration of DMSO should be equalized among all of the wells throughout the pilot and actual screening experiments. 6. Slower dispense mode (if available) is recommended to avoid the formation of air bubbles, which will disturb absorbance. If bubbles form, they can be removed by flash centrifugation without a plate sticker. 7. To dispense accurately using the automated 8-channel pipette, the cutting edge of the pipette tip should be in contact with the inner wall of the well during pipetting out. 8. Do not stack the plates during incubation at 37 °C. Stacked plates lead to slower thermal transfer and thereby result in unfavorable variability in the enzymatic reaction velocities between the plate center and edge regions within a single plate. 9. If the absorbance was gradually and/or regularly changed along the same row or column, it is quite likely to be a typical problem in handling of multi-samples on microplates (e.g., nonspecific binding due to insufficient priming of the dispenser flow path: clogged flow path, precipitation of the enzyme, substrate, and/or other buffer components) (Fig. 2). Since temperature is critical for the velocity of enzymatic reaction, utilize an incubator with good heat conduction to perform the reaction at a constant temperature. A direct-contact-type incubator has better thermal conductivity than that of a widely used air incubator. Another option is to perform the enzymatic reaction at room temperature for a longer time if the reaction can proceed under such a condition. 10. If any factor deviates from the appropriate range, the issue should be resolved before proceeding to the actual screening experiment. The S/B ratio can be raised by fine-tuning of the reaction composition (e.g., increase in the concentration of the allosteric effector and/or substrate, decrease in the enzyme concentration, prolonged/shortened reaction time). Since the CV value reflects dispensing/pipetting accuracy as well as plate handling, reconfirm the correction of the instrument first. Also, use nonbinding materials to deal with the reaction components (e.g., microplates, pipette tips, and dispenser flow path). The Z′-factor will be accordingly raised upon improvement of the S/B ratio and/or the CV value. 11. The number of library plates that are subjected to HTS can be increased or decreased according to the situation. It is possible to screen 22,400 compounds (320 compounds/plate) using 70 library plates.

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12. The isolated PTP domain should not be assessed in the primary screening because it consumes large amounts of materials. After excluding many compounds not of interest based on the results of primary screening, the inhibitory action of the hit candidates towards the isolated PTP domain alone should be examined in the secondary counter-screening.

Acknowledgements This work was supported by Grants-in-Aid for Scientific Research on Innovative Areas from MEXT/JSPS, Japan, under grant number 16 K19121 and 21 K07019 (to T. H.) and by Project for Cancer Research and Therapeutic Evolution (P-CREATE) from AMED, Japan, under grant number 21cm0106506h0006 (to M. H.). References 1. Cohen P, Cross D, J€anne PA (2021) Kinase drug discovery 20 years after imatinib: progress and future directions. Nat Rev Drug Discov 20:551–569 2. Pottier C, Fresnais M, Gilon M et al (2020) Tyrosine kinase inhibitors in cancer: breakthrough and challenges of targeted therapy. Cancers 12:731 3. Alonso A, Sasin J, Bottini N et al (2004) Protein tyrosine phosphatases in the human genome. Cell 117:699–711 4. Alonso A, Pulido R (2016) The extended human PTPome: a growing tyrosine phosphatase family. FEBS J 283:1404–1429 5. Hendriks WJ, Elson A, Harroch S et al (2013) Protein tyrosine phosphatases in health and disease. FEBS J 280:708–730 6. Verma S, Sharma S (2018) Protein tyrosine phosphatase as potential therapeutic target in various disorders. Curr Mol Pharmacol 11: 191–202 7. Kumar P, Munnangi P, Chowdary KR et al (2017) A human tyrosine phosphatase interactome mapped by proteomic profiling. J Proteome Res 16:2789–2801 8. Tartaglia M, Martinelli S, Stella L et al (2006) Diversity and functional consequences of germline and somatic PTPN11 mutations in human disease. Am J Hum Genet 78:279–290 9. Mohi MG, Neel BG (2007) The role of Shp2 (PTPN11) in cancer. Curr Opin Genet Dev 17: 23–30

10. Song Y, Zhao M, Zhang H et al (2022) Double-edged roles of protein tyrosine phosphatase SHP2 in cancer and its inhibitors in clinical trials. Pharmacol Ther 230:107966 11. Hof P, Pluskey S, Dhe-Paganon S et al (1998) Crystal structure of the tyrosine phosphatase SHP-2. Cell 92:441–450 12. Hayashi T, Senda M, Suzuki N et al (2017) Differential mechanisms for SHP2 binding and activation are exploited by geographically distinct Helicobacter pylori CagA oncoproteins. Cell Rep 20:2876–2890 13. Higashi H, Tsutsumi R, Muto S et al (2002) SHP-2 tyrosine phosphatase as an intracellular target of Helicobacter pylori CagA protein. Science 295:683–686 14. Hatakeyama M (2014) Helicobacter pylori CagA and gastric cancer: a paradigm for hitand-run carcinogenesis. Cell Host Microbe 15: 306–316 15. Lawrence HR, Pireddu R, Chen L et al (2008) Inhibitors of Src homology-2 domain containing protein tyrosine phosphatase-2 (Shp2) based on oxindole scaffolds. J Med Chem 51: 4948–4956 16. Zhang X, He Y, Liu S et al (2010) Salicylic acid based small molecule inhibitor for the oncogenic Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2). J Med Chem 53:2482–2493

Screening of Allosteric PTP Inhibitors 17. Chen L, Sung SS, Yip ML et al (2006) Discovery of a novel Shp2 protein tyrosine phosphatase inhibitor. Mol Pharmacol 70:562–570 18. Wu J, Zhang H, Zhao G et al (2020) Allosteric inhibitors of SHP2: an updated patent review (2015–2020). Curr Med Chem 28:3825– 3842 19. Chen YN, LaMarche MJ, Chan HM et al (2016) Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases. Nature 535:148–152 20. Luo Y, Li J, Zong Y et al (2023) Discovery of the SHP2 allosteric inhibitor 2-((3R,4R)-4-

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amino-3-methyl-2-oxa-8-azaspiro[4.5]decan8-yl)-5-(2,3-dichlorophenyl)-3-methylpyrrolo [2,1-f][1,2,4] triazin-4(3H)-one. J Enzyme Inhib Med Chem 38:398–404 21. LaRochelle JR, Fodor M, Ellegast JM et al (2017) Identification of an allosteric benzothiazolopyrimidone inhibitor of the oncogenic protein tyrosine phosphatase SHP2. Bioorg Med Chem 25:6479–6485 22. LaMarche MJ, Acker M, Argintaru A et al (2020) Identification of TNO155, an allosteric SHP2 inhibitor for the treatment of cancer. J Med Chem 63:13578–13594

Chapter 19 Intravital Imaging of Regulatory T Cells in Inflamed Skin Michael J. Hickey and M. Ursula Norman Abstract Regulatory T cells play key roles in skin homeostasis and inflammation and in regulating antitumor responses. Understanding of the biology of this cell type has been improved by the use of intravital microscopy for their visualization in various organs. Here we describe a multiphoton microscopy-based technique for intravital imaging of regulatory T cells in the skin. We provide a protocol for a model of antigen-dependent inflammation that induces robust regulatory T cell recruitment to the skin and describe the use of a regulatory T cell reporter mouse for visualization of these cells in inflamed skin. Key words Regulatory T cell, Treg, Skin, Contact hypersensitivity, Inflammation, Intravital microscopy

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Introduction Regulatory T cells (Tregs) are a subset of the CD4+ lymphocyte lineage recognized for their capacity to control autoimmune and inflammatory responses [1]. The critical role of these cells in tissue homeostasis is demonstrated in humans with the genetic condition immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome, in whom Tregs are absent or dysfunctional [2]. These patients, and mice with similar mutations, are affected by debilitating inflammation in multiple organs, including the skin, lung, and gut [3]. In mice and humans, the transcription factor Foxp3 is critical for Treg development and is commonly used to define these cells [4, 5]. In addition to their roles in the regulation of inflammation, evidence indicates that Tregs regulate the immune response against many forms of cancer, inhibiting the capacity of the host immune system to combat tumors [6, 7]. Given this, interest has grown in understanding the mechanisms of action of Tregs within and around tumors. One of the optimal ways for investigating immune cell function in vivo involves their direct microscopic visualization in target organs in live animals, via intravital microscopy

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[8, 9]. While this is readily applied to numerous immune cell subsets, using this technique to image Tregs in vivo in settings of inflammation and cancer has presented technical challenges. These primarily relate to the low frequency of these cells relative to other immune cell subsets and difficulties in differentiating Tregs from other subsets, particularly conventional CD4+ T cells. Nevertheless, over the last ~15 years, via the advent of the Foxp3-green fluorescent protein (GFP) Treg reporter mouse and the development of advanced forms of intravital imaging, particularly multiphoton microscopy (MP-IVM), researchers have made strides in in vivo imaging of Tregs in various inflammatory and tumor settings. This research has markedly improved our understanding of how these cells achieve their anti-inflammatory function [8, 10–14]. In this chapter, we describe the use of MP-IVM to investigate the actions of Tregs in vivo, in a model of antigen-induced skin inflammation [15, 16].

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Materials

2.1 Contact Hypersensitivity Model

1. Oxazolone (4-ethoxy-methylene-2-phenyl-2-oxazolin-5-one). 2. Acetone. 3. Olive oil. 4. Micropipette. 5. Small shaver.

2.2 Imaging and Surgical Materials and Instruments

1. Imaging platform with heated platforms for mouse and extended skin flap. 2. Dissecting microscope. 3. Multiphoton intravital microscope (see Note 1). 4. Catheter: PE10 polyethylene tubing, mounted on to a 30-gauge needle, pre-filled with saline in a 1-mL syringe. 5. Suture material—4-0 silk. 6. Tape—Transpore tape (3M) and Sleek tape (Leukoplast). 7. Depilatory cream (e.g., Nair). 8. Warmed saline solution. 9. Coverslip—22 × 50 mm. 10. Surgical instruments: Fine scissors (e.g., Lawton Delicate Scissors, 11.5 cm), straight Vanna-Tubingen Spring Scissors, fine round-end forceps (e.g., Graefe forceps—straight and curved), and self-closing fine forceps (Fine Science Tools). 11. Treg reporter mice: Foxp3-GFP strain (e.g., B6.129(Cg)Foxp3tm3(DTR/GFP)Ayr/J) (see Note 2)

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Methods

3.1 Contact Hypersensitivity (CHS) Model

1. Sensitization: Prepare solution of 5% oxazolone in acetone/ olive oil vehicle (4:1 vol/vol). Using Foxp3-GFP mice, shave a 1 cm2 region of back skin near the base of the tail. Use a micropipette to apply 50 μL of the oxazolone solution to the shaved skin (see Note 3). 2. Challenge: 5–7 days after sensitization, prepare 1% solution of oxazolone in acetone/olive oil vehicle (4:1 vol/vol). Using the previously sensitized mice, scruff the mouse and shave a 2 × 1 cm region of right flank/abdominal skin. While still holding the mouse, use a micropipette to apply 50 μL of the 1% oxazolone solution evenly across the shaved flank skin. If intending to measure skin swelling as an experimental readout (see Note 4), apply 20 μL of oxazolone solution to the right ear, and for control measurements, use a different pipette tip to apply 20 μL of acetone/olive oil vehicle to the left ear.

3.2 Preparation of Flank Skin for Multiphoton Imaging (See Note 5)

1. Anesthetize mouse. This can be done via intraperitoneal injection of a mixture of ketamine hydrochloride (150–180 mg/kg) and xylazine hydrochloride (10 mg/kg). Alternatively, inhaled anesthetic, e.g., isoflurane, can be used. 2. Reshave skin around challenge site to include entire abdominal area. 3. Catheterizing the jugular vein for intravenous access. This technique has been described in detail previously in this forum [17], and much of this previous description has been reproduced here for clarity. For delivery of reagents and additional anesthetic into the bloodstream, intravenous access is necessary and can be accomplished via cannulation of the jugular vein or tail vein. For jugular vein cannulation, secure anesthetized mouse on its back, taping down the forepaws and tail and immobilizing the head via a suture looped around the teeth. Expose the jugular vein via a 10-mm incision in the neck skin, directly above the vein. Using fine, round-ended forceps (e.g. Graefe forceps), use blunt dissection to free the vein from the surrounding connective tissue and fat and pass two 8–10-cm lengths of suture under the vein. Tie off the distal end of the vein and use the suture to immobilize the vessel by taping the suture to the operating surface. Use a 30-gauge needle to make a hole in the uppermost surface of the vein and pass the saline-filled cannula directly under the needle into the lumen of the vein. Pass the tip of the cannula ~5 mm past the second suture and tie a knot in the suture enclosing both the cannula and vein (Fig. 1a). Ensure the cannula is placed correctly by gently drawing back on the syringe. If blood does

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Fig. 1 Surgical preparation of abdominal/flank skin for MP-IVM. (a) Insertion and securing cannula in jugular vein. (b) Initial attachment of sutures to the edge of the skin incision on the side of the challenged skin. (c) Extension of skin on pedestal allowing visualization of skin blood supply. This allows lateral incisions to be made mobilizing the skin flap without disruption of blood supply. (d) Final positioning of the skin flap with the epidermis facing up, surrounded by vacuum grease and with coverslip in position (see Subheading 3.2)

not draw back into the cannula, reposition the cannula until this is achieved. Secure the catheter with a second knot, and also knot the suture at the distal end of the vein over the vessel and the cannula. As an alternative, a tail vein cannula can be generated using the tip of a 30-gauge needle secured in the end of the PE10 cannula. Insert this in the tail vein, using a temporary ligature to inflate the vein. Once backflow of blood into the cannula is achieved, tape the cannula securely to the tail. 4. Remove hair from the shaved region of challenged skin using a depilatory cream (see Note 6). 5. Place the mouse on its back on to the heated imaging platform. Make a 3–4-cm midline incision in the abdominal skin just to the side of the shaved area. Keep the exposed abdominal area

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moist with warm saline. Use self-closing fine forceps to grasp a small area of skin at the edge of the flap, starting at the lower end of the abdomen. Pass a length of 4–0 suture around the tips of the forceps and make a knot around the region of tissue, ensuring the knot is positioned just past the forceps tips. Tie the suture in place with two knots and release the forceps. Using the same technique, apply a total of 4–5 sutures along the periphery of the skin edge (Fig. 1b). Tape down the sutures using Transpore tape and gently apply tension to the sutures to move the skin edge away from the abdomen. Keep the exposed skin immersed in warm saline throughout the dissection period. 6. While visualizing the preparation using a dissecting microscope, use straight Vanna-Tubingen Spring Scissors to disconnect the loose connective tissue on the skin undersurface attaching the skin to the abdomen (Fig. 1c). Locate the skin vascular supply on the undersurface of the skin at both the cranial and caudal ends of the midline incision. Make small incisions across the flank in the axillary and inguinal regions at locations in between the major branches of these skinsupplying vessels, making sure to avoid these vessels and keep them intact, in order to create a skin flap incorporating the challenged skin. Remove the Transpore holding the sutures and reposition the mouse into a prone position, with the skin flap gently extended over the heated pedestal with the epidermal side facing up. Using the attached sutures, further extend the mobilized flank skin over the pedestal and immobilize the skin by taping the sutures in place using Sleek tape. 7. Surround the skin with a well of vacuum grease on the pedestal and immerse the exposed skin in saline. Apply a line of vacuum grease to the upper surface of one long edge of a 22 × 50 mm coverslip. With this line of grease adjacent to the mouse, place the coverslip down onto the well of grease on the pedestal and gently press down to hold the saline-immersed skin in place (Fig. 1d). 3.3 Multiphoton Intravital Microscopy of Skin Flank for Treg Visualization

1. Place mouse with immobilized skin flap on the stage of the multiphoton microscope (see Note 7) and use epifluorescence illumination and microscope eyepieces to bring the epithelial surface into focus. 2. Using multiphoton microscope and the appropriate channel for detection of GFP, find regions of interest containing Treg infiltration within the skin. 3. Image volumes of skin to required depth, acquiring signal from the necessary channels and for the appropriate duration to achieve the experimental aims (see Note 7). Typically, three

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Fig. 2 Representative MP-IVM image of inflamed skin of Foxp3-GFP mouse. Image shows region of skin 48 h after induction of contact hypersensitivity via oxazolone challenge in a sensitized mouse. Image includes Tregs, visible as GFP+ (green) cells, collagen visible via second-harmonic generation (blue), and hair follicles (autofluorescence—yellow)

different fields of view across the challenged area of the skin are recorded (Fig. 2). 4. Analyses: Data from MP-IVM recordings are typically analyzed using image analysis software (see Note 8). This is particularly pertinent when quantitating parameters relating to immune cell (e.g., Treg) migration and cell-cell interactions.

4

Notes 1. A wide variety of suppliers make multiphoton microscopy imaging systems, including but not limited to Olympus, Leica Microsystems, Zeiss, and Nikon. The key components for a highly functional multiphoton microscopy system include a high numerical aperture (NA) immersion lens, typically ≥1.0 NA, a tunable, pulsed two-photon laser and non-descanned (or “external”) detectors. The advanced lasers required for this work are typically acquired from specialized laser manufacturers, but are integrated into the microscope at installation. 2. Our experiments in imaging Tregs have been performed using this strain of Foxp3 reporter mouse [11–13, 15, 16, 18]. It is highly suitable for detection of Tregs. Other Treg reporters,

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e.g., the Foxp3rfp mouse, have been generated, but their suitability for Treg detection by MP-IVM needs to be confirmed on the imaging system intended for use. Also, if the interactions of Tregs with other subsets are of interest, mice in which cells express GFP can be used in combination with other suitable, nonoverlapping reporters for other cell types, e.g., cells labelled with DsRed, red fluorescent protein (RFP), or chemical fluorochromes such as the CellTracker dyes (Thermo Fisher Scientific). Appropriate combinations of fluorochrome reporters should be decided on based on the available emission filter setup(s) on the multiphoton microscope to be used. 3. Due to the volatile nature of acetone, the oxazolone solution tends to evaporate rapidly. As such, it is helpful to make up somewhat more volume than the exact amount needed for the number of mice being sensitized, e.g., if sensitizing six mice (requires 300 μL), make up 400 μL. 4. The ear is very commonly used in the study of CHS-associated inflammation, particularly as a site where skin swelling is readily assessed by measurement of the width of the ear using a micrometer [19]. However, we have found the abdominal flank skin more suitable for Treg imaging studies as the number of Tregs that can be visualized by MP-IVM is greater in the flank skin [15]. 5. For investigation of Treg behavior, MP-IVM of flank skin can be performed up to 5 days post-challenge. Our previous work has shown that in the dermis of uninflamed skin, a small number of Tregs are consistently visible by MP-IVM. Within 24 h of challenge, the number of Tregs per field of view has increased significantly with a further increase by 48 h and is maintained at high levels for at least 72 h. This increased abundance of Tregs is associated with increased Treg migration within the dermis [15, 16]. 6. Particularly in C57BL/6 background mice, thorough removal of hair is beneficial to the clarity of the imaging preparation. However, depilatory cream can cause skin irritation if left on the skin for prolonged periods. For the abdominal skin, we use two sequential 1-min applications of Nair, gently removing the cream in between applications using a piece of gauze soaked in warm saline. This achieves close to complete hair removal. The area of skin is gently patted dry after the last saline wash. To remove hair from the ear skin, a thin layer of Nair is applied to both sides of the ear and washed off with a Q-tip soaked in saline. 7. Setup of imaging experiments requires consideration of several variables and parameters. These include the following:

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(a) Laser excitation wavelength (multiphoton lasers can be tuned to different wavelengths in the range of ~700–1300 nm and different fluorochromes respond optimally at different excitation wavelengths. A commonly used approach is to perform imaging using a single wavelength that excites all of the fluorochromes used, although it is possible to use multiple excitation wavelengths either simultaneously or sequentially, depending on the microscope and laser capabilities). (b) Depth of imaging (typically in the range of 100–150 μm below the surface). (c) Z-step size (gap between individual X-Y planes in the volume of imaging, typically in the range of 2–5 μm). (d) Duration of imaging (as required for the experimental aims and typically determined according to the behaviors of the cells of interest and the state of the anesthetized animal). (e) Interval between time points of image acquisition (often in the range of 0.5–2 min, but must be longer than the duration of acquisition of a single imaging volume). (f) Number and nature of channels acquired (determined by the reporters/fluorochromes used in the experiment and the number of channels available on the microscope. A common feature of skin imaging is acquisition of a “second-harmonic” signal in which dermal collagen is visualized in the absence of exogenously applied stain. This signal is emitted at a wavelength half of the excitation wavelength, e.g., if 900 nm excitation is used, a secondharmonic signal will be emitted at 450 nm, requiring a corresponding filter set for detection). 8. Data generated in MP-IVM recordings is often multidimensional, involving x, y, and z data, plus time, as well as incorporating data from multiple fluorescence channels. Analysis of data of this complex nature typically requires powerful image analysis software such as Imaris (Bitplane), although some aspects of analysis can be performed using ImageJ/Fiji (free download available from https://imagej.net/software/fiji/) and openaccess software is also being developed to achieve these aims (e.g., https://www.immunemap.org/index.php/analysis-tool box). For analysis of immune cell migration, parameters including mean migration velocity (μm/min), track length (μm), displacement (straight line distance between start and finish points of migration—μm), and confinement (track length/displacement) are commonly assessed [16]. It is important to note that, due to slight movements in the preparation and differences in how the software defines the position of the cell at

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individual time points, analysis of truly static cells often generates a value for velocity of greater than the expected 0 μm/ min. To account for this, when defining a cell as migratory, it is common practice to impose an arbitrary velocity threshold— we have used >2 μm/min for this purpose [16].

Acknowledgements This work was supported by funding from the National Health and Medical Research Council of Australia (1042775, MJH) and the Monash University School of Clinical Sciences at Monash Health (MJH & MUN). References 1. Vignali DA, Collison LW, Workman CJ (2008) How regulatory T cells work. Nat Rev Immunol 8:523–532 2. Bennett CL, Christie J, Ramsdell F et al (2001) The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat Genet 27: 20–21 3. Brunkow ME, Jeffery EW, Hjerrild KA et al (2001) Disruption of a new forkhead/ winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat Genet 27:68–73 4. Hori S, Nomura T, Sakaguchi S (2003) Control of regulatory T cell development by the transcription factor Foxp3. Science 299: 1057–1061 5. Fontenot JD, Rasmussen JP, Williams LM et al (2005) Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity 22:329–341 6. Chattopadhyay S, Chakraborty NG, Mukherji B (2005) Regulatory T cells and tumor immunity. Cancer Immunol Immunother 54:1153– 1161 7. Zou W (2006) Regulatory T cells, tumour immunity and immunotherapy. Nat Rev Immunol 6:295–307 8. Mempel TR, Pittet MJ, Khazaie K et al (2006) Regulatory T cells reversibly suppress cytotoxic T cell function independent of effector differentiation. Immunity 25:129–141 9. Kitching AR, Hickey MJ (2022) Immune cell behaviour and dynamics in the kidney – insights from in vivo imaging. Nat Rev Nephrol 18:22–37

10. Bauer CA, Kim EY, Marangoni F et al (2014) Dynamic Treg interactions with intratumoral APCs promote local CTL dysfunction. J Clin Invest 124:2425–2440 11. Deane JA, Abeynaike LD, Norman MU et al (2012) Endogenous regulatory T cells adhere in inflamed dermal vessels via ICAM-1: association with regulation of effector leukocyte adhesion. J Immunol 188:2179–2188 12. Hickey MJ, Chow Z (2017) Viewing immune regulation as it happens: in vivo imaging for investigation of regulatory T-cell function. Immunol Cell Biol 95:514–519 13. Snelgrove SL, Abeynaike LD, Thevalingam S et al (2019) Regulatory T cell transmigration and intravascular migration undergo mechanistically distinct regulation at different phases of the inflammatory response. J Immunol 203: 2850–2861 14. Marangoni F, Zhakyp A, Corsini M et al (2021) Expansion of tumor-associated Treg cells upon disruption of a CTLA-4-dependent feedback loop. Cell 184:3998–4015.e19 15. Chow Z, Mueller SN, Deane JA et al (2013) Dermal regulatory T cells display distinct migratory behavior that is modulated during adaptive and innate inflammation. J Immunol 191:3049–3056 16. Norman MU, Chow Z, Snelgrove SL et al (2021) Dynamic regulation of the molecular mechanisms of regulatory T cell migration in inflamed skin. Front Immunol 12:655499 17. Norman MU, Hickey MJ (2020) Using intravital microscopy to study the role of MIF in leukocyte trafficking in vivo. In: Harris J, Morand EF (eds) Macrophage migration inhibitory

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factor, Methods in molecular biology. Humana Press, New York 18. Abeynaike LD, Deane JA, Westhorpe CL et al (2014) Regulatory T cells dynamically regulate selectin ligand function during multiple challenge contact hypersensitivity. J Immunol 193: 4934–4944

19. Lehtimaki S, Savinko T, Lahl K et al (2012) The temporal and spatial dynamics of Foxp3+ Treg cell-mediated suppression during contact hypersensitivity responses in a murine model. J Invest Dermatol 132:2744–2751

Chapter 20 Confocal Endomicroscopy Monitoring of Tumor Formation Adele Preaudet, Ka Yee Fung, and Tracy L. Putoczki Abstract The utilization of preclinical murine models of colorectal cancer (CRC) has been essential to our understanding of the onset and progression of disease. As the genetic complexity of these models evolves to better recapitulate emerging CRC subtypes, our ability to utilize these models to discover and validate novel therapeutic targets will also improve. This will be aided, in part, by the development of live animal imaging techniques, including confocal endomicroscopy for mice. Here in this chapter, we describe the combined use of standard white light endoscopy and confocal endomicroscopy thereby providing a method to rapidly image and assess changes in the colon of an individual live mouse in real time. These methods permit the generation of high-resolution cross-sectional images of the tumor microenvironment for immediate visualization of cells of interest, avoiding the need for euthanasia and tissue collection across multiple cohorts of mice. Key words Colon, Confocal, Crypt, Endoscopy, Mouse models, Reporter, Tumor

1

Introduction Colorectal cancer (CRC) is a devastating disease with increasing incidence; however, routine colonoscopies permit the removal of precancerous lesions resulting in a reduction in the incidence of advanced CRC [1]. In mice, colonoscopy can similarly be used to monitor the health and progression of gastrointestinal pathologies [2–6]. Here we describe a method to image the tumor microenvironment by coupling the CellVizio confocal microendoscopy system, which allows for imaging of fluorescent markers in live mice, together with the white light imaging capabilities of the Karl Storz endoscopy system, which allows for ease of maneuvering within the colon. Since confocal endoscopy is also used in the clinic [7], advances in our understanding of tumor biology made through imaging preclinical mouse models could be immediately translated to patients.

Brendan J. Jenkins (ed.), Inflammation and Cancer: Methods and Protocols, Methods in Molecular Biology, vol. 2691, https://doi.org/10.1007/978-1-0716-3331-1_20, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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The CellVizio laser scanning unit (class IIIB laser, 488-nm excitation) can be used together with a range of unique fiberoptic probes. Each fiber within a probe is a point detector, similar to the pinhole used in standard confocal microscopy imaging. The application of minimally invasive colonoscopy in mice means that pairing these two imaging systems provides an opportunity to closely monitor the colon microenvironment of live mice in real time [8]. This provides additional layers of information for the interpretation and analysis of murine CRC models, including an opportunity for the early identification of aberrant crypts and thus changes in disease onset and subsequent progression, observation of changes in the mucosal vasculature [8], monitoring the kinetics of immune cell populations [9] and their localization within and around a tumor, or even tracking the specificity, success, and in vivo half-life of therapeutics [10].

2

Materials

2.1

Mice

All animals should be bred in the same specific pathogen-free (SPF) facility on the same genetic background and age- and gendermatched (see Note 1).

2.2

Ethical Approval

All described procedures should only be performed following approval from the relevant Institutional Animal Ethics or Welfare Committee.

2.3

Reagents

1. Isoflurane. 2. Quantikit 488 (CellVizio). 3. Paper towels. 4. Beaker of water.

2.4

Equipment

The Coloview white light endoscopy and the CellVizio confocal endoscopy units should be assembled as per the manufacturer’s guidelines side by side on a work bench for ease of imaging (Fig. 1). 1. A clear workbench. 2. Karl Storz Coloview miniendoscopic system. (a) Endovision Tricam and 3-Chip camera head (cat # 20223011, 20221030). (b) Xenon 175 light source with antifog pump (cat # 2013501). (c) Hopkins straight forward telescope, diameter 1.9 mm (cat # 64301A). (d) Examination protection sheath (cat # 61209C).

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Fig. 1 The Karl Storz and CellVizio systems. The CellVizio confocal endoscopy system (a) is placed adjacent to the Karl Storz white light endoscopy system (b) on a clear bench. This allows for ease of running the two systems in parallel. A monitor allows for easy visualization of the colon throughout the procedure. A light source permits visualization of the colon, and a standard computer is used to record videos of the procedure for subsequent analysis. CellVizio software is used to monitor fluorescent signal. (c) A nose cone permits anesthesia administration. (d ) An endoscopy sheath allows for white light imaging in parallel to the use of a microprobe

(e) Fiber-optic light cable and air hose (cat # 495NL). (f) Computer and media player software. 3. CellVizio Dual Band confocal endoscopy system. (a) Confocal processor (cat # CDB-0001). (b) Proflex microprobe, Mini-Z, diameter 0.94 mm. 4. Isoflurane anesthetic machine, including an induction box and nose cone (Fig. 1).

3 Methods 3.1 Preparation of Equipment for Imaging

1. Turn on all equipment. 2. Clean and sterilize the Karl Storz endoscopy sheath using 70% (v/v) ethanol and an antibacterial agent. Use paper towel to pat dry. 3. Insert the CellVizio microprobe into the laser scanning unit as per the manufacturer instructions.

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4. Insert the fiber-optic probe down the working channel of the Karl Storz endoscopy sheath (see Note 2). 5. Initialize the CellVizio 488 laser scanning unit. 6. Turn on the confocal processor and launch the CellVizio Dual and Image Cell software. It will recognize the microprobe inserted into the laser scanning unit (see Note 3). 7. Initialize a standard desktop computer, and check camera visualization through the white light endoscopy in Quicktime Player, or a similar video software. Adjust the light levels as required for a clear image. 8. Use the Quantikit 488 (CellVizio) to clean and calibrate the microprobe as per the manufacturer instructions. 9. Anesthetize live mice in an induction chamber with 3% isoflurane and oxygen at a flow rate of 0.4 L/min (see Note 4). 10. Once breathing has stabilized, remove mouse from the induction chamber and place the head of the mouse in a nose cone, with the mouse positioned ventral side up. 11. Maintain the isoflurane at 0.5–2% flow rate for the endoscopy procedure. 12. Insert the endoscope sheath into water (as a lubricant) and then into the rectum and up to 3 cm into the colon. 3.2 Visualization of Colonic Tumors in Live Mice

1. Initiate video recording at any stage of the endoscopy procedure using standard media software, such as Quicktime Player or iMovies, run by a computer. 2. In order to guide entry to the colonic lumen of mice and identify regions of interest (i.e., tumor, tissue adjacent to tumor), the Karl Storz camera and thus light endomicroscopy images are used. 3. Use the Karl Storz images to guide appropriate placement of the confocal probe against the mucosal surface (Fig. 2a).

Fig. 2 Visualization of aberrant crypts and tumor cells. (a) White light endoscopy allows for ease of identification of regions to place the endoscopy sheath, allowing for the microprobe to make direct contact with the colonic mucosa (black arrow). (b, c) Identification of (b) normal crypts (white arrow) and (c) aberrant crypts and tumor cells (dotted lines) through visualization of YFP+ cells. Scale bars = 50 μm

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4. Place the sheath against the colonic mucosa for fluorescent imaging (see Note 5). 5. Initiate the 488 laser using the foot pedal (or manual computer controls). 6. Use the Image Cell software to record fluorescent videos in real time (see Note 6). 7. Once images are acquired (Fig. 2b), remove the sheath from the rectum. 8. Place the mouse back in its cage and monitor for recovery from anesthetic (see Note 7). 9. Recalibrate the system following the manufacturer protocols prior to imaging each individual mouse. 3.3 Cleaning the Endoscopy Units

1. Clean the Karl Storz endoscopy sheath, Karl Storz light probe and CellVizio fiber-optic probes thoroughly in accordance with manufacturer suggestions. 2. Disassemble and store the equipment as per the manufacturer suggestions.

4

Notes 1. A fluorescent reporter is required for visualization using the microprobe. In the experiments described, a Cdx2Cre; apc580s; RosaYFP mouse [11, 12] was utilized for epithelial specific expression of yellow fluorescent protein (YFP) allowing for imaging of colonic crypts using the 488 laser of the CellVizio unit. 2. Our results were generated using the Mini-Z series probe, which has a working distance of 50 μm and a lateral resolution of approximately 3.5 μm allowing for visualization of YFP+ epithelial cells. However, a number of probes are available with a range of 1.4–3.5 μm lateral resolution, which would permit the visualization of smaller cells. 3. The CellVizio laser requires approximately 20 min to initialize. 4. As an alternative, fluorescently labelled antibodies or other reagents can be used to image the colonic mucosal by confocal microendoscopy. 5. The microprobe must be placed directly against the wall to obtain a clear image. This can require manipulation of the animal to enable the turn of the sheath into the colonic wall. 6. In general, 9–50 frames per second are imaged up to a 1.4 μm lateral resolution, 10–70 μm optical section, and 225–600 μm field of view, with a z-axis signal integration of 200 μm surface

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depth as per CellVizio specifications. This can record in parallel to the light endoscopy videos as they are independent units running in tandem. 7. Animals recover from isoflurane anesthetic within approximately 10 min. The same animal can undergo repeat daily or weekly imaging to monitor disease progression (or localization of a labelled antibody/therapeutic).

Acknowledgements We thank the WEHI bioservices facility for routine animal care. The endoscopy procedure is performed by highly trained staff following protocols approved by the WEHI Animal Ethics Committee (2022.025). T.L.P is supported by a Sylvia and Charles Viertel Senior Medical Research Fellowship. We acknowledge the Victorian State Government Operational Infrastructure Support and Australian Government NHMRC Independent Research Institutes Infrastructure Support Scheme. References 1. Brenner H, Altenhofen L, Stock C et al (2014) Incidence of colorectal adenomas: birth cohort analysis among 4.3 million participants of screening colonoscopy. Cancer Epidemiol Biomark Prev 23:1920–1927 2. Waldner MJ, Wirtz S, Neufert C et al (2011) Confocal laser endomicroscopy and narrowband imaging-aided endoscopy for in vivo imaging of colitis and colon cancer in mice. Nat Protoc 6:1471–1481 3. Becker C, Fantini MC, Neurath MF (2006) High resolution colonoscopy in live mice. Nat Protoc 1:2900–2904 4. Bruckner M, Lenz P, Nowacki TM et al (2014) Murine endoscopy for in vivo multimodal imaging of carcinogenesis and assessment of intestinal wound healing and inflammation. J Vis Exp 90:e51875 5. Putoczki TL, Thiem S, Loving A et al (2013) Interleukin-11 is the dominant IL-6 family cytokine during gastrointestinal tumorigenesis and can be targeted therapeutically. Cancer Cell 24:257–271 6. Nguyen PM, Dagley LF, Preaudet A et al (2020) Loss of Bcl-G, a Bcl-2 family member, augments the development of inflammationassociated colorectal cancer. Cell Death Differ 27:742–757

7. Atreya R, Neumann H, Neufert C et al (2014) In vivo imaging using fluorescent antibodies to tumor necrosis factor predicts therapeutic response in Crohn’s disease. Nat Med 20: 313–318 8. Mielke L, Preaudet A, Belz G et al (2015) Confocal laser endomicroscopy to monitor the colonic mucosa of mice. J Immunol Methods 421:81–88 9. Varol C, Vallon-Eberhard A, Elinav E et al (2009) Intestinal lamina propria dendritic cell subsets have different origin and functions. Immunity 31:502–512 10. Oh G, Yoo SW, Jung Y et al (2014) Intravital imaging of mouse colonic adenoma using MMP-based molecular probes with multichannel fluorescence endoscopy. Biomed Opt Express 5:1677–1689 11. Hinoi T, Akyol A, Theisen BK et al (2007) Mouse model of colonic adenoma-carcinoma progression based on somatic Apc inactivation. Cancer Res 67:9721–9730 12. Srinivas S, Watanabe T, Lin CS et al (2001) Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev Biol 1:4

Chapter 21 Detection of Free Bioactive IL-18 and IL-18BP in Inflammatory Disorders Se´bastien Fauteux-Daniel, Charlotte Girard-Guyonvarc’h, Assunta Caruso, Emiliana Rodriguez, and Cem Gabay Abstract The interleukin (IL)-18 cytokine plays an important driver role in a range of autoimmune and inflammatory diseases, as well as cancer. IL-18 is a potent inducer of interferon gamma (IFN-γ), and the bioactivity of IL-18 is regulated by its natural soluble inhibitor, IL-18-binding protein (IL-18BP), which is present at high concentrations in the circulation. Many cell types have been described to secrete IL-18BP, constitutively or under the influence of IFN-γ, thus generating a negative feedback loop for IL-18. Therefore, solely measuring total IL-18 protein levels does not allow to evaluate its biological activity, especially in the context of systemic inflammatory diseases or other circumstances where IL-18BP is present (e.g., samples containing plasma, cells constitutively expressing IL-18BP). Considering there is a critical need to accurately measure the protein levels of both mature, biologically active IL-18 and IL-18BP as biomarkers of disease activity in patients and also stratification for potential anti-IL-18 therapy, in this chapter we provide the latest techniques to measure mature, free, and bioactive IL-18 and IL-18BP in different samples. Key words Inflammasome, Free IL-18, IL-18BP, Interferon gamma-inducing factor, ELISA, Western blot

1

Introduction IL-18, originally coined interferon gamma-inducing factor (IGIF), was first isolated from serum of LPS-challenged mice that could induce IFN-γ production by splenocytes [1]. IL-18 is a pro-inflammatory cytokine belonging to the IL-1 family. It is translated into a 192 amino acid pro-peptide devoid of a secretory signal peptide [2]. Accordingly, IL-18 remains sequestered into the cytosol where it is constitutively expressed until maturation [3]. Processing of pro-IL-18 into its bioactive form and release depends on cleavage by caspase-1, the catalytic subunit of the inflammasome [4, 5].

Brendan J. Jenkins (ed.), Inflammation and Cancer: Methods and Protocols, Methods in Molecular Biology, vol. 2691, https://doi.org/10.1007/978-1-0716-3331-1_21, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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In healthy conditions, bioactivity of IL-18 is rapidly buffered by its natural inhibitor IL-18-binding protein (IL-18BP), which is comparatively present in the circulation in large excess [6]. IL18BP forms a high affinity complex with IL-18 (Kd = 30–50 pM in human), preventing its binding to the cell surface IL-18 receptor (IL-18R) [7, 8]. IL-18BP expression was also shown to be strongly upregulated by IFN-γ, thus creating a negative feedback loop for IL-18 [8]. Numerous human diseases are associated with an elevation of mature IL-18, some of which can overwhelm the buffering capacities of IL-18BP (reviewed in [9]). While various inflammatory disorders present with high levels of IL-18, unbound free IL-18 (fIL-18) is only detected in a subset of autoinflammatory conditions, especially those that are prone to macrophage activation syndrome (MAS). In particular, systemic-onset juvenile idiopathic arthritis (SoJIA), its adult counterpart adult-onset Still’s disease, and some patients with NLRC4 gain-of-function mutations display detectable fIL-18. The pathogenic role of fIL-18 in MAS was corroborated in an experimental model where IL-18BP knockout (KO) mice presented with an aggravated CpG-induced MAS [10]. The role of IL-18 in cancer is controversial. Indeed, IL-18 is capable of potently promoting Th1 polarization and enhancing NK cell antitumor responses (reviewed in [11]), and recently, Zhou et al. have reported a promising effect of a synthetic mutated form of IL-18 (IL-18DR) that does not bind to IL-18BP and is thus fully potent to exert antitumoral effects in experimental models [12]. On the other hand, IL-18 also bears some pro-tumoral functions. For instance, IL-18 has been shown to be pro-angiogenic and induce adhesion molecules on endothelial cells (ICAM-1 and VCAM-1) [11, 13], and in a preclinical spontaneous mouse model for gastric cancer, IL-18 was shown to drive the formation of tumors downstream of the apoptosis-related speck-like protein containing a CARD (ASC) inflammasome adaptor [14]. However, preclinical models for other cancer types display opposing results, which may be influenced by the cell type expressing IL-18 (e.g., epithelial versus immune cell), as well as stage of disease in which IL-18 is produced [15]. In humans, recombinant IL-18 showed limited efficacy in patients with metastatic melanoma [16]. It has been hypothesized that the antitumoral activity of IL-18 may be limited locally by IL-18BP, which is highly expressed in cancer and could act as an immune checkpoint [12]. Therefore, the assessment of free IL-18 and IL-18BP levels, and considering the IL-18/IL-18BP balance in treatment strategy, may also be of interest in cancer. Solely measuring total levels of IL-18 may sometimes not be sufficiently informative to describe experimental situations. Particularly, in vivo experiments and assays under the influence of IFN-γ may benefit from being analyzed in relation with IL-18BP. More

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importantly, understanding the relevance of elevated levels of IL-18 can represent a challenge for physicians. Therefore, herein we describe a set of tools for the assessment of the IL-18/IL-18BP balance, which includes assessment of unbound, fIL-18 by ELISA in human or mice samples, detection of pro and mature forms of murine IL-18 protein by Western blot, detection of murine or human IL-18 using myeloid cell lines stably expressing IL-18Rα/ β, and detection of IL-18BP at a protein level by Western blot.

2 2.1

Materials Free IL-18 ELISA

1. Flat-bottom adsorbant 96-well plates. 2. Recombinant human IL-18BPa. 3. Phosphate-buffered saline (PBS). 4. Bovine serum albumin (BSA). 5. Tween 20. 6. Recombinant human or mouse IL-18 (R & D Systems, Cat. No. B001–5 or B004–5). 7. Biotinylated mouse monoclonal anti-human (or rat antimouse) IL-18 antibody (MBL, Nagoya, Japan). 8. Shaking platform. 9. Peroxidase-conjugated streptavidin. 10. 3,3′,5,5’-Tetramethylbenzidine (TMB). 11. 2 N H2SO4. 12. Microplate reader.

2.2 Organ Dissociation for Western Blot Analysis

1. Inox beads. 2. Bead mill. 3. RIPA buffer: (a) 150 mM NaCl. (b) 1.0% (v/v) Triton X-100. (c) 0.5% (w/v) sodium deoxycholate. (d) 0.1% (w/v) sodium dodecyl sulphate (SDS). (e) 50 mM Tris–HCl, pH adjusted to 8.0. (f) 5 mM EDTA. (g) 1 tablet of protease inhibitor per 10 mL (e.g., Roche, Cat. No. 04693159001).

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2.3 Western Blot Analysis for Recombinant IL-18 in Cell or Tissue Lysates

1. 4–12% Bis–Tris gel. 2. Molecular weight markers, 10–250 kDa. 3. Recombinant murine IL-18 pro-form amino acids (a. a.) 1–192 (MyBiosource, Cat. No. 966244 Proform). 4. Recombinant mature mIL-18 a.a. 36–192 (RnD, Cat. No. B004–5). 5. Running buffer: (a) 14.4% (w/v) glycine. (b) 3% (w/v) Tris base. (c) 1% (w/v) SDS. 6. Porablot 0.2-μm polyvinylidene fluoride (PVDF) membrane. 7. Polyclonal rabbit anti-IL-18 (Biovision, Cat. No. 5180R-100). 8. Goat anti-rabbit IgG horseradish peroxidase (HRP). 9. Tris-buffered saline (TBS): 10 mM Tris–HCl, pH adjusted to 7.4, 150 mM NaCl. 10. Triton X-100. 11. Laemmli sample buffer 5 ×: (a) 30% glycerol anhydrous. (b) 10% SDS. (c) 250 mM Tris–HCl pH adjusted to 7.4. (d) 0.02% bromophenol blue. 12. 1 M 1,4-dithiothreitol (DTT). 13. Horse Serum (PanBiotech, Cat. No. P30–0702). 14. Enhanced chemiluminescence No. 170–5060).

(ECL)

(Bio-Rad,

Cat.

15. PBS. 16. Stripping ReBlot Plus Strong (Merck Millipore, Cat. No. 2504). 2.4 Detection of Mouse Free IL-18 Bioassay

1. IL-18Rα/β + RAW264.7 cells (clone E12.3) (see Note 1). 2. IL-18Rα/β – RAW264.7 cells (clone E11) (see Note 1). 3. P10 petri dishes. 4. Nunc 96-well flat-bottom plate. 5. Complete medium: Dulbecco’s Modified Eagle Medium (DMEM) supplemented with Glutamax, 4.5 g/L glucose (Gibco), 10% fetal calf serum (FCS), and 1% penicillin/ streptomycin. 6. Cell culture medium: Complete medium supplemented with 300 μg/mL geneticin and 200 μg/mL hygromycin. 7. Lipopolysaccharide (LPS) (Sigma, Cat. No. LP2880).

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8. ELISA kit for mTNFα. 9. mIL-18 (R & D Systems, Cat. No. 9139-IL-050). 10. rhIL-18 (MBL, Cat. No. B001–5). 11. rmIL-18BPa (Creative Biomart, Cat. No. 767 M). 12. Blocking anti-IL-18Rα (R & D Systems, Cat. No. MAB12161, clone 112,624). 13. Anti-IL-18Rα – PE (Thermo Fisher, clone 12.5183.82). 2.5 Detection of Human Free IL-18 Bioassay

1. KG-1 cells (ATCC, Cat. No. CCL246) (see Note 2). 2. Culture medium: (a) Iscove’s Modified Dulbecco’s Media (IMDM). (b) 20% FCS. (c) 1% penicillin/streptomycin. (d) 0.05 mM 2β-mercaptoethanol. 3. Stimulation medium: (a) IMDM. (b) 20% FCS. (c) 1% penicillin/streptomycin. 4. T75 flasks. 5. Nunc 96-well flat-bottom plates. 6. rhIL-18 (RnD, Cat. No. B001–5). 7. rhTNFα (Peprotech, Cat. No. 300-01A). 8. rhIL-18BPa. 9. hIL-8 ELISA kit.

2.6 Detection of Murine IL-18BP by Western Blot

1. RIPA buffer: (a) 150 mM NaCl. (b) 1.0% (v/v) Triton X-100. (c) 0.5% (w/v) sodium deoxycholate. (d) 0.1% (w/v) SDS. (e) 50 mM Tris–HCl, pH adjusted to 8.0. (f) 5 mM EDTA. (g) Extemporaneously add 1 tablet of protease inhibitor per 10 ml (Roche, Cat. No. 04693159001). 2. 4–12% Bis–Tris gel. 3. Molecular weight markers, 10–250 kDa. 4. Running buffer:

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(a) 14.4% (w/v) glycine. (b) 3% (w/v) Tris base. (c) 1% (w/v) SDS. 5. Porablot 0.2 μm PVDF. 6. Polyclonal goat anti-mouse IL-18BP (RnD, Cat. No. AF122). 7. Donkey anti-goat IgG HRP (Santa Cruz, Cat. No. sc-2304). 8. TBS: 10 mM Tris–HCl, pH adjusted to 7.4, and 150 mM NaCl. 9. Tween 20. 10. Laemmli sample buffer 5 ×: (a) 30% glycerol anhydrous. (b) 10% SDS. (c) 250 mM Tris–HCl pH adjusted to 7.4. (d) 0.02% bromophenol blue. 12. 1 M DTT. 13. Horse Serum. 14. ECL. 15. PBS. 16. Stripping ReBlot Plus Strong (Merck Millipore, Cat. No. 2504).

3

Methods

3.1 Free IL-18 ELISA (See Note 3)

1. Coat plate overnight at 4 °C with recombinant human IL-18BPa in PBS. 2. Block nonspecific binding sites with 1% BSA in PBS-Tween 20 (0.1% v/v) at room temperature. 3. Prepare standard curve by serially threefold-diluting recombinant human (or mouse) IL-18 (for concentrations from 1000 to 1.37 pg/mL) in 1% BSA in PBS. 4. Add standards and sera at a fivefold dilution. 5. Incubate with biotinylated anti-human or mouse IL-18 antibody at room temperature on a shaking platform at 100 to 120 rpm for 2 h. 6. After washing, incubate with peroxidase-conjugated streptavidin for 30 min. 7. Thoroughly wash and then incubate with TMB for 20 min. 8. Stop the reaction by adding 2 N H2SO4. 9. Read plate with a microplate reader at an optical density (OD) of 450 nm using 570 nm as the reference wavelength.

Methods for IL-18/IL-18BP Balance Assessment

3.2 Organ Dissociation for Western Blot Detection of IL-18 or IL-18BP

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1. For organs, prepare 1 mL of RIPA buffer per organ with protease inhibitor (1 tablet for 10 mL). 2. Place 1 inox bead in a 2-mL snap-lock tube. 3. Add 700 μL of RIPA-protease inhibitor buffer per tube. Place on ice. 4. Transfer organs in dedicated tube and use a homogenizer to disrupt the tissue (for a bead mill: 30 Hz, 2 × 3 min). 5. Transfer lysates in new 1.5-mL tubes. 6. Centrifuge 15 min at 4 °C at 14000 × g. 7. Collect supernatant and measure protein concentration with a detergent compatible assay such as DC protein assay from Bio-Rad.

3.3 Western Blot Analysis for IL-18 (See Note 4)

1. Distribute 15 ng of recombinant proteins or 50–150 μg of organ lysates in snap-lock 1.5-mL tubes. 2. Add 6 μL of 5 × Laemmli buffer and 3 mL of 1 M DTT. 3. Complete the volume to 30 μL with PBS. 4. Heat samples for 5 min at 95 °C in a dry bath. 5. Assemble 4–12% Tris–Bis gels into electrophoresis unit and fill with running buffer. Wash wells with running buffer to ensure a straight migration. 6. Load 20 μL of samples or 6 μL of molecular weight markers per well. 7. Run electrophoresis at 100 V until the front of migration reaches stacking, and then increase to 120 V (see Note 5). 8. Blot on PVDF membrane using electrophoretic transfer equipment. 9. Rinse the membrane with TBS, 0.1% Triton X-100. 10. Block the PVDF membrane with TBS, 0.1% Triton X-100, and 5% Horse Serum for 1 h at room temperature under mild agitation. 11. Stain by placing the membrane in 0.5 μg/mL of anti-mIL-18 in TBS 0.1% Triton X-100, and 5% Horse Serum. Incubate overnight at 4 °C under mild agitation. 12. Wash the membrane three times by exchanging the previous solution with TBS, 0.1% Triton X-100 and incubating 5 min at room temperature under mild agitation. 13. Place the membrane in HRP-conjugated secondary antibody (0.04 μg/mL goat anti-rabbit IgG in TBS—0.1% Tween, 5% Horse Serum). Incubate 1 h at room temperature. 14. Wash three times as in step 12.

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15. Wash once with distilled water, and incubate 5 min at room temperature. 16. Reveal with ECL and acquire (see Note 6). 19. Wash twice for 5 min in TBS, 0.1% Triton X-100, and 5% Horse Serum at room temperature under mild agitation. 20. Perform housekeeping protein staining. 3.4 Detection of Mouse Free IL-18 Bioassay (See Note 7)

1. Verify that RAW264.7 cells are in good shape (round, monocytic-like, attached, and little spread). 2. Resuspend cells by pipetting the supernatant onto the cell layer. 3. Centrifuge at 320 × g for 5 min at 21 °C. 4. Discard supernatant (see Note 8). 5. Resuspend one petri dish in 1 mL of culture medium. Count. 6. Prepare a cell suspension of 5 × 104 cells/mL. 7. Distribute 200 μL per well in a 96-well plate (104 cells/well). 8. Incubate overnight (16 h) at 37 °C, 5% CO2. 9. The following day, cells are exposed to stimulation with IL-18 (see Note 9). 10. Cells are then incubated for 24 h at 37 °C, 5% CO2. 11. Centrifuge the 96-well plate at 320 × g for 5 min at 21 °C. 12. Harvest supernatant. 13. The readout is performed by measuring TNFα in cell supernatants using commercially available ELISA according to manufacturer’s instructions.

3.5 Detection of Human Free IL-18 Bioassay

1. Resuspend cells in culture medium by pipetting up and down using a serologic pipet (see Note 10). 2. Collect the cell suspension in a 50-mL tube and centrifuge at 320 × g for 5 min at 21 °C. 3. Discard the supernatant and resuspend in 1 mL of stimulation medium (exempt of 2β-mercaptoethanol), and then count. 4. Adjust the concentration to 5 × 105 cells/mL and seed 200 μL per well in a 96-well plate (105 cells/well). 5. Incubate overnight (16 h) at 37 °C, 5% CO2. 6. Centrifuge 5 min at 320 × g for 5 min at 21 °C. 7. Remove supernatant delicately and add 200 μL of IL-18 containing stimulation medium. rhIL-18 at 10 ng/mL as a positive control yields an IL-8 response in the range of 102 pg/mL, while stimulation with 1 ng/mL rhTNFα generates an IL-8 response 2–4-fold higher. 8. Incubate for 24 h at 37 °C, 5% CO2.

Methods for IL-18/IL-18BP Balance Assessment

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9. Centrifuge 5 min at 320 × g for 5 min at 21 °C. 10. Collect supernatant for downstream procedures. 11. The readout is generally performed by measuring hIL-8 supernatant using commercially available ELISA according to manufacturer’s instructions. 3.6 Western Blot Analysis for IL-18BP

1. Distribute 50–150 μg of organ lysates (see Note 11) in snaplock 1.5-mL tubes. 2. Add 6 μL of 5 × Laemmli sample buffer and 3 μL of 10 × DTT. 3. Complete the volume to 30 μL with PBS. 4. Heat samples at 95 °C for 5 min. 5. Assemble 4–12% Tris–Bis gels into electrophoresis unit and fill with running buffer. Wash wells with a long reaching tip to ensure straight migration. 6. Load 20 μL of samples or 6 μL of page ruler per well. 7. Run electrophoresis at 100 V until the front of migration reaches stacking, and then increase to 120 V. 8. Blot on PVDF membrane using electrophoretic transfer equipment. 9. Block the PVDF membrane with TBS—0.1% Tween, 5% Horse Serum—for 1 h at room temperature under mild agitation. 10. Stain by placing the membrane in 0.4 μg/mL of anti-mIL18BP in TBS—0.1% Tween, 5% Horse Serum. Incubate overnight at 4 °C under mild agitation. 12. Wash the membrane three times by exchanging the previous solution with TBS—0.1% Tween—and incubating 5 min at room temperature under agitation. 13. Place the membrane in HRP-conjugated secondary antibody (0.01 μg/mL donkey anti-goat IgG in TBS—0.1% Tween, 5% Horse Serum) and incubate 1 h at room temperature. 14. Wash three times (see step 12). 15. Wash three times with distilled water, and then incubate for 5 min at room temperature. 16. Develop blot with ECL (1:1) (see Note 6). 17. Wash twice for 5 min in TBS—0.1% Tween, 5% Horse Serum—at room temperature under mild agitation. 18. Perform housekeeping protein staining.

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Notes 1. Cells are kept in culture for 3’

AACCAGTTGA

AAGCAGTGGTATCAACGCAGAGTACAACCAGTTGANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AACCGGCGTA

AAGCAGTGGTATCAACGCAGAGTACAACCGGCGTANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AACCTAGTCC

AAGCAGTGGTATCAACGCAGAGTACAACCTAGTCCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AACTCTACAC

AAGCAGTGGTATCAACGCAGAGTACAACTCTACACNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AACTGTGTCA

AAGCAGTGGTATCAACGCAGAGTACAACTGTGTCANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AAGATGTCCA

AAGCAGTGGTATCAACGCAGAGTACAAGATGTCCANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AAGCATATGG

AAGCAGTGGTATCAACGCAGAGTACAAGCATATGGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AAGCTCACCT

AAGCAGTGGTATCAACGCAGAGTACAAGCTCACCTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AAGGCATGCG

AAGCAGTGGTATCAACGCAGAGTACAAGGCATGCGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AAGTTCCTTG

AAGCAGTGGTATCAACGCAGAGTACAAGTTCCTTGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AATACCGGTA

AAGCAGTGGTATCAACGCAGAGTACAATACCGGTANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AATCCATCTG

AAGCAGTGGTATCAACGCAGAGTACAATCCATCTGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AATCCGCTCC

AAGCAGTGGTATCAACGCAGAGTACAATCCGCTCCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AATCCTACCA

AAGCAGTGGTATCAACGCAGAGTACAATCCTACCANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AATCGTCCGC

AAGCAGTGGTATCAACGCAGAGTACAATCGTCCGCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AATGAGAGCA

AAGCAGTGGTATCAACGCAGAGTACAATGAGAGCANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AATGTCAGTG

AAGCAGTGGTATCAACGCAGAGTACAATGTCAGTGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACAACAGTCG

AAGCAGTGGTATCAACGCAGAGTACACAACAGTCGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACAACCATAC

AAGCAGTGGTATCAACGCAGAGTACACAACCATACNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACACAATCTC

AAGCAGTGGTATCAACGCAGAGTACACACAATCTCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN (continued)

Multiplex High-Throughput Sequencing

291

Table 2 (continued) Barcode

Primer sequence 5′ > 3’

ACACAGTGAA

AAGCAGTGGTATCAACGCAGAGTACACACAGTGAANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACACGGTCCT

AAGCAGTGGTATCAACGCAGAGTACACACGGTCCTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACACTTGCTG

AAGCAGTGGTATCAACGCAGAGTACACACTTGCTGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACCAGGACCA

AAGCAGTGGTATCAACGCAGAGTACACCAGGACCANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACCATAACAC

AAGCAGTGGTATCAACGCAGAGTACACCATAACACNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACCGGTACAG

AAGCAGTGGTATCAACGCAGAGTACACCGGTACAGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACCGTACTTC

AAGCAGTGGTATCAACGCAGAGTACACCGTACTTCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACCTGTCCGA

AAGCAGTGGTATCAACGCAGAGTACACCTGTCCGANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACCTTATGTG

AAGCAGTGGTATCAACGCAGAGTACACCTTATGTGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AATGAACACG

AAGCAGTGGTATCAACGCAGAGTACAATGAACACGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AATGACCTTC

AAGCAGTGGTATCAACGCAGAGTACAATGACCTTCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AATTAGGCCG

AAGCAGTGGTATCAACGCAGAGTACAATTAGGCCGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AATTGCGATG

AAGCAGTGGTATCAACGCAGAGTACAATTGCGATGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACAACGGAGC

AAGCAGTGGTATCAACGCAGAGTACACAACGGAGCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACAAGCGCGA

AAGCAGTGGTATCAACGCAGAGTACACAAGCGCGANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACACCGAATT

AAGCAGTGGTATCAACGCAGAGTACACACCGAATTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACACGCAGTA

AAGCAGTGGTATCAACGCAGAGTACACACGCAGTANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACAGTGCCAA

AAGCAGTGGTATCAACGCAGAGTACACAGTGCCAANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACATGTGTGC

AAGCAGTGGTATCAACGCAGAGTACACATGTGTGCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN (continued)

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Table 2 (continued) Barcode

Primer sequence 5′ > 3’

ACCGAACCGT

AAGCAGTGGTATCAACGCAGAGTACACCGAACCGTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACCGAGAGTC

AAGCAGTGGTATCAACGCAGAGTACACCGAGAGTCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACCTCCGACA

AAGCAGTGGTATCAACGCAGAGTACACCTCCGACANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACCTCTCTCC

AAGCAGTGGTATCAACGCAGAGTACACCTCTCTCCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACGAATGACA

AAGCAGTGGTATCAACGCAGAGTACACGAATGACANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACGCCTCAAC

AAGCAGTGGTATCAACGCAGAGTACACGCCTCAACNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACGCCTTCGT

AAGCAGTGGTATCAACGCAGAGTACACGCCTTCGTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACGCTGGATA

AAGCAGTGGTATCAACGCAGAGTACACGCTGGATANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACGTGCTGAT

AAGCAGTGGTATCAACGCAGAGTACACGTGCTGATNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACTCCAAGCC

AAGCAGTGGTATCAACGCAGAGTACACTCCAAGCCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACTTAACTGC

AAGCAGTGGTATCAACGCAGAGTACACTTAACTGCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACTTCATCAC

AAGCAGTGGTATCAACGCAGAGTACACTTCATCACNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACTTGAGGAA

AAGCAGTGGTATCAACGCAGAGTACACTTGAGGAANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACTTGTAAGG

AAGCAGTGGTATCAACGCAGAGTACACTTGTAAGGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AGACCGTTAT

AAGCAGTGGTATCAACGCAGAGTACAGACCGTTATNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AGACTAGCAT

AAGCAGTGGTATCAACGCAGAGTACAGACTAGCATNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AGAGTGTAAC

AAGCAGTGGTATCAACGCAGAGTACAGAGTGTAACNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AGAGTTCTGC

AAGCAGTGGTATCAACGCAGAGTACAGAGTTCTGCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AGCATGTCAT

AAGCAGTGGTATCAACGCAGAGTACAGCATGTCATNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN (continued)

Multiplex High-Throughput Sequencing

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Table 2 (continued) Barcode

Primer sequence 5′ > 3’

AGCCACTAGC

AAGCAGTGGTATCAACGCAGAGTACAGCCACTAGCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AGCGATAACG

AAGCAGTGGTATCAACGCAGAGTACAGCGATAACGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AGCGTACAAT

AAGCAGTGGTATCAACGCAGAGTACAGCGTACAATNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACGGTCCGTT

AAGCAGTGGTATCAACGCAGAGTACACGGTCCGTTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACGTAGGCAC

AAGCAGTGGTATCAACGCAGAGTACACGTAGGCACNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACTGGCGCAT

AAGCAGTGGTATCAACGCAGAGTACACTGGCGCATNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACTGGCTTCC

AAGCAGTGGTATCAACGCAGAGTACACTGGCTTCCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACTTCGTTGA

AAGCAGTGGTATCAACGCAGAGTACACTTCGTTGANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ACTTCTCCTG

AAGCAGTGGTATCAACGCAGAGTACACTTCTCCTGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AGAACCACGG

AAGCAGTGGTATCAACGCAGAGTACAGAACCACGGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AGAAGCAATC

AAGCAGTGGTATCAACGCAGAGTACAGAAGCAATCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AGAGATGCAG

AAGCAGTGGTATCAACGCAGAGTACAGAGATGCAGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AGAGCTTACA

AAGCAGTGGTATCAACGCAGAGTACAGAGCTTACANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AGATAGTGCT

AAGCAGTGGTATCAACGCAGAGTACAGATAGTGCTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AGCAATGCGC

AAGCAGTGGTATCAACGCAGAGTACAGCAATGCGCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AGCCAGAATA

AAGCAGTGGTATCAACGCAGAGTACAGCCAGAATANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AGCCAGCTCT

AAGCAGTGGTATCAACGCAGAGTACAGCCAGCTCTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AGCTATTCCA

AAGCAGTGGTATCAACGCAGAGTACAGCTATTCCANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AGCTCCTCAG

AAGCAGTGGTATCAACGCAGAGTACAGCTCCTCAGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN (continued)

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Table 2 (continued) Barcode

Primer sequence 5′ > 3’

AGGAGGCATA

AAGCAGTGGTATCAACGCAGAGTACAGGAGGCATANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AGGCGTCTGT

AAGCAGTGGTATCAACGCAGAGTACAGGCGTCTGTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AGTAACTCAC

AAGCAGTGGTATCAACGCAGAGTACAGTAACTCACNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AGTAAGCGTT

AAGCAGTGGTATCAACGCAGAGTACAGTAAGCGTTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AGTCTGTACG

AAGCAGTGGTATCAACGCAGAGTACAGTCTGTACGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AGTGCAATGT

AAGCAGTGGTATCAACGCAGAGTACAGTGCAATGTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ATAAGGTGCA

AAGCAGTGGTATCAACGCAGAGTACATAAGGTGCANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ATACACGACA

AAGCAGTGGTATCAACGCAGAGTACATACACGACANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ATAGGCCATT

AAGCAGTGGTATCAACGCAGAGTACATAGGCCATTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ATATCCGCAT

AAGCAGTGGTATCAACGCAGAGTACATATCCGCATNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ATCCAATACG

AAGCAGTGGTATCAACGCAGAGTACATCCAATACGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ATCCGCTGTG

AAGCAGTGGTATCAACGCAGAGTACATCCGCTGTGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ATCGCGATTA

AAGCAGTGGTATCAACGCAGAGTACATCGCGATTANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ATCGGTAGGC

AAGCAGTGGTATCAACGCAGAGTACATCGGTAGGCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ATGACTCAGT

AAGCAGTGGTATCAACGCAGAGTACATGACTCAGTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ATGCACCGGA

AAGCAGTGGTATCAACGCAGAGTACATGCACCGGANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AGGTATCCTC

AAGCAGTGGTATCAACGCAGAGTACAGGTATCCTCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AGGTCACCAA

AAGCAGTGGTATCAACGCAGAGTACAGGTCACCAANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

AGTCCACGTA

AAGCAGTGGTATCAACGCAGAGTACAGTCCACGTANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN (continued)

Multiplex High-Throughput Sequencing

295

Table 2 (continued) Barcode

Primer sequence 5′ > 3’

AGTCTCGGCA

AAGCAGTGGTATCAACGCAGAGTACAGTCTCGGCANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ATAACGCCTC

AAGCAGTGGTATCAACGCAGAGTACATAACGCCTCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ATAAGAGGTC

AAGCAGTGGTATCAACGCAGAGTACATAAGAGGTCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ATACCTCCGG

AAGCAGTGGTATCAACGCAGAGTACATACCTCCGGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ATAGCAGTGC

AAGCAGTGGTATCAACGCAGAGTACATAGCAGTGCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ATCAGCACTT

AAGCAGTGGTATCAACGCAGAGTACATCAGCACTTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ATCAGCGAGG

AAGCAGTGGTATCAACGCAGAGTACATCAGCGAGGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ATCCGTCCAT

AAGCAGTGGTATCAACGCAGAGTACATCCGTCCATNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ATCGACGGCT

AAGCAGTGGTATCAACGCAGAGTACATCGACGGCTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ATCTAAGGAG

AAGCAGTGGTATCAACGCAGAGTACATCTAAGGAGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ATGACGGTAA

AAGCAGTGGTATCAACGCAGAGTACATGACGGTAANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ATGCGGACTG

AAGCAGTGGTATCAACGCAGAGTACATGCGGACTGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ATGCTTCCTA

AAGCAGTGGTATCAACGCAGAGTACATGCTTCCTANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ATGGACCAAC

AAGCAGTGGTATCAACGCAGAGTACATGGACCAACNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ATGGTCTTAG

AAGCAGTGGTATCAACGCAGAGTACATGGTCTTAGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ATTATCGGAC

AAGCAGTGGTATCAACGCAGAGTACATTATCGGACNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ATTCGGAACA

AAGCAGTGGTATCAACGCAGAGTACATTCGGAACANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CAAGATGAGG

AAGCAGTGGTATCAACGCAGAGTACCAAGATGAGGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CAAGCCAACG

AAGCAGTGGTATCAACGCAGAGTACCAAGCCAACGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN (continued)

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Table 2 (continued) Barcode

Primer sequence 5′ > 3’

CACGAGTCTG

AAGCAGTGGTATCAACGCAGAGTACCACGAGTCTGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CACGCTCCAA

AAGCAGTGGTATCAACGCAGAGTACCACGCTCCAANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CAGTGCTCTT

AAGCAGTGGTATCAACGCAGAGTACCAGTGCTCTTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CAGTTAAGCA

AAGCAGTGGTATCAACGCAGAGTACCAGTTAAGCANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CATGTACGCC

AAGCAGTGGTATCAACGCAGAGTACCATGTACGCCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CATTACACTG

AAGCAGTGGTATCAACGCAGAGTACCATTACACTGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CCAAGGAGTT

AAGCAGTGGTATCAACGCAGAGTACCCAAGGAGTTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CCAATTGTTC

AAGCAGTGGTATCAACGCAGAGTACCCAATTGTTCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CCATAACTTG

AAGCAGTGGTATCAACGCAGAGTACCCATAACTTGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CCATACTGAC

AAGCAGTGGTATCAACGCAGAGTACCCATACTGACNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ATGGTGAGCG

AAGCAGTGGTATCAACGCAGAGTACATGGTGAGCGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

ATGTGGAAGC

AAGCAGTGGTATCAACGCAGAGTACATGTGGAAGCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CAACAATCCA

AAGCAGTGGTATCAACGCAGAGTACCAACAATCCANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CAAGAAGCAT

AAGCAGTGGTATCAACGCAGAGTACCAAGAAGCATNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CAAGTGGATC

AAGCAGTGGTATCAACGCAGAGTACCAAGTGGATCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CACAGTTCAT

AAGCAGTGGTATCAACGCAGAGTACCACAGTTCATNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CACTGAGCAC

AAGCAGTGGTATCAACGCAGAGTACCACTGAGCACNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CAGATCAATG

AAGCAGTGGTATCAACGCAGAGTACCAGATCAATGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CATAGCTATC

AAGCAGTGGTATCAACGCAGAGTACCATAGCTATCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN (continued)

Multiplex High-Throughput Sequencing

297

Table 2 (continued) Barcode

Primer sequence 5′ > 3’

CATCACCACC

AAGCAGTGGTATCAACGCAGAGTACCATCACCACCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CATTCGACGA

AAGCAGTGGTATCAACGCAGAGTACCATTCGACGANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CCAACTATGG

AAGCAGTGGTATCAACGCAGAGTACCCAACTATGGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CCACAAGTGC

AAGCAGTGGTATCAACGCAGAGTACCCACAAGTGCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CCAGCTTAGT

AAGCAGTGGTATCAACGCAGAGTACCCAGCTTAGTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CCATAGATCA

AAGCAGTGGTATCAACGCAGAGTACCCATAGATCANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CCATGTGCTT

AAGCAGTGGTATCAACGCAGAGTACCCATGTGCTTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CCATTCAGCG

AAGCAGTGGTATCAACGCAGAGTACCCATTCAGCGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CCGAACAAGC

AAGCAGTGGTATCAACGCAGAGTACCCGAACAAGCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CCGAATAGTG

AAGCAGTGGTATCAACGCAGAGTACCCGAATAGTGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CCGACTTCTC

AAGCAGTGGTATCAACGCAGAGTACCCGACTTCTCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CCGCGTTATG

AAGCAGTGGTATCAACGCAGAGTACCCGCGTTATGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CCGCTAGCTT

AAGCAGTGGTATCAACGCAGAGTACCCGCTAGCTTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CCGGTCTCTA

AAGCAGTGGTATCAACGCAGAGTACCCGGTCTCTANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CCGTACGATG

AAGCAGTGGTATCAACGCAGAGTACCCGTACGATGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CCTAGTTGAG

AAGCAGTGGTATCAACGCAGAGTACCCTAGTTGAGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CCTATTCTGT

AAGCAGTGGTATCAACGCAGAGTACCCTATTCTGTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CCTGATGCCA

AAGCAGTGGTATCAACGCAGAGTACCCTGATGCCANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CCTGCAATAC

AAGCAGTGGTATCAACGCAGAGTACCCTGCAATACNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN (continued)

298

Xiang Mark Li et al.

Table 2 (continued) Barcode

Primer sequence 5′ > 3’

CGAGGAACAA

AAGCAGTGGTATCAACGCAGAGTACCGAGGAACAANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CGATAACCGC

AAGCAGTGGTATCAACGCAGAGTACCGATAACCGCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CGCCAGTGTT

AAGCAGTGGTATCAACGCAGAGTACCGCCAGTGTTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CGCCTTGTAC

AAGCAGTGGTATCAACGCAGAGTACCGCCTTGTACNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CCGAACCTAA

AAGCAGTGGTATCAACGCAGAGTACCCGAACCTAANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CCGAAGACCT

AAGCAGTGGTATCAACGCAGAGTACCCGAAGACCTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CCGATCCACT

AAGCAGTGGTATCAACGCAGAGTACCCGATCCACTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CCGATGATAC

AAGCAGTGGTATCAACGCAGAGTACCCGATGATACNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CCGGAGTATC

AAGCAGTGGTATCAACGCAGAGTACCCGGAGTATCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CCGGCCAATT

AAGCAGTGGTATCAACGCAGAGTACCCGGCCAATTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CCGTCAGAAC

AAGCAGTGGTATCAACGCAGAGTACCCGTCAGAACNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CCTAGACACG

AAGCAGTGGTATCAACGCAGAGTACCCTAGACACGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CCTCAACCGA

AAGCAGTGGTATCAACGCAGAGTACCCTCAACCGANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CCTCCATAAG

AAGCAGTGGTATCAACGCAGAGTACCCTCCATAAGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CCTTGTATTC

AAGCAGTGGTATCAACGCAGAGTACCCTTGTATTCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CGAGATCTCT

AAGCAGTGGTATCAACGCAGAGTACCGAGATCTCTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CGATCCTGTG

AAGCAGTGGTATCAACGCAGAGTACCGATCCTGTGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CGCCAACCAT

AAGCAGTGGTATCAACGCAGAGTACCGCCAACCATNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CGCGGATTCA

AAGCAGTGGTATCAACGCAGAGTACCGCGGATTCANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN (continued)

Multiplex High-Throughput Sequencing

299

Table 2 (continued) Barcode

Primer sequence 5′ > 3’

CGCTTAAGGC

AAGCAGTGGTATCAACGCAGAGTACCGCTTAAGGCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CGCTTACTAA

AAGCAGTGGTATCAACGCAGAGTACCGCTTACTAANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CGCTTCTTGG

AAGCAGTGGTATCAACGCAGAGTACCGCTTCTTGGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CGGAGATTGG

AAGCAGTGGTATCAACGCAGAGTACCGGAGATTGGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CGGAGCTCAA

AAGCAGTGGTATCAACGCAGAGTACCGGAGCTCAANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CGGCAACTTA

AAGCAGTGGTATCAACGCAGAGTACCGGCAACTTANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CGGCTCATCA

AAGCAGTGGTATCAACGCAGAGTACCGGCTCATCANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CGTAACGGAT

AAGCAGTGGTATCAACGCAGAGTACCGTAACGGATNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CGTAAGATTC

AAGCAGTGGTATCAACGCAGAGTACCGTAAGATTCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CGTCCTAGGA

AAGCAGTGGTATCAACGCAGAGTACCGTCCTAGGANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CGTCGGCAAT

AAGCAGTGGTATCAACGCAGAGTACCGTCGGCAATNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CTAACTTCAG

AAGCAGTGGTATCAACGCAGAGTACCTAACTTCAGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CTAATAGCGT

AAGCAGTGGTATCAACGCAGAGTACCTAATAGCGTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CTATGAACGG

AAGCAGTGGTATCAACGCAGAGTACCTATGAACGGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CTCAAGGACC

AAGCAGTGGTATCAACGCAGAGTACCTCAAGGACCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CTCGCAACGT

AAGCAGTGGTATCAACGCAGAGTACCTCGCAACGTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CTCGTGCCTA

AAGCAGTGGTATCAACGCAGAGTACCTCGTGCCTANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CGGAAGCTGT

AAGCAGTGGTATCAACGCAGAGTACCGGAAGCTGTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CGGAATACAC

AAGCAGTGGTATCAACGCAGAGTACCGGAATACACNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN (continued)

300

Xiang Mark Li et al.

Table 2 (continued) Barcode

Primer sequence 5′ > 3’

CGGATCGGTA

AAGCAGTGGTATCAACGCAGAGTACCGGATCGGTANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CGGATTCTAG

AAGCAGTGGTATCAACGCAGAGTACCGGATTCTAGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CGGTCGTATT

AAGCAGTGGTATCAACGCAGAGTACCGGTCGTATTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CGGTGACATC

AAGCAGTGGTATCAACGCAGAGTACCGGTGACATCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CGTACTGTAA

AAGCAGTGGTATCAACGCAGAGTACCGTACTGTAANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CGTAGAAGAC

AAGCAGTGGTATCAACGCAGAGTACCGTAGAAGACNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CGTGAGTTAT

AAGCAGTGGTATCAACGCAGAGTACCGTGAGTTATNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CGTGTCAAGC

AAGCAGTGGTATCAACGCAGAGTACCGTGTCAAGCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CTACACCAGG

AAGCAGTGGTATCAACGCAGAGTACCTACACCAGGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CTAGCACAAT

AAGCAGTGGTATCAACGCAGAGTACCTAGCACAATNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CTCACCTGTC

AAGCAGTGGTATCAACGCAGAGTACCTCACCTGTCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CTCCTATTGT

AAGCAGTGGTATCAACGCAGAGTACCTCCTATTGTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CTGGATTGAC

AAGCAGTGGTATCAACGCAGAGTACCTGGATTGACNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CTGTAGTCAG

AAGCAGTGGTATCAACGCAGAGTACCTGTAGTCAGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CTGTCGCTTC

AAGCAGTGGTATCAACGCAGAGTACCTGTCGCTTCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CTGTCTGTGT

AAGCAGTGGTATCAACGCAGAGTACCTGTCTGTGTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CTTCATATCG

AAGCAGTGGTATCAACGCAGAGTACCTTCATATCGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CTTGCTGACG

AAGCAGTGGTATCAACGCAGAGTACCTTGCTGACGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GAAGGATTAG

AAGCAGTGGTATCAACGCAGAGTACGAAGGATTAGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN (continued)

Multiplex High-Throughput Sequencing

301

Table 2 (continued) Barcode

Primer sequence 5′ > 3’

GAATCGAGCC

AAGCAGTGGTATCAACGCAGAGTACGAATCGAGCCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GACCATCTAA

AAGCAGTGGTATCAACGCAGAGTACGACCATCTAANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GACGACCACA

AAGCAGTGGTATCAACGCAGAGTACGACGACCACANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GAGACATCTT

AAGCAGTGGTATCAACGCAGAGTACGAGACATCTTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GAGCGAGTCA

AAGCAGTGGTATCAACGCAGAGTACGAGCGAGTCANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GATAGACTGT

AAGCAGTGGTATCAACGCAGAGTACGATAGACTGTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GATAGAGGCG

AAGCAGTGGTATCAACGCAGAGTACGATAGAGGCGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GATCTCATTC

AAGCAGTGGTATCAACGCAGAGTACGATCTCATTCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GATCTGGTCG

AAGCAGTGGTATCAACGCAGAGTACGATCTGGTCGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GATGTGACAG

AAGCAGTGGTATCAACGCAGAGTACGATGTGACAGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GATTAAGTCC

AAGCAGTGGTATCAACGCAGAGTACGATTAAGTCCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CTGTGCAACA

AAGCAGTGGTATCAACGCAGAGTACCTGTGCAACANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CTTATGTTGC

AAGCAGTGGTATCAACGCAGAGTACCTTATGTTGCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

CTTGGATCTT

AAGCAGTGGTATCAACGCAGAGTACCTTGGATCTTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GAAGAGTTCT

AAGCAGTGGTATCAACGCAGAGTACGAAGAGTTCTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GAATCTTCTC

AAGCAGTGGTATCAACGCAGAGTACGAATCTTCTCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GAATTACGGC

AAGCAGTGGTATCAACGCAGAGTACGAATTACGGCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GAGAACGAAG

AAGCAGTGGTATCAACGCAGAGTACGAGAACGAAGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GAGACAAGGC

AAGCAGTGGTATCAACGCAGAGTACGAGACAAGGCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN (continued)

302

Xiang Mark Li et al.

Table 2 (continued) Barcode

Primer sequence 5′ > 3’

GAGTAGACCA

AAGCAGTGGTATCAACGCAGAGTACGAGTAGACCANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GATACGCTTA

AAGCAGTGGTATCAACGCAGAGTACGATACGCTTANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GATAGGTCAA

AAGCAGTGGTATCAACGCAGAGTACGATAGGTCAANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GATATCAGGA

AAGCAGTGGTATCAACGCAGAGTACGATATCAGGANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GATGAGTGAC

AAGCAGTGGTATCAACGCAGAGTACGATGAGTGACNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GATGGATACA

AAGCAGTGGTATCAACGCAGAGTACGATGGATACANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GATTGCACGC

AAGCAGTGGTATCAACGCAGAGTACGATTGCACGCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GCAAGCGAAT

AAGCAGTGGTATCAACGCAGAGTACGCAAGCGAATNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GCAATGTAAG

AAGCAGTGGTATCAACGCAGAGTACGCAATGTAAGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GCACACTATA

AAGCAGTGGTATCAACGCAGAGTACGCACACTATANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GCACTTAATC

AAGCAGTGGTATCAACGCAGAGTACGCACTTAATCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GCAGGAGATG

AAGCAGTGGTATCAACGCAGAGTACGCAGGAGATGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GCATCCGATC

AAGCAGTGGTATCAACGCAGAGTACGCATCCGATCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GCCAAGTACA

AAGCAGTGGTATCAACGCAGAGTACGCCAAGTACANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GCCATATCGA

AAGCAGTGGTATCAACGCAGAGTACGCCATATCGANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GCCGTCAATA

AAGCAGTGGTATCAACGCAGAGTACGCCGTCAATANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GCTATTATCC

AAGCAGTGGTATCAACGCAGAGTACGCTATTATCCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GCTCAGTAAT

AAGCAGTGGTATCAACGCAGAGTACGCTCAGTAATNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GGACGATGCT

AAGCAGTGGTATCAACGCAGAGTACGGACGATGCTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN (continued)

Multiplex High-Throughput Sequencing

303

Table 2 (continued) Barcode

Primer sequence 5′ > 3’

GGCATCGTGA

AAGCAGTGGTATCAACGCAGAGTACGGCATCGTGANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GGCGCTATAA

AAGCAGTGGTATCAACGCAGAGTACGGCGCTATAANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GGCGTTAAGT

AAGCAGTGGTATCAACGCAGAGTACGGCGTTAAGTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GGTAATGTGT

AAGCAGTGGTATCAACGCAGAGTACGGTAATGTGTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GGTGGTTGGA

AAGCAGTGGTATCAACGCAGAGTACGGTGGTTGGANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GCACTCGGAA

AAGCAGTGGTATCAACGCAGAGTACGCACTCGGAANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GCACTGCGTT

AAGCAGTGGTATCAACGCAGAGTACGCACTGCGTTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GCAGTACTGG

AAGCAGTGGTATCAACGCAGAGTACGCAGTACTGGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GCATATGAGT

AAGCAGTGGTATCAACGCAGAGTACGCATATGAGTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GCCACGATTC

AAGCAGTGGTATCAACGCAGAGTACGCCACGATTCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GCCATAGGTT

AAGCAGTGGTATCAACGCAGAGTACGCCATAGGTTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GCCTGGACAT

AAGCAGTGGTATCAACGCAGAGTACGCCTGGACATNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GCGTAATTAC

AAGCAGTGGTATCAACGCAGAGTACGCGTAATTACNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GCTGCTTATA

AAGCAGTGGTATCAACGCAGAGTACGCTGCTTATANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GGAATAAGCA

AAGCAGTGGTATCAACGCAGAGTACGGAATAAGCANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GGCATTATTG

AAGCAGTGGTATCAACGCAGAGTACGGCATTATTGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GGCCGAGATT

AAGCAGTGGTATCAACGCAGAGTACGGCCGAGATTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GGCTATTGAT

AAGCAGTGGTATCAACGCAGAGTACGGCTATTGATNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GGCTGCTACT

AAGCAGTGGTATCAACGCAGAGTACGGCTGCTACTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN (continued)

304

Xiang Mark Li et al.

Table 2 (continued) Barcode

Primer sequence 5′ > 3’

GGTGTTCACC

AAGCAGTGGTATCAACGCAGAGTACGGTGTTCACCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GGTTAGATCT

AAGCAGTGGTATCAACGCAGAGTACGGTTAGATCTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GGTTATGGCG

AAGCAGTGGTATCAACGCAGAGTACGGTTATGGCGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GGTTCACTGG

AAGCAGTGGTATCAACGCAGAGTACGGTTCACTGGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GTAACCTTGG

AAGCAGTGGTATCAACGCAGAGTACGTAACCTTGGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GTAAGAACCT

AAGCAGTGGTATCAACGCAGAGTACGTAAGAACCTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GTATTGTGGA

AAGCAGTGGTATCAACGCAGAGTACGTATTGTGGANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GTCCGCATCA

AAGCAGTGGTATCAACGCAGAGTACGTCCGCATCANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GTCGGTGACA

AAGCAGTGGTATCAACGCAGAGTACGTCGGTGACANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GTCTCGAGTG

AAGCAGTGGTATCAACGCAGAGTACGTCTCGAGTGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GTGACTATAC

AAGCAGTGGTATCAACGCAGAGTACGTGACTATACNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GTGGTTAATG

AAGCAGTGGTATCAACGCAGAGTACGTGGTTAATGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GTTCATTGCC

AAGCAGTGGTATCAACGCAGAGTACGTTCATTGCCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GTTCCGGTGA

AAGCAGTGGTATCAACGCAGAGTACGTTCCGGTGANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GTTGATCCGC

AAGCAGTGGTATCAACGCAGAGTACGTTGATCCGCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GTTGTATGCT

AAGCAGTGGTATCAACGCAGAGTACGTTGTATGCTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TAAGGTACGG

AAGCAGTGGTATCAACGCAGAGTACTAAGGTACGGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TACGGACATA

AAGCAGTGGTATCAACGCAGAGTACTACGGACATANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GGTTGTGCAA

AAGCAGTGGTATCAACGCAGAGTACGGTTGTGCAANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN (continued)

Multiplex High-Throughput Sequencing

305

Table 2 (continued) Barcode

Primer sequence 5′ > 3’

GTAACCAGTA

AAGCAGTGGTATCAACGCAGAGTACGTAACCAGTANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GTAAGGCTCC

AAGCAGTGGTATCAACGCAGAGTACGTAAGGCTCCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GTAATCCACG

AAGCAGTGGTATCAACGCAGAGTACGTAATCCACGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GTCCTTCGGT

AAGCAGTGGTATCAACGCAGAGTACGTCCTTCGGTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GTCGCTCTCT

AAGCAGTGGTATCAACGCAGAGTACGTCGCTCTCTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GTCTCTTAAG

AAGCAGTGGTATCAACGCAGAGTACGTCTCTTAAGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GTCTTCCGAG

AAGCAGTGGTATCAACGCAGAGTACGTCTTCCGAGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GTGTGCCTGT

AAGCAGTGGTATCAACGCAGAGTACGTGTGCCTGTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GTGTGTGTCC

AAGCAGTGGTATCAACGCAGAGTACGTGTGTGTCCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GTTCGTCGAA

AAGCAGTGGTATCAACGCAGAGTACGTTCGTCGAANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

GTTGAATTGG

AAGCAGTGGTATCAACGCAGAGTACGTTGAATTGGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TAACCGTAGC

AAGCAGTGGTATCAACGCAGAGTACTAACCGTAGCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TAACGTCGAT

AAGCAGTGGTATCAACGCAGAGTACTAACGTCGATNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TACTACCGCC

AAGCAGTGGTATCAACGCAGAGTACTACTACCGCCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TACTGTCAAG

AAGCAGTGGTATCAACGCAGAGTACTACTGTCAAGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TAGCGAACGC

AAGCAGTGGTATCAACGCAGAGTACTAGCGAACGCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TAGCGCCAAC

AAGCAGTGGTATCAACGCAGAGTACTAGCGCCAACNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TAGTAGTCTC

AAGCAGTGGTATCAACGCAGAGTACTAGTAGTCTCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TAGTCCGCTG

AAGCAGTGGTATCAACGCAGAGTACTAGTCCGCTGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN (continued)

306

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Table 2 (continued) Barcode

Primer sequence 5′ > 3’

TATCGTTACG

AAGCAGTGGTATCAACGCAGAGTACTATCGTTACGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TCAAGTGCAG

AAGCAGTGGTATCAACGCAGAGTACTCAAGTGCAGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TCACGCCACT

AAGCAGTGGTATCAACGCAGAGTACTCACGCCACTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TCACGTTGGC

AAGCAGTGGTATCAACGCAGAGTACTCACGTTGGCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TCCACGGTCA

AAGCAGTGGTATCAACGCAGAGTACTCCACGGTCANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TCCACTCGCT

AAGCAGTGGTATCAACGCAGAGTACTCCACTCGCTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TCCTAAGAGA

AAGCAGTGGTATCAACGCAGAGTACTCCTAAGAGANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TCCTCTAGTA

AAGCAGTGGTATCAACGCAGAGTACTCCTCTAGTANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TCGCACTTGA

AAGCAGTGGTATCAACGCAGAGTACTCGCACTTGANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TCGCCTACTG

AAGCAGTGGTATCAACGCAGAGTACTCGCCTACTGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TCTACATCCG

AAGCAGTGGTATCAACGCAGAGTACTCTACATCCGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TCTCCACATT

AAGCAGTGGTATCAACGCAGAGTACTCTCCACATTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TAGGACGCCT

AAGCAGTGGTATCAACGCAGAGTACTAGGACGCCTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TAGGTTGCAA

AAGCAGTGGTATCAACGCAGAGTACTAGGTTGCAANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TAGTGGAACT

AAGCAGTGGTATCAACGCAGAGTACTAGTGGAACTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TATCATGCAG

AAGCAGTGGTATCAACGCAGAGTACTATCATGCAGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TCACAGATAC

AAGCAGTGGTATCAACGCAGAGTACTCACAGATACNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TCACCGCCTA

AAGCAGTGGTATCAACGCAGAGTACTCACCGCCTANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TCATTGTCCA

AAGCAGTGGTATCAACGCAGAGTACTCATTGTCCANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN (continued)

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Table 2 (continued) Barcode

Primer sequence 5′ > 3’

TCCACACTAG

AAGCAGTGGTATCAACGCAGAGTACTCCACACTAGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TCCGACTAAC

AAGCAGTGGTATCAACGCAGAGTACTCCGACTAACNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TCCGTTATCT

AAGCAGTGGTATCAACGCAGAGTACTCCGTTATCTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TCGAAGCATT

AAGCAGTGGTATCAACGCAGAGTACTCGAAGCATTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TCGAGAGAGC

AAGCAGTGGTATCAACGCAGAGTACTCGAGAGAGCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TCGCGTAGCA

AAGCAGTGGTATCAACGCAGAGTACTCGCGTAGCANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TCGGCGTTAA

AAGCAGTGGTATCAACGCAGAGTACTCGGCGTTAANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TCTCTCCTAT

AAGCAGTGGTATCAACGCAGAGTACTCTCTCCTATNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TCTTGCTCGG

AAGCAGTGGTATCAACGCAGAGTACTCTTGCTCGGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TGAACTAACC

AAGCAGTGGTATCAACGCAGAGTACTGAACTAACCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TGAAGAAGGT

AAGCAGTGGTATCAACGCAGAGTACTGAAGAAGGTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TGAGCGTTCC

AAGCAGTGGTATCAACGCAGAGTACTGAGCGTTCCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TGAGTACGTA

AAGCAGTGGTATCAACGCAGAGTACTGAGTACGTANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TGGAATGGAG

AAGCAGTGGTATCAACGCAGAGTACTGGAATGGAGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TGTCATTCGC

AAGCAGTGGTATCAACGCAGAGTACTGTCATTCGCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TGTGCTTCAG

AAGCAGTGGTATCAACGCAGAGTACTGTGCTTCAGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TGTTCAGGAT

AAGCAGTGGTATCAACGCAGAGTACTGTTCAGGATNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TTACACACGT

AAGCAGTGGTATCAACGCAGAGTACTTACACACGTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TTACTGTGAC

AAGCAGTGGTATCAACGCAGAGTACTTACTGTGACNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN (continued)

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Table 2 (continued) Barcode

Primer sequence 5′ > 3’

TTATGCCGCG

AAGCAGTGGTATCAACGCAGAGTACTTATGCCGCGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TTCACGGAAG

AAGCAGTGGTATCAACGCAGAGTACTTCACGGAAGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TTCGAGTGAT

AAGCAGTGGTATCAACGCAGAGTACTTCGAGTGATNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TTCTGTACCT

AAGCAGTGGTATCAACGCAGAGTACTTCTGTACCTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TTGGTAACAG

AAGCAGTGGTATCAACGCAGAGTACTTGGTAACAGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TTGGTCAGTA

AAGCAGTGGTATCAACGCAGAGTACTTGGTCAGTANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TGAAGATCCA

AAGCAGTGGTATCAACGCAGAGTACTGAAGATCCANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TGACCTGAGA

AAGCAGTGGTATCAACGCAGAGTACTGACCTGAGANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TGCGCTCATT

AAGCAGTGGTATCAACGCAGAGTACTGCGCTCATTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TGCGTGCTCA

AAGCAGTGGTATCAACGCAGAGTACTGCGTGCTCANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TGTGACGTGC

AAGCAGTGGTATCAACGCAGAGTACTGTGACGTGCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TGTGCACTAA

AAGCAGTGGTATCAACGCAGAGTACTGTGCACTAANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TGTTGTGACT

AAGCAGTGGTATCAACGCAGAGTACTGTTGTGACTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TTAACGCTGA

AAGCAGTGGTATCAACGCAGAGTACTTAACGCTGANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TTATAGGAGG

AAGCAGTGGTATCAACGCAGAGTACTTATAGGAGGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TTATCGCGTT

AAGCAGTGGTATCAACGCAGAGTACTTATCGCGTTNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TTCAGGAGTA

AAGCAGTGGTATCAACGCAGAGTACTTCAGGAGTANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TTCCATCGAG

AAGCAGTGGTATCAACGCAGAGTACTTCCATCGAGNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TTGGCAATTC

AAGCAGTGGTATCAACGCAGAGTACTTGGCAATTCNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN (continued)

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Table 2 (continued) Barcode

Primer sequence 5′ > 3’

TTGGCTCCAC

AAGCAGTGGTATCAACGCAGAGTACTTGGCTCCACNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TTGTCGGCCA

AAGCAGTGGTATCAACGCAGAGTACTTGTCGGCCANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

TTGTGTTCGA

AAGCAGTGGTATCAACGCAGAGTACTTGTGTTCGANNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTVN

Table 3 Pre-amplification PCR thermal cycler protocol Cycle step

Temperature

Time

Cycles

Initial denaturation

96 °C

00:01:00

1

Denaturation Annealing Extension

98 °C 58 °C 72 °C

00:00:20 00:04:00 00:06:00

5

Denaturation Annealing Extension

98 °C 60 °C 72 °C

00:00:20 00:00:30 00:06:00

7

Final extension

72 °C

00:10:00

1

1

1

Hold

2.7 PreAmplification PCR and Cycling

4 °C

1. Pre-amplification PCR mix (30 μL): 25 μL KAPA HiFi HotStart ReadyMix (2 ×) and 5 μL pre-amplification primer (10 μM). 2. Pre-amplification PCR thermal cycler protocol (see Table 3 for details).

2.8 End Preparation Mix and Cycling

1. End preparation mix (10 μL): 3 μL NEBNext Ultra II End Prep Enzyme Mix and 7 μL NEBNext Ultra II End Prep Reaction Buffer. 2. End preparation mix PCR thermal cycler protocol (see Table 4 for details).

2.9 Adaptor Ligation Mix

1. Adaptor ligation mix (33.5 μL): 2.5 μL NEBNext Adaptor for Illumina (Neat), 30 μL NEBNext Ultra II Ligation Master Mix, and 1 μL NEBNext Ligation Enhancer.

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Table 4 End preparation mix PCR thermal cycler protocol Cycle step

Temperature

Time

Cycles

End repair

20 °C

00:30:00

1

Heat inactivation

65 °C

00:30:00

1

1

1

Cycles

Hold

4 °C

Table 5 Index PCR thermal cycler protocol Cycle step

Temperature

Time

Initial denaturation

98 °C

00:00:30

1

Denaturation Extension Final extension

98 °C 65 °C 65 °C

00:00:10 00:01:15 00:05:00

10*

Hold

4 °C

1

1 1

Fig. 4 Examples of D5000 TapeStation results for amplified cDNA of varying quality. The TapeStation traces are representative of cDNA samples that are excellent (i), good (ii), and poor (iii) quality. Excellent-quality samples have predominantly high-molecular-weight cDNA of greater than 1.5 kb (indicated by the red vertical line) and good-quality samples have the majority of cDNA greater than 1 kb (red line). Poor-quality samples usually lack high-molecular-weight cDNA, comprising significantly of molecules that are less than 500 bp (red line). Poor-quality samples require two additional rounds of amplification during indexing PCR (see Subheading 3.19). Y-axis varies in magnitude and is displayed per TapeStation output 2.10 Index PCR Mix and Cycling

1. A 35 μL of index PCR mix consists of 25 μL NEBNext Ultra II Q5 Master Mix, 5 μL P5 PCR primer (10 μM), and 5 μL NEBNext Index Primer/i7 Primer (10 μM). 2. Index PCR thermal cycler protocol (see Table 5 for details). Samples which generate “poor” quality cDNA require two additional cycles (Fig. 4).

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2.11 NextSeq 500 Sequencing (See Note 4)

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1. Read: Read 1, 26 cycles. 2. Read: Index 1 (i7), 6 cycles. 3. Read: Index 2 (i5), 0 cycles. 4. Read: Read 2, 60 cycles.

2.12 Consumables and Equipment

1. T25 cm2 tissue culture flasks. 2. 384-well Corning® black-walled, clear-bottom plate. 3. 384-well V-bottom plate. 4. 384-well PCR plate. 5. JANUS® G3 Automated workstation. 6. BioTek EL406 washer dispenser. 7. Cytation C10 confocal imaging reader. 8. TapeStation 4200. 9. NanoDrop 2000 UV-Vis Spectrophotometer. 10. NextSeq 500.

3

Methods All experimental methods (up to indexing PCR) should be carried out under sterile conditions.

3.1 Automated Cell Dispensing in 2D

1. Using an automated liquid handling workstation (EL406, BioTek), dispense a cell suspension comprising 500 cells (see Note 5) in 45 μL media into each well of a black-walled, clearbottom 384-well plate. 2. Incubate cells in a LiCONiCs STX220 incubator at 37 °C/5% CO2 for 24 h before addition of drugs and for the duration of the experiment (in this experiment, end point is 24 h after drug treatment).

3.2 Automated Cell Seeding for 3D Cells in Matrigel

1. Using a JANUS® G3 liquid handling robot with a cooled stage (PerkinElmer), configure the VariSpan™ arm to aspirate and dispense viscous liquid Matrigel using 200-μL sterile conductive tips. Enable liquid sensing for the VariSpan™ arm to retract the tip during dispensing to minimize disturbance to the Matrigel. Aliquot all Matrigel working solutions into 2-mL pre-cooled microcentrifuge tubes and placed onto cold blocks during cell seeding [11] (see Note 6). 2. First dispense a 10 μL base layer of 80% Matrigel (see Notes 6 to 9) in each well of a black-walled, clear-bottom 384-well plate.

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3. Pulse centrifuge the plate (to reach a minimum 60 × g) and place in the incubator at 37 °C/5% CO2 to allow base layer polymerization. 4. Remove cells by adding trypsin, and resuspend MCF7 cells at a ratio of 2000 cells per 10 μL (see Note 5) in Matrigel at 2% or 80% final concentration diluted in media. Then dispense the Matrigel cell suspension on top of the solidified base layer using the JANUS® G3 liquid handling robot as described above, with a dispense height of 1 mm above well bottom. 5. Following a pulse centrifugation step (to 60 × g), allow the Matrigel to set for 15–30 min (see Note 9) at 37 °C/5% CO2 followed by addition of 25 μL media using the EL406 (see Note 10). Incubate cells in the LiCONiCs incubator at 37 ° C/5% CO2 for the duration of the experiment. 6. On day 3 of culture, aspirate 20 μL media using the EL406 (aspiration height 56–7.11 mm above carrier bottom; see Note 10), leaving 5 μL media as dead volume and the full 20 μL Matrigel plug intact. Dispense fresh media (20 μL) via the EL406. 7. Next, researchers have the freedom to customize the addition of perturbations and staining steps to match their highthroughput imaging needs. We outline three optional methods for consideration (see Subheading 3.3, 3.4, and 3.5). 3.3 Compound Library Preparation and Delivery (Optional)

1. Assay-ready compound plates: (a) Prepare compound master plates or obtain from library providers (e.g., Compounds Australia; Griffith University, Queensland). Hydrate compounds using the EL406 by dispensing the desired volumes of cell culture media to make up the intended final concentrations. We recommend screening multiple doses rather than replicates to ensure you find an effective dose. (b) Configure the Modular Dispense Technology (MDT) head of the JANUS® G3 robot with filtered 30-μL tips (part number 6001299) to transfer compounds from the rehydrated compound library plates to experimental plates. Typically, we transfer 5 μL of compound to 45 μL of cells in the experimental plate (see Note 11). (c) Heat-seal and store the compound plate at -80 °C if it is being used for repeated dosing (see Note 12). We recommend using hydrated compound plates within a week. 2. Self-curated compounds: (a) Researchers who construct compound or drug libraries in-house should be cautious of plate storage time and usage (see Note 13).

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(b) A D300e Digital Dispenser (Tecan) (see Note 14) provides ultimate flexibility for dispensing compounds over long dose ranges and enables any combination and random patterning. 3.4 Automated Dye Dispensing (Optional)

1. When working with adherent 2D or 3D cells, we recommend capturing a live, bright-field (BF) image of the well prior to sequencing. Staining cells with fluorescent dyes to detect cell viability (such as Hoechst) or phenotypic changes (such as dead cell stains) adds valuable additional information and does not impact sequencing quality (see Note 15). 2. Use a D300e Digital Dispenser (Tecan) to dispense fluorescent dyes (see Note 14).

3.5 Fixing and Staining (Optional)

1. To compare the transcript profile with a phenotypic end point, we recommend setting up parallel experimental plates to label cells with fluorophore-conjugated antibodies. This allows a direct evaluation of cells at the point of sequencing or at the longer-term phenotypic end point. This can be as basic as measuring cell viability (Hoechst or DAPI) or as complex as large-scale multiplexed panels staining cell machinery and targets of interest for cell painting campaigns (see Note 16). 2. For 2D experiments, use the EL406 to aspirate the media at height 36 (~4.57 mm above carrier bottom; see Note 10) and then fix the cells by dispensing 25 μL of 4% pre-filtered paraformaldehyde (PFA) in PBS-/-. Incubate for 10 min at room temperature. 3. Use the EL406 to aspirate the PFA, and then wash wells with 50 μL of PBS-/-. 4. Use the EL406 to aspirate the PBS-/-, and then dispense 25 μL of staining solution containing 0.1% Triton X-100. 5. Seal and incubate overnight at 4 °C. 6. For 3D experiments, fixing Matrigel-embedded cells requires extra caution. Please refer to our published protocol [12].

3.6 Automated Imaging

1. For 2D experiments, 24 h after drug treatment, perform live cell imaging via BF using the Cytation C10 Cell Imaging Multi-Mode reader (BioTek) at 4 × magnification (wide field of view) one field/well, at fixed imaging focal height (see Note 15). This generates a reference point for cell viability comparisons. 2. For 3D experiments, 24 h after drug treatment, live image spheroids via BF using the Cytation C10 at 4x magnification (wide field of view) (one field/well, average focus stacking projection of 3 z-heights at 100 μm apart). A fixed imaging

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focal height will speed up image acquisition. This generates a reference point for cell viability comparisons. 3. If measuring whole well viability using CTG, use a parallel plate as CTG is lytic. 3.7 Cell Sample Storage for 2D Assay

1. At the appropriate time point, use the EL406 to aspirate the media and wash once with 50 μL of PBS-/-. 2. Aspirate the PBS-/- (residual PBS -/- does not interfere with downstream processing), heat-seal the plate, and store at -80 ° C before RNA-seq processing (see Note 17).

3.8 Cell Recovery in 3D Assay

Matrigel can prohibit direct RNA extraction and downstream library preparation; therefore, we provide optimized protocols and considerations for high-throughput cell recovery (see Notes 18 and 19). 1. Use the EL406 liquid handling workstation to aspirate media and replace with 40 μL PBS-/-. 2. Aspirate PBS-/- and add 40 μL of 1 × neat TrypLE to each well. Place the plate inside Cytation C10 and activate orbital shaking at 425 cpm (orbit diameter 3 mm) at 37 °C/5% CO2 for a minimum of 30 min (see Note 20). 3. Configure the JANUS® G3 liquid handling robot with the MDT head fitted with 30 μL filter tips (see Subheading 3.3, step 1b). 4. Remove extraction reagent: Pulse centrifuge the plate to a minimum 60 × g (see Note 21) and remove 30 μL of the dissociation solution (Cell Recovery Solution or TrypLE) using the JANUS® G3 MDT head (performs two aspirations of 15 μL each). Set the aspiration height to 3 mm above the well bottom to avoid disrupting the Matrigel structures. 5. Sample mixing: Add 20 μL of PBS-/- to each well and mix over three cycles of aspiration and dispense using the JANUS® G3 MDT head. To account for non-disassociated Matrigel pieces, increase the MDT aspiration and dispensing speed to 150 μL per sec (default at 30 μL per sec) (see Note 22). Set the aspiration height to 0.5 mm above the well bottom and the dispense height to 2 mm above the well bottom. 6. Sample transfer: Transfer samples from the 384-well Corning plate to a 384-well V-bottom plate using the JANUS® G3 MDT head (three transfers, 20 μL each round). 7. Sample harvesting: Centrifuge the 384-well V-bottom plate (931 × g/200 rpm) for 2 min to pellet cells before returning to the JANUS® G3 for supernatant removal.

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8. Supernatant removal: Configure the MDT head with 30-μL filter tips and perform a three-step aspiration. At each step, remove 20 μL of supernatant at a speed of 30 μL per sec and serially decrease the aspiration height from 4 mm above well bottom to 2 mm to 1 mm. Any residual PBS-/- does not interfere with downstream processing. 9. For cells embedded in 80% Matrigel, the increased stiffness requires more rigorous cell removal. Repeat the extraction one more time by repeating the cell recovery steps 5 to 8 (see Note 23). 10. Seal the plate and store at -80 °C (see Note 17). 11. To evaluate the cell recovery efficiency, take one whole-well BF image to have a quick check and a permanent record to compare back to if necessary. At least 50% of the cells in the well should be recovered before proceeding to the next step cell lysis. 3.9 Cell Lysis and Reverse Transcription

Unless otherwise stated, all liquid handling steps use the JANUS® G3 MDT head fitted with 30 μL filter tips with system default aspiration and dispense height and speed. 1. Retrieve the cell plate from -80 °C storage and place on ice until ready to proceed. 2. Retrieve the primer library plate from -20 °C storage and thaw on ice (see Note 24). 3. Prepare lysis buffer (see Subheading 2.5). Vortex and centrifuge briefly. 4. Add 17 μL of lysis buffer to each well of the cell plate (see Note 25). Seal the cell plate, vortex at room temperature for 15 min at 2000 rpm (see Note 26), and pulse centrifuge. 5. Prepare reverse transcription (RT) master mix on ice (see Subheadings 2.6). Vortex and centrifuge briefly. 6. Dispense 7.5 μL of the RT master mix into each well of a new 384-well Eppendorf® twin.tec PCR plate. 7. Transfer 12.5 μL of the lysate from each well of the cell plate into corresponding wells of the PCR plate prepared in step 5 (see Note 26). 8. Use the JANUS® G3 MDT head configured with the 384-well Pin Tool (100 nL, part number 70229690) (see Note 27) to stamp transfer the primers from the primer library plate (see Subheading 2.4, step 1) to the RT master mix—lysate PCR plate. 9. Repeat step 5 a total of three times (see Notes 28 and 29).

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10. Seal the plate, mix briefly by vortexing at 2000 rpm for 10 sec, and pulse centrifuge. 11. Incubate at 42 °C for 2 h. 3.10 Pooling, First0Strand cDNA Synthesis, and Cleanup

1. Use a multichannel pipette to combine the entire RT reaction volume from each well into a reservoir containing Zymo Research® DNA binding buffer (see Subheading 2.3) in a 1:7 ratio (sample to buffer). Mix briefly with a serological pipette. 2. Prepare four Zymo-Spin™ V Columns with Reservoir assemblies (see 2.3). 3. Divide the combined RT reaction/DNA binding buffer mixture into four aliquots of equal volume directly into the Column with Reservoir assemblies. 4. Follow the Zymo DNA Clean & Concentrator-100 Kit instructions (see Note 30) to purify and concentrate each aliquot separately. 5. Pool the four aliquots of eluted cDNA. 6. Add 420 μL of AMPure XP beads to the pool and pipette to mix. Incubate for 5 min at room temperature (see Note 31). 7. Use a magnetic stand to pellet the beads for 5 min or until the solution clears. Remove and discard the supernatant. 8. Add 1 mL of 80% ethanol to the pellet. Wait 30 s. Remove and discard the ethanol. 9. Repeat step 8 for a total of two washes. 10. Centrifuge briefly and place back on the magnetic stand. Remove and discard any residual ethanol and air-dry for 2 min. 11. Remove the tube from the magnetic stand and add 22 μL of nuclease-free water. Pipette to resuspend the beads and incubate for 2 min at room temperature. 12. Use a magnetic stand to pellet the beads for 30 s or until the solution clears. 13. Transfer 20 μL of the purified pool to a standard PCR strip tube.

3.11 PreAmplification PCR

1. Prepare the pre-amplification PCR mix on ice (see Subheading 2.7). Vortex and centrifuge briefly. 2. Add 30 μL of the pre-amplification PCR mix to the sample from Subheading 3.10, step 13, and mix. 3. Incubate the PCR mix in a thermal cycler using protocol settings (see Subheading 2.7). 4. Pre-amplified cDNA can be stored at 4 °C for 24 h or up to a week at -20 °C.

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3.12

cDNA Cleanup

317

1. Add 40 μL of AMPure XP beads to each pre-amplified sample. Pipette to mix. 2. Incubate for 5 min at room temperature. 3. Use a magnetic stand to pellet the beads for 5 min or until the solution clears. Remove and discard the supernatant. 4. Add 200 μL of 80% ethanol to the pellet. Wait 30 sec. Remove and discard the ethanol. 5. Repeat step 4 for a total of two washes. 6. Centrifuge briefly and place back on the magnetic stand. 7. Remove and discard any residual ethanol and air-dry for 2 min. 8. Remove from the magnetic stand and add 53 μL of nucleasefree water. Pipette to resuspend the beads. Incubate for 2 min at room temperature. 9. Use a magnetic stand to pellet the beads until the solution clears. 10. Transfer 50 μL of the purified pool to a PCR strip tube. 11. Safe stopping point. Samples can be stored at 4 °C overnight, or up to 2 weeks at -20 °C.

3.13 cDNA QC and Quantification

1. If D5000 ScreenTapes- are unavailable, the 2100 Bioanalyzer can be used as an alternative means of QC. 2. Run 1 μL of the purified cDNA on an Agilent D5000 ScreenTape following the manufacturer’s instructions. Refer to Fig. 4 for expected results.

3.14

Shearing

The steps below describe how to prepare 300–350 base pair (bp) DNA fragments from full-length cDNA using the Covaris ME220 platform (see Note 32). Alternative DNA shearing platforms can be used; however, conditions must be optimized to generate fragments of similar length. 1. Dilute 100–500 ng of pre-amplified cDNA in 50 μL of nuclease-free water. Transfer the diluted cDNA to a Covaris Snap-Cap microTUBE with AFA fiber (see Subheading 2.3). 2. Configure the Covaris ME220 with the following conditions: Duty Factor 20%, Peak Power 70 W, Cycles per Burst 1000, and Duration 45 sec. 3. Load the sample tubes in the Covaris and run the shearing program. 4. After shearing is complete, transfer 50 μL of sheared cDNA to a standard PCR tube.

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End Preparation

1. Prepare the end preparation mix on ice (see Subheading 2.8) and vortex and centrifuge briefly. 2. Add 10 μL of the end preparation mix to the sheared sample from Subheading 3.14, step 4, and mix. 3. Incubate the end preparation mix in a thermal cycler using protocol settings (see Subheading 2.8).

3.16 Adaptor Ligation

1. Prepare the adaptor ligation mix on ice (see Subheading 2.9) and vortex and centrifuge briefly. 2. Immediately add 33.5 μL of the adaptor ligation mix to the end preparation reaction mix from Subheading 3.15, step 3. Vortex and centrifuge briefly. 3. Incubate at 20 °C for 15 min. 4. Add 3 μL of USER Enzyme (see Subheading 2.3) to the ligation mixture. Vortex and centrifuge briefly. 5. Incubate at 37 °C for 15 min.

3.17 Size Selection of Adaptor-Ligated DNA

1. Add 25 μL of AMPure XP beads and pipette to mix. 2. Incubate for 5 min at room temperature. 3. Use a magnetic stand to pellet the beads for 5 min or until the solution clears. 4. Transfer 125 μL of the supernatant to a new PCR tube and discard the beads. 5. Add 10 μL of AMPure XP beads and pipette mix. 6. Incubate for 5 min at room temperature. 7. Use a magnetic stand to pellet the beads for 5 min or until the solution clears. Remove and discard the supernatant. 8. Add 200 μL of 80% ethanol to the pellet. Wait 30 sec. Remove and discard the ethanol. 9. Repeat step 8 for a total of two washes. 10. Centrifuge briefly and place back on the magnetic stand. 11. Remove and discard any residual ethanol and air-dry for 2 min. 12. Remove from the magnetic stand and add 17 μL of nucleasefree water. Pipette to resuspend the beads. 13. Incubate for 2 min at room temperature. 14. Use a magnetic stand to pellet the beads until the solution clears. 15. Transfer 15 μL of the purified pool to a PCR strip tube. 16. Safe stopping point. Samples can be stored at 4 °C overnight, or up to 2 weeks at -20 °C.

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3.18

Index PCR

319

1. Prepare the index PCR mix on ice (see Subheading 2.10) and vortex and centrifuge briefly. 2. Add 35 μL of the index PCR mix to the sample from Subheading 3.14, step 15. Pipette to mix. 3. Incubate the PCR mix in a thermal cycler using the protocol settings detailed in Subheading 2.10. 4. Indexed libraries can be stored at 4 °C for 24 h or up to a week at -20 °C.

3.19 Index PCR Cleanup

1. Add 45 μL of AMPure XP beads and pipette to mix. 2. Incubate for 5 min at room temperature. 3. Use a magnetic stand to pellet the beads for 5 min or until the solution clears. Remove and discard the supernatant. 4. Add 200 μL of 80% ethanol to the pellet. Wait 30 sec. Remove and discard the ethanol. 5. Repeat step 4 for a total of two washes. 6. Centrifuge briefly and place back on the magnetic stand. 7. Remove and discard any residual ethanol and air-dry for 2 min. 8. Remove from the magnetic stand and add 33 μL of nucleasefree water. Pipette to resuspend the beads. 9. Incubate for 2 min at room temperature. 10. Use a magnetic stand to pellet the beads for 30 s or until the solution clears. 11. Transfer 31 μL of the purified pool to a PCR strip tube.

3.20 Library QC and Quantification: TapeStation

1. If D1000 ScreenTapes are unavailable, the 2100 Bioanalyzer can be used as an alternative means of QC.

3.21 Library QC and Quantification: qPCR

1. Quantify the Purified Library Using the KAPA Library Quantification Kit (Illumina) (See Subheading 2.3), Following the manufacturer’s Instructions (See Notes 33 and 34)

3.22

1. Prepare the quantified library for sequencing following the “NextSeq 500 and 550 System Denature and Dilute Libraries Guide” (see Note 35). Spike the denatured and diluted library with 20% PhiX (see Subheading 2.3).

Sequencing

2. Run 1 μL of the purified cDNA on an Agilent D1000 ScreenTape following manufacturer’s instructions. Refer to Fig. 4 for expected results.

2. Add 5.16 μL of 100 μM custom read 1 primer to well 20 of the Illumina, NextSeq 500/550 High Output Kit v2.5 (75 Cycles) reaction cartridge (see Subheading 2.3). Pipette to mix. 3. Load the final sequencing pool on the NextSeq 500 using the conditions detailed in Subheading 2.11.

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3.23 Bioinformatic Analysis

4

1. Raw read counts are generated using the STARsolo analysis package (see Note 36). The gene count matrix is analyzed using the R package Seurat.

Notes 1. Items 12–16 (see Subheading 2.3) can be purchased together in a single kit (Zymo Research® DNA Clean & Concentrator®-100, Cat# D4030). 2. Items 23 to 27 (see Subheading 2.3) can be purchased together in a single kit (NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®, Cat# E7645S). 3. Items 28 to 30 (see Subheading 2.3) can be purchased together in a single kit (NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 1), Cat# E7335S). 4. If only sequencing one library, the index 1 (i7) read can be omitted and the read 2 sequencing length increased to 66 cycles. 5. Prior to performing MAC-seq, researchers should optimize the cell seeding density in their 2D and 3D models. The number suggested in this protocol is optimized for MCF7 cells. Researchers can refer to Fig. 2b to estimate the cell number required for RNA-seq. We have chosen the seeding density for the 2D and 3D experiments based on our established growth conditions which are defined by appropriate phenotypic end points at 72 h and 7 days, respectively. Optimization is critical to determine the appropriate confluency (2D) or spheroid size (3D) before introducing perturbations (chemical or generic) for desired effects. 6. When using the JANUS® G3 or other robotic liquid handlers to dispense Matrigel, caution should be taken to ensure the appropriate working environment temperature, so the Matrigel stays in liquid form. We use a cold block stored at -20°C. A 20% dead volume (768 μL for a 10 μL gel layer dispense for one 384-well plate) is required for the Matrigel reservoir (in this case a 2-mL microcentrifuge tube) to be compatible with the VariSpan™ arm with liquid sensing tips. Aspiration and dispensing speed should be considered to balance a fast workflow with gentle handling given the viscosity of the Matrigel. Avoid liquid blowout to reduce bubble formation. Use liquid sensing tips if possible. Centrifugation after Matrigel dispensing helps to reduce air bubbles. Small air bubbles tend to disappear after a couple days.

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7. For 3D cell cultures, we lay an 80% Matrigel (v/v in cell culture media) base layer to prevent cells migrating to the well bottom and growing in a 2D monolayer. 8. Our standardized working concentration of Matrigel is 9.3 mg/ml (defined as 100%). 9. Depending on the cell type, the percentage of Matrigel can vary from 2% to 80% or higher. We recommend a minimum of 15-min incubation at 37 °C/5% CO2 to allow high-percentage (50% and above) Matrigel polymerization and 30 min for low-percentage gels. We recommend pre-cooling media on ice when diluting Matrigel to the desired percentage to prevent Matrigel polymerization (especially for low Matrigel percentages). 10. We recommend optimizing media dispense and aspirate conditions for 2D and 3D cultures to match with assay plate dimension and liquid handling hardware. With the EL406, the dispensing speed is set to “high,” with an X offset 20 (~0.91 mm off well center) so the liquid will hit the well wall and avoid disturbing cells. For media aspiration, the EL406 has a 96-pin manifold aspirator. For 2D cultures the aspiration height is set to 36 (~4.57 mm above carrier bottom) for the Corning 384-well plate. For 3D cultures with a 20 μL Matrigel plug, the aspiration height is set to 56 (~7.11 mm above carrier bottom). All liquid dispensing and aspiration by EL406 used in this protocol are the same parameters unless otherwise stated. 11. When using the JANUS® G3 MDT head, a wash reservoir of PBS can be used in between stamp transfer to reduce tip usage. Placing an air gap between different aspirations is encouraged. 12. We use the Agilent PlateLoc Thermal Microplate Sealer unless otherwise stated. If not available, researchers can use aluminum adhesive seals and manually seal plates. For live cell and fixed sample staining, we recommend optimizing the labelling time and temperatures for the dye or antibodies to be used. 13. Be cautious to track compound lot numbers throughout projects and ensure the correct solvents and appropriate controls are used. Meticulous recording of batch-logged daughter plates, date and usage tracking, and controls for solvent (DMSO, ethanol, or aqueous) is required. 14. When using the Tecan D300e digital dispenser, the cassette can only accommodate DMSO as a compound vehicle. Warning: Dispensing any aqueous solutions in the Tecan requires addition of Triton-X-100 which can be toxic to cells even at a very low concentration. If using Tecan to dispense stains, pulse spin the staining solution stock vial before loading stain to Tecan cassette. Avoid pipetting from the bottom of the vial.

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15. For live cell imaging, parameters will vary depending on the imaging system used and the staining applied. Using a low-magnification lens means less fields are needed to capture a whole well image, therefore speeding up the acquisition. In 3D experiments, we take BF images in addition to the end point fluorescent images, at the beginning (day 3) and end of drug treatment (day 4) and after cell recovery steps. These images are important to help assess cell seeding and growth, to relate cell viability data derived from CTG, and to determine recovery efficiency. During an experiment, researchers can make stop/go decisions based on the recovery efficiency estimated from BF images. More accurate calculation can be performed retrospectively via in-depth image analysis. 16. We have tested Hoechst and ImageIT dyes, and staining does not interfere with downstream RNA-seq. We recommend caution using fixed samples and optimization is required. In our test with 2% PFA-fixed 3D samples, although the TapeStation indicated good RNA quality, the RNA-seq failed to produce any reads. 17. If the sample count is less than 384, we recommend storing independent samples/experiments in sealed plates at -80 °C until 384 are accumulated. We recommend performing lysis on all samples at the same time, so if samples are collected from different time points in the same experiment, we suggest multiple plates are set up. Samples should be washed with PBS before storage. 18. For 3D cell recovery, we have tested different Enzymes (Dispase® II, TrypLE™, collagenase A, collagenase type II). They all required considerably longer time (30 min to 1 h) to dissociate Matrigel (even at 2% v/v) than their conventional use in low-density plate formats. We recommend optimizing cell recovery for each specific matrix model prior to MAC-seq screening. 19. We have evaluated the nonenzymatic Cell Recovery Solution to Matrigel at a ratio of 2:1. Our 3D model has a total Matrigel volume of 20 μL, therefore requiring a minimum of 40 μL Cell Recovery Solution. In our tests, the efficiency of Cell Recovery Solution to disassociate Matrigel in a 384-well plate is much less compared to a 96-well plate, possibly due to the reduced contact area. It takes even longer for the Cell Recovery Solution to disassociate high-percentage Matrigel. Our tests indicated a minimum of 2.5 h to achieve 80–90% cell recovery in Matrigel ranging from 2% to 80%. Researchers intending to use Cell Recovery Solution should be cautious about the cold temperature–induced cell stress (Fig. 3d).

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20. In this step, we have used the incubation and shaking function that comes with the Cytation microscope. Researchers can adapt this step to their own instrumentation. A 30-min minimum incubation at 37 °C with gentle shaking is recommended, and researchers can extend the incubation to 1 h. 21. Avoid high-speed centrifugation of the 3D samples prior to cell extraction. This might force spheroids to embed firmly into the 80% Matrigel base, making it harder for subsequent recovery. 22. At our recommended volume and time conditions (40 μL, 2.5 h for Cell Recovery Solution at 4 °C or 30 min for TrypLE at 37 °C), we have found MCF7 spheroids can maintain their structural integrity even after extraction (Fig. 4). Small spheroids, and those treated with cytotoxic agents, tend to fall apart during the extraction process. 23. Cells embedded in low-percentage Matrigel are easier to recover. We recommend running the cell extraction procedure at least two times for cells embedded in high-percentage Matrigel (50% and above) to achieve a good recovery yield. 24. Primer library plates containing oligonucleotides at working concentration should only be thawed three times before being discarded. 25. MDT aspiration height is set to 0.5 mm above well bottom and dispensing height is 2 mm above well bottom. The tip touching function is enabled post dispensing at 7 mm above well bottom and offset to 40% well width to ensure liquid droplets detach from tips. 26. Observe the cells under microscope to ensure sufficient lysis. 27. To fit the JANUS® G3 MDT head with 384-well Pin Tool, first fit the 96-well, P50 head attachment. The Pin Tool liquid aspiration step is set to 1 mm above well bottom, with a tip retraction distance of 10 mm at a speed of 5 mm per sec, with 5 cycles to ensure tip loading. The Pin Tool liquid dispensing step is set to 5 mm above well bottom, with a tip retraction distance of 12 mm at a speed of 200 mm per sec, with 5 cycles to ensure liquid exchange. 28. When using the Pin Tool, be mindful of possible volume variations and individual pin accuracy. We evaluated our Pin Tool using a dye absorbance assay and determined a minimum of three transfers is sufficient for liquid delivery to all 384 positions. 29. The Pin Tool should be washed with water and then ethanol and then touch-dried on lint-free blotting paper between stamping transfers. We recommend using a 384-well plate as a carrier for water and ethanol wash stations to minimize cross contamination.

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30. Zymo DNA Clean & Concentrator-100 Kit protocol:url: https://files.zymoresearch.com/protocols/_d4029_d4030_ dna_clean_concentrator-100_kit.pdf. 31. Bring AMPure XP beads to room temperature prior to use. 32. The method is also compatible with tagmentation-based library preparation, if a DNA shearing platform is not available [5, 6]. However, our observation of tagmentation libraries suggests higher variability in final results. 33. KAPA Library Quantification Kit Illumina® platforms: url: h t t p s : // r o c h e s e q u e n c i n g s t o r e . c o m / w p - c o n t e n t / uploads/2017/10/KAPA-Library-Quantification-Kit.pdf. 34. The library concentration calculated by TapeStation and qPCR are often discordant. Use the qPCR-derived concentration to calculate sequencing loading concentration. 35. NextSeq 500 and 550 System Denature and Dilute Libraries Guide url: https://sapac.support.illumina.com/downloads/ nextseq-500-denaturing-diluting-libraries-15048776.html. 36. STARsolo—mapping, demultiplexing, and quantification for single-cell RNA-seq url: https://github.com/alexdobin/ STAR/blob/master/docs/STARsolo.md#starsolo-mappingdemultiplexing-and-quantification-for-single-cell-rna-seq version 2.7.10a. Barcode list is provided in Table 2. Data aligned to human genome assembly GRcH38.

Acknowledgements We thank members of the Molecular Genomics Core facility and Victorian Centre for Functional Genomics for their valuable input into the development of these methodologies. The Victorian Centre for Functional Genomics (KJS) is funded by the Australian Cancer Research Foundation (ACRF), Phenomics Australia, through funding from the Australian Government’s National Collaborative Research Infrastructure Strategy (NCRIS) program and the Peter MacCallum Cancer Centre Foundation. This project has been supported by specific project funding through Phenomics Australia (KJS) and separate Peter MacCallum Foundation grants to GMA and SR. References 1. Ye C, Ho DJ, Neri M et al (2018) DRUG-seq for miniaturized high-throughput transcriptome profiling in drug discovery. Nat Commun 9:4307 2. Li J, Ho DJ, Henault M et al (2021) DRUGseq provides unbiased biological activity

readouts for drug discovery. Biorxiv 2021(06):07.447456 3. Alpern D, Gardeux V, Russeil J et al (2019) BRB-seq: ultra-affordable high-throughput transcriptomics enabled by bulk RNA barcoding and sequencing. Genome Biol 20:71

Multiplex High-Throughput Sequencing 4. Moyerbrailean GA, Davis GO, Harvey CT et al (2015) A high-throughput RNA-seq approach to profile transcriptional responses. Sci Rep 5: 14976 5. Todorovski I, Feran B, Fan Z et al (2022) RNA decay defines the therapeutic response to transcriptional perturbation in cancer. Biorxiv 2022(04):06.487057 6. Kong IY, Trezise S, Light A et al (2022) Epigenetic modulators of B cell fate identified through coupled phenotype-transcriptome analysis. Cell Death Differ 29:2519–2530 7. So J, Lewis AC, Smith LK et al (2022) Inhibition of pyrimidine biosynthesis targets protein translation in acute myeloid leukemia. EMBO Mol Med 14:e15203 8. Wang Y, Jeon H (2022) 3D cell cultures toward quantitative high-throughput drug screening. Trends Pharmacol Sci 43:569–581

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9. Szyman´ski P, Markowicz M, Mikiciuk-Olasik E (2011) Adaptation of high-throughput screening in drug discovery—toxicological screening tests. Int J Mol Sci 13:427–452 10. Hughes RE, Elliott RJR, Dawson JC et al (2021) High-content phenotypic and pathway profiling to advance drug discovery in diseases of unmet need. Cell Chem Biol 28:338–355 11. Choo N, Ramm S, Luu J et al (2021) Highthroughput imaging assay for drug screening of 3D prostate cancer organoids. Slas Discov 26: 1107–1124 12. Ramm S, Vary R, Gulati T et al (2022) Highthroughput live and fixed cell imaging method to screen Matrigel-embedded organoids. Organoids 2:1–19

Chapter 23 Primary Intestinal Fibroblasts: Isolation, Cultivation, and Maintenance Abhimanu Pandey, Melan Kurera, and Si Ming Man Abstract Intestinal fibroblasts maintain homeostasis and contribute to inflammatory responses and the development of cancer. Intestinal fibroblasts express pattern recognition receptors which can mount an immune response. Since intestinal fibroblasts interact with diverse immune and nonimmune cells, further insights into the biology of intestinal fibroblasts could expand our knowledge of the development, homeostasis, and pathophysiology of the intestine. Here, we describe a simple protocol for the isolation, cultivation, and maintenance of primary fibroblasts from the mouse colon. These cells express α-smooth muscle actin, a characteristic of specialized contractile fibroblasts called myofibroblasts. We also outline the use of these colonic fibroblasts for immunoblotting and immunofluorescence assays with or without stimulation with a growth factor. Key words Intestinal fibroblasts, Myofibroblasts, Inflammation, Cancer, Intestinal homeostasis, Primary cell culture

1

Introduction Intestinal fibroblasts are mesenchymal cells that contribute to the maintenance of tissue structure and integrity [1, 2]. During homeostasis, intestinal fibroblasts secrete extracellular matrices and structural proteins, such as collagen and elastin, to maintain intestinal homeostasis [3, 4]. Intestinal fibroblasts have also emerged as a key cell type in initiating an immune response against infection and cancer [4]. For instance, intestinal fibroblasts from patients with chronic intestinal inflammation or mice treated with the inflammatory agent dextran sulfate sodium express increased levels of inflammatory markers such as interleukin (IL)-1α and prostaglandin E2 [5]. These immune responses are largely initiated by germline-encoded pattern recognition receptors (PRRs) located

Brendan J. Jenkins (ed.), Inflammation and Cancer: Methods and Protocols, Methods in Molecular Biology, vol. 2691, https://doi.org/10.1007/978-1-0716-3331-1_23, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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on the cell membrane and/or in the cytoplasm of intestinal fibroblasts [6–9], leading to the production of cytokines, chemokines, and growth factors [4, 6–8]. In mouse colonic fibroblasts, activation of the PRR nucleotide-binding oligomerization domain (NOD) 2 promotes the production of the chemokine CCL2, which drives the recruitment of monocytes and clearance of the pathogenic bacterium Citrobacter rodentium [10]. Activation of NOD-like receptor pyrin domain-containing protein NLRP6 in mouse colonic fibroblasts can promote reepithelialization during colonic inflammation and inflammation-associated colorectal cancer [11]. However, the precise mechanisms of how intestinal fibroblasts initiate intestinal repair but promote intestinal inflammation and cancer remain elusive. Following intestinal injury, tissue-resident intestinal fibroblasts can become myofibroblasts, a type of specialized contractile fibroblast which express α-smooth muscle actin (α-SMA) [3, 4]. Myofibroblasts secrete stimulatory molecules such as Wnt ligands and epithelial growth factors, promoting intestinal cell proliferation and tumor development [12]. Given their critical role in the development and progression of intestinal cancer, myofibroblasts are considered an emerging target for anticancer drugs [13, 14]. Here, we describe the isolation, cultivation, and maintenance of primary mouse colonic fibroblasts which express α-SMA characteristic of myofibroblasts. This protocol can also be used to isolate and culture fibroblasts from the mouse small intestine. The protocol was modified from previously reported methods of culturing mouse fibroblasts [15–18], which is now faster (less than 3 h of sample preparation) and simple to use. We observed that primary colon fibroblasts could be passaged up to four times, with further passages resulting in a loss of cell morphology and proliferation. We also outline the use of colonic fibroblasts for immunoblotting and immunofluorescence assays. This protocol could also be used to culture primary colon fibroblasts from genetically modified mice, such as those lacking genes encoding members of the PRR family [6, 7] for research into the biology of PRRs in intestinal fibroblasts. In addition, these cells could be co-cultured with immune cells such as intestinal macrophages and intestinal T cells to study cell-cell interactions.

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Materials Prepare all solutions using distilled water and reagents of analytical grade and in the biosafety cabinet unless otherwise mentioned. Follow disposal procedures as outlined by the local institution.

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2.1 Extraction Solution (Volume per Colon)

1. 57.8 mL sterile DPBS + 600 μL fetal bovine serum (FBS) + 750 μL 0.5 M ethylenediaminetetraacetic (EDTA) + 750 μL 1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) + 75 μL 1 M dithiothreitol (DTT) (see Note 1).

2.2 Digestion Media (Volume per Colon)

1. Dissolve 8 mg collagenase D + 5 mg pronase in 2 mL of complete media (see Subheading 2.3). 2. Filter sterilize the solution using a 0.2-μm filter and prewarm (see Note 2) before use.

2.3 Culture Media (Also Known as Complete Media)

1. For 500 mL, add 100 mL FBS, 5 mL 1 M HEPES, 5 mL 100 mM sodium pyruvate, 5 mL 100× penicillin-streptomycin glutamine, and 500 μL 55 mM 2-mercaptoethanol in 384.5 mL Roswell Park Memorial Institute (RPMI) 1640 (see Note 3).

2.4 AmmoniumChloride-Potassium (ACK) Lysis Buffer

1. For 500 mL, add 4.15 g ammonium chloride (NH4Cl), 0.5 g potassium bicarbonate (KHCO3), and 18.6 mg disodium salt (Na2) EDTA in 400 mL distilled water. Adjust the pH to 7.4 and make up the volume to 500 mL using distilled water. Store the solution at 4 °C. 2. Filter sterilize 10 mL solution (volume per colon) using a 0.2-μ m filter and prewarm before use.

2.5 Tissue Processing

1. Scissors and forceps. 2. Biosafety cabinet. 3. Dulbecco phosphate-buffered saline (DPBS). 4. Microscopy slide. 5. Falcon tubes. 6. 70-μm sterile cell strainer. 7. 0.2-μm sterile filter. 8. 18-gauge needle. 9. 5-mL syringe. 10. 2-mL sterile cryotube. 11. Parafilm. 12. Shaking incubator at 250 rpm at 37 °C. 13. Centrifuge. 14. T-175 flask.

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Methods

3.1 Extraction of Mouse Colon and Denudation of Epithelium

1. Ethically cull mice following local institute procedures and harvest the colon (see Note 4). 2. Clean the exterior of the tissue by removing residual fat and membranes. Using an 18-gauge needle attached to a 5-mL syringe, flush the lumen with ice-cold DPBS up to three times to remove the fecal content. 3. Cut open the tissue longitudinally and place it in ice-cold DPBS. Scrape the mucus from the luminal side by slowly passing the entire tissue under a microscope slide. 4. Cut the tissue into 2–5-mm lengths and place the dissected tissue pieces in a 50-mL Falcon tube containing 20 mL of ice-cold DPBS. 5. Allow the tissue pieces to settle at the bottom of the Falcon tube and remove the DPBS. 6. Add 20 mL of prewarmed extraction solution to the tissue pieces and shake rigorously at 250 rpm for 15 min at 37 °C. 7. Allow the tissue pieces to settle at the bottom of the Falcon tube and remove the extraction solution. 8. Repeat steps 6 and 7 twice.

3.2 Isolation of Colon Fibroblasts

1. Filter the extraction media containing the tissue fragments (see Subheading 3.1, step 8) through a 70-μm cell strainer into a 50-mL Falcon tube. Rinse the cell strainer twice with 5 mL of sterile prewarmed DPBS. 2. Using sterile forceps, place the tissue pieces into a 2-mL cryotube containing 1.8 mL of prewarmed digestion media (see Note 5). Secure the lid using parafilm and shake rigorously at 250 rpm for 30 min at 37 °C (see Note 6). 3. Filter the digested tissue through a 70-μm cell strainer into a 50-mL Falcon tube. Use a sterile syringe plunger to grind the remaining tissue within the cell strainer. Wash the cell strainer twice with 5 mL of room temperature complete media. 4. Centrifuge the filtered solution at 500×g for 5 min at 4 °C. 5. Remove the supernatant and resuspend the cell pellet in 10 mL of prewarmed ACK lysis buffer. 6. Centrifuge again at 500×g for 5 min at 4 °C. 7. Remove the supernatant and resuspend the cell pellet in 10 mL of prewarmed complete media. Filter the solution into a T-175 flask using a 70-μm cell strainer. Wash the cell strainer with prewarmed 10 mL complete media and incubate the cells at 37 °C in a humidified 5% CO2 incubator.

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a

Day 1

Day 3

Day 7

Day 10

b

P1 Day 5

P2 Day 5

P3 Day 5

P4 Day 5

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Fig. 1 Microscopic images of colon fibroblasts from a 10-week-old C57BL/6 mouse. (a) The growth and proliferation of colon fibroblasts over 10 days. (b) Cell confluency of 1 × 106 colon fibroblasts on day 5 after passage (P) 1 to 4 in a T-175 flask. Colon fibroblasts were imaged using the Leica DMi1 microscope. Scale bar, 100 μm

8. After 4 h, remove the media and replace it with fresh 20 mL prewarmed complete media. 9. Incubate the cells at 37 °C in a humidified 5% CO2 incubator. The growth and proliferation of colon fibroblasts over 10 days can be observed under a bright-field microscope (Fig. 1a). 3.3 Passaging of Colon Fibroblasts

1. Once the colon fibroblasts reach 70–90% cell confluency, remove the media, and wash the cells with 10 mL of prewarmed DPBS. 2. Add 5 mL 0.25% Trypsin-EDTA and incubate the cells at 37 °C for 5 min (see Note 7). 3. Transfer the cells to a 50-mL Falcon tube. 4. Wash the flask with 10 mL prewarmed complete media to dislodge the remaining cells and transfer the cells to the same 50-mL Falcon tube in step 3, Subheading 3.3. 5. Centrifuge at 500×g for 5 min at 4 °C. 6. Remove the supernatant and resuspend the cell pellet in 10 mL prewarmed complete media. 7. Count cells using a cell counter or a similar method. Passage the cells for expansion. We recommend seeding 1 × 106 cells in a T-175 flask and passage when the cell confluency reaches 70–90%. We notice that, after four passages, the colon fibroblasts lose viability (Fig. 1b).

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DAPI

α - SMA + DAPI

100X

40X

α - SMA

Fig. 2 Immunofluorescence staining of α-smooth muscle actin (α-SMA) and DAPI in mouse colon fibroblasts after passage 1 (see Fig. 1b). Colon fibroblasts were seeded on a coverslip at a density of 1.25 × 105 cells per well in a 12-well plate. Colon fibroblasts were imaged using the Zeiss Axio Observer microscope. Scale bars, 20 μm (top panel) and 10 μm (bottom panel)

3.4 Immunofluorescence Staining and Immunoblotting of Colon Fibroblasts

1. Following cell counting outlined in step 7, Subheading 3.3, colon fibroblasts can be used for experimental work. Seed 1.25 × 105 cells on a sterilized coverslip placed inside a 12-well plate. For the staining of α-SMA and 4′,6-diamidino2-phenylindole (DAPI), follow the immunofluorescence staining protocol as described previously [17] (Fig. 2). 2. For protocols involving stimulation of colonic fibroblasts with growth factors such as insulin growth factor (IGF)-1, starve the cells in reduced growth media (see Note 8) for 48 h to reduce the basal phosphorylation of molecules in the growth factor signalling pathway, including the phosphorylation of extracellular signal-regulated kinase (ERK). 3. Following starvation, incubate the colonic fibroblasts in prewarmed complete media (see Note 3) with or without growth factor for up to 60 min. Proceed with immunoblotting (see Note 9) and immunofluorescence staining as described previously [17] (Fig. 3a, b).

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b P-ERK

IGF-1

DAPI

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P-ERK β-actin+DAPI

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Untreated

Time (min)

β-actin

P-ERK

IGF-1

α-Tubulin

T-ERK

Fig. 3 Immunoblotting and immunofluorescence staining of mouse colon fibroblasts after passage 1 (see Fig. 1b). (a) Immunoblot of the indicated proteins on the cell lysate of colon fibroblasts serum-starved (in OptiMEM) for 48 h and then left untreated or pre-treated with insulin growth factor (IGF)-1 for the indicated time. Following serum starvation, colon fibroblasts were kept in complete media containing 10% FBS with or without IGF-1. “P-” indicates phosphorylated protein and “T-” indicates total protein. (b), Immunofluorescence staining of P-ERK, β-actin, and DAPI of colon fibroblasts serum-starved (in Opti-MEM) for 48 h and left untreated or treated with IGF-1 for 60 min. Following serum starvation, colon fibroblasts were kept in complete media containing 10% FBS with or without IGF-1. Colon fibroblasts were imaged using the Zeiss Axio Observer microscope. Scale bar, 20 μm

4

Notes 1. Prepare 1 M solution of DTT in distilled water and store in 100 μL aliquots at -20 °C. Thaw at room temperature before use. 2. Prewarmed refers to the incubation of solution or media at 37 ° C in a humidified 5% CO2 incubator for 30 min prior to use. 3. Reduce the volume of FBS to 50 mL (10%) when seeding cells for stimulation. 4. In our experience, colons from mice aged 8–12 weeks provide the best yield of fibroblasts. 5. Depending on the amount of tissue, adjust the volume of the media with a minimum total volume of 1.5 mL and a maximum total volume of 1.8 mL. To allow thorough mixing and efficient digestion, do not fill the tube to the top. If using the whole small intestine, double the volume of digestion media. 6. Place the cryotube horizontally to allow thorough mixing. Check every 10 min and briefly vortex the tube to break up any tissue clumps.

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7. Depending on the cell confluency, gentle scrapping using a cell scraper to detach the cells might be required. 8. We recommend using Opti-MEM for the starvation of colonic fibroblasts. If using RPMI, starve the cells for 48 h followed by an additional 1 h of starvation in DPBS before proceeding to stimulation. 9. Following the collection of colon fibroblasts for immunoblotting analysis, normalize the protein levels using the BCA protein normalization kit or a similar assay. After treatment with insulin growth factor (IGF)-1, we have observed variable levels of β-actin despite protein normalization; therefore, we suggest using the reference protein marker α-tubulin or GAPDH as a loading control.

Acknowledgements The authors acknowledge the facilities and the scientific and technical assistance of Microscopy Australia at the Centre for Advanced Microscopy (ANU, Australia), a facility that is funded by the University and the Federal Government. The authors acknowledge the National Collaborative Research Infrastructure Strategy (NCRIS) via Phenomics Australia. A.P. and M.K. are supported by the Gretel and Gordon Bootes Medical Research Foundation. M.K. is supported by the John Curtin School of Medical Research PhD Scholarship. S.M.M. is supported by the Australian National University, the National Health and Medical Research Council of Australia (under Project and Ideas Grants APP1146864 and APP2002686), and a CSL Centenary Fellowship. References 1. Koliaraki V, Prados A, Armaka M et al (2020) The mesenchymal context in inflammation, immunity and cancer. Nat Immunol 21:974– 982 2. Uccelli A, Moretta L, Pistoia V (2008) Mesenchymal stem cells in health and disease. Nat Rev Immunol 8:726–736 3. Koliaraki V, Dotto GP, Buckley CD et al (2022) Mesenchymal cells in health and disease. Nat Immunol 23:1395–1398 4. Davidson S, Coles M, Thomas T et al (2021) Fibroblasts as immune regulators in infection, inflammation and cancer. Nat Rev Immunol 21:704–717

5. Onfroy-Roy L, Hamel D, Malaquin L et al (2021) Colon fibroblasts and inflammation: sparring partners in colorectal cancer initiation? Cancers (Basel) 13:1749 6. Li D, Wu M (2021) Pattern recognition receptors in health and diseases. Signal Transduct Target Ther 6:291 7. Man SM, Jenkins BJ (2022) Contextdependent functions of pattern recognition receptors in cancer. Nat Rev Cancer 22:397– 413 8. Pandey A, Shen C, Feng S et al (2021) Cell biology of inflammasome activation. Trends Cell Biol 31:924–939

Culturing Primary Intestinal Fibroblasts 9. Bautista-Hernandez LA, Go´mez-Olivares JJ, Buentello-Volante B et al (2017) Fibroblasts: the unknown sentinels eliciting immune responses against microorganisms. Eur J Microbiol Immunol (Bp) 7:151–157 10. Kim YG, Kamada N, Shaw MH (2011) The Nod2 sensor promotes intestinal pathogen eradication via the chemokine CCL2dependent recruitment of inflammatory monocytes. Immunity 34:769–780 11. Normand S, Delanoye-Crespin A, Bressenot A et al (2011) Nod-like receptor pyrin domaincontaining protein 6 (NLRP6) controls epithelial self-renewal and colorectal carcinogenesis upon injury. Proc Natl Acad Sci U S A 108: 9601–9606 12. Chalkidi N, Paraskeva C, Koliaraki V (2022) Fibroblasts in intestinal homeostasis, damage, and repair. Front Immunol 13:924866

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13. Chen X, Song E (2019) Turning foes to friends: targeting cancer-associated fibroblasts. Nat Rev Drug Discov 18:99–115 14. Deng L, Jiang N, Zeng J et al (2021) The versatile roles of cancer-associated fibroblasts in colorectal cancer and therapeutic implications. Front Cell Dev Biol 9:733270 15. Khan M, Gasser S (2016) Generating primary fibroblast cultures from mouse ear and tail tissues. J Vis Exp 107:53565 16. Khalil H, Nie W, Edwards RA et al (2013) Isolation of primary myofibroblasts from mouse and human colon tissue. J Vis Exp 80: 50611 17. Karki R, Man SM, Malireddi RKS et al (2016) NLRC3 is an inhibitory sensor of PI3K-mTOR pathways in cancer. Nature 540:583–587 18. Seluanov A, Vaidya A, Gorbunova V (2010) Establishing primary adult fibroblast cultures from rodents. J Vis Exp 44:2033

Chapter 24 Lipid Nanoparticle-Mediated Delivery of miRNA Mimics to Myeloid Cells Elaine Kang and Marcin Kortylewski Abstract MicroRNA (miRNA) dysregulation is known to be associated with a variety of human diseases, including cancers and immune disorders. MiR146a represents one of the best characterized regulators of the immune response, as well as cellular survival through the negative feedback inhibition of nuclear factor-kappa B (NF-ĸB) signaling in myeloid cells. Restoration of miR146a levels would be an attractive therapeutic strategy for reducing exaggerated immune responses or to prevent certain types of blood cancers. However, delivery of synthetic miRNA mimics to target myeloid cells remains challenging. Here, we describe an optimized lipid nanoparticle (LNP) strategy for the delivery of miRNA mimics to myeloid immune cells and provide detailed protocols for characterization of LNP complexes and their biological activity. The encapsulation of miR146a within a lipid complex protects the nucleic acid from nuclease degradation, while allowing for rapid uptake by target myeloid immune cells. The strategy results in an efficient inhibition of target interleukin (IL) 1 receptor associated kinase 1 (IRAK1) and tumor necrosis factor receptor associated factor 6 (TRAF6) protein expression, thereby resulting in reduced NF-ĸB activity in mouse macrophages in vitro. The LNP-encapsulated miR146a effectively inhibits expression of IL-6, a major proinflammatory mediator downstream from NF-ĸB. This LNP-based strategy is suitable for testing of other miRNAs or RNA therapeutics targeting myeloid immune cells. Key words MicroRNA, miR-146a, NF-ĸB, Lipid nanoparticles, Inflammation, Oligonucleotides

1

Introduction MicroRNAs (miRNAs)are endogenous, small noncoding RNAs that are responsible for regulating expression of multiple target genes [1]. These miRNAs regulate gene expression through different modes of action, including sequence-specific binding to 3′ untranslated regions of target gene mRNA to repress target protein expression, or by upregulating target gene expression through increased mRNA stability [2]. Over 2000 miRNAs have been discovered, and they are involved in the regulation of various biological pathways, including inflammation [3]. Moreover, dysregulation of miRNAs has been associated with many immune

Brendan J. Jenkins (ed.), Inflammation and Cancer: Methods and Protocols, Methods in Molecular Biology, vol. 2691, https://doi.org/10.1007/978-1-0716-3331-1_24, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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disorders and with tumorigenesis [4]. miR146a is one of the most well-characterized miRNAs, and its dysregulation can lead to various severe consequences such as autoimmune disorders [5]. However, the delivery of miR146a to its target cells posts a challenge. Previously, we have demonstrated that direct conjugation of CpG oligonucleotides with miRNAs can enhance the delivery to myeloid cell populations and resolve the acute inflammation response in mice [6]. Here, we describe a strategy for enhanced delivery of miR146a mimic to immune cells through encapsulation in modified lipid nanoparticles (LNPs). LNPs have emerged as an attractive tool for gene therapy as underscored by recent rapid approvals of COVID-19 mRNA vaccines and by an earlier approval of Onpattro, which was the first LNP (siRNA) product approved in the market [7]. LNPs are generally composed of four different components, which are helper lipids, polyethene glycol, cholesterol, and an ionizable/cationic lipid [8]. Ionizable lipid forms a complex with negatively charged oligonucleotides and alongside other components to forms a stable, monodisperse lipid complex [9]. These lipid complexes have been characterized and studied in preclinical and clinical settings [10–12]. Here, we demonstrate that miR146a-encapsulated LNPs can be readily taken up by myeloid cells and induce a cargo-specific response in macrophage cell lines as well as in splenocytes.

2

Materials

2.1 Encapsulation of MiRNA Mimic (miR146a) into Lipid Nanoparticles with Quantification and Validation

1. Commercially Available Lipids (Avanti, MedChemExpress) (a) L-α-Phosphatidylcholine, hydrogenated (Soy) (HSPC). (b) Cholesterol. (c) D-Lin-MC3-DMA (MC3). (d) 1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG). 2. NanoAssemblr benchtop unit (Precision Nanosystem). 3. MiR146a sequence: (a) miR146a-mimic (guide)—5’ CAUGGGUU 3′.

UGAGAACUGAAUUC

(b) miR146a-mimic (passenger)—5’ CCCAUGGAAUUCA GUUCUCAAA 3′. 4. DNase-/RNase-free H2O. 5. Ethanol 200 proof. 6. Amicon ultracentrifugation tubes with10-kDa cutoff (EMD Millipore).

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7. Quantification of the encapsulated oligonucleotide using Quant-iT Ribogreen RNA Assay kit (Thermo Fisher). 8. Nanoparticle tracking analysis using NS300 (Malvern). 2.2 Polyacrylamide (PAGE) Gel Reagents

1. 10 × Tris-borate-EDTA buffer, BioReagent. 2. 40% acrylamide/bis-acrylamide solution (19:1). 3. Gel red dye (Biotium). 4. Tetramethylethylenediamine. 5. Double-stranded RNA molecular weight marker. 6. RNA loading dye (2 ×). 7. DNA loading dye (6 ×).

2.3 Western Blotting Reagents

1. Western blot assembly unit. 2. Sample protein concentrations were determined using Pierce BCA protein analysis kit. 3. PageRuler prestained protein ladder. 4. Pierce™ PVDF transfer membrane. 5. Immobilon blotting filter paper. 6. Rabbit IRAK1-specific antibody (e.g., Cat. No. D51G7, Cell Signaling Technology). 7. Rabbit TRAF6-specific antibody (e.g., Cat. No D21G3, Abcam). 8. Goat anti-rabbit IgG H & L. 9. Monoclonal antibody.

β-actin-specific

and

peroxidase-conjugated

10. SuperSignal West Femto Maximum ECL detection reagent. 11. Oligofectamine™ transfection reagent. 12. Proteinase inhibitor (cOmplete). 13. Phosphatase inhibitors (PhosSTOP). 14. Wash buffer: Tris-buffered saline containing 1% Tween 20 (TBST). 15. Blocking solution: 5% milk in TBST. 2.4 NF-KB Reporter Assay Using RAWBlue™ Cells

1. Quanti-Blue solution for the colorimetric enzyme assay. 2. Absorbance reading at 625 nm on Cytation 3 (BioTek). 3. Clear-bottom 96-well plate. 4. Gibco™ Dulbecco’s Modified Eagle’s Medium (DMEM), high glucose. 5. Fetal bovine serum (FBS).

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6. Antibiotic-antimycotic (100 ×). 7. GlutaMAX™ supplement. 2.5 LNP (miR146aCy3) Uptake by Primary Mouse Splenocytes

1. Mice: 8–12-week-old C57BL/6 J mice (e.g., Jackson Laboratory) (see Note 1). 2. DNAse I grad II, from bovine pancreas. 3. Collagenase D. 4. 70 μm cell strainer. 5. 1 mL syringe plunger. 6. Red blood cell lysis buffer: NH4Cl (150 mM), KHCO3 (10 mM), Na2EDTA dissolved in H2O (pH 7.4). 7. Single-cell suspension of fresh splenocytes in DMEM supplemented with 10% FBS, 1% glutamine, 1% antibioticantimycotic at 37 °C. 8. Collection tube: Round-bottom polystyrene tubes. 9. Staining buffer: 1 × phosphate-buffered solution (PBS), 2% FBS, 0.1% NaN3. 10. CD16/CD32 monoclonal antibody. 11. Fixable viability dye (e.g. Fixable Aqua), mouse CD11b, CD11c, CD3, CD19. 12. Flow cytometer (e.g., Attune, Thermo Fisher).

2.6 The Assessment of IL-6 Splenocyte Cytokine Levels

1. MiR146KO Laboratory).

mice—B6.Cg-Mir146tm1.1Bal/J

(Jackson

2. Lipopolysaccharide (LPS) from Salmonella enterica serotype. 3. IL-6 mouse uncoated ELISA kit. 4. STOP buffer: Add 1 mL concentrated sulfuric acid (H2SO4) to 17.2 mL of double-distillated water.

3

Methods

3.1 Formulation of MiR146 into Lipid Nanoparticles

1. Reconstitute lyophilized pellet of miR146a with DNase-/ RNase-free water to make 1 mM stock solution; mix well at 37 °C for 5 min. 2. Hybridize the miR146a (guide/passenger duplex) by mixing an equal molar amount of each oligo and incubate at 80 °C using dry bath for 5 min, and then let the solution cool down to room temperature. 3. Quantify the oligo concentration using Cytation 3 multi-plate reader.

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Table 1 LNP composition (for 250 μL total volume) M.W.

Concentration(mg/mL)

Mol %

HSPC

783

10

10

28.4

Cholesterol

386.66

10

38.5

53.6

MC3

642

10

50

PEG-DMG

2805

10

Ethanol

1.5

Volume(μL)

115.5 15.1 37.25

4. Prepare each component of lipids by dissolving measured lipids (>5 mg) in ethanol and heat at 50 °C for 15 min; pipette up and down in between to allow better heat distribution and solubilization. 5. Prepare the combined lipid formulation (Table 1) by mixing all lipid components in ethanol (prepare extra based on the oligonucleotide amount desired to encapsulate). 6. Prepare the oligonucleotide solution by diluting it to 50 mM in citrate buffer at 0.133 mg/mL (250 μL lipid mixture and 750 μL oligonucleotide in citrate buffer for total of 1 mL of LNPs). 7. Prepare the LNP formulation using NanoAssemblr benchtop unit with total flow rate 9 mL/min, total flow rate ratio 3:1, aqueous phase at 1.5 mL, and organic phase at 0.5 mL; initial waste is 0.25 mL and end waste is 0.05 mL. 8. Upon completion of mixing of lipid with oligonucleotide components, add an excess amount of 1 × PBS to the collection tube (top up to 15 mL). 9. Transfer the LNP(miR146) product into a 10-kDa Amicon microtube and centrifuge at 2000 × g for 20–30 min at 16 ° C. Discard the flow through and add 14 mL PBS to the top unit; repeat the centrifuge process. Collect the product, LNP (miR146a) into a 1.5-mL Eppendorf tube and store at 4 °C (Fig. 1a) (see Note 2). 3.2 Characterization of LNP Formulation

1. Extract the miR146a from LNPs using 10 μL of 2% Triton-X for each 10 μL of LNP(miR146a) and incubate at 37 °C for 15 min. The released oligonucleotides should be diluted 10 × using DNase-/RNase-free water and then diluted 200 × in TE buffer. 2. Prepare standards with naked miR146a by first diluting the oligo stock to 200 μg/mL, and then add 10 μL of 2% TritonX together with 10 μL miR146a. Dilute the mixture to 50–100 ng/mL with 1 × TE buffer.

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Fig. 1 Generation and characterization of LNP(miR146). (a) LNP production process. (b) Nanotracking analysis (NTA) was performed of LNP variants using Nanosight; shown are representative average LNP size distributions in three individual measurements. (c) LNPs formulated with miR146 were examined using gel electrophoresis on 15% PAGE comparing a reference miR146a oligonucleotide, LNP(miR146a) formulation, and miR146a extracted from LNPs

3. Pipette 100 μL standards (serial diluted) and testing sample into a black well/clear-bottom plate and add 100 μL Quant-iT solution (1:2000 in TE buffer) to all wells. Incubate in the dark for up to 5 min and then read using excitation/emission spectra of 480/520 nm. 4. Calculate concentration of the LNP-encapsulated oligonucleotide based on a standard curve. 5. Prepare sample by diluting stock LNP (approx. 2.5 mg/mL of lipid) 400 × with PBS. 6. Analyze the LNP size and particle concentration obtained by performing three 60 s runs at a continuous flow rate of syringe pump speed (30) NTA analysis (NS300 Malvern) (Fig. 1b). 3.3 Validation of MiR146 Encapsulation in LNPs Using Gel Electrophoresis

1. Assemble the gel plates in the gel casting chamber, prepare 15% PAGE gel (see Table 2) and pour quickly between gel plates using a pipette, insert comb, and leave to polymerize for about 30 min. 2. Place the gel in the electrophoresis apparatus according to manufacturer’s instruction and fill the chamber with 1 × TBE. 3. Repeat Subheading 3.2, step 1. Then add 20 μL of 2 × RNA loading buffer (released cargo). 4. Dilute miR146a to 200 μg/mL and mix with an equal amount of 2 × RNA loading buffer. 5. Mix 10 μL of LNP(miR146a) with 10 μL DNA loading dye. 6. Load 2 μL of each testing sample together with doublestranded RNA molecular marker and run the gel at 110 V for 1 h.

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Table 2 Fifteen percent PAGE gel Volume Acrylamide/bis-acrylamide 40%

5.6 mL

H2O

7.7 mL

10 × TBE

1.5 mL

10% APS

200 μL

TEMED

10 μL

7. Remove the gel and transfer into a dish containing 1 × TBE with gel red staining solution (diluted 1:1000 in TBE buffer), incubate for 10–15 min, and then examine the gel using ChemiDoc MP imaging system (Fig. 1c) (see Notes 3 and 4). 3.4 Functional Verification of LNP (miR146) in Mouse Macrophages by Western Blot

1. Plate and culture RAW264.7 macrophages on a 6-well plate overnight at a desired density (105 cells/well) in DMEM supplemented with 10% FBS, 1% glutamine, and 1% antibiotics/ antimycotics. 2. Next day, add pre-complexed Oligofectamine/miR146a to cultured wells for 6 h and replace with fresh medium. Add a desired concentration of LNP(miR146a) (usually 50–200 nM). 3. On the day of collection, carefully collect the cells after incubation on ice for 10 min to allow cell detachment, and pipette the medium to facilitate cell detachment. 4. Spin the cells down at 500 × g for 3 min, wash the cell pellet with 1 × PBS, and repeat centrifugation. 5. Resuspend cell pellet in RIPA lysis buffer supplemented with proteinase (cOmplete) and phosphatase inhibitors (PhosSTOP) (Table 3) at a desired volume (e.g., 100 μL), and allow for complete lysis of the cells by incubating on ice for 30 min. 6. Remove the DNA pellet by centrifugation at maximal speed for 25 min at 4 °C and remove the pellet using a pipette. 7. Read protein concentration by loading 5 μL of standards (e.g., Pierce BCA protein analysis kit) or samples in duplicate to a 96-well plate and add 195 μL of reading solution. 8. Incubate at 37 °C for 20 min and read absorbance at 560 nm. 9. Assemble the gel plates in the gel casting chamber and prepare SDS-PAGE gels (Table 4).

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Table 3 RIPA buffer Stock solution

For 100 mL

20 mM Tris pH 8.2

2M

1 mL

158 mM NaCl

5M

3.16 mL

1% triton X-100

100%

1 mL

1% sodium deoxycholate

1g

0.1% SDS

20%

0.5 mL

5 mM EDTA

0.5 M

1 mL

Table 4 8% SDS-PAGE gel for Western blotsa For two gels (μL) Acrylamide/bis-acrylamide 40%

2000

Tris pH 8.8

2500

SDS 20% H2O APS (10%) TEMED Total volume

50 5400 100 5 10,000

a

Resolving gel (8%); please leave ~3.0-cm space above the gel and then overlay with isopropanol

10. Fill the gel chamber with Tris–HCl running buffer, load 5 μg of protein sample (denatured with 20% β-mercaptoethanol in 4 × loading dye) to each well. 11. Run the gel at 90 V for 30 min and then 110 V for 90 min. 12. Prepare transfer membrane by activating in methanol for 30 s and then soak in transfer buffer. 13. Prepare transfer cassette and carefully layer gel, blotting membrane, and blotting paper. 14. Run the transfer with 110 V for 1 h at 4 °C. 15. Add blocking solution to the blotting membrane for 30 min. 16. Cut the gel based on the protein ladder position, and incubate with primary antibodies (anti-IRAK1 1:3000; anti-β-actin 1: 100,000 in TBST with 2.5% milk) overnight at 4 °C.

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17. Add wash buffer to the blotting membrane with rinsing over five times, and then incubate with secondary antibodies for 40 min. 18. Repeat washing steps and image the membranes. 19. Analyze the target bands relative to control (β-actin) using ImageLab software (see Fig. 2a). 3.5 In Vitro RAWBlue™ Assay to Verify on-Target Activity of LNP (miR146a)

1. Plate 1–2 × 104 RAW-Blue™ cells in 96-well plates with appropriate medium. 2. On the next day, carefully remove the supernatant and replace with treatment medium. 3. After desired incubation time (24–48 h), remove the supernatant and replace with LPS-containing medium (100 ng/mL) at 200 μL per well. 4. After desired stimulation time (4–8 h), remove 20 μL of supernatant to a new 96-well plate, and add 180 μL of QuantiBlue solution. 5. Use untreated cell supernatant as negative control and LPS-treated only cell supernatant as positive control (see Note 5). 6. Incubate for 1–3 h until visible color differences (purple to deep blue color transition). 7. Read the plate at 625 nm and analyze as fold activation compared to untreated control (see Fig. 2b).

3.6 Cell-Selective Uptake of LNP (miR146a) by Primary Mouse Immune Cells

1. Harvest spleens from C57BL/6 mice, and transfer to ice-cold culture medium. 2. Prepare 70 μm cell strainer and place on top of a 50-mL conical tube. 3. Transfer harvested spleen into Petri dishes and wash twice with Hank’s Balanced Salt Solution. 4. Add 5–10 mL of collagenase D (400 U/mL) and DNase I (1 mg/mL) and cut spleen into small cubes (~1–2 mm in diameter) using surgical scissors, and incubate for 15–30 min at 37 °C. 5. Neutralize the reaction by adding excess medium, and carefully push through the digested tissue through a cell strainer with a syringe plunger. 6. Centrifuge the splenocytes at 400 × g for 5 min at 4 °C. 7. Resuspend the pellet in 3–5 mL of red blood cell lysis buffer and incubate at room temperature for 5 min. 8. Quench the reaction by adding the same volume of medium.

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Fig. 2 In vitro activity and anti-inflammatory effect of LNP(miR146a). (a) RAW 264.7 macrophages were incubated with LNP(miR146a) or transfected with miR146a using Oligofectamine™ at the same concentration (200 nM) for the indicated times, and target proteins were analyzed using Western blotting Untreated, U.T. (b) RAW-Blue™ cells were treated using various concentrations of LNP(miR146a) for 24 or 48 h and then stimulated with LPS (100 ng/mL) for 4 h. The supernatants were later collected for colorimetric assessment of the NF-ĸB-driven SEAP activity. Shown are representative results for experiments repeated twice in triplicates; means ± standard deviation

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9. Remove red blood cell lysis buffer from splenocytes by centrifugation at 400 × g for 5 min at 4 °C twice. 10. Resuspend the pellet with 5 mL of DMEM culture medium supplemented with 10% FBS, 1% L-glutamine. 11. Count splenocytes and plate 2–3 × 106 cells per well (48-well plate) in 800 μL medium. 12. Add labeled oligonucleotide formulations at desired concentrations LNP(miR146aCy3) (200–500 nM). 13. Incubate 4–8 h. 14. Collect cells, and resuspend each sample in 100 μL of PBS containing 1 μL Fixable Aqua (or other viability dye). 15. Stain cells for 15–30 min on ice, wash once with 2 mL PBS, and resuspend in the staining buffer containing Fcγ block (CD16/ 32) and specific antibody cocktail. 16. Stain cells for 30 min on ice, and wash twice with 2 mL staining buffer. 17. Analyze cell uptake using flow cytometry (see Fig. 3a). 3.7 Cytokine Assay to Verify Biological Activity of LNP (miR146a)

1. Harvest spleens from miR146a KO mice, and transfer to ice-cold culture medium. 2. Acquire single-cell suspension splenocytes (see Subheading 3.6, steps 2–10). 3. Count the splenocytes and plate 2–3 × 106 cells per well (48-well plate) in 800 μL medium.

Fig. 3 Cell-selective internalization and activity of LNP(miR146a) on primary mouse splenocytes. (a) Wild-type C57BL/6 mouse splenocytes were incubated with various concentrations of fluorescently labeled LNP (miR146aCy3) for 8 h and then uptake by different immune cell subsets was assessed using flow cytometry. MACs, macrophages. DCs, dendritic cells. Untreated, U.T. (b) Splenocytes from miR146aKO mice were preincubated overnight with LNP(miR146a) or control LNP(scrRNA). Cells were then stimulated using LPS (100 ng/mL) for the indicated times and supernatants were collected for ELISA analysis of mouse IL-6 secreted post stimulation. ELISA was performed according to manufacturer’s protocol; means ± standard deviation (n = 3). ** P < 0.01. ns, not significant. Two-way ANOVA with multiple comparison of each individual time point

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4. Add oligonucleotide formulations at desired concentrations LNP(miRNA) 50–200 nM. 5. Incubate cells overnight. 6. Stimulate the splenocytes by directly adding LPS (100 ng/mL). 7. Collect culture supernatant (50 μL) at 8, 12, or 24 h after stimulation and immediately store at -80 °C until ready to run ELISA (Fig. 3b) (see Notes 6 and 7). 8. Refer to manufacturer’s instructions to perform ELISA, and read absorbance at 450 nm (IL-6 levels) and 570 nm (for background subtraction).

4

Notes 1. All experiments performed with mice need to be approved by an institutional animal experimentation ethics committee and be conducted in compliance with the guidelines of the country’s governmental bodies that regulate animal experimentation. 2. The protocol focuses on the use of the NanoAssemblr benchtop equipment for standardized LNP preparation. However, LNPs can also be prepared using more labor-intensive, manual methods if a microfluidics device is not available as described by others [13]. 3. Freshly prepared LNPs can be filtered through a 0.2-μm filter to sterilize the stock solution before use or storage. Generally, LNP formulations generated using the described method can be stored at 4 °C for up to 3 months without significant loss of miRNA functionality. Small-size LNPs (