Lung Cancer: Methods and Protocols 9781071612774, 9781071612781

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Methods in Molecular Biology 2279

Pedro G. Santiago-Cardona Editor

Lung Cancer Methods and Protocols

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-bystep 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.

Lung Cancer Methods and Protocols

Edited by

Pedro G. Santiago-Cardona Biochemistry and Cancer Biology Divisions, Ponce Health Sciences University-Ponce Research Institute, Ponce, Puerto Rico

Editor Pedro G. Santiago-Cardona Biochemistry and Cancer Biology Divisions Ponce Health Sciences University-Ponce Research Institute Ponce, Puerto Rico

ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-0716-1277-4 ISBN 978-1-0716-1278-1 (eBook) https://doi.org/10.1007/978-1-0716-1278-1 © Springer Science+Business Media, LLC, part of Springer Nature 2021 This work is subject to copyright. All rights are reserved 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. Cover Illustration Caption: For more information, see Figure 3 from Chapter 4 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 Lung cancer is characterized by an aggressive nature and a poor patient survival. This is in spite of the recent significant advances in the understanding of the genetics, histology, and molecular and cellular biology of the disease, as well as its clinical aspects. These knowledge gains have resulted in improved detection, diagnosis, and patient treatment. Treatment in particular has evolved to include targeted therapies based on particular tumor genetics and molecular biology. Yet, in spite of this progress, the prognosis for lung cancer patients remains relatively poor, with still low 5-year survival rates when compared to other cancer types. Therefore, research aimed at furthering our understanding of this fatal disease is more than warranted, at the basic, translational, and clinical levels. Together, the chapters of this book have the overarching goal to serve as a laboratory manual that contains protocols and in-depth discussion for commonly used experimental approaches for the characterization of several aspects of lung tumor biology. In the handling of many lung cancers cases, information about diagnosis, tumor grading, staging, and histological sub-classification is obtained from the analyses of tumor biopsy or resection specimens. Characterization and detection of clinically informative biomarkers in such biological specimens is thus a priority for lung cancer management. Analysis of biomarkers in lung tumor tissues guides clinical interventions and helps to assess the histological origin of the tumor, probability of response to targeted therapy, metastatic potential, probability of disease recurrence, and probability of acquiring resistance to therapy. Along these lines, Chapters 1–3 of this book describe the protocols for the immunohistochemistry (IHC) detection of TTF-1, P40, and NAPSIN-A, respectively. These three protein biomarkers have proven extremely useful in sub-classifying non–small cell lung carcinomas (NSCLC) into adenocarcinomas or squamous cell carcinomas (SCC) when detected by IHC in lung tumor biopsy samples. Another clinically valuable biomarker is PD-L1. Assessing the expression of PD-L1 in tumoral tissue may help clinicians to determine which patients can benefit from immunotherapy using immune checkpoint inhibitors. Lung cancer patients with strong tumoral PD-L1 expression may show a better response to immunotherapy, and therefore, assessing PD-L1 expression in tumor biopsies by IHC can help clinicians to stratify patients based on the likelihood of favorable outcomes from immunotherapy. Chapter 4 describes a detailed protocol for the IHC detection of PD-L1 expression in clinical samples, while Chapter 5 describes a protocol that can be used to validate the specificity of anti-PD-L1 antibodies by immunoblot analysis, a validation that is extremely important to assess whether a particular PD-L1 antibody is suitable for clinical applications. These chapters dealing with immunological detection of clinically relevant biomarkers are followed by two chapters dealing with the optimization aspects of immunological detection of antigens. Chapter 6 describes the optimization of the immunohistochemistry procedure using lung cancer cell line–derived tridimensional spheroids with a tumor-like tissue architecture. Using these spheroids for protocol optimization purposes will avoid the use of valuable human lung tumor tissue samples in the optimization stage. Some clinically informative biomarkers are phosphorylation of specific proteins, and the immunologic detection of these phospho-proteins presents the additional challenge of

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ensuring that the used antibody is able to detect specifically the phosphorylated version of the protein. Chapter 7 describes a method for the immunoblot validation of the phosphospecificity of antibodies using lung cancer cell lines. Chapters 8–12 are devoted to the topic of the genetic and molecular characterization of lung cancer biological samples. This is a central topic in lung cancer biology due, first, to the variety of mutated alleles that have been found to have a strong oncogenic driver effect in lung cancer and, second, to the importance of tumor genetics in determining many aspects of tumor biology, as well as many aspects of the clinical interventions and management of lung cancer. Aspects such as response to targeted therapy, acquisition of resistance to anticancer treatments, and disease prognosis can be strongly influenced by tumor genetics and molecular biology. The experimental approaches in this group of chapters include the detection of oncogenic gene fusions, splice variants, and abnormal gene expression profiles using NanoString technology (Chapter 8), protocols for PCR-based approaches for the detection of mutations in EGFR , KRAS , and BRAS genes (Chapters 9 and 10), a PCR-based approach to detect MET exon skipping (Chapter 11), and an immunocytochemical approach for detecting ALK and ROS1 rearrangements in lung cancer cytological samples (Chapter 12). The following group of chapters of this book switch their focus to protocols for the generation of research tools and preclinical lung cancer models that can be extremely valuable to achieve a better understanding of lung tumor biology. Chapter 13 describes a procedure for the generation of patient-derived xenografts by implanting human lung tumor tissue into immunodeficient mice. This mouse-humanized xenograft model can prove extremely valuable as a preclinical model to study various aspects of lung tumor biology. Chapter 14 describes the generation of carcinogen-induced mouse cell lines mimicking traits of lung adenocarcinomas. Being derived from mice exposed to tobacco smoke carcinogens, these cell lines are extremely useful since their origin recapitulates the etiology of the human disease. Many aspects of the aggressive nature of lung cancer, including relapse and resistance against therapy, have been attributed to the presence of tumoral cancer stem cells. Chapter 15 describes a procedure for the in vitro enrichment of mouse lung cancer stem cells, together with a protocol for their characterization using a whole transcriptome analysis. Chapter 16 describes an in vivo imaging procedure to monitor tumor growth and progression in an orthotopic lung cancer model in mice. This chapter demonstrates the power of such a model to monitor tumor response to chemotherapy. The disruption of apoptotic pathways is one among the many traits associated with the cancer state, and understanding how this breakdown occurs in cancer cells is still the topic of intense research. Chapter 17 describes an annexin V/propidium iodide–based staining protocol to assess apoptosis in lung cancer cells. It is generally accepted that aldehydes and carcinogens in tobacco smoke have the capacity to react with DNA bases, creating mutagenic adducts. Such mutagenic adducts play an important etiological role in lung cancer and can be considered biomarkers for aldehyde exposure. Chapter 18 describes a high-performance liquid chromatography-tandem mass spectrometry–based protocol to detect such DNA adducts with specificity and sensitivity. Last but not least, the book closes with a chapter addressing the very important topic of the effectivity of anti-cancer drug delivery. Chapter 19 describes a method for cisplatin encapsulation in liposomes, with the aim of increasing drug delivery while reducing toxicity. Taken together, we hope the chapters of this book give the reader a global perspective of the research efforts related to lung cancer, while allowing them to experimentally probe the different aspects of lung cancer research in their laboratories, including the experimentally

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relevant tests used in the establishment of the diagnosis and prognosis of lung cancer. It is hoped that the book serves as a guide to assist the molecular cancer biologists in their search for the understanding of the molecular aspects of this disease. Ponce, Puerto Rico

Pedro G. Santiago-Cardona

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

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1 Automated TTF-1 Immunohistochemistry Assay for the Differentiation of Lung Adenocarcinoma Versus Lung Squamous Cell Carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Rosa Ve´lez Cintro n, Andre´s J. Martı´nez, Jo Ann Jusino, Marı´a Conte-Miller, and Adalberto Mendoza 2 Immunohistochemical Detection of p40 Expression in Lung Cancer Clinical Samples. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Aruna Nambirajan and Deepali Jain 3 Automated Immunohistochemistry Assay for the Detection of Napsin-A in Formalin-Fixed Paraffin-Embedded Tissues from Lung Tumors . . . . . . . . . . . . 23 Rosa Ve´lez Cintro n, Jo Ann Jusino, Marı´a Conte-Miller, Andre´s J. Martı´nez, and Adalberto Mendoza 4 Detection of Programmed Cell Death Ligand 1 Expression in Lung Cancer Clinical Samples by an Automated Immunohistochemistry System . . . . . 35 Edwin Roger Parra and Sharia Herna´ndez Ruiz 5 Western Blot as a Support Technique for Immunohistochemistry to Detect Programmed Cell Death Ligand 1 Expression . . . . . . . . . . . . . . . . . . . . . 49 Edwin Roger Parra and Sharia Herna´ndez Ruiz 6 Creation of Formalin-Fixed, Paraffin-Embedded 3D Lung Cancer Cellular Spheroids for the Optimization of Immunohistochemistry Staining Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 Jennifer Caba´n-Rivera, Camille Chardon-Colon, Alberto Pedraza-Torres, Yoan E. Rodrı´guez, ˜ ones-Alvarado, Raymond Quin and Pedro G. Santiago-Cardona 7 Immunoblot Validation of Phospho-Specific Antibodies Using Lung Cancer Cell Lines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 Wilfredo M. Pedreira-Garcı´a, Jaileene Pe´rez-Morales, Camille Chardon-Colon, Jennifer Caba´n-Rivera, and Pedro G. Santiago-Cardona 8 Detection of Non-Small Lung Cell Carcinoma-Associated Genetic Alterations Using a NanoString Gene Expression Platform Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 Johan Staaf, Mats Jo¨nsson, and Anna F. Karlsson 9 A PCR-Based Approach for Driver Mutation Analysis of EGFR, KRAS, and BRAF Genes in Lung Cancer Tissue Sections. . . . . . . . . . . . . 109 Rodrigo de Oliveira Cavagna, Leticia Ferro Leal, Fla´via Escremim de Paula, Gustavo Noriz Bernardinelli, and Rui Manuel Reis

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Contents

6-Color Crystal Digital PCRTM for the High-Plex Detection of EGFR Mutations in Non-Small Cell Lung Cancer . . . . . . . . . . . . . . . . . . . . . . . . Jordan Madic, Ce´cile Jovelet, Imane Dehri, and Allison C. Mallory Detection of MET Exon 14 Skipping Alterations in Lung Cancer Clinical Samples Using a PCR-Based Approach . . . . . . . . . . . . . . . . . . . . . . Jane S. Y. Sui, Stephen P. Finn, and Steven G. Gray Immunocytochemical Detection of ALK and ROS1 Rearrangements in Lung Cancer Cytological Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diane Frankel, Elise Kaspi, and Patrice Roll A Method for the Establishment of Human Lung Adenocarcinoma Patient-Derived Xenografts in Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Joanne Lundy, Brendan J. Jenkins, and Mohamed I. Saad A Method for the Establishment and Characterization of Mouse Lung Adenocarcinoma Cell Lines that Mimic Traits of Human Adenocarcinomas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Magda Spella, Ioannis Lilis, and Georgios T. Stathopoulos Whole Transcriptome Sequencing Analysis of Cancer Stem/ Progenitor Cells Obtained from Mouse Lung Adenocarcinomas. . . . . . . . . . . . . . Ansam Sinjab, Reem Daouk, Wassim Abou-Kheir, and Humam Kadara In Vivo Imaging of Orthotopic Lung Cancer Models in Mice . . . . . . . . . . . . . . . . Peng Liu, Liwei Zhao, Laura Senovilla, Oliver Kepp, and Guido Kroemer An Annexin V-FITC—Propidium Iodide-Based Method for Detecting Apoptosis in a Non-Small Cell Lung Cancer Cell Line . . . . . . . . . . . . . Robin Kumar, Ankit Saneja, and Amulya K. Panda Detection of DNA Adduct Formation in Rat Lungs by a Micro-HPLC/MS/MS Approach. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ange´lica B. Sanchez, Camila C. M. Garcia, Paolo Di Mascio, and Marisa H. G. Medeiros A Method for Liposome Co-encapsulation of Phenethyl Isothiocyanate and Cisplatin and Determining Its Toxicity Against Lung and Lung Cancer Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mengwei Sun and Anthony J. Di Pasqua

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors WASSIM ABOU-KHEIR • Department of Anatomy, Cell Biology and Physiological Sciences, Faculty of Medicine, American University of Beirut, Beirut, Lebanon GUSTAVO NORIZ BERNARDINELLI • Center of Molecular Diagnoses, Barretos Cancer Hospital, Barretos, Brazil JENNIFER CABA´N-RIVERA • Epidemiology Program, School of Public Health, Ponce Health Sciences University-Ponce Research Institute, Ponce, Puerto Rico CAMILLE CHARDO´N-COLO´N • Biochemistry and Cancer Biology Divisions, Ponce Health Sciences University-Ponce Research Institute, Ponce, Puerto Rico ROSA VE´LEZ CINTRO´N • Southern Pathology Services Inc., Ponce, Puerto Rico; Pathology Division, Ponce Health Science University, Ponce, Puerto Rico MARI´A CONTE-MILLER • Southern Pathology Services Inc., Ponce, Puerto Rico; Pathology Division, Ponce Health Science University, Ponce, Puerto Rico REEM DAOUK • Department of Anatomy, Cell Biology and Physiological Sciences, Faculty of Medicine, American University of Beirut, Beirut, Lebanon RODRIGO DE OLIVEIRA CAVAGNA • Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, Brazil FLA´VIA ESCREMIM DE PAULA • Center of Molecular Diagnoses, Barretos Cancer Hospital, Barretos, Brazil IMANE DEHRI • Stilla Technologies, Villejuif, France PAOLO DI MASCIO • Departamento de Bioquı´mica, Instituto de Quı´mica, Universidade de Sa˜o Paulo, Sa˜o Paulo, SP, Brazil ANTHONY J. DI PASQUA • Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, Binghamton University, Johnson City, NY, USA STEPHEN P. FINN • Thoracic Oncology Research Group, Trinity Translational Medicine Institute, St. James’s Hospital, Dublin, Ireland; Department of Histopathology, Cancer Molecular Diagnostics, Labmed Directorate, St. James’s Hospital, Dublin, Ireland; Department of Histopathology and Morbid Anatomy, Trinity College Dublin, Dublin, Ireland DIANE FRANKEL • Aix Marseille Univ, APHM, INSERM, MMG, Hoˆpital la Timone, Service de Biologie Cellulaire, Marseille, France CAMILA C. M. GARCIA • Departamento de Bioquı´mica, Instituto de Quı´mica, Universidade de Sa˜o Paulo, Sa˜o Paulo, SP, Brazil; Nu´cleo de Pesquisas em Cieˆncias Biologicas & Departamento de Cieˆncias Biologicas, Instituto de Cieˆncias Exatas e Biologicas, Universidade Federal de Ouro Preto, Ouro Preto, MG, Brazil STEVEN G. GRAY • Thoracic Oncology Research Group, Trinity Translational Medicine Institute, St. James’s Hospital, Dublin, Ireland; Department of Clinical Medicine, Trinity College Dublin, Dublin, Ireland; School of Biological Sciences, Dublin Institute of Technology, Dublin, Ireland SHARIA HERNA´NDEZ RUIZ • Department of Translational Molecular Pathology, Translational Molecular Pathology Immunoprofiling Laboratory, The University of Texas MD Anderson Cancer Center, Houston, TX, USA DEEPALI JAIN • Department of Pathology, All India Institute of Medical Sciences, New Delhi, India

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Contributors

BRENDAN J. JENKINS • Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, VIC, Australia; Department of Molecular and Translational Sciences, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, VIC, Australia MATS JO¨NSSON • Division of Oncology and Pathology, Department of Clinical Sciences Lund, Lund University, Lund, Sweden ´ CECILE JOVELET • Stilla Technologies, Villejuif, France JO ANN JUSINO • Southern Pathology Services Inc., Ponce, Puerto Rico HUMAM KADARA • Department of Translational Molecular Pathology, University of Texas MD Anderson Cancer Center, Houston, TX, USA ANNA F. KARLSSON • Division of Oncology and Pathology, Department of Clinical Sciences Lund, Lund University, Lund, Sweden ELISE KASPI • Aix Marseille Univ, APHM, INSERM, MMG, Hoˆpital la Timone, Service de Biologie Cellulaire, Marseille, France OLIVER KEPP • Metabolomics and Cell Biology Platforms, Gustave Roussy Comprehensive Cancer Institute, Villejuif, France; Equipe 11 labellise´e Ligue contre le Cancer, Centre de Recherche des Cordeliers, INSERM UMR 1138, Paris, France; Sorbonne Universite´, Paris, France; Universite´ of Paris, Paris, France GUIDO KROEMER • Metabolomics and Cell Biology Platforms, Gustave Roussy Comprehensive Cancer Institute, Villejuif, France; Equipe 11 labellise´e Ligue contre le Cancer, Centre de Recherche des Cordeliers, INSERM UMR 1138, Paris, France; Sorbonne Universite´, Paris, France; Universite´ of Paris, Paris, France; Suzhou Institute for Systems Medicine, Chinese Academy of Sciences, Suzhou, China; Department of Women’s and Children’s Health, Karolinska Institute, Stockholm, Sweden; Poˆle de Biologie, Hoˆpital Europe´en Georges Pompidou, AP-HP, Paris, France ROBIN KUMAR • Product Development Cell, National Institute of Immunology, New Delhi, India LETICIA FERRO LEAL • Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, Brazil; Barretos School of Medicine Dr. Paulo Prata – FACISB, Barretos, Brazil IOANNIS LILIS • Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece PENG LIU • Metabolomics and Cell Biology Platforms, Gustave Roussy Comprehensive Cancer Institute, Villejuif, France; Equipe 11 labellise´e Ligue contre le Cancer, Centre de Recherche des Cordeliers, INSERM UMR 1138, Paris, France; Sorbonne Universite´, Paris, France; Universite´ of Paris, Paris, France JOANNE LUNDY • Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, VIC, Australia; Department of Molecular and Translational Sciences, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, VIC, Australia; Department of Surgery, School of Clinical Sciences at Monash Health, Monash University, Clayton, VIC, Australia JORDAN MADIC • Stilla Technologies, Villejuif, France ALLISON C. MALLORY • Stilla Technologies, Villejuif, France ANDRE´S J. MARTI´NEZ • School of Public Health, Ponce Health Science University, Ponce, Puerto Rico MARISA H. G. MEDEIROS • Departamento de Bioquı´mica, Instituto de Quı´mica, Universidade de Sa˜o Paulo, Sa˜o Paulo, SP, Brazil ADALBERTO MENDOZA • Southern Pathology Services Inc., Ponce, Puerto Rico; Pathology Division, Ponce Health Science University, Ponce, Puerto Rico

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ARUNA NAMBIRAJAN • Department of Pathology, All India Institute of Medical Sciences, New Delhi, India AMULYA K. PANDA • Product Development Cell, National Institute of Immunology, New Delhi, India EDWIN ROGER PARRA • Department of Translational Molecular Pathology, Translational Molecular Pathology Immunoprofiling Laboratory, The University of Texas MD Anderson Cancer Center, Houston, TX, USA ALBERTO PEDRAZA-TORRES • Biochemistry and Cancer Biology Divisions, Ponce Health Sciences University-Ponce Research Institute, Ponce, Puerto Rico WILFREDO M. PEDREIRA-GARCI´A • Biochemistry and Cancer Biology Divisions, Ponce Health Sciences University-Ponce Research Institute, Ponce, Puerto Rico JAILEENE PE´REZ-MORALES • Biochemistry and Cancer Biology Divisions, Ponce Health Sciences University-Ponce Research Institute, Ponce, Puerto Rico RAYMOND QUIN˜ONES-ALVARADO • Biochemistry and Cancer Biology Divisions, Ponce Health Sciences University-Ponce Research Institute, Ponce, Puerto Rico RUI MANUEL REIS • Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, Brazil; Center of Molecular Diagnoses, Barretos Cancer Hospital, Barretos, Brazil; Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal; ICVS/3B’s—PT Government Associate Laboratory, Braga/Guimaraes, Portugal YOAN E. RODRI´GUEZ • Biochemistry and Cancer Biology Divisions, Ponce Health Sciences University-Ponce Research Institute, Ponce, Puerto Rico PATRICE ROLL • Aix Marseille Univ, APHM, INSERM, MMG, Hoˆpital la Timone, Service de Biologie Cellulaire, Marseille, France MOHAMED I. SAAD • Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, VIC, Australia; Department of Molecular and Translational Sciences, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, VIC, Australia ANGE´LICA B. SANCHEZ • Departamento de Bioquı´mica, Instituto de Quı´mica, Universidade de Sa˜o Paulo, Sa˜o Paulo, SP, Brazil; Nu´cleo de Pesquisas em Cieˆncias Biologicas & Departamento de Cieˆncias Biologicas, Instituto de Cieˆncias Exatas e Biologicas, Universidade Federal de Ouro Preto, Ouro Preto, MG, Brazil ANKIT SANEJA • Product Development Cell, National Institute of Immunology, New Delhi, India PEDRO G. SANTIAGO-CARDONA • Biochemistry and Cancer Biology Divisions, Ponce Health Sciences University-Ponce Research Institute, Ponce, Puerto Rico LAURA SENOVILLA • Metabolomics and Cell Biology Platforms, Gustave Roussy Comprehensive Cancer Institute, Villejuif, France; Equipe 11 labellise´e Ligue contre le Cancer, Centre de Recherche des Cordeliers, INSERM UMR 1138, Paris, France; Sorbonne Universite´, Paris, France; Universite´ of Paris, Paris, France ANSAM SINJAB • Department of Translational Molecular Pathology, University of Texas MD Anderson Cancer Center, Houston, TX, USA MAGDA SPELLA • Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece JOHAN STAAF • Division of Oncology and Pathology, Department of Clinical Sciences Lund, Lund University, Lund, Sweden GEORGIOS T. STATHOPOULOS • Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece;

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Comprehensive Pneumology Center (CPC) and Institute for Lung Biology and Disease (iLBD), University Hospital, Ludwig-Maximilians University and Helmholtz Center Munich, Member of the German Center for Lung Research (DZL), Munich, Germany JANE S. Y. SUI • Thoracic Oncology Research Group, Trinity Translational Medicine Institute, St. James’s Hospital, Dublin, Ireland; Department of Medical Oncology, Mater Misericordiae University Hospital, Dublin, Ireland MENGWEI SUN • Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, Binghamton University, Johnson City, NY, USA LIWEI ZHAO • Metabolomics and Cell Biology Platforms, Gustave Roussy Comprehensive Cancer Institute, Villejuif, France; Equipe 11 labellise´e Ligue contre le Cancer, Centre de Recherche des Cordeliers, INSERM UMR 1138, Paris, France; Sorbonne Universite´, Paris, France; Universite´ of Paris, Paris, France; Universite´ Paris-Saclay, Villejuif, France

Chapter 1 Automated TTF-1 Immunohistochemistry Assay for the Differentiation of Lung Adenocarcinoma Versus Lung Squamous Cell Carcinoma Rosa Ve´lez Cintro´n, Andre´s J. Martı´nez, Jo Ann Jusino, Marı´a Conte-Miller, and Adalberto Mendoza Abstract Due to therapeutic advances, the subclassification of non-small cell lung carcinomas (NSCLC) between the adenocarcinomas and squamous cell carcinomas subtypes is essential for the practice of personalized and targeted medicine. The clinical management for these two NSCLC subtypes is different due to their different molecular properties and histological origins. Immunohistochemistry (IHC) markers such is TTF-1 play a key role in the differentiation of lung adenocarcinomas and squamous cell carcinomas. However, immunohistochemistry is a complex process involving many critical steps and the reliability of results depends on the standardization of the assay as well as the appropriate interpretation. Different laboratories use different reagents and different IHC approaches for the detection of TTF-1 in lung cancer tumors. Here we describe an automated IHC protocol used in our laboratory for the detection of TTF-1 in formalin-fixed, paraffin-embedded (FFPE) tissue sections from lung tumors. Key words Immunohistochemistry, Lung, Cancer, TTF-1, Adenocarcinoma, Squamous cell carcinoma

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Introduction Lung cancer has been described as the second most diagnosed cancer type around the world and one of the main causes of cancer associated deaths [1, 2]. Approximately 85% of lung cancers are non-small cell lung cancers (NSCLC) of which 40% are of the adenocarcinoma subclassification and between 20% and 30% are of the squamous cell carcinoma subtype [1]. In 2015 the World Health Organization (WHO) reinforced the use of immunohistochemistry (IHC) markers in the pathology practice for the subclassification of NSCLC [3]. In view of therapeutic advances, NSCLC subclassification between adenocarcinomas and squamous cell carcinomas is essential for the implementation of precision medicine; a

Pedro G. Santiago-Cardona (ed.), Lung Cancer: Methods and Protocols, Methods in Molecular Biology, vol. 2279, https://doi.org/10.1007/978-1-0716-1278-1_1, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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Rosa Ve´lez Cintro´n et al.

new form of medical practice where the treatment decisions are based on the molecular and histological properties of the tumor [3]. The clinical management of lung adenocarcinomas and squamous cell carcinomas is different due to their molecular properties. Molecular hallmarks found in lung adenocarcinomas such as the presence of EGFR mutations and of ALK and ROS1 gene rearrangements, are associated to the patient’s positive response to existing targeted therapies such as tyrosine kinase inhibitors (TKIs) [4– 6]. Targeted therapies against these alterations are efficient only in advanced lung adenocarcinomas but they possess adverse effects and are contraindicated in squamous cell carcinomas, hence the importance of being able to distinguish between these two NSCLC subtypes. On the other hand, targeted treatments specific for squamous cell carcinomas are also available. Two protein markers, Napsin-A and Thyroid Transcription Factor 1 (TTF-1) have demonstrated a sensitivity of 80% in differentiating lung adenocarcinomas from squamous cell carcinomas [7–11], and they are both readily detectable by IHC of formalinfixed paraffin-embedded (FFPE) lung tumor tissue. The IHC technique involves the use of antibodies to detect specific protein markers in FFPE tissue sections [12]. There are several variations in the IHC procedure used among different laboratories. One of the most commonly used IHC procedures for TTF-1 IHC detection in FFPE lung tumor tissues is described in this chapter. The IHC reaction starts when a primary antibody binds the antigen of interest. Then, a secondary antibody linked to an enzyme binds the primary antibody. A substrate that reacts with the enzyme in the secondary antibody is added at the end of the process to promote the formation of a colored insoluble precipitate that is readily observed under a bright light microscope (Fig. 1). In the postanalytical phase of the IHC, the interpretation of the staining intensity and localization is as important as the analytical phase, which is performing the IHC per se. It is important to know the expected localization and pattern of the signal. Immunohistochemistry is widely used by many clinical and research laboratories. Different laboratories implement different IHC approaches for the detection of TTF-1 based on the commercially available reagents and antibodies. Here we describe an automated IHC protocol used in our laboratory for the detection of TTF-1 in FFPE lung tumor tissue sections, as part of our pathology workflow to distinguish between the adenocarcinomas and squamous cell carcinomas NSCLC subtypes.

Automated TTF-1 Immunohistochemistry

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Fig. 1 Summary of the workflow of the immunohistochemistry technique. (a) A primary antibody binds the epitope in the target protein. (b) A secondary antibody linked to an enzyme binds to the primary antibody in a highly specific way. (c, d) The enzyme substrate, when added to the tissue under study, is converted by the enzyme into an insoluble, colored precipitate that may be visualized under a bright light microscope

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Materials Always follow the product manufacturer’s instructions regarding storing conditions, reagent stability, safety procedures, and waste disposal.

2.1 Ready to Use Reagents (see Note 1)

1. Deionized and distilled water. 2. Ethanol 100%, and 70%, 85%, and 90% ethanol dilutions. Dilutions can be prepared from 100% ethanol diluted with water to the desired concentration. 3. Paraffin, pre-warmed at 60  C. 4. Xylene. 5. Clarify Clearing Agent Xylene Substitute (this procedure was optimized using the American Master Tech; Cat. # CACLELT).

2.2 Immunohistochemistry and Histology Reagents

1. TTF-1 Monoclonal Antibody (Dako, Cat. No. IR056). 2. Immunohistochemistry staining kit. We use the EnVision FLEX, High pH Kit (Dako, Cat. No. K8000). The kit includes all the reagents required for the procedure, including the EnVision FLEX Peroxidase-Blocking Reagent, EnVision FLEX/HRP, EnVision FLEX DAB + Chromogen, and the EnVision FLEX Substrate Buffer. Some solutions from this kit require preparation or dilution previous to starting the IHC procedure (strictly follow the kits instructions, EnVision

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TM FLEX Package Insert from Agilent Technologies). Prepare the 1 EnVision FLEX Target Retrieval High pH Solution (Dako Omnis GV804) by diluting the concentrated 50 EnVision FLEX Target Retrieval Solution 1:50 in distilled or deionized water. The resulting solution is 1. 3. Hematoxylin staining solution (this procedure was optimized using Hematoxylin Dako Omnis, Dako; Cat. #GC808). 4. Eosin stain. 5. Neutral-Buffered 10% formalin solution (available ready to use). 6. Wash buffer, we use the Dako Omnis 20 Wash Buffer (Dako; Cat. No. GC807). Before use, prepare a 1 solution by diluting 1 mL of 20 Wash Buffer in 20 mL of distilled or deionized water. 2.3 Additional Equipment and Materials

1. Water bath or flotation bath, set to 45  C, have a thermometer at hand to monitor the temperature. 2. Microtome. 3. Microscope slides, we use FLEX IHC Microscope Slides (Dako; Cat. No. K8020), but other alternatives can be used. 4. Cover slipping Film. We use Tissue-Tek Sakura (Cat. No. 4770), together with the Sakura Tissue-Tek Automatic Cover slipper. Cover slipping can also be done manually if an automatic cover slipper is not available. 5. Racks for histological slides. 6. Absorbent paper. 7. Cold plate or ice sheet. 8. Laboratory oven (capable of reaching 60  C). 9. Dako Omnis Automated IHC Staining System. This procedure is optimized for this staining system. Other staining systems can be used, such as the Dako Auto-Stainer Link 48, but some procedure parameters will have to be optimized. 10. Standard bright light microscope. 11. Standard histology embedding cassettes with lids. 12. Embedding chamber, console or station. There are several models available, all of them acceptable. Follow any instructions by the manufacturer. 13. Forceps or tweezers, for careful handling of tissues. 14. KP Marker plus pen, or equivalent, for delineating the tumor are in the tissue.

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Methods

3.1 Tissue Fixation and Processing

1. Fix lung tumor tissue biopsies for 6–72 h in the 10% neutralbuffered formalin solution (see Note 2). After fixation of the tissue, it is ready for paraffin embedding and creation of tissue blocks, as described in the following steps. Tissue processing to create the paraffin block can be done manually or in a tissue processor. 2. Dehydrate tissues in a series of ethanol washes as follows, each wash lasting 45 min: 70%, 85%, 90%, and 100% ethanol. The final 100% ethanol wash must be performed three times. 3. Clear the tissue by incubating it in xylene for 3 min. 4. Infiltrate tissues by immersion in molten paraffin, this will create the paraffin blocks that will be cut (see next section). Immerse the tissues in paraffin at 60  C for 45 min. Do a second immersion for 75 min. 5. Embed the tissues to form the paraffin blocks. For this, place tissues in standard histology embedding cassettes and pour molten paraffin into the embedding station. The paraffin should be pre-warmed to 60  C for several hours. Submerge the cassette containing the tissue into the molten paraffin in the embedding station. Use pre-warmed forceps to handle the tissue carefully inside the cassette. Place the tissue with the side that was originally cut (during biopsy specimen collection) facing downwards in the cassette, in such a manner that this side of the tissue is the one closest to the surface of the paraffin from which the first tissue sections will be produced. Ensure that the paraffin, the embedding cassettes, the embedding station and all tools to handle the tissue have all been pre-warmed at 60  C. We recommend starting with the pre-warming of all the materials early in the morning so that when the embedding is started the paraffin and all materials have reached the appropriate temperature. Maintain the tissues in the cassettes immersed in paraffin for 5 min. 6. Place the embedded tissue in the cold plate to solidify the paraffin.

3.2 Tissue Sectioning

1. Set up the water bath to 45  C. Use a thermometer to monitor the temperature in the water. 2. Place the tissue block over a cold plate or ice sheet. 3. Using the microtome, cut tissue sections at 4μm thick (see Note 3 and Fig. 2). 4. As you cut with the microtome, carefully handle the resulting paraffin ribbons containing the tissues with tweezers and float them in the water bath immediately after cutting them. Always

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Fig. 2 Cutting of tissue sections from the paraffin block. (a) Tissue sections are cut to 4 microns thick tissue sections using a microtome. (b) The cut ribbons containing the paraffin tissue sections are transferred to a water bath to allow the sections to stretch. (c) Each tissue section is mounted on a slide

verify that the water in the water bath has reached 45  C prior to transferring the tissue sections to it (see Notes 4 and 5 and Fig. 2). 5. Allow the sections to stretch. If needed, use a stick to help stretch the tissue. Select one tissue section from the ribbon and place it over a FLEX IHC microscope slide (see Note 6 and Fig. 2). 6. Drain the excess of water from the slide by pressing the edges of the slide against an absorbent paper. 7. Place the slide in a rack for histological slides and oven-bake it for 1 h at 60  C (see Note 7). 3.3 Immunohistochemistry Staining in the Dako Omnis IHC Staining System

All the steps in this section will be performed by placing the slides in the Dako Omnis IHC Staining System using the parameters described in the appropriate step below. 1. Deparaffinize the slides by allowing the slide to interact with the Xylene substitute at 25  C. Incubation time should be between 10 s and 1 min (see Note 8). Perform this step twice. 2. Wash with deionized water for 5 s at room temperature. Perform this step twice. 3. Perform the antigen retrieval by incubating the slides 30 min at 97  C with the EnVision FLEX Target Retrieval High pH 1 Solution (see Note 9). 4. Wash the slides in 1 Wash Buffer Omnis during exactly 2.40 min at room temperature (see Note 10). 5. Incubate the slides with the primary antibody TTF-1 Monoclonal Antibody for 20 min at room temperature (see Note 11). 6. Wash the slides in 1 Wash Buffer Omnis during 2 min at room temperature.

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7. Incubate the slides for 3 min at room temperature with the EnV FLEX Peroxidase-Blocking Reagent (see Note 12). 8. Wash the slides in 1 Wash Buffer Omnis for 2 min at room temperature. 9. Incubate the slides for 2 min at room temperature with the EnV FLEX + Mouse LINKER. 10. Incubate the slides for 20 min at room temperature with the EnV FLEX/HRP. 11. Wash the slides in 1 Wash Buffer Omnis for 2 min at room temperature. Repeat this wash a second time. 12. Wash the slides in deionized water during exactly 31 s at room temperature. 13. Wash the slides in 1 Wash Buffer Omnis for 2 min at room temperature. 14. Incubate the slides for 5 min with the EnV FLEX Substrate Working Solution (containing the DAB chromogen) at room temperature. 15. Wash the slides in 1 Wash Buffer Omnis for 2 min at room temperature. 16. Wash the slides in deionized water for exactly 31 s at room temperature. 17. Wash the slides in 1 Wash Buffer Omnis for 2 min at room temperature. 3.4

Counterstaining

1. Incubate the slides for 6 min with Hematoxilin staining solution at room temperature (see Note 13). 2. Wash the slides in deionized water for 31 s at room temperature. 3. Wash the slides in 1 Wash Buffer Omnis for 2 min at room temperature.

3.5

Cover Slipping

1. Remove the slides from the Dako Omnis System, place them in a rack for histological slides and allow them to air dry. 2. Place the rack with the slides in the SakuraTissue-Tek system for automatic cover slipping. Alternatively, this step may be performed manually if an automatic cover slipping device is not available.

3.6 Staining Evaluation and Interpretation

1. Under a bright light microscope, check out for a positive TTF-1 staining signal, which is characterized by a brown nuclear staining similar to the one illustrated in Fig. 3. 2. It is important to evaluate the TTF-1 signal in the tumor cells and not in the non-tumoral adjacent cells. It is recommended that an experienced pathologist assists you in identifying the tumor area within the tissue section.

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Fig. 3 Representative staining for TTF-1 in lung tumor tissue sections. (a) Negative staining for TTF-1. (b) Positive staining for TTF-1 is characterized by a brown signal in the nucleus (see arrows)

3. Stain an additional tissue section slide obtained from the same tissue block under evaluation with Hematoxylin and Eosin (H&E) routine staining, to identify the tumor or the region of interest (i.e., apparent lesion). Mark the region with the marker pen. 4. Superpose the IHC and the H&E slides to identify the region of interest in the IHC slide. 5. Screen the entire slide at 10 to find the brown signal. If the brown signal is present (Fig. 3) this may be indicative of TTF-1 positive expression. 6. Increase the magnification to 40 or 100 to confirm the expected staining pattern for TTF-1 (nuclear staining). Use quality control slides (Table 1) to distinguish nonspecific signal or background from true signal. Quality control slides are recommended to ensure the reliability of the results. 7. A positive expression of TTF-1 is indicative of a lung adenocarcinoma, as opposed to squamous cell carcinoma. Additional IHC stains may be included to support the diagnosis.

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Notes 1. Although ready to use reagents are usually more expensive, they offer the benefit of obtaining consistent and reproducible results even when the assays are performed by different laboratory technologists. Minimizing the reagents that need to be prepared will also minimize the potential of errors and the variability of results among the scientific population. Using reagents classified as for In Vitro Diagnostic for research purposes facilitates the transfer of the results obtained during research studies to a clinical setting which is the goal of a biomedical or a clinical investigation.

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Table 1 Appropriate positive and negative controls recommended for the validation of TTF-1 staining in lung tissue sections Appropriate controls for Napsin-A immunohistochemistry Control type Description

Information provided

Positive control

This control will provide information about A section of lung cancer adenocarcinoma the appropriate performance of the assay which is known to be positive for TTF-1 and the stability of the reagents. Any should be processed in exactly the same technical problem in the assay can be way as described in the method’s section, identified if an unexpected result is parallel to the unknown sample under obtained in the positive control evaluation

Negative reagent control (NRC)

This control will provide information about A section of the sample under evaluation nonspecific staining or background should be processed in exactly the same signal. Comparing the signal obtained in way as described in the method’s section, the section under evaluation against the except that the primary antibody is signal obtained in the NRC will allow to omitted discriminate between the nonspecific and the specific (true) signal for TTF-1

Positive internal control

This control will specifically help to A section of the sample under evaluation distinguish a true negative from a false should be processed in exactly the same negative result caused by inappropriate way as described in the method’s section, pre-analytical conditions. The positive but the primary antibody must be directed expression of the control protein will against a protein known to be expressed in confirm a true negative result in a tissue the lung tissue that lacks TTF-1 expression

2. Fixation conditions are critical in IHC. The gold standard fixative is the 10% neutral-buffered formalin solution with a pH of 7.0—7.4. This fixative prevents autolysis, is a process in which the tissue is degraded by the enzymes that are present in the tissue. Fixation also preserves the tissue morphology. The proportion of tissue to fixative should be between 1:1 and 1:20. Use of other types of fixatives may interfere with the IHC staining and is usually contraindicated in IHC. The time from the tissue collection (extraction from the patient) to the tissue fixation is also critical. This time is known as ischemic time and should be less than 1 h. Prolonged ischemic times promote the irreversible degradation of the antigens in the cells. This may result in absence of staining, nonspecific staining and poor definition of the IHC staining. The fixation time is also critical during the fixation process. It is recommended a fixation time between 6 and 72 h. In our experience, small biopsies may be fixed between 6 and 48 h to avoid overfixation. Insufficient fixation time as well as prolonged fixation time (more than 72 h) interferes with the IHC reaction. Particularly, the excess of fixation will mask the antigens in the cells avoiding the detection of the target protein by the antibody.

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3. Thick tissue sections may yield high background making difficult the analysis and interpretation of the TTF-1 and other IHC markers. The recommended thickness of the tissue sections to be used in IHC should be between 3 and 5μm. It is also recommended to use freshly cut sections. Some antigens can be lost after prolonged storage conditions (i.e., more than 2 months). 4. Floating the paraffin sections or ribbons in a water bath will allows tissue to stretch. With these steps wrinkles and folds are removed before placing the tissue sections on the slide. It is very important to keep the temperature of the water in the flotation bath within 5–10  C below the melting point of the paraffin being used. Forty to fifty degrees Celsius (40–50  C) is usually an optimal temperature for the majority of paraffin types. Always verify the paraffin melting point to set up the appropriate temperature for your flotation bath. Very low temperatures will not remove the folds in the tissue section. Very high temperatures (close to or above the paraffin melting temperature) will melt the paraffin in the section causing alterations in the morphology of the tissue. 5. It is important to use distilled water in the flotation bath. Tap water or low-quality water usually contains impurities that may affect the IHC reaction. It may also interfere in the adhesion of the tissue section to the slide causing the tissue to detach. The water reservoir must be emptied and cleaned daily using a laboratory-grade wipe. Avoid the presence of particulate in the flotation bath reservoir by covering it when not in use. 6. The quality of the slides is very important to allow the adherence of the tissue to the slide. The FLEX IHC microscope slides are coated with an additive that helps in this process. Other type of slides may be used but its ability to keep the tissue attached must be evaluated by the laboratory. Examples of slides additives that may help in the tissue adherence to the slide are those that add positive charges to the slide surface. Positive charges in the slides react with the negative charges in the tissue promoting adherence of the tissue to the slide. 7. Oven-baking slides is also very important to allow tissue adhesion to the slide. The temperature of the oven must be equal or slightly above the melting point of the paraffin. However, very high temperatures or prolonged incubation time may destroy the TTF-1 target antigen. This may yield false negative results. 8. Incomplete removal of paraffin may result in poor TTF-1 staining or lack of TTF-1 signal in positive control tissues, where TTF-1 is expected to be expressed.

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9. Antigen retrieval is necessary to unmask the TTF-1 antigens. Tissue over-fixation may mask the antigens. If the antigen retrieval step is not included in the procedure, the interaction between the antibody and the TTF-1 antigen may fail and a false negative result may be obtained. It is important to optimize the antigen retrieval conditions (incubation time, temperature, and pH according to the fixation conditions). The temperature and the pH are important variables that need to be well controlled during the retrieval process. High temperatures are needed to unmask the antigens in the cells. 10. Washes are important to avoid nonspecific binding and background signal. 11. It is important to optimize the incubation time and temperature with the primary antibody. Also, the concentration of the antibody must be determined by titration experiments in order to obtain the conditions that fit better with the rest of your protocol (particularly with the pre-analytical process). 12. This step will block the endogenous peroxidase activity to eliminate the background staining caused by it. This step is important when using horseradish peroxidase-conjugated antibodies. 13. Hematoxylin and eosin counterstaining allows the visualization of the morphology or architecture of the tissue. This process allows the identification of the nucleus and other important structures in the tissue. The counterstaining provides a visual direction to the observer in order to determine the localization of the IHC signal (i.e., nuclear vs. cytoplasmic). References 1. American Cancer Society (2019) Key Statistics for Lung Cancer. https://www.cancer.org/can cer/non-small-cell-lung-cancer/about/keystatistics.html. Accessed 10 July 2019 2. Torre LA, Bray F, Siegel RL et al (2015) Global cancer statistics 2012. CA Cancer J Clin 65 (2):87–108 3. Travis WD, Brambilla E, Nicholson AG et al (2015) The 2015 World Health Organization classification of lung tumors: impact of genetic, clinical and radiologic advances since the 2004 classification. J Thorac Oncol 10 (9):1243–1260 4. Zhou C, Wu Y-L, Chen G et al (2010) Efficacy results from the randomized phase III OPTIMAL (CTONG 0802) study comparing firstline erlotinib versus carboplatin (CBDCA) plus gemcitabine (GEM) in Chinese advanced non-small cell lung cancer (NSCLC) patients (PTS) with EGFR activating mutations. Ann Oncol 21(Suppl. 8):viii1–viii12

5. Maemondo M, Inoue A, Kobayashi K et al (2010) Gefitinib or chemotherapy for nonsmall-cell lung cancer with mutated EGFR. N Engl J Med 362:2380–2388 6. Inamura K (2017) Lung cancer: understanding its molecular pathology and the 2015 WHO classification. Front Oncol 7:193 7. Loo PS, Thomas SC, Nicolson MC et al (2010) Subtyping of undifferentiated non-small cell carcinomas in bronchial biopsy specimens. J Thorac Oncol 5:442–447 8. Nicholson AG, Gonzalez D, Shah P et al (2010) Refining the diagnosis and EGFR status of non-small cell lung carcinoma in biopsy and cytologic material, using a panel of mucin staining, TTF-1, cytokeratin 5/6, and P63, and EGFR mutation analysis. J Thorac Oncol 5:436–441 9. Travis WD, Rekhtman N, Riley GJ et al (2010) Pathologic diagnosis of advanced lung cancer

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based on small biopsies and cytology: a paradigm shift. J Thorac Oncol 5:411–414 10. Turner BM, Cagle PT, Sainz IM et al (2012) Napsin A, a new marker for lung adenocarcinoma, is complementary and more sensitive and specific than thyroid transcription factor 1 in the differential diagnosis of primary pulmonary carcinoma: evaluation of 1674 cases by tissue microarray. Arch Pathol Lab Med 136:163–171

11. Gurdus D, Grigoras ML, Motoc AG et al (2019) Clinical relevance and accuracy of p63 and TTF-1 for better approach of small cell lung carcinoma versus poorly differentiated nonkeratinizing squamous cell carcinoma. Romanian J Morphol Embryol 60(1):139–143 12. National Cancer Institute. NCI Dictionary of Cancer Terms. https://www.cancer.gov/ publications/dictionaries/cancer-terms/def/ immunohistochemistry. Accessed 20 July 2019

Chapter 2 Immunohistochemical Detection of p40 Expression in Lung Cancer Clinical Samples Aruna Nambirajan and Deepali Jain Abstract Immunohistochemistry is the technique by which antigens in tissues are detected by means of antigen–antibody reaction. The p40 antibody is directed against the ΔN domain of the ΔNp63 isoform of p63 and is a highly specific marker for the squamous cell carcinoma subtype of non-small cell lung carcinomas (NSCLC). As such, immunohistochemical detection of this antigen in NSCLC biopsies is extremely valuable to assess tumor histological subtype. Herein we describe a manual procedure for performing p40 immunohistochemistry on formalin-fixed paraffin-embedded tissue sections by the indirect polymerbased two-step technique using hydrogen peroxide and 3–3’diaminobenzidine detection system. Key words p40, p40 antibody 5–17 clone, Lung squamous cell carcinoma, Immunohistochemistry, Formalin-fixed paraffin-embedded tissue sections, Horse radish peroxidase, 3–30 diaminobenzidine

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Introduction Immunohistochemistry (IHC) is an immunological technique for identifying antigens in tissues by their specific recognition and binding with specific antibodies. IHC enables the detection and cellular localization (i.e., nuclear, cytoplasmic, membranous, Golgi, etc.) of the antigen of interest in tissue sections. The signal generated by the technique can be observed under a bright field microscope. While the antigen is primarily bound by polyclonal or monoclonal antibodies, the latter being more specific, the visualization by light microscopy is facilitated by the use of enzyme labels conjugated with either the primary antibody itself (direct IHC method) or with secondary antibodies directed against the primary antibody (indirect IHC method). The conjugated enzymes react with a chromogenic substrate to produce colored reaction products that can be seen in tissues under the microscope. The most popular

Pedro G. Santiago-Cardona (ed.), Lung Cancer: Methods and Protocols, Methods in Molecular Biology, vol. 2279, https://doi.org/10.1007/978-1-0716-1278-1_2, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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enzyme-substrate currently used in IHC applications is the enzyme horseradish peroxidase (HRP), and its chromogen substrate, 3,3α-diaminobenzidine tetrahydrochloride (DAB), which produces a stable brown insoluble reaction product readily visualized by light microscopy [1]. The IHC method is suitable for the evaluation of lung cancer clinical samples such as small biopsies, resection specimens and cytology specimens, all of which can be used for IHC analysis with appropriate technical modifications (see Note 1). In general, tissue fixed in 10% neutral buffered formalin is preferred for IHC due to better preservation of both antigen epitopes and tissue morphology. The most popular technique for IHC currently established in routine diagnostic practice on both automated and manual platforms is the two-step indirect method utilizing polymer secondary antibodies, wherein a large number of immunoglobulins and HRP molecules are conjugated on a dextran polymer backbone [1]. The broad analytical steps of this method on formalin-fixed paraffin-embedded (FFPE) tissue sections include: (a) deparaffinization to remove paraffin and rehydration of sections, (b) antigen retrieval, either by heat induced or proteolytic methods or both, to break the crosslinking methylene bridges in tissue elements induced by formalin fixation, and to facilitate access of the primary antibody to the antigen of interest, (c) quenching of endogenous enzymes in tissue (e.g., peroxidase in neutrophils) that may act on chromogen substrates to produce nonspecific colored reaction products, (d) serum blocking to prevent non-specific binding of primary and secondary antibodies with plasma proteins and charged connective tissue components, (e) primary antibody incubation, (f) polymer secondary antibody incubation, (g) chromogen substrate application and reaction to generate the insoluble detectable product, (h) counterstaining with hematoxylin, (i) dehydration and clearing, and (j) mounting and cover slipping [1]. The p40 antibody is directed against the ΔN domain of the ΔNp63 isoform of p63 [2, 3]. This isoform is specifically expressed in the basal and progenitor cell layers of stratified epithelia, myoepithelial cells, thymic epithelial cells, trophoblasts and is consistently expressed in squamous cell carcinomas of various sites, including those of lung [2, 3]. The antigen identified by p40 antibody is localized in the cell nucleus and is visualized as brown homogenous nuclear staining in cells. In this chapter, we describe in detail the manual two-step indirect IHC method using polymer secondary antibodies and HRP-DAB enzyme-chromogen pair for detecting p40 on FFPE tissue sections [4].

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Materials

2.1 IHC Buffer Solutions and Histology Reagents

When applicable, prepare solutions using distilled water, and always use analytical grade reagents. 1. 0.05 M Tris buffered saline (TBS). In 8 L of distilled water, dissolve 85 g of NaCl and 60.5 g of Tris (hydroxymethyl) aminomethane. Adjust pH to 7.6 with concentrated hydrochloric acid, and complete volume to 10 L with distilled water. 2. 1 Phosphate-Buffered Saline (PBS), pH 7.2. To prepare, dissolve 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4.2 H2O, and 0.24 g of KH2PO4, in 800 mL of water. Adjust the pH to 7.2 with HCl and add distilled water to complete the volume to 1 L. Pre-made, ready-to-use PBS can also be purchased. 3. Heat-mediated antigen retrieval (AR) solution. This is a trisodium citrate buffer. Prepare by dissolving 19.7 g of trisodium citrate in 4 L of distilled water. Adjust pH to 6.0 with 1 M hydrochloric acid and complete volume to 5 L. 4. Hydrogen peroxide (H2O2) endogenous enzyme quenching solution. This solution eliminates tissue endogenous peroxidase activity, thus minimizing background signal. To prepare, mix 4 mL of 30% H2O2 with 96 mL of absolute methanol. You must prepare this solution fresh before each procedure. 5. Xylene. 6. Alcohol 50%, 70% and 95% solutions. Prepare by diluting absolute or 95% ethanol with distilled water. 7. Hematoxylin solution. 8. Coverslips. 9. Mounting medium (we use DPX mounting medium, but other alternatives are acceptable).

2.2 Antibodies and Detection Kits

1. p40 primary antibody. This protocol was optimized with the 5–17 clone (Calbiochem, Darmstadt, Germany) diluted 1:3000 in distilled water. 2. IHC detection reagents. There are many commercially available IHC HRP detection kits and reagents. This protocol was optimized with the UltraVision™ Quanto Detection System HRP (Thermo Fisher Scientific, United Kingdom). This kit includes the following reagents: UltraVision™ protein block, Primary antibody Amplifier Quanto, HRP Polymer Quanto, and the DAB Quanto chromogen and substrate. These are all the reagents needed to develop the IHC signal.

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2.3 Laboratory Equipment

1. Domestic microwave oven. 2. Moist incubation chamber with capacity to hold tissue slides. This can be commercially purchased, but it can also be prepared in the laboratory. We use a lidded staining trough with Perspex strips running along the length on which slides are placed separated from each other. We use a wet sponge or a layer of wet filter paper kept at the bottom of the chamber to maintain moisture. We use a wax pen to outline the tissue sections on the slide to ensure that reagents and solutions are retained on top of the tissue section and does not spill outside the tissue area. 3. Slides holders or racks. You need at least one rack that should be microwaveable to be used in the antigen retrieval step. 4. Slide staining jars. 5. Light microscope.

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Methods All individual steps, except the antigen retrieval procedure, are carried out at room temperature. For handling and washing of tissue sections, it is recommended that you place the tissue slides that are going to be stained in a slide holder and use the staining jars for all the washes (see Note 2). The indicated incubation steps are carried out in a moist incubation chamber to avoid drying of tissues. 1. Deparaffinise tissues by immersing them in xylene, 3 dips  5 min each (see Note 3). 2. Rehydrate tissues with sequential ethanol washes of decreasing ethanol concentrations as follows: do 95%, 70%, and 50% alcohol washes, 3 min each. 3. Wash slides with TBS 2–3 times. 4. Place slides in a microwave resistant staining rack, and place the rack in a microwave resistant plastic container filled with an adequate amount of Tris-sodium citrate antigen retrieval (AR) solution (~600 mL for 25 slides). The amount of AR solution should be sufficient to completely immerse the slides. Heat the container in the microwave oven for 20 min at 800 W with the lid sufficiently loose to ensure escape of steam. Check the AR solution volume midway through the incubation and add distilled water if the volume of AR solution has decreased due to evaporation. Ensure that the sections do not dry out (see Note 4). 5. Remove the container from the microwave oven and allow it to cool to room temperature.

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6. Transfer the slides from the cooled AR solution to the moist incubation chamber. 7. Wash with PBS 2–3 times. 8. Incubate slides in the H2O2 quenching solution for 10 min. 9. Wash with PBS 2–3 times. 10. Incubate slides with UltraVision™ Protein Block for 5 min (see Notes 5 and 6). 11. Remove excess blocking solution by gently blowing or by slightly inclining the slide and absorbing the liquid by placing an absorbent paper at the edge of the tissue section. Do not wash with PBS. 12. Apply the diluted primary antibody on top of the tissue sections and incubate for 1 h (see Notes 7–9). 13. Wash with PBS 2–3 times. 14. Apply Primary Antibody Amplifier Quanto and incubate for 10 min (see Notes 10 and 11). 15. Wash with PBS 2–3 times. 16. Apply HRP Polymer Quanto and incubate for 10 min. 17. Wash with PBS 2–3 times. 18. Apply 1 drop of DAB Quanto Chromogen (30 μL) to 1 mL of DAB Quanto Substrate, mix by swirling and apply to slide, incubate for 5 min with monitoring (see next step). 19. Monitor the progression of the reaction by checking how the colored precipitate forms under a light microscope. As soon as a strong and distinct nuclear signal can be appreciated, stop the reaction by incubating the slides in water. This incubation should not exceed 7 min, in order to avoid background signal. 20. Wash with distilled water. 21. Counterstain with hematoxylin for 1 min and coverslip with DPX mounting medium. Incubation time may be increased when using older stocks of hematoxylin. A representative preparation is shown in Fig. 1 (see Notes 12–14).

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Notes 1. Among the pre-analytical factors that influence the success and validity of IHC, adequate fixation of tissue in formalin is the most important. For best results, fresh tissue removed from patient should be immersed in ten times volume of neutral buffered 10% formalin as soon as possible (delay of more than 1 h significantly deteriorates antigens and invalidates IHC

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Fig. 1 p40 immunohistochemistry in formalin fixed paraffin embedded sections. (a) Microphotograph of hematoxylin and eosin stained section from an endobronchial biopsy showing features of a non-small cell lung carcinoma. (b) p40 immunohistochemistry shows diffuse and strong nuclear staining in tumor cells diagnostic of squamous cell carcinoma. Arrow shows nuclear staining in the basal cells of respiratory epithelium (internal positive control)

results), and the recommended duration of fixation is between 6 and 24 h for small biopsies and 24–72 h for resections [5]. The formalin fixed tissue is subject to subsequent dehydration, clearing, and infiltration during processing and is finally embedded in tissue molds as paraffin blocks. Four-micron thick tissue sections cut from these blocks are the standard samples for IHC in routine diagnostic pathology practice [5]. 2. Tissue sections may come off the slide due to the repeated washing steps involved in IHC. It is therefore recommended that the tissue sections are placed on charged or adhesivecoated glass slides. To ensure good IHC staining, tissue sections should be preferably cut fresh, fixed overnight in a 37  C incubator or placed in a hot oven (60  C) for 30 min [1]. 3. Deparaffination of tissue sections is essential to ensure adequate penetration of reagents into the sections. Incomplete deparaffination can lead to false negative staining. Using xylene pre-warmed to 37  C in an incubator for deparaffination improves completeness of wax removal [1]. 4. The pH of the antigen retrieval solution is important for successful IHC. Alteration in the pH of the designated buffer solution during antigen retrieval has been found to alter the epitope structure leading to altered staining patterns or complete absence of staining [1]. For p40 IHC using the 5–17 clone, heat mediated antigen retrieval at pH 6 (citrate) is optimal.

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5. Endogenous peroxidase blocking is performed by treating the section to hydrogen peroxide in methanol. This step can be performed at any stage prior to secondary antibody application, although some authors prefer to perform this step after primary antibody incubation as methanol may interfere with labile antigenic epitopes [1]. 6. Serum block is primarily the application of an innocuous protein solution that neutralizes charged sites on the sections and prevents the antibody from binding to non-antigenic sites such as collagen, reticulin, or fibrinogen [1]. Following application of serum block, the section should not be washed with PBS. This step is performed prior to application of primary antibody. 7. The most commonly used p40 antibody is the 5–17 clone (Calbiochem). This is a rabbit polyclonal IgG antibody raised against a synthetic peptide corresponding to amino acids 5–17 of human p40 [6]. 8. Among the different types of primary lung cancer, the p40 antibody labels tumor cells showing squamous differentiation and is diffusely positive in lung squamous cell carcinoma (>50% tumor cells), focally positive in the squamous component of adenosquamous carcinoma, NUT (nuclear antigen in testis) carcinoma, and thymic neoplasms. Focal weak staining may be seen in adenocarcinoma and is usually interpreted as negative [6]. 9. Revalidation of IHC procedure using adequate number of positive and negative controls is necessary whenever there is a change in the clone or company of the primary antibody, staining platforms or detection kits [6]. 10. The sensitivity of IHC depends upon the degree of signal amplification, which in turn depends upon the enzyme label: antigen ratio. Indirect IHC methods are more sensitive than direct methods due to an additional layer of secondary antibodies which are conjugated with enzyme labels resulting in higher enzyme label: antigen ratio. The use of polymer secondary antibodies (e.g., EnVisionTM, Dako), micropolymer secondary antibodies (e.g., Ultravision™ Quanto detection system, ThermoScientific™) as in the method described above, multimer secondary antibodies (e.g., ultraVIEW, Ventana Medical Systems), tyramide treatment after secondary antibody application (OptiView Amplifier, Ventana Medical Systems), etc. are all improvements to the IHC technique that increase the ratio of enzyme label: antigen resulting in increased sensitivity. Primary antibody concentration and duration of incubation varies with different antibody clones and detection systems and needs to be standardized accordingly [1].

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11. The specificity of IHC is inversely proportional to the degree of nonspecific and background staining seen in the tissue sections. Nonspecific signals in IHC occur mainly due to any of the following reasons: (a) primary antibody cross-reacting with other antigens, (b) primary or secondary antibodies nonspecifically binding to plasma proteins and charged connective tissue components, (c) presence of endogenous enzymes in tissue (e.g., peroxidase in neutrophils) that act on chromogen substrates to produce colored reaction products, and (d) drying of the tissue sections during the procedure, which leads to nonspecific antibody binding. The use of monoclonal primary antibodies with single epitope specificity, blocking with serum prior to primary antibody incubation to adsorb and neutralize all charged plasma and connective tissue elements, quenching of endogenous enzyme activity prior to secondary antibody application, thorough washing with PBS to remove unbound reagents after every step of IHC, and protecting sections from drying out minimizes nonspecific staining and improves specificity [1]. 12. The use of controls that have been processed in the same way as the test specimen under study is essential while performing IHC for quality assurance [5]. Positive staining in the controls indicates that the test is valid. Positive controls can be internal in the form of positive staining elements within the test sections (e.g., basal cells of respiratory epithelium normally stain strongly for p40 and serve as internal positive controls when present, see Fig. 1) or external when a known positive control is run as an extra section on the test slide itself or on a separate slide. While internal positive controls are better in terms of being subjected to similar pre-analytical processing conditions, they may not always be present in the test section and running an external positive control with a known pattern and intensity of staining is recommended for quality control. A negative control can be run by either omitting the application of the primary antibody or by using an antibody directed against an unrelated antigen [1]. 13. Use of automated staining platforms improves uniformity in IHC procedure and staining, this is particularly important to ensure reproducibility across different laboratories [1]. 14. Cytology specimens including alcohol-fixed smears may be used for IHC, however, these will need to be validated with adequate number of positive and negative controls prepared in the same manner as the test sample. For instance, p40 immunocytochemistry on alcohol fixed smears can be validated by using alcohol fixed smears prepared from squamous cell carcinoma aspirates as positive controls, and smears from lymphoma and adenocarcinoma aspirates as negative controls. The

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Fig. 2 p40 immunocytochemistry in alcohol-fixed cytology smears. (a, b) Papanicolaou stained smears from lung aspirate shows fragments from a carcinoma. (c) p40 immunocytochemistry shows diffuse and strong nuclear staining in tumor cells indicative of squamous differentiation

duration of antigen retrieval, primary antibody concentration and duration of incubation may need to be modified for immunocytochemistry [7]. In our laboratory, we are using the same protocol for alcohol-fixed smears as for IHC on formalin-fixed sections with excellent concordance (Fig. 2). References 1. Sanderson T, Wild G, Cull AM et al (2019) Immunohistochemical and immunofluorescent techniques. In: Suvarna SK, Layton C, Bancroft JD (eds) Bancroft’s theory and practice of histological techniques, 8th edn. Elsevier, Amsterdam 2. Bishop JA, Teruya-Feldstein J, Westra WH et al (2012) p40 (ΔNp63) is superior to p63 for the diagnosis of pulmonary squamous cell carcinoma. Mod Pathol 25:405–415 3. Pelosi G, Fabbri A, Bianchi F et al (2012) ΔNp63 (p40) and thyroid transcription factor1 immunoreactivity on small biopsies or cellblocks for typing non-small cell lung cancer: a novel two-hit, sparing-material approach. J Thorac Oncol 7:281–290 4. Walia R, Jain D, Madan K et al (2017) p40 & thyroid transcription factor-1

immunohistochemistry: a useful panel to characterize non-small cell lung carcinoma-not otherwise specified (NSCLC-NOS) category. Indian J Med Res 146:42–48 5. Thunnissen E, Allen TC, Adam J et al (2018) Immunohistochemistry of pulmonary biomarkers: a perspective from members of the pulmonary pathology society. Arch Pathol Lab Med 142:408–419 6. Hung YP, Sholl LM (2018) Diagnostic and predictive immunohistochemistry for non-small cell lung carcinomas. Adv Anat Pathol 25:374–386 7. Jain D, Nambirajan A, Borczuk A et al (2019) Immunocytochemistry for predictive biomarker testing in lung cancer cytology. Cancer Cytopathol 127:325–339

Chapter 3 Automated Immunohistochemistry Assay for the Detection of Napsin-A in Formalin-Fixed Paraffin-Embedded Tissues from Lung Tumors Rosa Ve´lez Cintro´n, Jo Ann Jusino, Marı´a Conte-Miller, Andre´s J. Martı´nez, and Adalberto Mendoza Abstract Immunohistochemistry (IHC) enables the selective detection of proteins in cells of formalin-fixed-paraffinembedded (FFPE) tissue sections. This technique plays a key role in the identification and classification of primary lung cancer tumors through the evaluation of the expression of the aspartic proteinase Napsin-A. However, immunohistochemistry is a complex process involving many critical steps and the lack of standardization as well as inappropriate analytical conditions may contribute to inconsistent results between laboratories. Automated immunohistochemistry addresses this issue by ensuring the quality and the reproducibility of the results among different laboratories. Here we describe an automated IHC protocol used in our laboratory for the detection of Napsin-A in FFPE lung tissue sections. Key words Immunohistochemistry, Lung adenocarcinoma, Napsin-A, Biomarkers, Clinical pathology, Lung cancer diagnosis

1

Introduction Immunohistochemistry (IHC) is a laboratory technique that uses antibodies to detect specific markers in formalin-fixed, paraffinembedded (FFPE) tissue sections [1]. There are several variations of the IHC technique. The most commonly used IHC method involves the utilization of a primary antibody that binds the epitope or antigen (antibody’s target region) in the protein of interest followed by the addition of a secondary antibody that binds to the primary antibody in a highly specific way. The secondary antibody is usually linked to an enzyme or molecule that is activated when the appropriate substrate is added. The interaction between the enzyme and the substrate promotes the formation of a precipitate that may be observed and scored through the microscope (Fig. 1).

Pedro G. Santiago-Cardona (ed.), Lung Cancer: Methods and Protocols, Methods in Molecular Biology, vol. 2279, https://doi.org/10.1007/978-1-0716-1278-1_3, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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Fig. 1 Steps in the process of immunohistochemistry. (a) A primary antibody binds to the epitope in the target protein. (b) A secondary antibody linked to an enzyme binds to the primary antibody in a highly specific way. (c, d) The enzyme substrate is converted by the enzyme into an insoluble product that precipitates in the tissue and can be observed through the microscope

Immunohistochemistry is currently well accepted by many pathology laboratories worldwide and it is also widely used in research to analyze molecules of interest in order to study their roles in both healthy and malignant cells [2, 3]. However, the reliability of the results in the clinical and research settings depends on the level of standardization and reproducibility among different laboratories [4, 5]. Automated IHC propitiates a sufficiently controlled environment allowing the reproducibility of the results [6]. IHC has contributed to tremendous advances in the clinical pathology and oncology fields by promoting the discovery of target biomarkers with diagnostic, therapeutic, or prognostic value [7]. This is the case of the aspartic proteinase Napsin-A which is expressed in the cytoplasm of the parenchyma cells in the lung. Detection of Napsin-A through IHC has been demonstrated to be an excellent tool to distinguish primary lung adenocarcinomas from other carcinomas during the pathological diagnosis [8]. Also, Napsin-A has demonstrated a sensitivity of 87.25% and a specificity of 95.02% for lung adenocarcinomas [9]. Here we describe an automated IHC protocol used in our laboratory for the detection of Napsin-A in FFPE tissue sections from lung.

2

Materials Always follow the manufacturer’s instructions regarding storing conditions, reagent stability, safety procedures, and waste disposal.

2.1 Standard Histology Reagents

As much as possible, and when applicable, we recommend that you buy solutions that are ready-to-use, in commercially pre-made or pre-mixed form (see Note 1).

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1. Ethanol 100%, and 70%, 85%, and 90% ethanol dilutions. Dilutions can be prepared from 100% ethanol diluted with water to the desired concentration. 2. Paraffin, pre-warmed at 60  C. 3. Xylene. 4. Clarify Clearing Agent Xylene Substitute (this procedure was optimized using the American Master Tech; Cat. # CACLELT). 5. Hematoxylin stain (this procedure was optimized using Hematoxylin Dako Omnis, Dako; Cat. #GC808). 6. Eosin stain. 7. Dako Omnis IHC 20 wash buffer. 8. Neutral-buffered 10% formalin solution. (available ready to use). 9. Deionized and distilled water. 2.2 Immunohistochemistry Kits, Antibodies and Reagents

1. Napsin-A Prediluted, ready-to-use, Polyclonal Antibody (we use Biocare Medical; Cat. # PP 434 AA). 2. Immunohistochemistry kit. There is a variety of products for developing the IHC, in our laboratory we optimized this procedure using the EnVision FLEX, High pH Kit (Dako, Cat. # GV800). The EnVision FLEX Kit includes the peroxidaseblocking reagent, the horse-radish peroxidase (HRP, this is an HRP-conjugated a goat secondary antibody against rabbit or mouse primary antibodies), the DAB + Chromogen, and the substrate buffer. In order to prepare the substrate buffer working solution, mix the DAB and the substrate buffer following the kit’s instruction (EnVision TM FLEX Package Insert from Agilent Technologies). 3. EnVision FLEX Target Retrieval Low pH 50 X Solution (Dako; Cat. # GV805).

2.3 Laboratory Equipment and Consumables

1. Water bath or flotation bath. Have a thermometer at hand to monitor the temperature. 2. Microtome. 3. Microscope slides. 4. Cover slipping film. We use Tissue-Tek (Sakura Cat. # 4770), together with the Sakura Tissue-Tek Automatic Cover slipper. Cover slipping can also be done manually if an automatic cover slipper is not available. 5. Racks for histological slides. 6. Absorbent paper. 7. Cold plate or ice sheet.

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8. Laboratory oven, capable of reaching 60  C. 9. Dako Omnis Automated IHC Staining System (this procedure is optimized for this system). Other staining systems can be used, such as the Dako Auto Stainer Link 48, but some procedure parameters will have to be optimized. 10. Standard bright light microscope. 11. Standard tissue embedding cassettes with lids. 12. Embedding chamber, console or station. There are several models available, all of them acceptable. Follow any instructions by the manufacturer. 13. Forceps or tweezers, for careful handling of tissues. 14. KP Marker plus pen, or equivalent.

3

Methods

3.1 Reagent Preparation

1. Following the manufacturer’s instructions, dilute the EnVision FLEX Target Retrieval Low pH 50 X Solution to 1:50 in distilled or deionized water. The resulting solution is 1 and its pH must be 6.0. 2. Following the manufacturer recommendations, prepare a 1 wash solution by diluting the 20 Dako Omnis Wash Buffer to prepare a 1solution. Prepare this by diluting 1 mL of the 20 wash buffer in 20 mL of distilled or deionized water.

3.2 Tissue Fixation and Processing

1. Fix lung tissue biopsies in 10% Neutral-Buffered Formalin solution for 6–72 h (see Note 2). After fixation of the tissue, it is ready for paraffin embedding and creation of tissue blocks, as described in the following steps. Tissue processing to create the paraffin block can be done manually or in a tissue processor. 2. Dehydrate tissues in a series of ethanol washes as follows, each wash lasting 45 min: 70%, 85%, 90%, and 100% ethanol. The final 100% ethanol wash must be performed three times. 3. Clear the tissue by incubating it in xylene for 3 min. 4. Infiltrate tissues by immersion in molten paraffin, this will create the paraffin blocks that will be cut (see next section). Immerse the tissues in paraffin at 60  C for 45 min. Do a second immersion for 75 min. 5. Embed the tissues to form the paraffin blocks. For this, place tissues in standard histology embedding cassettes and pour molten paraffin into the embedding station. The paraffin should be pre-warmed to 60  C for several hours. Submerge the cassette containing the tissue into the molten paraffin in the embedding station. Use pre-warmed forceps to handle the tissue carefully inside the cassette. Place the tissue with the

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side that was originally cut (during biopsy specimen collection) facing downwards in the cassette, in such a manner that this side of the tissue is the one closest to the surface of the paraffin from which the first tissue sections will be produced. Ensure that the paraffin, the embedding cassettes, the embedding station, and all tools to handle the tissue have all been pre-warmed at 60  C. We recommend starting with the pre-warming of all the materials early in the morning so that when the embedding is done the paraffin and all materials have reached the appropriate temperature. Maintain the tissues in the cassettes immersed in paraffin for 5 min. 6. Place the embedded tissue in the cold plate to solidify the paraffin. 3.3 Tissue Sectioning

1. Set up the water bath to 45  C, using an external thermometer to monitor the water temperature. 2. Place the tissue block over a cold plate or ice sheet. 3. Using the microtome, cut tissue sections at 4 μm thick (see Note 3 and Fig. 2). 4. As you cut with the microtome, carefully handle the resulting paraffin ribbons containing the tissues with tweezers and float them in the water bath immediately after cutting them. Always verify that the water in the water bath has reached 45  C prior to transferring the tissue sections to it (see Notes 4 and 5 and Fig. 2). 5. Allow the sections to stretch. If needed, use a stick to stretch the tissue. Select one tissue section from the ribbon and place it over a microscope slide (see Note 6 and Fig. 2).

Fig. 2 Cutting tissue sections in the microtome. (a) Tissue sections are cut in the microtome to a thickness of 4 μm. (b) The cut ribbons containing the paraffin tissue sections are transferred to a water bath to allow the sections to stretch. (c) Each tissue section is mounted in a slide

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6. Drain the excess water from the slide by pressing the edges of the slide against an absorbent paper. 7. Place the slide in a rack for histological slides and bake it in the oven for 1 h at 60  C (see Note 7). 3.4 Immunohistochemistry Staining in the Dako Omnis IHC Staining System

All the steps in this section will be performed by placing the slides in the Dako Omnis IHC Staining System using the parameters described in the appropriate step below. 1. Deparaffinize the slides by allowing the slide to interact with the Clarify Clearing Agent Xylene Substitute at 25  C for 10 s (incubation top) and 1 min (incubation bottom) (see Note 8). Perform this step twice. 2. Wash with deionized water for 5 s at room temperature. Repeat this step twice. 3. For the antigen retrieval step, incubate the slides for 30 min at 97  C with the EnVision FLEX Target Retrieval Low pH 1 Solution (see Note 9). 4. Wash the slides in Wash Buffer Omnis 1 during exactly 2.40 min at room temperature (see Note 10). 5. Incubate the slides with the primary anti-Napsin-A Prediluted Polyclonal Antibody for 25 min at room temperature (see Note 11). This incubation should be 30 min if using the Dako Auto Stainer Link 48 and should be optimized if any other staining system is used. We recommend that you prepare negative control tissue sections by having some sections incubated with an irrelevant mouse monoclonal antibody (not expected to recognize Napsin-A). Be sure to include 0.015 mol/L sodium azide in the negative control solution. 6. Wash the slides in Wash Buffer Omnis 1 for 2 min at room temperature. 7. Incubate the slides for 3 min at room temperature with the EnVision FLEX Peroxidase-Blocking Reagent (see Note 12). 8. Wash the slides in Wash Buffer Omnis 1 for 2 min at room temperature. 9. Incubate the slides for 20 min at room temperature with the EnVision FLEX-HRP, or for 25 min if using the Dako Auto Stainer Link 48. 10. Wash the slides in Wash Buffer Omnis 1 for 2 min at room temperature, repeat wash for a second time. 11. Wash the slides in deionized water for exactly 31 s at room temperature. 12. Wash the slides in Wash Buffer Omnis 1 for 2 min at room temperature.

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13. Incubate the slides for 5 min with the EnVision FLEX Substrate Working Solution (containing the DAB chromogen) at room temperature. Repeat this step one more time if using the Dako Auto Stainer Link 48 system. 14. Wash the slides in Wash Buffer Omnis 1 for 2 min at room temperature. 15. Wash the slides in deionized water for 31 s at room temperature. 16. Wash the slides in Wash Buffer Omnis 1 for 2 min at room temperature. 17. Counterstain the slides by incubating them for 6 min with Hematoxylin Dako Omnis at room temperature. 18. Wash the slides in Wash Buffer Omnis 1 for 2 min at room temperature. 19. Wash the slides in deionized water for exactly 31 s at room temperature. 3.5

Cover Slipping

1. Remove the slides from the Dako Omnis System and place them in a rack for histological slides and allow them to air dry. 2. Place the rack with the slides in the SakuraTissue-Tek system for automatic cover slipping. Perform this step manually if an automatic cover slipper is not available.

3.6 Staining Evaluation and Interpretation

The Napsin-A positive signal is characterized by a brown granular cytoplasmic staining (Fig. 3). 1. Using a tissue section from the same paraffin block that has been stained with hematoxylin and eosin (H&E) using a routine staining protocol, identify the tumor area or the region of interest (i.e., apparent lesion) within the tissue. Mark the region with the KP Marker Plus pen or equivalent. 2. Superpose the IHC-stained and the H&E-stained slides to identify the region of interest in the IHC-stained slide, which should be the tumor area. 3. Screen the entire slide under the microscope at 10to find the brown signal (as shown in Fig. 3). If the brown signal is present, this is indicative of a Napsin-A positive expression. 4. Increase the magnification to 40 or 100 to confirm that the expected staining pattern for Napsin-A (granular cytoplasmic staining) is present and to exclude the possibility of background or antibody nonspecific staining. The presence of nonspecific staining is suggested if there is a brown signal in the negative control slides that were processed without the addition of the primary antibody.

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Fig. 3 IHC staining for Napsin-A. (a) Negative control in which the primary antibody was omitted from the staining procedure. (b) Positive staining for Napsin-A is characterized by a brown cytoplasmic staining signal

5. A positive expression of Napsin-A in lung cancer tissue may confirm the presence of a primary adenocarcinoma originated in the lung. Additional IHC stains with other biomarkers may be conducted to support the diagnosis. For quality control, in addition to the negative control described above, Table 1 shows several control treatments that are recommended to ensure the reliability of the results.

4

Notes 1. Although ready-to-use reagents are usually more expensive, they offer the benefit of yielding reproducible results even when the assays are performed by different laboratory technologists. Minimizing the reagents that need to be prepared will also minimize the potential for errors and the variability of results among laboratories and researchers. Using reagents classified as For In Vitro Diagnostic for research purposes facilitates the transfer of the results obtained during research studies to a clinical setting, which is the goal of biomedical or clinical investigations. 2. Fixation conditions are critical in IHC. The use of 10% Neutral Formalin prevents autolysis and preserves the tissue morphology. The proportion of tissue to fixative should be between 1:1 and 1:20. Use of other types of fixatives may interfere with the IHC staining and is usually contraindicated in IHC. 3. Thick tissue sections may yield high background, making it difficult to analyze and interpret the Napsin-A IHC staining. It is also recommended to use freshly cut sections. Some antigens can be lost after prolonged storage conditions (i.e., more than 2 months).

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Table 1 Positive and negative controls that can be used to ensure staining specificity Appropriate controls in Napsin-A immunohistochemistry Control type Description

Information provided

Positive control

This control will provide information about A section of lung cancer adenocarcinoma the appropriate performance of the assay which is known to be positive for Napsinand the stability of the reagents. Any A should be processed in exactly the same failure in the assay can be identified if an way as the unknown sample (sample under unexpected result is obtained in the evaluation) positive control

Negative reagent control (NRC)

This control will provide information about A section of the sample under evaluation nonspecific staining or background should be processed in exactly the same signal. Comparing the signal obtained in way as described in the method’s section the section under evaluation against the but without adding the primary antibody signal obtained in the NRC will allow to discriminate between the nonspecific and the specific (true) signal for Napsin-A

Positive internal control

This control will specifically help to A section of the sample under evaluation distinguish a true negative from a false should be processed in exactly the same negative result caused by inappropriate way (as described in the method’s section) pre-analytical conditions. The positive but the primary antibody must be directed expression of the control protein will against a protein known to be expressed in confirm a true negative result in a tissue the lung tissue with lack of Napsin-A expression

4. Floating the paraffin sections or ribbons in a water bath will allows tissue to stretch by removing wrinkles and folds before placing sections on slides. It is very important to keep the temperature of the water in the floatation bath within 5–10  C below the melting point of the paraffin being used. Forty to fifty degrees Celsius (40–50  C) is usually an optimal temperature for the majority of paraffin types. Always verify the paraffin melting point to set up the appropriate temperature for your flotation bath. Very low temperatures will not remove the folds in the tissue section. Very high temperatures (close to or above the paraffin melting temperature) will melt the paraffin in the section causing alterations in the morphology of the tissue. 5. It is important to use distilled water in the flotation bath. Tap water or low-quality water usually contains impurities that may affect the IHC reaction. It may also interfere in the adhesion of the tissue section to the slide causing the tissue to detach from the slide. The water reservoir must be emptied and cleaned daily using a laboratory-grade wipe. Avoid the presence of particulate in the flotation bath reservoir by covering it when not in use.

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6. The quality of the slides is very important to allow the adherence of the tissue to the slide. We use the FLEX IHC Microscope slides which are coated with an additive that helps in this process. Other types of slides may be used but their ability to keep the tissue attached must be evaluated by the laboratory. 7. Baking the slide is also very important to allow tissue adhesion to the slide. The temperature of the oven must be equal or slightly above the melting point of the paraffin. However, very high temperatures or prolonged incubation time may destroy the Napsin-A target antigen. This may yield false negative results. 8. Incomplete removal of paraffin may result in poor staining or lack of Napsin-A expression when it is expected to be expressed. 9. Antigen retrieval is necessary to unmask the epitope in the Napsin-A antigen. The fixation of the tissue may mask the target molecules. If an antigen retrieval step is not included, the interaction between the antibody and the Napsin-A antigen may fail and a false negative result may be obtained. It is important to optimize the antigen retrieval conditions (incubation time, temperature and pH according to the fixation conditions) [10]. 10. Wash steps are critical to avoid nonspecific binding and background staining. 11. A slight modification to the manufacturer’s recommended protocol was performed. The manufacturer recommends an incubation time of 30 min. Our protocol demonstrated a better performance using an incubation of 25 min with the Napsin-A primary antibody. 12. This step will block the endogenous peroxidase activity to eliminate the background staining caused by it. References 1. National Cancer Institute Dictionary of Cancer Terms. https://www.cancer.gov/ publications/dictionaries/cancer-terms/def/ immunohistochemistry. Accessed 20 July 2019 2. Taylor CR (1986) Principles of immunomicroscopy. In: Taylor CR, Cote RJ (eds) Immunomicroscopy: a diagnostic tool for the surgical pathologist, 3rd edn. Saunders Elsevier, Philadelphia 3. Duraiyan J, Govindarajan R, Kaliyappan K et al (2012) Applications of immunohistochemistry. J Pharm Bioall Sci 4(Suppl S2):307–309 4. O’ Hurley G, Sjo¨stedt E, Rahman A et al (2014) Garbage in, garbage out: a critical

evaluation of strategies used for validation of immunohistochemical biomarkers. Mol Oncol 8(4):783–798 5. Gambella A, Porro L, Pigozzi S et al (2017) Section detachment in immunohistochemistry: causes, trouble-shooting and problem-solving. Histochem Cell Biol 148(1):95–101 6. Sukswai N, Khoury JD (2019) Immunohistochemistry innovations for diagnosis and tissuebased biomarker detection. Curr Hematol Malig Rep 14(5):368–375 7. Leong AS, Wright J (1987) The contribution of immunohistochemical staining in tumor diagnosis. Histopathology 11:1295–1305

Automated Napsin-A Immunohistochemistry 8. Bradley M, Turner PT, Cagle IM et al (2012) Napsin-A, a new marker for lung adenocarcinoma, is complementary and more sensitive and specific than thyroid transcription factor 1 in the differential diagnosis of primary pulmonary carcinoma: evaluation of 1674 cases by tissue microarray. Arch Pathol Lab Med 136 (2):163–171 9. Jin L, Liu Y, Wang X, Qi X (2018) Immunohistochemical analysis and comparison of

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Napsin-A, TTF1, SPA and CK7 expression in primary lung adenocarcinoma. Biotech Histochem 93(5):364–372 10. Shi SR, Key ME, Kalra KL (1991) Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 39(6):741–748

Chapter 4 Detection of Programmed Cell Death Ligand 1 Expression in Lung Cancer Clinical Samples by an Automated Immunohistochemistry System Edwin Roger Parra and Sharia Herna´ndez Ruiz Abstract Programmed cell death 1 (PD-1) plays an important role in subsiding immune responses, in promoting self-tolerance through suppressing the activity of T-cells, and in promoting differentiation of regulatory T-cells. One of its ligands, programmed cell death ligand 1 (PD-L1) acts as a checkpoint regulator in immune cells and is also expressed in a wide range of cancer types. Anti-PD therapy modulates immune responses at the tumor site, targets tumor-induced immune defects, and repairs ongoing immune responses. Since drugs that target the PD-1/PD-L1 pathways became available as a cancer treatment, there is need for the use of different antibodies to detect the presence of these proteins in tumoral samples by immunohistochemistry or other assays. Because the detection of these antigens in tumor samples is highly clinically informative for guiding treatment decisions, especially to establish the aptness of a patient to receive anti-PD therapy, it is necessary to have a validation process that guaranties that the test results obtained when using antibodies against these proteins are specific, selective, reproducible, and conducive to quantification of antigen abundance in cancer tissue sections. Here we describe an automated immunohistochemistry staining procedure that can be applied for the validation of multiple anti-PD-L1 antibody clones when used for the staining of formalin-fixed, paraffin-embedded lung cancer tissue sections. Key words PD-L1, Immunohistochemistry

1

Antibody

optimization,

Cell

lines,

Immune

cell

expression,

Introduction The PD-1 receptor is a transmembrane protein expressed in a variety of activated immune cells such as helper T-cells CD4+, cytotoxic T-cells CD8+, B-cells, natural killer T-cells, and double negative T-cells CD4–CD8, present in the thymus, activated monocytes, dendritic cells (DCs), macrophages, and immature Langerhans cells [1, 2]. This receptor has two ligands called programmed cell death
1 and
2 (PD-L1 and PD-L2), and when the T-cell PD-1 receptor binds to its ligands on antigen presenting cells (APCs), an immune inhibitory pathway is activated leading to

Pedro G. Santiago-Cardona (ed.), Lung Cancer: Methods and Protocols, Methods in Molecular Biology, vol. 2279, https://doi.org/10.1007/978-1-0716-1278-1_4, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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T-cells suppression [3]. Both PD-1 and PD-L1 expression can be detected in a broad number of cells such as hematopoietic cells including T-cells, B-cells, macrophages, DCs, and mast cells, and non-hematopoietic healthy tissue cells such as vascular endothelial cells, keratinocytes, pancreatic islet cells, astrocytes, placenta syncytiotrophoblast cells, and corneal epithelial and endothelial cells [4]. Multiple solid tumor types including melanoma, renal cell carcinoma, sarcomas, non-small cell lung carcinomas, thymomas, ovarian, and colorectal cancers co-opt this immune shield by expressing PD-L1 to generate an immunosuppressive tumor microenvironment and avoid T-cell cytolysis [5–8]. Historically, immunohistochemistry (IHC) has been used to determine the presence or absence of a given protein antigen in a tissue [9]. In clinical pathology, assessing the presence of an antigen by IHC serves as a diagnostic, prognostic, and predictive tool, and IHC signal intensity quantification directly influences the management of patients in the clinical setting. For example, the assessment of estrogen receptor-α (ER- α), and human epidermal growth factor receptor 2 (HER2) by IHC in breast cancer tumors is the definitive test to determine whether or not a patient will receive targeted therapies that can cost as much as $100,000 per year [10, 11]. IHC assays using different primary antibodies, and antibody-specific scoring pathology approaches (1%, 5%, 10%, 50%, H-score or combined malignant cells and lymphocytes expression) have been reported to assess the prevalence of PD-L1 positivity expression thought different tumor types as non-small cell lung cancer (NSCLC), melanomas, sarcomas, renal cell carcinomas, bladder carcinomas, pancreatic, colorectal, and thymic tumors [11–14]. Evaluation of PD-L1 immunohistochemical staining may be difficult. Macrophages often exhibit membranous staining and may be misinterpreted as cancer cells in tissue samples if they infiltrate cancer cells. Also, unspecific cytoplasmic staining may occur, and weak partial membranous staining of tumors cells count as positive but may be difficult to interpret as positive expression. The mentioned difficulties are especially troublesome when a very limited proportion of tumor cells such as 1% is sufficient for a positive test, as is the case when using some PD-L1 clones such as the 28-8 clone, a situation that may lead to significant interobserver variation [15]. Also, several factors such as the quantity of tissue or cellular material available from small biopsies or cytology samples, how stable are the epitopes detected by the various antibodies, use of stored, pre-cut sections, and pre-analytical issues such as tissue fixation and processing, can have a major impact on the outcomes of immunohistochemical reactions, thus contributing to difficulties in the assessment of PD-L1 expression [16]. PD-L1 induction in tumors results from either oncogenic signaling, leading to constitutive widespread expression, or in response to IFN-γ release by

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effector T-cells during their immune response to the tumor, leading to variable PD-L1 expression observed across different cells and tissues [17]. As PD-L1 IHC protocols have been independently developed for specific anti–PD-1/PD-L1 therapies using different PD-L1 diagnostic assays (primary antibody clone plus immunostaining platform/protocol), each clone potentially can demonstrate distinct staining properties, which could prohibit the interchangeability of their clinical use. This would pose a significant challenge for pathology laboratories to offer PD-L1 testing, both from the laboratory resources and the budgetary points of view [18]. Recent publications have revealed both significant concordance and discordance between different PD-L1 diagnostic clones [15]. Seeking to harmonize the use of different anti-PD-L1 antibody clones, the blueprint study and a German ring trial showed varying degrees of tumor proportion score (TPS) concordance between the PD-L1 IHC clone 22C3 pharmDx kit and three other companion or complementary kits [18, 19]. To ensure that treatment decisions based on PD-L1 expression are consistent and objective, standards for PD-L1 testing need to be established [20] and there is a need to harmonize the staining procedures for analysis proposes. The automated IHC procedure described in this chapter can be used to optimize, validate, and compare different available commercial anti-PD-L1 antibody clones and to identify which ones can be reliably used by surgical pathologists to evaluate the PD-L1 expression on formalin-fixed, paraffin-embedded (FFPE) tumor samples, using an IHC workflow.

2

Materials We have optimized the procedure described below using Leica Biosystems Bond™ Tissue Staining Platform, which is a fully automated IHC staining system that improves efficiency, quality and speed of the IHC staining procedure. This is of great importance to ensure standardization of the procedure and reproducibility of the results. We are providing the catalog numbers for specific products that have been tested to perform optimally with Leica Bond™ automated platforms. Unless otherwise specified, all products below are from Leica Biosystems, and are designed to be compatible with the Leica Bond™ automated platforms. We also provide the catalog numbers of the specific anti-PD-L1 antibody clones that have been optimized with this automated platform, these are from different companies and sources as indicated after the antibody.

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2.1 IHC Reagents and Antibody Clones

1. Bond™ Dewax Solution, supplied as ready-to-use (Cat. No. AR9222). 2. Bond™ Epitope Retrieval 1 (supplied as ready-to-use, citratebased pH 6.0) (Cat. No. AR9961). 3. Bond™ Epitope Retrieval 2 (EDTA-based pH 9.0) (Cat. No. AR9640). 4. Bond™ Wash Solution, supplied as 10 concentrate, dilute to 1 with distilled water before use (Cat. No. AR9590). 5. Bond™ Polymer Refine Detection kit, which includes the peroxide block, post primary (polymer-HRP anti-mouse), polymer reagent, DAB Refine chromogen and hematoxylin counterstain (Cat. No. DS9800). 6. Anti PD-L1 antibody clones (see Note 1): EPR1161-2(dilution 1:100; Epitomics-Abcam Burlingame, CA, cat#ab174838); E1L3N (dilution 1:100; Cell Signaling Technology, cat#13684); clone E1J2J (dilution 1:100; Cell Signaling Technology, cat#15165); 7G11 (dilution 1:40; Gordon Freeman Laboratory, Boston University, Boston, MA, 1 aliquot kindly donated by the Gordon Freeman Laboratory); SP142 (dilution 1:100; Spring Bioscience, cat#M4424); rabbit polyclonal ab58810 (dilution 1:200; Abcam, cat#ab58810); 28-8 (dilution 1:400; Abcam, cat#ab205921). 7. PowerVision Poly-HRP Anti-Rabbit IgG (Cat. No. PV6121) (see Note 2).

2.2

IHC Equipment

1. Leica Biosystems BOND-MAX™ fully automated, advanced staining system. 2. BOND™ Universal Covertiles S21.4583 or S21.4611).

(Cat.

Nos.

S21.2001,

3. BOND™ Mixing Stations (Cat. No. S21.1971). 4. Leica Biosystems Aperio ScanScope AT2 slide scanner. 5. Aperio ImageScope Pathology Slide Viewing Software, downloadable from the Leica Biosystem webpage. 6. Aperio Image Toolbox analysis software, Leica Biosystems webpage. 7. Leica BOND™ Plus Slides (Cat. No. S21.2113). 8. BOND™ Slide Label and Print Ribbon (Cat. No. S21.4564). 2.3 Standard Reagents and Additional Equipment

The following reagents are standard solutions used in immunohistochemistry and do not need to be purchased from a specific vendor. 1. Ethanol, absolute and 75% and 95%. 2. Xylene (or xylene substitutes).

PD-L1 Immunohistochemistry in Lung Cancer

39

3. Hydrogen peroxide, 3%solution. 4. Distilled or deionized water. 5. Basic histology glassware and staining jars. 6. Drying oven capable of maintaining 60  C stable temperature. 7. Cover glasses, 24  40 mm. 8. Mounting medium, we use Thermo Fisher Cytoseal, but equivalent products are acceptable.

3

Methods

3.1 Automated PD-L1 Immunohistochemistry Staining

For IHC staining, we cut 4-μm-thick sections and stain them with the Leica BOND-MAX™ system. This is the procedure described in this section, which we perform exactly as described in the system’s instruction manual [21]. 1. On the BOND-MAX™ instrument, ensure the bulk and hazardous waste containers have enough capacity to perform the required staining runs. 2. Ensure there is adequate alcohol, distilled or deionized water, BOND™ Dewax Solution, BOND™ Epitope Retrieval Solution 1 and BOND™ Wash Solution in the bulk reagent containers to perform the required staining runs. 3. Install a clean BOND™ Mixing Station. 4. Turn on the BOND-MAX™ fully automated, advanced staining system. 5. Turn on the BOND™ Controller attached to the BONDMAX™ system, open the BOND™ software. 6. Program the staining system using the parameters shown in Table 1. Ensure to add all the solutions indicated in the table in the proper reservoir in the staining system. Note from Table 1 that while steps 1 to 20 are performed in automated form in the staining system, steps 22 to 24 are manually performed. Use alcohol 100% for the alcohol rinse step 3. For the dehydration step (Step 24), use ethanol washes as follows: 2 washes with 75% ethanol, 2 min each; 2 washes with 95% ethanol, 2 min each; 2 washes with 100% absolute ethanol, 2 min each. 7. After performing all steps in Table 1, perform three changes in xylene or xylene substitute, 1 min each.

3.2 Image Acquisition, Digital Analysis and Staining Analysis

1. Using the Leica Biosystems Aperio ScanScope AT2 slide scanner, digitally scan the stained slides from positive and negative controls (see Note 3) and tumor microarray (TMA) cases (see Note 4). Perform the digital scanning capturing the image

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Edwin Roger Parra and Sharia Herna´ndez Ruiz

Table 1 Parameters used to program the automated staining system Step

Reagent

Time

Temp ( C)

1

Bake (in oven, no reagent)

30 min

60

2

Bond Dewax solution

3

72

3

Alcohol rinse

3

4

Bond wash

3 (5 min each)

5

Epitope retrieval #1 (citrate buffer ph 6)

20 min

Epitope retrieval #2 (Tris-EDTA buffer)

100 100

6

Bond wash

4 (3 min each)

7

Peroxide block (3.0% hydrogen peroxide)

5 min

8

Bond wash

5

35

Pretreatment (protein block, enzyme treatment) 9

PD-L1 antibody

15 min

10

Bond wash

5

11

Post primary (polymer-HRP anti-mouse)

8 min

12

Bond wash

5 (2 min each)

13

Polymer (poly-HRP anti-rabbit IgG)

8 min

14

Bond wash

5

15

Deionized water

1

16

DAB refine

10 min

17

Deionized water

3

18

Hematoxylin

8 min

19

Deionized water

1

20

Bond wash

2

22

Print label and place on slides

(Manual)

23

Remove covertiles and rinse with deionized water

(Manual) 10 dips  3

24

Dehydrate slides and coverslip with cytoseal

(Manual)

RT

X: indicates the number of times the step is done by the machine. RT: Room temperature

with a 20 objective. After scanning, the images are visualized using ImageScope software, and analyzed using the Aperio Image Toolbox analysis software. 2. Separately analyze membranous PD-L1 expression in the tumor compartment and in tumor-associated inflammatory cells (TAICs) in the stroma compartment, using the same cell membrane algorithm for each antibody clone (see Notes 5 and 6).

PD-L1 Immunohistochemistry in Lung Cancer

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3. Score the staining intensity as 0 (no staining), 1+ (weak staining), 2+ (moderate staining), or 3+ (strong staining). Determine extension (percentage) of expression in both the tumor and stroma compartments (see Note 7).

4

Notes 1. During the antibody optimization, the optimal antibody concentration, which gives the best staining with minimum background, must be determined experimentally for each clone, and it is usually determined by using a series of dilutions in a titration experiment. For example, if the antibody product datasheet suggests using a 1:200 dilution, it is recommended to make dilutions of lower than recommended, 1:50, 1:100, and higher than the recommend 1:400 and 1:500 [22]. Figure 1 shows IHC staining using different dilutions of the antiPD-L1 clone 22C3. 2. The conventional method of developing humanized monoclonal antibodies (mAbs) uses proprietary antibodies sourced from mice or rabbits. Table 2 shows a comparison between poly-, monoclonal, and recombinant antibodies. The characteristics of the antibody are important, since, depending on their origin, their performance could be different and this must be considered at the time of optimization [23].

Fig. 1 Microphotographs of representative IHC staining with different dilutions of PD-L1 clone 22C3 (DAKO, Cat. No. AS480) in commercial HDLM-2 control cell lines (a, b) and in placenta (c, d). Dilutions are 1:50 (a, c) and 1:100 (b, d). Magnification is 200

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Table 2 Comparison of characteristics between polyclonal vs monoclonal and recombinant antibodies Properties

Monoclonal antibody

Polyclonal antibody

Epitope selectivity

Generated by a single B-cell Mixture of antibodies that all Antibodies crated to line and thus recognize recognize different recognize a specific only a single epitope of a epitopes of the protein of epitope of a protein of protein of interest. interest. Less sensible to interest Changes in epitopes affect changes in the epitopes the function of the Ab

Source

Mice or rabbit

Variety of species including mice, rabbit, goat, sheep, and donkey

Recombinant antibody

Entirely animal-free production process

High reproducibility and Reproducibility More reproducible Prone to batch to batch guaranteed continuity generated immortal B-cell variability (produced from of availability without hybridomas which are animal sera). Quantity of any dependence on constant and renewable Abs obtained is limited by animal immunization resources the size of the animal and its lifespan Cross-reactivity Less likely to cross-react with May contain nonspecific antibodies and other proteins, yields background staining lower background

No background staining

Specificity/ sensitivity

Highly specific and Highly specific due to single More sensitive due to sensitivity targeting multiple target epitope but less epitopes of an antigen but sensitive because often less specific than unable to detect masked monoclonal antibodies antigen

Challenges

More challenging to work with when looking at low-abundance proteins or proteins that show variability. More expense and time (up to a year). Highly susceptible

Last resort due to their Generated much more higher cost rapidly, at less expense, and with less technical skill (months) but poor choice for long-running studies. More stable (pH and salt concentration)

3. The use of cell lines can also prove beneficial for validating antibodies by IHC. Cells can be allowed to grow to confluence in a cell plate and then detached, centrifuged to form a cell pellet, and the cell pellet can be processed by fixation, embedding, and sectioning just like a piece of tissue. Validation of antibodies and protocol optimization using cell pellets is beneficial in particular since it saves valuable tissues. Antibody validation using cells is particularly advantageous when cells can be manipulated by transfection to introduce different ‘dose’ levels of the target protein in otherwise weakly positive or negative cell lines as controls [24]. Transfection efficiency rarely reaches 100%, a proportion of the cells should remain negative or weakly stained for the target in question which can be useful

PD-L1 Immunohistochemistry in Lung Cancer

43

Fig. 2 Microphotographs of representative examples of PD-L1 IHC marker in tonsil tissue. (a) PD-L1 stained tonsil at low magnification. (b) Superficial epithelium used as an internal negative control. (c) Positive reticulated epithelium. (d) Positive macrophages of a germinal center

in differentiating IHC signal from background noise. However, as protein expression levels are often modulated in disease, it may be important to include a range of pathologies and, preferably, matched normal tissue [25]. In our experience we have found sample tissues from placenta and tonsil to be useful controls [26]. As can be seen in Fig. 2, IHC staining of commercial control samples and cell lines is becoming increasingly available, and these can be used as alternatives to control tissue samples. In our experience, as an example, we have used the HEK293 cell line as negative control, and HEK293transfected with PD-L1 human gene as positive controls (the presence of absence of PD-L1 expression is confirmed by western blots after transfection). In addition, cell signaling commercializes the cell line HDLM-2 as PD-L1 positive, and PC3 cells as PD-L1 negative, (SignalSlide #13747; Cell Signaling Technology, Danvers, MA) [27]. Properly validated cell lines may, in fact, be superior to histological tissue as controls in some cases for quantitative IHC assays, especially if combined with image analysis [28]. Commercial controls should also be cut onto the same slide for IHC processing as the clinical samples [20]. 4. In addition, for validation purposes and based on the titers for the specifics antibodies obtained with the IHC validations, we have also stained a TMA set of 9 slides containing non–small

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Edwin Roger Parra and Sharia Herna´ndez Ruiz

cell lung carcinomas (NSCLC), staged I to III without neoadjuvant therapy administered to the patient, (n ¼ 185; 122 adenocarcinomas or ADCs and 63 squamous cell carcinomas or SCCs) to compare PD-L1 IHC expression between different anti-PD-L1 clones. The TMA sections were prepared using three 1.0 mm tissue cores obtained from the center, middle, and periphery of the tumor. 5. In the last years, supporting evidence for the value of identifying multiple markers in the same tissue section using double IHC [29] or multiplex immunofluorescence (mIF) staining [30–32] have emerged as potential tools to help a deeper understanding of the distribution of these molecules, providing a unique insight into spatial, cell-type, and even phenotype co-localization–type specific distribution, trying to avoid the confusions observed when using a simple staining method. Our experience has shown that mIF staining performed in clinical specimens paraffin sections, using the same tissue section to probe for several targets, provides a useful tool to identify PD-L1 expression in a variety of cells in the tumor microenvironment (Fig. 3), minimizing the error observed when using a single IHC staining. The implementation of mIF in the same tissue section and the flexibility to create panels to different targets, offers many opportunities for innovative digital image analysis approaches (such as inflammatory tumor infiltration, cell phenotyping, proximity, 3D-reconstruction), increasing the novelty of this methodology [32]. In addition, we believe that application of this type of methodology to answer scientific research question or testing hypothesis of clinical importance could provide answers to different questions [33]. 6. In order to make an accurate evaluation of the performance of the different antibody clones in the tissue and cell line controls, it is important to assess the correct staining by the clones, a positive staining being defined as tumoral cells exhibiting partial or complete membranous staining [34] as defined in the Blueprint Phase 2 Project [18]. 7. We consider the uniformity and a clearly defined membrane staining pattern within tissues and cell lines to be accurate and positive. The staining pattern is comparable to the noncommercial clone 5H1 (dilution 1:40; generated by Lieping Chen, Yale University), to the PD-L1 antibody clone 22C3 (Code AS480, DAKO, shown in Fig. 1) stained with the DAKO Autostainer Link 48, and the SP263 (ready to use, Ventana Medical System Inc.); using previously optimized IHC conditions and performed according to the standard automated protocols, as references of well-validated antibodies in the literature.

PD-L1 Immunohistochemistry in Lung Cancer

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Fig. 3 Microphotograph of multiplex immunofluorescence using a panel of immune marker antibodies, with the Opal7 color Kit (Akoya/PerkinElmer, Waltham, MA) in the same tissue section. Lung adenocarcinoma tissue showing PD-L1 expression by tumor cells (white arrow) and negative in malignant cells (yellow arrow). Pancytokeratin indicates malignant cells (Cyan). PD-L1 (Orange); 40 ,6-Diamidino-2-Phenylindole (DAPI), nuclear staining (Blue); CD68 (yellow); CD3 (Red); CD8 (pink); and PD-1 (green). Multiplex immunofluorescence magnification is 200

Acknowledgments The authors would like to acknowledge the people that work in the Translational Molecular Pathology Immunoprofiling Laboratory, Luisa Solı´s, Mei Jang, Tong Li, Auriole Tamegnon, Barbara Mino, Wei Lu, and Jianling Zhou and the pathologists team that works in the image analysis, for their dedication to provide high quality data.

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References 1. Okazaki T, Honjo T (2007) PD-1 and PD-1 ligands: from discovery to clinical application. Int Immunol 19(7):813–824 2. Salmaninejad A, Valilou SF, Shabgah AG et al (2019) PD-1/PD-L1 pathway: basic biology and role in cancer immunotherapy. J Cell Physiol 234(10):16824–16837 3. Callea M, Pedica F, Doglioni C (2016) Programmed death 1 (PD-1) and its ligand (PD-L1) as a new frontier in cancer immunotherapy and challenges for the pathologist: state of the art. Pathologica 108(2):48–58 4. Sun C, Mezzadra R, Schumacher TN (2018) Regulation and function of the PD-L1 checkpoint. Immunity 48(3):434–452 5. Blank C, Gajewski TF, Mackensen A (2005) Interaction of PD-L1 on tumor cells with PD-1 on tumor-specific T cells as a mechanism of immune evasion: implications for tumor immunotherapy. Cancer Immunol Immunother 54(4):307–314 6. Iwai Y, Ishida M, Tanaka Y (2002) Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade. Proc Natl Acad Sci U S A 99(19):12293–12297 7. Blank C, Mackensen A (2007) Contribution of the PD-L1/PD-1 pathway to T-cell exhaustion: an update on implications for chronic infections and tumor evasion. Cancer Immunol Immunother 56(5):739–745 8. Patel SP, Kurzrock R (2015) PD-L1 expression as a predictive biomarker in cancer immunotherapy. Mol Cancer Ther 14(4):847–856 9. Rimm DL, Han G, Taube JM et al (2017) A prospective, multi-institutional, pathologistbased assessment of 4 immunohistochemistry assays for PD-L1 expression in non-small cell lung cancer. JAMA Oncol 3(8):1051–1058 10. Wolff AC, Hammond ME, Schwartz JN et al (2007) American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J Clin Oncol 25(1):118–145 11. Bordeaux J, Welsh A, Agarwal S et al (2010) Antibody validation. BioTechniques 48 (3):197–209 12. Igarashi T, Teramoto K, Ishida M et al (2016) Scoring of PD-L1 expression intensity on pulmonary adenocarcinomas and the correlations with clinicopathological factors. ESMO Open 1(4):e000083 13. Kulangara K, Zhang N, Corigliano E et al (2019) Clinical utility of the combined positive

score for programmed death ligand-1 expression and the approval of Pembrolizumab for treatment of gastric cancer. Arch Pathol Lab Med 143(3):330–337 14. Sunshine JC, Nguyen PL, Kaunitz GJ et al (2017) PD-L1 expression in melanoma: a quantitative immunohistochemical antibody comparison. Clin Cancer Res 23 (16):4938–4944 15. Brunnstrom H, Johansson A, WestbomFremer S et al (2017) PD-L1 immunohistochemistry in clinical diagnostics of lung cancer: inter-pathologist variability is higher than assay variability. Mod Pathol 30(10):1411–1421 16. Kerr KM, Tsao MS, Nicholson AG et al (2015) Programmed death-ligand 1 immunohistochemistry in lung cancer: in what state is this art? J Thorac Oncol 10(7):985–989 17. Koppel C, Schwellenbach H, Zielinski D et al (2018) Optimization and validation of PD-L1 immunohistochemistry staining protocols using the antibody clone 28-8 on different staining platforms. Mod Pathol 31 (11):1630–1644 18. Tsao MS, Kerr KM, Kockx M et al (2018) PD-L1 immunohistochemistry comparability study in real-life clinical samples: results of blueprint phase 2 project. J Thorac Oncol 13 (9):1302–1311 19. Roge R, Vyberg M, Nielsen S (2017) Accurate PD-L1 protocols for non-small cell lung cancer can be developed for automated staining platforms with clone 22C3. Appl Immunohistochem Mol Morphol 25(6):381–385 20. Cree IA, Booton R, Cane P et al (2016) PD-L1 testing for lung cancer in the UK: recognizing the challenges for implementation. Histopathology 69(2):177–186 21. Biosystems L. Bond™ Oracle™ HER2 IHC System for Leica BOND-MAX System Instructions For Use 2014 [Manual for use on Leica Biosystems’ BOND-MAX fully automated, advanced staining system.]. https:// drp8p5tqcb2p5.cloudfront.net/fileadmin/ downloads_lbs/Oracle_HER2_Bond_IHC_ System_USA_Breast_Only/User_Manuals_ IFUs/Bond_Oracle_HER2_IHC_System_ TA9145_EN-US_Rev_B.pdf 22. Abcam. Antibody dilutions and titer. https:// www.abcam.com/protocols/antibodydilutions-and-titer 23. Lipman NS, Jackson LR, Trudel LJ et al (2005) Monoclonal versus polyclonal antibodies: distinguishing characteristics, applications, and information resources. ILAR J 46(3):258–268

PD-L1 Immunohistochemistry in Lung Cancer 24. Stadler C, Hjelmare M, Neumann B et al (2012) Systematic validation of antibody binding and protein subcellular localization using siRNA and confocal microscopy. J Proteome 75(7):2236–2251 25. Howat WJ, Lewis A, Jones P et al (2014) Antibody validation of immunohistochemistry for biomarker discovery: recommendations of a consortium of academic and pharmaceutical based histopathology researchers. Methods 70 (1):34–38 26. Parra ER, Uraoka N, Jiang M et al (2017) Validation of multiplex immunofluorescence panels using multispectral microscopy for immune-profiling of formalin-fixed and paraffin-embedded human tumor tissues. Sci Rep 7(1):13380 27. Parra ER, Villalobos P, Mino B (2018) Comparison of different antibody clones for immunohistochemistry detection of programmed cell death ligand 1 (PD-L1) on non-small cell lung carcinoma. Appl Immunohistochem Mol Morphol 26(2):83–93 28. Torlakovic EE, Nielsen S, Vyberg M et al (2015) Getting controls under control: the time is now for immunohistochemistry. J Clin Pathol 68(11):879–882

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29. Taylor CR (2014) Predictive biomarkers and companion diagnostics. The future of immunohistochemistry: “in situ proteomics,” or just a “stain”? Appl Immunohistochem Mol Morphol 22(8):555–561 30. Nghiem PT, Bhatia S, Lipson EJ et al (2016) PD-1 blockade with Pembrolizumab in advanced Merkel-cell carcinoma. N Engl J Med 374(26):2542–2552 31. Yuan J, Hegde PS, Clynes R et al (2016) Novel technologies and emerging biomarkers for personalized cancer immunotherapy. J Immunother Cancer 4:3 32. Parra ER, Francisco-Cruz A, Wistuba I (2019) State-of-the-art of profiling immune contexture in the era of multiplexed staining and digital analysis to study paraffin tumor tissues. Cancers 11(2):247 33. Parra ER (2018) Novel technology to assess programmed death-ligand 1 expression by multiplex immunofluorescence and image analysis. Appl Immunohistochem Mol Morphol 26(2):e22–ee4 34. Hirsch FR, McElhinny A, Stanforth D et al (2017) PD-L1 immunohistochemistry assays for lung cancer: results from phase 1 of the blueprint PD-L1 IHC assay comparison project. J Thorac Oncol 12(2):208–222

Chapter 5 Western Blot as a Support Technique for Immunohistochemistry to Detect Programmed Cell Death Ligand 1 Expression Edwin Roger Parra and Sharia Herna´ndez Ruiz Abstract Antibody selection and optimization are crucial to guarantee accurate and reproducible results when using such antibodies for applications such as western blot analysis and immunohistochemistry (IHC). This is especially important when selecting good candidate antibodies that will be used for cancer immunotherapy diagnostics and research. In this chapter, we describe a Western Blot technique as support methodology for the selection and validation of Programmed Cell Death Ligand 1 (PD-L1) antibodies that can be subsequently used in immunohistochemistry applications. Western Blot is a sensitive, specific, and widely available protein characterization technique, used for the detection of specific antigens. PD-L1 is a major immune checkpoint protein that mediates antitumor immune suppression and response, which is routinely detected using IHC in formalin-fixed and paraffin-embedded tissues as part of cancer clinical diagnostic workflows. For this reason, it is critical to define and select the best antibody clones and validate them using different techniques in order to have a reliable detection of positive staining when these antibodies are used in IHC. Key words Western Blot, PD-L1, Antibody optimization, Cell lines, Immune cell expression, Immunohistochemistry

1

Introduction Western blot (sometimes called immunoblot) is a widely employed analytical technique used for analysis and detection of specific proteins in a complex mixture extracted form cells. It uses gel electrophoresis to separate the proteins by size and charge. The proteins are then transferred and immobilized to a carrier membrane (typically nitrocellulose, nylon, or PVDF). The target protein of interest is then detected by a primary antibody, which in turn is detected by a secondary antibody conjugated to a fluorescent label or to an enzyme which generates an insoluble precipitate upon addition of its substrate. The detected bands become visible through color or emission of light, after a reaction with an applied reagent [1, 2].

Pedro G. Santiago-Cardona (ed.), Lung Cancer: Methods and Protocols, Methods in Molecular Biology, vol. 2279, https://doi.org/10.1007/978-1-0716-1278-1_5, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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Western blots are in wide use across a broad range of scientific and clinical disciplines. This method is a powerful tool to detect and characterize a multitude of proteins, especially those that are of low abundance, thanks to the great sensitivity of the technique. It is able to show the presence of a specific protein through the binding of an antibody, which is useful to identify the expression of that protein from various tissues, or for monitoring how protein expression changes as a response to disease progression or drug treatment [3, 4]. Immunoblotting is very valuable, especially for testing the specificity of antibodies that are to be subsequently used in immunohistochemistry (IHC) experiments. This is due to the immunoblot’s ability to provide simultaneous resolution of multiple immunogenic antigens. Moreover, since IHC is a semi quantitative technique, quantitative assessment of protein expression can be better accomplished by Western blot analysis [3, 5]. An appropriate validation and evaluation of PD-L1 expression on tissues is important, since there are different PD-L1 clones developed in different assays, as target for immunotherapy. Using lung cancer as a model, the procedure described below can be used to compare commercially available PD-L1 clones by Western Blot analysis, and thus to identify which ones can be reliably used by IHC in formalin-fixed paraffin-embedded (FFPE) tissues to support the surgical pathologist in the evaluation by IHC PD-L1 expression.

2

Materials

2.1 Gel Electrophoresis Separation

1. Sample buffer, 4. This buffer can be obtained commercially as a ready-to-use solution. We use NuPAGE™ LDS 4 Sample Buffer, but equivalents are acceptable. 2. 20 NuPAGE™ MES SDS Running Buffer. We buy this buffer as a commercially available pre-mixed, ready-to-use solution. Alternatively, you can also use 20 MOPS SDS running buffer, also commercially available from many vendors. Before using, dilute to 1 by mixing 50 mL of 20 NuPAGE™ MES or 20 MOPS SDS running buffer with 950 mL of deionized water. 3. Precast NuPAGE™ Novex 4–12% Bis-Tris polyacrylamide gradient electrophoresis gel cassettes. We use precast gels, but gels prepared in the laboratory can also be used. 4. Mini gel electrophoresis apparatus, including power supply. We routinely conduct this protocol using the XCell SureLock™ Mini-Cell system. But other electrophoresis systems can be used following the manufacturer’s instructions.

Immunoblot Detection of PD-L1

51

5. Protein marker ladder. Be sure the marker of choice provides good resolution in the size range in which your protein of interest is expected to migrate. In this case, for PD-L1 you should choose a marker that provides good resolution in the 40–50 kDa range. 2.2

Protein Transfer

1. Transfer buffer. We use the NuPAGE™ 20 Bis-Tris transfer buffer. Dilute to 1 with distilled water before using. 2. Gel transfer apparatus with its components, including power supply. Follow manufacturer’s instructions on how to assemble the transfer apparatus. 3. Nitrocellulose western blot transfer membranes, 0.45 μm pore size.

2.3 Membrane Blocking, Immunolabeling, Washing and Development

1. Tris Buffered Saline with Tween-20® (TBST) buffer, 1: 50 mM Tris–HCl, 150 mM NaCl, 0.05% Tween-20, pH 7.4. To prepare 1 L, dissolve in 800 mL of distilled water 8.8 g of NaCl, 0.2 g of KCl, and 3.0 g of Tris base. Add 500 μL of Tween-20®. Adjust the pH to 7.4 with HCl and complete the volume to 1 L with distilled water. Sterilize by filtration or autoclaving. 2. Blocking solution. To prepare, add 5 g of non-fat dry milk powder to 100 mL of the TBST buffer described above. This is also called NFDM buffer, and it is 5% (w/v) milk. 3. Wash buffer. This is TBST 1 prepared as described above, but with 0.1% Tween-20®. 4. Antibody incubation buffer. This is TBST with 5% (w/v) bovine serum albumin (BSA). 5. Primary antibodies. We have tested this procedure with the following PD-L1 antibodies and dilutions: EPR1161-2, dilution 1:2000 (Epitomics-Abcam, Burlingame, CA, cat#ab174838); E1L3N, dilution 1:2000 (Cell Signaling Technology, Beverly, MA, cat#13684); clone E1J2J, dilution 1:2000 (Cell Signaling Technology, cat#15165); 7G11, dilution 1:2000 (generated in the Gordon Freeman Laboratory, Boston University, Boston, MA); SP142, dilution 1:2000 (Spring Bioscience, Pleasanton, CA, cat#M4424); PD-L1 rabbit polyclonal, dilution 1:2000 (Abcam, cat#ab58810); 28–8, dilution 1:2000 (Abcam, Cambridge, MA, cat#ab205921); SP263, dilution 1:500 (Ventana Medical System Inc., Tucson, AZ, cat#790-4905); 1H5, dilution1:1000 (generated in the Lieping Chen Laboratory, Yale University, New Haven, CT). All these antibodies should generate a band between of 40–50 kDa molecular weight. As a control load antibody, we use β actin at a dilution of 1:2000 (Chemicon International, Temecula, CA).

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6. Secondary antibody. Use an anti-mouse or an anti-rabbit secondary antibody, depending of the primary antibody. In our case, we use a secondary antibody conjugated to horseradish peroxidase, which is provided in the SuperSignal Chemiluminescence Kit (see item 8 below). 7. Stripping solution. We use the Re-Blot Plus stripping solution from Chemicon International, but equivalents from other vendors are acceptable. This is required when re-blotting the same membrane with a different antibody, and the first antibody needs to be stripped from the membrane. 8. Signal development kit. We use SuperSignal Chemiluminescence Kit from Pierce Biotechnology, but other kits can be used as well, following manufacturer’s instructions. Regarding the SuperSignal Chemiluminescence kit, there are separate kits for detection of mouse and rabbit secondary antibodies. The kit contains all the reagents required for signal development, including a luminol enhancer solution for increased sensitivity. 2.4 Cell Lines and Tissues

For this procedure you need previously prepared protein extracts from tissues and cell lines, using your extraction and lysis method of choice (see Note 1). Protein concentration in the extracts should have been previously determined by the method of your choice. 1. Tissues. We usually include in this protocol a human tonsil tissue lysate as a positive control. 2. We use the following human lung-derived or lung adenocarcinomas cell lines: H23, H157, H461, H4006, H1171, and H193. 3. HEK293 cells, a human embryonic kidney cell line. This is a highly transfectable cell line, and we use non-transfected cells as well as cells transfected with the PD-L1 gene, as negative and positive controls, respectively.

2.5 Additional Materials

1. X-ray films. We use Kodak Biomax MR X-ray films. 2. Micro-pipettes with tips, different volumes. 3. Screw cap tubes, or sealable plastic bags (for membrane blocking and incubating the membranes with antibodies). 4. Rotor or shaker platform. 5. Plastic wrap. 6. Film casette.

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53

Methods

3.1 Sample Preparation, Electrophoresis, and Transfer

1. . Using 2 μg of protein from the lysates from cell lines or tissues, prepare the sample by mixing 2.5 μL of the 4 NuPAGE™ LDS Sample Buffer, the volume of protein sample required for 2 μg of protein, and deionized water to complete a final volume of 10 μL. 2. Assemble the gel electrophoresis apparatus following manufacturer’s instructions, including the precast gels. At this point you need to remove the combs and the white tape that seals the bottom of the gel cassette. Place the gels in the tank, and add 1 running buffer to the gel tank, this will take approximately 400 mL of running buffer to completely fill each chamber. If using the XCell SureLock™ Mini-Cell, add 600 mL of running buffer to the lower chamber and 200 mL of running buffer to the upper chamber (for reduced samples, use running buffer with antioxidant in the upper chamber). Rinse the gel wells three times using 1 running buffer. Ensure that the gel, including its lanes, are fully submerged in the running buffer. 3. Load your 10 μL samples into the wells, including the protein ladder marker. Avoid overloading the wells in order to avoid cross- contamination of the samples (see Note 2). 4. Start the gel run. Optimal run times vary depending on gel percentage and power supply used for electrophoresis. However, when using the XCell SureLock™ Mini-Cell set-up with MES running buffer, we run the gel for 35 min at 200 V constant voltage. If using the MOPS running buffer, we run the gel for 50 min at 200 V constant (see Note 3). 5. After the electrophoresis, disassemble the electrophoresis apparatus. Carefully retrieve the gel, and use it to assemble the transfer set-up, including the nitrocellulose membrane, according to the manufacturer’s instructions. Fill the transfer tank in 1 Bis-Tris transfer buffer, and transfer following the transfer apparatus instructions.

3.2 Immunolabeling and Signal Development

1. Disassemble the transfer apparatus, carefully removing the membranes. Handle the membranes with gloves or tweezers, never allowing contact with bare hands. 2. Place the membranes in conical tubes or sealable plastic bags. Keep membranes moist at all times. Block membranes in blocking solution (NFDM buffer) for 1 h at room temperature. Use continuous shaking or rotation. 3. Briefly rinse membranes with wash buffer.

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4. Incubate membranes overnight at 4  C in the antibody incubation buffer using any of the anti-PD-L1 primary antibodies described in the previous section at the indicated dilution. Dilute the antibody first into the antibody incubation buffer before adding the membrane. Use continuous shaking or rotation for the incubation. 5. Wash the membranes in wash buffer, 2–3 times, 5 min per wash. 6. Incubate membranes in the secondary antibody at a dilution of 10 ng/mL in the antibody incubation buffer. Incubate for 20 min at room temperature with continuous shaking or rotation. 7. Wash the membranes in wash buffer, 3 times, 5 min per wash. 8. Develop the western blot signal on the membrane using the SuperSignal Chemiluminescence kit (Pierce Biotechnology), or any other kit of your choice, strictly adhering to the kit’s instructions. When incubating the membrane with the Working Solution (provided with the kit), we use approximately 0.1 mL per cm3 of membrane area, and incubate for 5 min. 9. After completing the steps indicated in the signal detection kit, drain excess liquid from the membrane (but keep it moist), and place the membrane in a plastic sealable bag or cover with clear plastic wrap, avoiding bubbles between the membrane and the plastic. 10. Place the wrapped membrane inside the film cassette. Fixing it to the cassette with tape is recommended to avoid displacement of the film during exposure. 11. In a dark room, insert an X-ray film inside the cassette and tightly close the cassette. Never expose films to white light (see Notes 4 and 5). Expose the film for 1 min (this time can be optimized) and proceed to develop the film with the method of choice, ensuring it is compatible with the film type. 12. If needed, the same membrane can be re-probed with another antibody. In this case, the previous antibody needs to be stripped from the membrane. For this purpose, we use the Re-Blot Plus stripping solution (Chemicon International) according to the manufacturer’s protocols [6, 7], but other solutions and methods can be used. It is recommended that the membrane is exposed to a film after stripping to ensure complete removal of the previously bound antibodies (see Note 6).

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Notes 1. A wide selection of samples is very important for antibody validation. Your selection of samples can vary from the one we use for this protocol, however, it is always important to include samples that you know express high levels of PD-L1, to be used as positive controls, as well as samples in which PD-L1 protein expression is absent, to be used as negative controls. 2. Always load your protein ladder in the same sequence in the appropriate well from your gel to avoid mistakes during the analysis of your antibodies. 3. Depending on whether we use MES or MOPS running buffer, we need to check the time and the volts to obtain good results with all your antibodies tested. 4. Detection of signals is the last step and the molecular weight of the protein can be estimated by comparison with marker proteins, and the amount of protein can be determined as this is related to band intensity. In most applications, it is enough to confirm protein presence and roughly estimate the amount. 5. Depending on the antibody used and its dilution, we observed that we need to test several exposure times to detect the signal. In general, we start short exposure times (a few seconds), and if band intensity is inadequate, we progressively increase exposure time until band intensity is satisfactory, being careful never to expose the film for more than 1 min. 6. While antibody optimization for western blots is useful as a first step to select the best antibodies, it only guarantees that the chosen antibody will provide accurate results for western blot analysis. If the goal is to use the antibody for IHC, for example, then the user must demonstrate that the antibody is also able to specifically recognize its target when used in those other application [8]. Figure 1 shows representative western blot results when different PD-L1 antibody clones are tested in a panel of lung cancer cell lines.

Acknowledgments The authors would like to acknowledge the people that work in the Translational Molecular Pathology Immunoprofiling Laboratory for their dedication to provide high quality data.

Edwin Roger Parra and Sharia Herna´ndez Ruiz

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A

E

HEK293

H193

H1171

H4006

H157

H461

H23

Tonsil

MW

HEK293Trans. (+)

PD-L1 (22C3)

HEK293Trans. (+)

H193

H4006

H1171

H461

H157

H23

Tonsil

MW

HEK293

PD-L1 (E1L3N)

50KDa

50KDa

40KDa

40KDa

B

F

HEK293

HEK293Trans. (+) HEK293Trans. (+)

H193

H1171

H4006

H461

H23

HEK293

50KDa

H157

MW Tonsil

HEK293

H193

H1171

H4006

H461

H157

H23

Tonsil

MW

PD-L1 (SP263)

HEK293Trans. (+)

PD-L1 (E1J2J)

50KDa

40KDa

40KDa

G

PD-L1 (5H1)

50KDa

50KDa

40KDa

40KDa

D

H193

H1171

H4006

H461

H157

H23

MW Tonsil

H1171

H193

H461

H4006

H157

H23

Tonsil

MW

HEK293

PD-L1 (SP142)

HEK293Trans. (+)

C

50KDa 40KDa

HEK293Trans. (+)

HEK293

H193

H1171

H4006

H461

H157

MW

H23

β actin Tonsil

H193

H1171

H4006

H461

H157

Tonsil H23

MW

HEK293

PD-L1 (28-8)

HEK293Trans. (+)

H

50KDa 40KDa

Fig. 1 Representative western blots using different anti-PD-L1 antibody clones tested in a panel of human lung cancer cell lines. Cell lines tested were H23, H157, H461, H4006, H1171, and H193. Human tonsil extracts are used in all the blots as a positive control. Untransfected HEK293 cells served in all panels as negative controls, while the HEK293-PD-L1-transfected cells were used as positive controls. Notice the absence of signal in the untransfected HEK293 cells, while there is a very strong band in the HEK293-PD-L1-transfected cells. The anti-PD-L1 clones used were the E1L3N (a), E1J2J (b), SP142 (c), 28-8 (d), 22C3 (e, this is from Dako, Carpinteria, CA, Kit cat#SK006), SP263 (f), and 5H1 (g). Membranes were also probed with β actin to show protein loading in the lanes (h). The PD-L1 molecular weight (MW) looks similar with all the PD-L1 clones. The 22C3 clone (e) did not show any bands when tested in western blots

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References 1. Eslami A, Lujan J (2010) Western blotting: sample preparation to detection. J Vis Exp 44:e2359 2. Kramer DK. Western blotting (immunoblot): Gel electrophoresis for proteins 2013. https:// www.antibodies-online.com/resources/17/ 1224/western-blotting-immunoblot-gel-elec trophoresis-for-proteins/ 3. Kurien BT, Dorri Y, Dillon S et al (2011) An overview of Western blotting for determining antibody specificities for immunohistochemistry. Methods Mol Biol 717:55–67 4. Moore C. Introduction to Western Blotting Endeavour House, Langford Business Park, Langford Lane, Kidlington, Oxford OX5 1GE, UK.: MorphoSys UK Ltd; 2009. http://www. spacesrl.com/wp-content/uploads/2011/03/ WesternBlottingBrochure.pdf 5. Graham A, Nothnick WB (2020) Concurrent immunohistochemical localization and Western

Blot analysis of the MIF receptor, CD74, in formalin-fixed, paraffin-embedded tissue. Methods Mol Biol 2080:123–134 6. Thermo Fisher Scentific. NuPAGE Bis-Tris Gels 2019 [Pub. No. MAN0007891:[Manual]. https://assets.thermofisher.com/TFS-Assets/ LSG/manuals/MAN0007891_NuPAGE_ BisTris_MiniGels.pdf 7. Parra ER, Villalobos P, Mino B et al (2018) Comparison of different antibody clones for immunohistochemistry detection of programmed cell death ligand 1 (PD-L1) on non-small cell lung carcinoma. Appl Immunohistochem Mol Morphol 26(2):83–93 8. Bordeaux J, Welsh A, Agarwal S et al (2010) Antibody validation. BioTechniques 48 (3):197–209

Chapter 6 Creation of Formalin-Fixed, Paraffin-Embedded 3D Lung Cancer Cellular Spheroids for the Optimization of Immunohistochemistry Staining Procedures Jennifer Caba´n-Rivera, Camille Chardo´n-Colo´n, Alberto Pedraza-Torres, Yoan E. Rodrı´guez, Raymond Quin˜ones-Alvarado, and Pedro G. Santiago-Cardona Abstract In an era of precision medicine important treatment decisions are dictated by expression of clinically informative tumor protein biomarkers. These biomarkers can be detected by immunohistochemistry (IHC) performed in tumor tissue sections obtained from biopsies or resections. Like all experimental procedures, IHC needs optimization for several of its steps. However, the investigator must avoid optimizing the IHC procedure using valuable human biopsy samples which may be difficult to obtain. Ideally, valuable biopsy samples should only be subjected to IHC once the IHC protocol has been optimized. In this chapter, we describe a procedure for IHC optimization using tri-dimensional (3D) cellular spheroids created from cultured cells. In this approach, cultured cells are pelleted into 3D spheroids, which are then processed just like a tissue sample, namely, fixed, embedded, sectioned, mounted on slides, and stained with IHC just like a human tissue sample. These 3D cellular spheroids have a tissue-like architecture and cellularity resembling a tumor section, and both cellular and antigen structure are preserved. This method is therefore acceptable for IHC optimization before proceeding to the IHC staining of human tumor samples. Key words Spheroids block, Immunohistochemistry optimization, p39, Lung cancer, Cell lines, Formalin-fixed-paraffin-embedded tissues

1

Introduction Clinically relevant protein biomarkers are needed to improve lung cancer management. Many important clinical decisions regarding disease management are guided by the detection by immunohistochemistry (IHC) staining of these biomarkers in tumor tissue

Jennifer Caba´n-Rivera, Camille Chardo´n-Colo´n, Alberto Pedraza-Torres, and Yoan E. Rodrı´guez contributed equally to the protocol optimization. Pedro G. Santiago-Cardona (ed.), Lung Cancer: Methods and Protocols, Methods in Molecular Biology, vol. 2279, https://doi.org/10.1007/978-1-0716-1278-1_6, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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obtained from biopsies or resections. For example, expression of several isoforms of cytokeratin, as detected by IHC, have diagnostic value for non-small cell lung carcinomas (NSCLC) [1]. As another example, a panel of IHC markers is routinely used in pathology laboratories for the subclassification of NSCLC into squamous cell carcinomas or adenocarcinomas subtypes [2, 3]. In the context of basic and translational cancer research, IHC staining for any given protein should ideally be preceded by extensive optimization of the IHC workflow in order to ensure a strong staining signal that is specific, reproducible and of sufficient quality for publication. This involves optimizing parameters such as antibody concentration and incubation time, conditions for effective antigen retrieval, blocking to minimize background, number and duration of washes, signal development conditions, etc. Even when these parameters are optimized, they may still have to be re-checked when changing the batch of the antibody or of any other IHC reagent. The optimization stage may involve a certain degree of trial and error. Therefore, it is strongly recommended that valuable human tumor tissue samples are not used during this stage. Pre-made tumor microarrays (TMAs) are commercially available for many cancer types, but these can be too expensive and also too valuable to be used for optimization purposes. Therefore, regardless of their origin, valuable human samples should ideally only be subjected to IHC analysis once the IHC protocol has been optimized. In this chapter, we describe a procedure for IHC optimization using tridimensional (3D) cellular spheroids created from cultured cells. This approach is relatively inexpensive since it starts with cultured cells. Briefly, cells are grown in culture to form confluent monolayers, then they are detached from culture plates, centrifuged, and pelleted. The cell pellet is then processed like a tissue biopsy would normally be, namely, fixed, embedded, sectioned into tissue sections, and mounted on glass slides that can then be used for IHC staining just like any other tissue biopsy. The resulting 3D cellular spheroids have a tissue-like architecture and cellularity resembling a tumor section, and the cellular structure and antigen availability are preserved during their preparation. They are therefore acceptable as an inexpensive biological material that can be used for IHC optimization. We have successfully used the protocol described in this chapter to optimize an IHC staining procedure to detect p39 protein expression in lung cancer cell culture-derived tridimensional (3D) spheroids. We have previously reported elevated p39 expression as a biomarker for advanced stage lung squamous cell carcinomas (SCC) [4], and slides prepared from 3D spheroids have been instrumental in our laboratory to optimize the IHC protocol for p39 and for other antigens. After optimizing the p39 IHC staining with the 3D spheroids, we successfully used the optimized protocol

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to stain lung cancer TMAs for p39 expression by IHC [4]. It is important that the cell line chosen to prepare the 3D spheroids matches the tumor tissue type that will be eventually studied. In our case, we created the 3D spheroids using the NSCLC cell line H520. This cell line is of the SCC NSCLC subtype and is patient-derived from SCC tumors. We have identified p39 as part of a metastasisassociated proteomic biomarker signature in SCC [4] and therefore we optimized the IHC procedure using H520 3D spheroids before actually conducting the IHC p39 analyses using human lung TMAs. Therefore, choice of the appropriate cell line to prepare the 3D spheroids is of great importance. It is important to keep in mind that 3D spheroid models may not be suitable if preserving the heterogeneity of the tumor microenvironment is of the essence. The spheroids produced with the procedure herein described are relatively homogeneous in terms of cellular composition and lack the cellular variety that typically represents the complex interactions between the tumor and its microenvironment. Therefore, these spheroid structures may not be ideal to reliably assess protein expression that is strongly affected by intercellular signaling events. Neither they provide a suitable model to address the issue of tumor multiclonal heterogeneity. However, it must be kept in mind that the purpose of this approach is not to study oncogenesis-related biological process, but it is rather aimed at the optimization of the immunological detection of an epitope within the context of a 3D tumor-like tissue architecture, and to mimic as much as possible the mechanical aspects of antigen accessibility in the context of 3D tissue structures. This chapter describes the complete procedure, from the formation of the 3D spheroids from cell cultures, preparation of spheroid sections, staining of the sections with hematoxylin and eosin to verify their cellular density, and finally, IHC staining of the spheroid sections for p39 protein expression.

2

Materials All the solutions should be prepared using distilled water, unless otherwise specified. Reagents and solutions should be stored at room temperature, unless otherwise specified. Follow all waste disposal regulations for waste materials.

2.1 Cell Lines and Cell Culture Reagents

1. Cell lines. We optimized this protocol using the H520 non-small cell lung carcinoma (NSCLC) cell line, which is of the squamous cell carcinoma subtype. Keep in mind that the purpose of the procedure described in this chapter is to use cell lines to optimize the IHC protocol that will then be applied to formalin-fixed, paraffin-embedded (FFPE) tissue sections. Therefore, the cell lines for the optimization of the IHC

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protocol should be as histologically equivalent as possible to the tumor tissue that will be subsequently tested. For example, H520 and H1666 cell lines originated from squamous cell lung carcinomas and from lung adenocarcinomas, respectively, therefore they can be used for IHC optimization for these lung tumor types. We culture cells in 100 mm culture plates or T75 flasks. 2. Cell culture medium. This depends of the cell type of your choice. We culture H520 cells in RPMI medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin. 3. Phosphate-Buffered Saline (PBS), 1. You can prepare 1 PBS as follows: dissolve 8.0 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4.2 H2O and 0.24 g of KH2PO4, in of 800 mL of water. Adjust the pH to 7.2 with HCl (start with concentrated HCL and then use more diluted HCL as you approach the desired pH) and add distilled water to complete the volume to 1 L. Alternatively, ready-to-use commercially available PBS is also acceptable. Use cold. 4. 0.25% Trypsin-EDTA solution. We buy in ready-to-use pre-mixed form. We use this solution to detach cells from the culture flasks or plates. Alternatively, you can scrape cells with a rubber scraper or spatula. 2.2 Histology Reagents and Supplies

1. Histology tissue embedding and processing cassettes. 2. 10% neutral buffered formaldehyde solution. We purchase this as a ready-to-use pre-mixed solution, it is available from several vendors. 3. Ethanol. You need absolute (100%) ethanol, anhydrous, histological grade, and you also need 95%, 90%, 85%, 80%, and 70% ethanol dilutions in distilled water (dH2O). 4. Xylene or xylene substitute. 5. Paraffin or tissue embedding medium, we use Paraplast X-TRA®, but other alternatives are acceptable. Check the manufacturer’s instruction for melting temperature. 6. Tissue embedding base metal molds, with dimensions of 32  25  12 mm. 7. Embedding rings, be sure they fit into the embedding base molds. 8. Microscope glass slides, 25  75 mm, including coverslips of the appropriate size. 9. Bluing reagent solution, ready-to-use solution.

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10. Acid alcohol, 1%. Prepare a solution that is 1% hydrochloric acid in 70% ethanol. You can dilute 2 mL of concentrated 12.1 M HCl in 200 mL 70% ethanol. 11. Eosin staining solution, we use Eosin Y solution 1%, alcoholic, ready-to-use. 12. Hematoxylin stain, we use Harris Modified Hematoxylin. 13. Mounting medium, we use Cytoseal 60, but other alternatives can be used. 14. Standard slide racks and histology staining and washing jars. 15. HistoCore Arcadia C-Cold Plate from Leica Biosystems, or any other histology cold plate. 16. HistoCore Arcadia H-Heated Paraffin Embedding Station, or any other heated histology embedding station. 17. Microtome with blade (follow all necessary precautions when handling sharp microtome blades). 2.3 Immunohistochemistry Reagents

1. Citrate antigen retrieval solution. Prepare by dissolving 1.92 g of trisodium citrate dehydrate and 0.74 g of EDTA in 800 ml of H2O, adjust pH to 6.2 with 1 N HCl, add 0.5 ml of Tween 20® and complete to a final volume of 1 L. This solution can be stored at 4  C for up to 6 months. 2. Hydrogen peroxide solution, 3%. Start with 30% hydrogen peroxide (H2O2) and dilute to 3% with distilled water. 3. PAP pen, or any other hydrophobic pen or marker, this is needed for drawing hydrophobic barriers around the 3D spheroid section in microscope slides. 4. Primary antibody of your choice, depending on the antigen you want to test. We optimized this protocol using a rabbit monoclonal antibody against p39, clone EPR5074 from Abcam. (Cat. No. 124896) (see Note 1). For a 1:50 dilution add 20 μL of antibody to 980 μL of 1 PBS. 5. Super sensitive Link-Label IHC kit from BioGenex (LP000ULE). This immunohistochemistry kit includes a biotinlabeled anti-rabbit secondary antibody and a streptavidin conjugated horseradish peroxidase (HRP). Use a 1:20 dilution for the secondary antibody and a 1:10 dilution for the streptavidin. Other immunohistochemistry detection kits are acceptable but be sure to use according to manufacturer’s instructions, and that the secondary antibody should be compatible with your primary antibody (see Note 2). 6. Diaminobenzidine (DAB) reagent. This is the substrate for the HRP, it produces a dark brown precipitate when it is oxidized by the HRP. We use the BioGenex Two Components DAB-Pack (HK542-XAKE), following product instructions.

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To prepare 1 mL of DAB reagent solution, add 2 drops of DAB reagents to 1 mL of DAB buffer, vortex the solution, and store at 4  C (see Note 3). 2.4 General Laboratory Equipment

1. Incubator set at 37  C, 5% CO2. 2. Humid chamber. This is to prevent the slides from drying up due to evaporation during antibody incubations. It can be commercially purchased or can be prepared in the laboratory using a tight sealing container. Humidity inside the chamber can be maintained by including absorbent paper soaked in distilled water. 3. Microscope with camera, and suitable image acquisition software. 4. Lint-free tissue paper. 5. Water bath, set to 80–85  C. 6. Tabletop centrifuge with a rotor capable of holding 50 mL tubes, preferably refrigerated. We use an Eppendorf 5810R, but equivalents are acceptable. 7. Conical tubes, 50 mL. 8. Filter paper, 180 μm. 9. Lens paper. Wax paper or weighting paper can also be used. 10. Oven, set to 37  C, then to 64  C.

3

Methods

3.1 Spheroid Preparation from Cell Cultures

For the preparation of 3D spheroid blocks with tissue-like organization from lung cancer H520 cell line (or the cell line of your choice), you should start with at least 5–6 confluent T75 flasks or 100 mm cell culture plates for each cell line in order to obtain a good-sized cell pellet from which the spheroid will be formed. Procedures related to spheroid fixation and preparation of the spheroid blocks (described in detail in Subheadings 3.1 and 3.2) are adaptations of previously published protocols [5, 6]. 1. Collect cells by scraping them from the culture plate in 5 mL of 1 PBS. We recommend this volume for T75 flasks; you should adjust the volume of 1 PBS when using plates. It is important that the entire surface of the bottom of the flask or plate is covered with thin layer of 1 PBS. Alternatively, detach cells from the plate by incubating the cultures with a 0.25% Trypsin-EDTA solution at 37  C for 5–10 min. 2. Transfer the cell suspension to a 50 mL tube and pellet the cells by centrifugation (up to 300  g) for 5 min, at room temperature (RT). If the cells were detached by trypsinization, dilute

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the trypsin solution 1:10 with fresh culture medium to ensure inactivation of the trypsin. Do this dilution before the centrifugation step. Keep in mind that at this point the size of the pellet will affect the cellularity of the spheroid. If you end up with spheroid tissue sections in which cells are sparse, you need to increase the number of culture plates in order to obtain a larger pellet. 3. Carefully remove the supernatant after the centrifugation, being careful not to disrupt the cell pellet. 4. Resuspend the cell pellet in 20–25 mL cold 1 PBS and centrifuge at 300  g for 10 min at RT. 5. Remove the supernatant, then carefully add 20–25 mL of the 10% neutral buffered formalin (NBF). Pour the NBF down the tube’s inner wall to avoid disrupting the cell pellet which will constitute the spheroid. 6. To produce the fixed spheroid, fix the cell pellet by incubating in NBF overnight at 4  C. 7. After overnight fixation carefully remove the NBF (see Note 4). 8. Resuspend the spheroid in 10–15 mL cold 1 PBS and centrifuge at 300  g for 10 min at RT. Repeat this step for a total of three 1 cold PBS washes. 3.2 Preparation of Spheroid Paraffin Blocks

1. Carefully transfer the spheroids from 50 mL tube to a piece of filter paper. You can use a spatula or a pipette, as long as you do not break the spheroid. Allow the spheroids to air-dry on the filter paper. During this step the filter paper will remove the excess moisture from the spheroid. If there are any remnants of cells in the tube you can wash the tube with cold 1 PBS using a pipette and deposit the wash in the filter paper. 2. Collect spheroids from the filter paper by gently scraping the paper with a flat spatula. Be careful not to break the filter paper to avoid losing sample, or to scrape too hard that pieces of the filter paper could detach together with the spheroid. 3. Prepare a small envelope or pocket using lens paper (Fig. 1), prepare one of these for each spheroid. You can also use wax paper or weighing paper. Carefully transfer the dry spheroid to the lens paper envelope and place it into a tissue processing cassette. 4. Dehydrate and clear the spheroids by following the schedule shown in Table 1. Use glass jars for the washes indicated in the table. Carefully label each glass jars with each solution. Place the tissue cassette into the corresponding glass jar for the indicated time. Proceed to the following paraffin embedding steps below in this section.

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Fig. 1 Paraffin embedding of the cell spheroid. The spheroid cell pellet is placed inside an envelope made of lens paper (the envelope can be appreciated inside the paraffin in the mold in the left panel), which in turn is submerged in the molten paraffin in the metal mold. After a few minutes at room temperature the paraffin solidifies in the mold. The spheroid can be seen in the paraffin block (arrow in the right panel) Table 1 Steps for the dehydration and clearing of cell 3D spheroids formed from cell pellets. All these steps are performed at room temperature Procedure

Steps

Solution

Time

Dehydration

1 2 3 4

70% alcohol 80% alcohol 95% alcohol 100% alcohol

15 15 15 15

Clearing

1 2 3 4

Xylene Xylene Xylene Xylene

10 10 10 10

5. Heat the HistoCore Paraffin Embedding station. To determine the appropriate temperature, look for the melting temperature recommended for the specific paraffin or embedding medium of your choice, as indicated in the product instructions. Melt the paraffin or embedding medium and add to a heated metal mold, ensuring to cover the entire bottom of the mold. Place and keep the mold in the hot surface. 6. Using tweezers, carefully remove the spheroid from the cassette (leave it in the paper envelope) and place it in the molten paraffin within the metal mold. Allow it to cool down. 7. Once the paraffin solidifies (Fig. 1, left panel), remove the block from the metal mold and cut around the spheroid using a scalpel, in such way that you obtain a piece of paraffin containing inside the embedded cell spheroid. 8. Using the trimmed embedded spheroid, repeat step 6 (immersion in molten paraffin) and let the paraffin to slightly cool down, but not to become entirely solid (Fig. 1. center and right panels).

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Fig. 2 Final embedding set-up of the cell spheroid to obtain the paraffin block that will be subsequently cut. The block is put again in molten paraffin (left), and before the paraffin solidifies the O-ring or an embedding cassette without the lid is placed (center), and the whole set-up is allowed to solidify to create the final block that will be cut in the microtome (right)

9. Before the paraffin entirely solidifies, place a tissue embedding cassette without the lid (or use an O-ring) over the metal mold and fill with molten paraffin. This will create a paraffin block in which the cassette encases and holds the embedded spheroid together. This set-up is illustrated in Fig. 2. 10. Allow the paraffin block to solidify by placing it in the cold plate for 1–2 min. 11. Leave the block to continue solidification at room temperature overnight (see Note 5). 3.3 Microtome Sectioning of Spheroid Paraffin Blocks

1. Carefully remove the spheroid block from the metal mold. Keep the paraffin-embedded spheroid block on ice at all times before sectioning (see Note 6). 2. Using a microtome, cut the spheroid blocks to get ribbon spheroid sections (Fig. 3). The sections should have a thickness of 5 μm (see Note 7). 3. Mount the spheroid sections on glass slides.

3.4 Hematoxylin and Eosin (H&E) Staining

The spheroid sections generated in Subheading 3.3 will be used for immunohistochemistry (IHC) staining as described in Subheading 3.5 below. However, before proceeding with the IHC some of the sections must first be stained with Hematoxylin & Eosin (H&E). The purpose of the H&E staining is to allow you to observe under the microscope if the spheroid sections have the appropriate cellular density and architecture to resemble a tumor tissue section. Do not proceed to the IHC staining steps described in Subheading 3.5 until you have achieved spheroid sections with sufficient cellularity. 1. Place slides in a slide rack and incubate at 37  C overnight in an oven or incubator. 2. Next day, and right before staining, incubate the slides at 64  C for 1 h.

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Fig. 3 The final block shown in Fig. 2 is cut into paraffin ribbons 5 μm thick

3. Inside a fume hood, submerge the slide rack in xylene substitute for 2 min. Do two additional xylene substitute submersions, also 2 min each, for a total of 3. Use fresh xylene for each submersion. This and the remaining steps in this section must be performed inside the fume hood. 4. Hydrate the slides by submerging the slide rack into the jars that contain a series of ethanol solutions as follows: two consecutive washes in 100% alcohol, 1 min each; one 95% ethanol, 1 min; and a final rinse in tap water for 30 s. Do this rinse carefully to avoid the spheroid sections to detach from the slide. 5. Submerge the slide rack in the jar that contains hematoxylin for 2–4 min. 6. Rinse the slides with tap water for 30 s. Do this rinse carefully to avoid the spheroid sections to detach from the slide. 7. Submerge the slide rack into a jar containing 1% acid alcohol for 15–20 s. 8. Rinse slides with tap water for 30 s. Do this rinse carefully to avoid the spheroid sections to detach from the slide. 9. Submerge the slide rack in a jar containing Bluing reagent for 30 s. 10. Carefully rinse the slides with tap water for 30 s, always avoiding direct contact with the tissue section. 11. Dehydrate the spheroid sections by submerging the slide rack into jars with ethanol washes as follows: 70% alcohol for 1 min, then 95% alcohol for 1 min. 12. Submerge the slide rack into a jar that contains the Eosin solution for 2 min. 13. Submerge the slide rack into a jar with 95% alcohol for 10 s, followed by a 100% alcohol wash for 1 min.

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Fig. 4 H&E staining of a spheroid slide prepared from H520 cells, observed under light microscope at magnification of 100. The figure shows the preservation of cellular structure with intact nuclei and cell membranes, and a monolayer that resembles a tissue-like organization

14. Submerge the slide rack into a jar containing xylene substitute for 2 min. Repeat this step a second time. 15. Drain the xylene from the slides and allow the slides to dry inside the hood. 16. Add 2–3 drops of mounting media on top of each spheroid section and carefully place a cover glass on top of the section, avoiding the formation of air bubbles (see Note 8). 17. Analyze the slides under a light microscope. Capture images at various magnifications. Figure 4 illustrates a spheroid section with good cellular density. The cellular density should be as similar as possible to the cellularity of a tumor tissue and should have similar architecture in terms of cell-to-cell contacts. 3.5 Immunohistochemistry (IHC) of Spheroid Sections

Once the H&E staining has allowed you to determine that the spheroid sections are of sufficient cellularity, you can proceed to stain with IHC some of the slides generated in Subheading 3.3. 1. Place the slides in a slide rack and incubate at 37  C overnight. Omit this step if you have performed this previously as described in step 1 of Subheading 3.4. 2. Incubate slides at 64  C for 1 h. Omit this step if you have performed this previously as described in step 2 of Subheading 3.4. 3. Remove the paraffin from the spheroid sections by submerging slides in a slide rack in xylene for 5 min. Remember that this step involving xylene and all volatile solvents should be performed inside the fume hood.

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4. Hydrate slides by performing the following ethanol washes: 100% alcohol for 3 min, 95% ethanol for 3 min, and 70% ethanol for 2 min. 5. Perform a 1-h antigen retrieval step with the antigen retrieval citrate buffer heated to 80–85  C. The slides should be completely submerged in the buffer, and the jar should in turn be tightly covered and submerged into a water bath set at 80–85  C (see Note 9). 6. Remove the whole jar from the water bath and allow it to cool down to room temperature for 20–30 min (see Note 10). 7. Transfer the slides to 1 PBS, incubate for 10 min. 8. Place slides flat in the humid chamber. From this point forward, prevent the slides form drying out. In the subsequent steps, ensure that the tissue sections are completely soaked in indicated solutions. 9. Cover the spheroid sections in the slides with a layer of 3% hydrogen peroxide block to inactivate endogenous peroxide activity. Incubate for 30 min at room temperature. 10. Rinse sections with 1 PBS for 10 min. 11. Carefully dry the slides with a lint-free absorbent paper. Dry only in the areas surrounding the spheroid sections, do not touch directly the sections with the absorbent paper. 12. After drying the areas surrounding the sections, use the PAP pen or any hydrophobic pen to make a hydrophobic barrier surrounding the sections. Again, be careful not to damage the spheroid sections. 13. Apply the primary antibody solution by adding a drop sufficiently large cover the entire area of the spheroid section. At this step the slides should be in the humid chamber. 14. Incubate with the primary antibody overnight or between 12 and 18 h at 4–8  C. Remember to perform negative control sections in which the primary antibody is omitted. The negative control sections are incubated in 1 PBS instead of the antibody solution. 15. Remove the primary antibody and rinse the slides in 1 PBS for 5 min. Remove excess PBS with a lint-free absorbent paper. Remember that sections must never be allowed to get dry. 16. Incubate slides in the secondary antibody solution, ensuring you cover the whole section area with antibody solution. Incubation should be in the humid chamber at room temperature for 30 min. 17. Remove the secondary antibody solution and wash the slides in 1 PBS for 5 min. Remove the excess liquid with an absorbent paper.

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18. Add the streptavidin-HRP mix to each section and incubate in the humid chamber for 30 min at room temperature. 19. Remove the streptavidin-HRP mix and wash the slides with 1 PBS for 5 min. Remove excess PBS with an absorbent paper. 20. Prepare the DAB solution and add a drop of solution to each section. Incubate for a maximum of 2 min (see Note 11). 21. Stop the DAB reaction by submerging the slides in two consecutive washes with distilled water. 22. Place the slides under running tap water for 5 min, being careful that the water does not directly touch the sections. 23. Dehydrate the slides with ethanol washes in the following order: 85%, 90%, 95%, and absolute 100% ethanol for 2 min each wash. 24. Incubate the slides in xylene for 2 min, inside the fume hood. 25. Drain the xylene from the slides and let them to air-dry inside the fume hood. 26. Add 3 drops of mounting media on top of each section and carefully place a cover glass on top of the section, avoiding the formation of air bubbles inside the sample (see Note 8). 27. Analyze the slides under a light microscope. Take images at 10, 20, 40 up to 100 magnification. A positive p39 staining signal is appreciated as a brown precipitate (Fig. 5). The localization of the staining (nuclear, cytoplasmic, membrane, etc.) depends on the cellular localization of the antigen you are studying.

Fig. 5 Spheroid section from H520 cells immunohistochemically stained using an anti-p39 antibody. (a) Negative control section in which the primary antibody was omitted. (b) Primary antibody at a 1:50 dilution and at 40 magnification. (c) Primary antibody at a 1:50 dilution and at 100 magnification. Staining is observed as a strong brown signal (b and c), which is absent from the negative control (a)

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Notes 1. The appropriate dilution or concentration should be empirically determined for each antibody. We recommend that you start using the dilution or concentration recommended by the manufacturer, and depending on the results, you can use the antibody more concentrated if the staining is too weak, or more diluted, if background staining is too strong. When using antibodies against phosphorylated proteins, it is recommended that you use freshly cut tissue sections. 2. Many immunohistochemistry kits are commercially available. You have to ensure that the secondary antibody that is included with the kit matches the primary antibody that you are using (e.g., a goat anti-rabbit secondary antibody if you are using a rabbit polyclonal primary antibody, or a goat anti-mouse when using a mouse monoclonal primary antibody). 3. The DAB reagent and solution are photosensitive, prepare the solution in amber colored tubes or bottles, or wrap the tube with aluminum foil to avoid its exposure to light. Solution should be prepared right before using it and used fresh for the procedure to work optimally. Using a DAB solution that has been stored for days or weeks can result in poor staining intensity. When troubleshooting for weak staining signal, preparing a fresh DAB solution may be a good place to start. 4. Make sure you carefully remove the NBF, preferably by decanting or by slowly suctioning with a pipette. Try not to disrupt the spheroid. If the spheroid breaks apart during the removal of the NBF, spin again at 300  g for 10 min at RT. 5. If the block is placed at 20  C for storage or to speed up solidification, cracks might form in the paraffin. This could complicate the sectioning process. Therefore, complete solidification is best conducted at room temperature overnight. 6. Try not to cover the blocks with the ice. Instead, place them with the paraffin facing down just over the ice. This will keep the surface cool and humid. 7. If the sections are to be placed in a water bath, make sure the temperature is not too high. If the water bath is too hot, the cell spheroid could detach from the paraffin resulting in loss of the sample. We used a room temperature water bath. 8. Gently slide the tip of a pencil into the cover glass to remove any air bubbles that might have formed over the sample section. 9. Citrate buffer should be pre-heated in a water bath (or microwave). Pre-heat the buffer before initiating the antigen retrieval step, do not submerge the slides in the buffer until it has reached the desired temperature.

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10. At this point the jar can be slightly opened to allow the vapors to be released and ensure the buffer is cooled down to room temperature. Do this step inside a fume hood. 11. When we use the anti-p39 primary antibody diluted to 1:50, we noticed that the optimal DAB incubation time is around 1 min. You must determine the optimal DAB incubation time for your cell lines and antibody of choice but bear in mind that prolonged reaction times can lead to high background.

Acknowledgments Work in our laboratory is supported by the U54 Moffitt Cancer Center-Ponce Health Sciences University Partnership NIH-NCI (#2U54CA163071-06), the NIMHD- NIAID funded Puerto Rico Clinical & Translational Research Consortium (#U54MD007587), the Molecular Genomics (MAGIC) Core (MBCL-RCMI Grant RR003050 MD007579) and its staff, the PHSU RCMI Program (Award Number #5G12MD007579-33 from The National Institute on Minority Health and Health Disparities), the PHSU-Moffitt Cancer Center Summer Research Program 2U54CA163071.07, and the PHSU RISE program under NIH Grant 2R25GM096955. Jennifer Caba´n-Rivera, Camille Chardo´n-Colo´n, Alberto Pedraza-Torres, and Yoan E. Rodrı´guez all contributed equally to the protocol optimization. References 1. Chen Y, Cui T, Yang L et al (2011) The diagnostic value of cytokeratin 5/6, 14, 17, and 18 expression in human non-small cell lung cancer. Oncology 80(5–6):333–340 2. Rekhtman N, Ang DC, Sima CS et al (2011) Immunohistochemical algorithm for differentiation of lung adenocarcinoma and squamous cell carcinoma based on large series of whole-tissue sections with validation in small specimens. Mod Pathol 24(10):1348–1359 3. Righi L, Vavala` T, Rapa I et al (2014) Impact of non–small-cell lung cancer-not otherwise specified immunophenotyping on treatment outcome. J Thorac Oncol 9(10):1540–1546 4. Pe´rez-Morales J, Mejı´as-Morales D, RiveraRivera S et al (2018) Hyper-phosphorylation of

Rb S249 together with CDK5R2/p39 overexpression are associated with impaired cell adhesion and epithelial-to-mesenchymal transition: implications as a potential lung cancer grading and staging biomarker. PLoS One 13(11): e0207483. https://doi.org/10.1371/journal. pone.0207483 5. Mathew EP, Nair V (2017) Role of cell block in cytopathologic evaluation of image-guided fine needle aspiration cytology. J Cytol 34 (3):133–138 6. Poojan S, Han-Seong K, Ji-Woon Y et al (2018) Determination of protein expression level in cultured cells by immunocytochemistry on paraffin-embedded cell blocks. J Vis Exp 135: E57369. https://doi.org/10.3791/57369

Chapter 7 Immunoblot Validation of Phospho-Specific Antibodies Using Lung Cancer Cell Lines Wilfredo M. Pedreira-Garcı´a, Jaileene Pe´rez-Morales, Camille Chardo´n-Colo´n, Jennifer Caba´n-Rivera, and Pedro G. Santiago-Cardona Abstract The cancer phenotype is usually characterized by deregulated activity of a variety of cellular kinases, with consequent abnormal hyper-phosphorylation of their target proteins. Therefore, antibodies that allow the detection of phosphorylated versions of proteins have become important tools both preclinically in molecular cancer research, and at the clinical level by serving as tools in pathological analyses of tumors. In order to ensure reliable results, validation of the phospho-specificity of these antibodies is extremely important, since this ensures that they are indeed able to discriminate between the phosphorylated and unphosphorylated versions of the protein of interest, specifically recognizing the phosphorylated variant. A recommended validation approach consists in dephosphorylating the target protein and assessing if such dephosphorylation abrogates antigen immunoreactivity when using the phospho-specific antibody. In this chapter, we describe a protocol to validate the specificity of a phospho-specific antibody that recognizes a phosphorylated variant of the Retinoblastoma (Rb) protein in lung cancer cell lines. The protocol consists in the dephosphorylation of the Rb-containing protein lysates by treating them with bovine intestinal phosphatase, followed by assessment of the dephosphorylation by immunoblot. Key words Retinoblastoma protein, Phospho-protein, Immunoblot, Lung cancer, Cell lines, Phosphorylation, Bovine intestinal phosphatase

1

Introduction The cancer phenotype is usually characterized by overactivation of signal transduction pathways that include components with kinase activity. As a consequence of the overactivation of such pathways, abnormally high levels of phosphorylation of several intracellular proteins occurs, and this hyper-phosphorylation usually has a disruptive effect on the normal regulation of protein function, which is in turn conducive to the cancer state. For example, the MAPK pathway can be abnormally overactive in cancer cells, an overactivation that can arise as a consequence of several mutational events,

Pedro G. Santiago-Cardona (ed.), Lung Cancer: Methods and Protocols, Methods in Molecular Biology, vol. 2279, https://doi.org/10.1007/978-1-0716-1278-1_7, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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such as amplification or activation of receptor tyrosine kinases or gain-of-function mutations in the Ras protein [1–3]. Overactivation of the MAPK pathway in the context of cancer or of rapidly diving cells, results in phosphorylation and activation of MAPK intermediaries such as Erk as part of an aberrant cancer-associated signaling that leads to uncontrolled cell proliferation [4]. As another example, the retinoblastoma (Rb) protein, one of the most important cellular tumor suppressors, is inactivated by hyper-phosphorylation in rapidly dividing cells, including cancer cells [5–8]. These, and many other examples of proteins whose function is regulated by phosphorylation has created a need to develop phospho-specific antibodies to specifically detect the phosphorylated variants of proteins whose function is regulated at the phosphorylation level. Expectedly, these phospho-specific antibodies have become important tools for molecular studies of cancer cells and tissues, especially when hyper-phosphorylation of key proteins is associated with the cancer phenotype, as well as in the clinic in the context of pathological assessment of tumors. Phospho-specific antibodies can be used to detect phosphorylated proteins in tissue sections by immunohistochemistry and by immunoblotting of protein lysates from cancer cell lines and cancer tissue biopsies. Obtaining reproducible results with these antibodies relies in great measure on their preliminary validation, including ensuring that they are indeed able to discriminate between the phosphorylated and unphosphorylated versions of the protein of interest, specifically recognizing the phosphorylated variant. Determining this can be relatively straightforward, and the approach consists in dephosphorylating the target protein and assessing if such dephosphorylation abrogates immunoreactivity in either an immunoblot or in an immunohistochemistry assay, or if it generates an electrophoretic mobility shift consistent with dephosphorylation as visualized in an immunoblot. In this chapter, we describe a protocol to dephosphorylate protein lysates obtained from lung cancer cell lines by treating them with a phosphatase enzyme, specifically with bovine intestinal phosphatase. This is followed by subjecting such dephosphorylated lysates to immunoblot analyses using a phospho-specific antibody, in our case against Rb phosphorylated in Serine 249 (S249). We have recently identified this phosphorylation as a biomarker predicting poor prognosis in lung cancer [9]. In an immunoblot assay, successful dephosphorylation should be observed in the form of an altered electrophoretic mobility of the protein of interest, since removal of phosphate groups affect a protein’s molecular weight, or as a complete abrogation of immunoreactivity when using the phospho-specific antibody in either immunoblots or immunohistochemistry.

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Materials All the solutions were prepared using distilled water, unless otherwise stated. Reagents and solutions were stored at room temperature, unless specified. Follow all waste disposal regulations when disposing waste materials.

2.1 Cell Lysis and Protein Extraction and Quantification

1. Cell lines of your choice. The procedure described below requires that the user has cell cultures ready for protein extraction. We optimized the procedure using a variety of lung cancer cell lines to detect total and phosphorylated Rb in them, but this protocol is adaptable to any cell line that expresses the phospho-protein of interest. It is important that you grow your cell cultures in the appropriate medium such that they are at approximately 90–95% confluence at the moment of protein extraction. 2. 1
Phosphate-Buffered Saline (PBS), pH 7.2. To prepare, dissolve 8.0 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4.2 H2O and 0.24 g of KH2PO4, in 800 mL of water. Adjust the pH to 7.2 with HCl and add distilled water to complete the volume to 1 L. Premade, ready-to-use PBS can also be purchased. 3. Trypsin-EDTA solution. This is for detaching cells from culture plates. We purchase this solution in premixed, ready-touse form. Alternatively, cells can also be mechanically detached using a rubber scraper. 4. RIPA lysis buffer. For convenience, we use a commercially available premixed 10
RIPA buffer, which is diluted to 1
when using. This solution can also be prepared in the laboratory using the standard recipe, which consists of 10 mM Tris– HCL, pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% (v/v) NP-40 (or 1% Triton X-100, if NP-40 is not available), 0.5% (v/v) sodium deoxycholate, 0.1% (v/v) SDS, and 150 mM NaCl. 5. Broad specificity protease inhibitor cocktail, use according to manufacturer’s specifications (see Note 1). 6. Protein concentration determination assay reagents. We perform a standard protein quantification using Bradford Assay, following its instructions, and using BSA as a quantification standard. We use Bio-Rad’s Protein Assay Dye Reagent Concentrate, but other substitute assays can be used.

2.2 Alkaline Bovine Intestinal Phosphatase (BIP) Treatment of Protein Lysates

1. Alkaline phosphatase from bovine intestinal mucosa (bovine intestinal phosphatase or BIP). We have optimized this protocol with the one provided by Sigma-Aldrich (Cat. No. P011410KU), provided at a specific activity of 6694 DEA Units/mg

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protein, and at a concentration of 123 Units/μL. Other commercially available phosphatases can be used as long as the manufacturer’s indications are followed. 2. 10
Dephosphorylation Buffer. This is the buffer for the BIP reaction. To prepare, dissolve 6.07 g of Tris base (50 mM), 5.84 g of NaCl (100 mM), 0.97 g of MgCl2 (10 mM) and 0.16 g of DTT (1 mM) in 800 mL distilled water, stir the solution until the reagents are dissolved. Adjust the pH to 7.9 and complete to a final volume of 1 L with distilled water. Numbers in parentheses are the molar concentrations of each component in the final solution. Use Table 1 as a reference for the components and their final concentrations in the working solution. 3. 2
BIP inhibitor cocktail. To prepare, dissolve 0.042 g of sodium fluoride (NaF), 0.092 g of sodium orthovanadate (Na3VO4) and 0.013 g of sodium pyrophosphate decahydrate 99% (NaPP) in 4 mL of distilled water in a 15 mL conical tube. Vortex the contents to ensure complete dissolution of the components, complete volume with distilled H2O to 5 mL. Use Table 2 as a reference for the components and their final concentrations in the working solution. This should be a 2
solution with concentrations of 50 mM Na3VO4, 5 mM Table 1 Amounts and final working concentrations of each of the reagents used for the preparation of the 10
dephosphorylation buffer

Reagent

Molecular weight (g/mol)

Amount to weight (g)

Expected final concentration (mmol/L)

Tris

121.14

6.07

50

NaCl

58.44

5.84

100

MgCl2

95.21

0.97

10

DTT

154.25

0.16

1

Table 2 Amounts and final working concentrations of each of the reagents used for the preparation of the 2
phosphatase inhibitors solution

Reagent

Molecular weight (g/mol)

Amount to weight (g)

Expected final concentration (mmol/L)

Sodium fluoride

42.0

0.042

200

Sodium orthovanadate

265.9

0.092

100

Sodium pyrophosphate

183.9

0.013

10

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NaPP, and 100 mM NaF that, when brought down to 1
in the final reaction mixture should be enough to inhibit the activity of 100 Units of BIP [10–14]. 2.3 SDS Polyacrylamide Gel Electrophoresis

1. 1.5 M Tris–HCl, pH 8.8. Dissolve 181.7 g of Tris base in 800 mL H2O. Adjust pH to 8.8 with concentrated HCl (use less concentrated HCL as you approach the desired pH). Add H2O to complete the volume to 1 L. Store at 4  C. 2. 0.5 M Tris–HCl, pH 6.8. To prepare, dissolve 60.6 g of Tris base in 800 mL H2O. Adjust pH to 6.8 with concentrated HCl (use less concentrated HCL as you approach the desired pH). Add H2O to complete the volume to 1 L. Store at 4  C. 3. 30% acrylamide/Bis-acrylamide solution, can be purchased in ready-to-use form (see Note 2). 4. Ammonium persulfate (APS) 10% (w/v) in water (see Note 3). Prepare by dissolving 0.5 g of ammonium persulfate in 5 mL of H2O. 5. Tetramethylethylenediamine (TEMED). 6. 10% SDS. Dissolve 10 g of SDS in 80 ml H2O. Complete volume to 100 mL. This solution can be kept at room temperature for up to 6 months. 7. 2
SDS sample loading buffer: 100 mM Tris–HCl pH 6.8, 4% (w/v) sodium dodecyl sulfate (or SDS, electrophoresis grade), 0.2% bromophenol blue, 20% (v/v) glycerol and 200 mM dithiothreitol (DTT). 8. 10
SDS-PAGE running buffer. Dissolve 30.0 g of Tris base, 144.0 g of glycine and 10.0 g of SDS in 1 L of H2O. Check that the pH is 8.3 but is expected that minimal or no pH adjustment will be required. Store the running buffer at room temperature and dilute to 1
before use. 9. Ethanol 70% (for cleaning electrophoresis glass plates). 10. Protein ladder molecular weight marker. When blotting for Rb, we use Bio-Rad Precision Plus Protein Kaleidoscope (Cat. No. 1610-375). You can use any other protein ladder provided it has sufficient markers in the molecular weight range of your protein of interest, in our case, around 110 kDa, which is the molecular weight of Rb.

2.4

Transfer

1. Nitrocellulose blotting membranes. We use 0.45 μm pore size for immunoblotting Rb, but a smaller pore size may be recommended should you want to adapt this protocol for low molecular weight proteins. Choose membrane pore size according to the molecular weight of the protein of interest.

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2. 1
transfer buffer: Dissolve 3.03 g of Tris base and 14.4 g of glycine in 500 ml of H2O. Add 200 ml of methanol, and complete to a final volume of 1 L with H2O. 3. Tris-buffered saline with Tween-20 (TBST): First prepare a 10
TBS stock by dissolving 24.2 g of Tris base and 87.6 g of NaCl in 800 ml of H2O, adjust to pH 7.6 with 1 M HCL, and complete to a final volume of 1 L. To prepare the TBST, add 1 mL of Tween-20 to 1 L of 1
TBS. 4. Ponceau-S membrane staining solution. We recommend that you use this dye to stain the membrane after transfer to verify the presence of protein in the membrane. This gives an indication of how effective the transfer was. Prepare a 0.5% (w/v) of Ponceau-S in a 1% acetic acid solution. To remove the Ponceau-S stain from the membrane before blotting, you need TBST with 5% dry milk. 5. Filter paper. 2.5 Immunoblotting Reagents

The protocols described in this chapter were optimized specifically for the antibodies described below and using lung cancer cell lines. The protocol can be adaptable to other phospho-specific antibodies, but additional optimization could be required, specifically in the antibody dilution and incubation times. 1. Blocking solution. Dissolve 0.5 g of bovine serum albumin (BSA) in 10 mL of 1
TBST. 2. Primary antibody against phosphorylated serine 249 in Rb (anti-Rb Phospho-Ser249). We purchase this rabbit polyclonal antibody from Sigma-Aldrich (Cat. No. SAB1305397) and use it at a dilution of 1:500 in TBST. We usually prepare primary antibody solutions in TBST that can be stored for several months at 4  C. To prepare such antibody solutions, first dissolve 0.5 g of BSA in 10 ml of TBST and add 30 μL of a 20% Sodium azide stock solution. Mix well and add the antibody at the indicated dilution (see Note 4). 3. Primary antibody against total Rb (mouse monoclonal 4H1, Cell Signaling Cat. No. 9309). In order to validate a phosphorantibody, you need to blot the protein lysate with both antibodies, one against the phosphorylated form of the protein and the other against the total protein. We use this antibody at a dilution of 1:1000 in TBST, and we prepare it exactly as described above for the antibody against phospho-S249. It is important to blot for total Rb, as the extent of Rb phosphorylation is assessed as the ratio of phosphorylated Rb to total Rb protein. 4. Secondary antibodies: we use horse-radish peroxidase (HRP)conjugated secondary antibodies. For mouse monoclonal primary antibodies, we use an HRP-conjugated, affinity-purified

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horse anti-mouse IgG (Cell Signaling, Cat. No. 7076S). For rabbit polyclonal primary antibodies, we use an HRP-conjugated, affinity-purified goat anti-rabbit IgG (Cell Signaling, Cat. No. 7074S). For both of these secondary antibodies, we prepare a TBST solution of the antibody exactly as described above for primary antibodies (except that we omit the sodium azide since we prepare fresh for each use), with the antibody diluted to 1:5000. 5. Supersignal West Pico Plus™ Chemilumiscent Kit. This kit is compatible with HRP-conjugated secondary antibodies. The selection of the kit to develop the chemiluminescent signal is dictated by the enzyme conjugated to the secondary antibody (HRP, versus alkaline phosphatase, for example). Other available kits are acceptable, provided they are compatible with your choice of antibodies. Use strictly following the kit’s instructions. 6. ChemiDoc imaging system and software, or other equivalent imaging system compatible with chemiluminescent signals. 2.6 Additional Laboratory Equipment and Plasticware

1. Tabletop centrifuge with capacity for 15 mL tubes, preferably refrigerated. 2. Conical tubes, 15 and 50 mL volumes. 3. Culture plates or bottles, 35 mm or other of your preference. 4. Gel electrophoresis system, including power source. Assemble and use as per manufacturer’s instructions. 5. Transfer system, including power source. Assemble and use as per manufacturer’s instructions. 6. 1.5 mL microcentrifuge tubes. 7. Refrigerated centrifuge for microcentrifuge tubes. 8. Ice bucket, ice. 9. Heat plate, with capacity to hold 1.5 mL microcentrifuge tubes. To be used at 30  C, 70  C, and 95–100  C. 10. Glass or plastic Pasteur pipettes.

3

Methods

3.1 Cell Lysis and Preparation of Protein Extracts

1. Ensure that you start with cells cultured at approximately 90–95% confluence. Collect cells by scraping them from the culture plate in 1–2 mL of 1
PBS. We culture cells in 35 mm culture plates, you should adjust the volume of PBS depending on your culture plate, but it is important to use the minimum volume of PBS to cover the entire plate surface with a thin PBS layer. Alternatively, detach cells from the plate by incubating in trypsin-EDTA solution at 37  C for 5 min (see Note 5).

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2. Transfer the cell suspension to a 15 ml tube and pellet cells by low speed centrifugation (5 min at 300–400
g). If you detach the cells using the trypsin-EDTA solution, before the centrifugation step you need to dilute it 1:10 with culture medium to ensure inactivation of trypsin. Remove the supernatant after the centrifugation. 3. Lyse cells by resuspending the cell pellet in RIPA buffer supplemented with the protease inhibitor cocktail (ensure that the cocktail does not include phosphatase inhibitors, since these will inhibit subsequent steps). Use the minimal possible volume of RIPA buffer to ensure adequate protein concentration in the lysate. Transfer the cell suspension to a 1.5 mL microcentrifuge tube. 4. Incubate at 4  C or on ice for 30 min to allow lysis to proceed. 5. Centrifuge tube for 10 min at 1400
g at 4  C. Transfer supernatant to a fresh microcentrifuge tube. 6. Quantify the protein in your cell lysate using your method of choice, using a BSA concentration curve to determine protein concentration in your sample (see Note 6). 3.2 Protein Lysate Dephosphorylation with Alkaline Bovine Intestinal Phosphatase (BIP)

1. Once the amount of protein in the lysate has been quantified, proceed to prepare the dephosphorylation reaction in a 1.5 mL microcentrifuge tube, following the indications shown in Table 3. Notice that you need to prepare three reactions for each protein lysate you wish to analyze: one reaction with protein lysate with only the BIP buffer (this acts as a negative control since the phosphorylation of lysate proteins should remain unaffected); a second reaction with protein lysate, BIP buffer and the BIP enzyme; and a third reaction to which you will add the BIP inhibitor cocktail in addition to the components of the second reaction. You can dephosphorylate between 400–500 μg of total protein with 100 Units of BIP [10–13]. Given the 123 U/μL activity of the BIP enzyme we use, we can use 1 μL of enzyme to treat 400–500 μg of total protein. You can prepare a final reaction volume of 50 μL. It is recommended that you aim to obtain highly concentrated protein extracts (at least 50 μg/μL) in order to be able to perform the reaction in such a small volume. 

2. Incubate reaction mixtures for 30 min at 30 C (if using the Sigma-Aldrich BIP), or as indicated by the manufacturer if using a BIP from other vendors (see Note 7). 3. Stop the reaction by transferring the reaction mixture to ice. 4. Take an aliquot from each reaction mixture, the volume should contain 20–30 μg of total dephosphorylated protein. Mix with an equal volume of 2
SDS-PAGE sample loading buffer. The remaining reaction mixture can be stored at -20  C.

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Table 3 Amounts of the components used in the setup of the BIP dephosphorylation reaction Amount of Protein (400–500 μg)

Amount of 10
Amount of Dephosphorylation Phosphatase Buffer (123 Units/μL)

Amount of 2
Phosphatase Inhibitor H2 O

Protein lysate + buffer

10 μL

5 μL

–

–

35 μL

Protein lysate + buffer + BIPa

10 μL

5 μL

1 μL

–

34 μL

Protein lysate + buffer + BIPa + BIP Inh.b

10 μL

5 μL

1 μL

25 μL

9 μL

Reaction

Final reaction volume can be 50 μL. Try to obtain concentrated protein lysates with a protein concentration of at least 50 μg/μL. This will allow you to use a volume of approximately 10 μL in the final 50 μL reaction a BIP ¼ Bovine Intestinal Alkaline Phosphatase b BIP Inh. ¼ Bovine Intestinal Alkaline Phosphatase Inhibitor

5. Denature proteins by heating the sample at 95–100  C for 5 min, or at 70  C for 30 min. Proceed to the SDS-PAGE separation of the sample described in the next section. 3.3 SDS Polyacrylamide Gel Electrophoresis and Transfer

1. Assemble your gel electrophoresis apparatus following manufacturer’s instructions. At this point you will only need to assemble the gel casting system needed to pour the gels. We use a standard Bio-Rad gel electrophoresis apparatus with its accompanying gel casting system. Be sure to clean thoroughly all glass plates with 70% ethanol. This will decrease the risk of forming air bubbles while pouring the gel into the glass plates. 2. Prepare the separating gel as follows: in a 50 mL conical tube, mix 7.9 mL of distilled H2O, 7 mL of the 30% acrylamide, bis-acrylamide mix, 5.0 mL of 1.5 M Tris–HCl pH 8.8 and 0.2 mL of 10% SDS. Add then 200 μL of 10% APS and 8 μL of TEMED. Gently mix avoiding the formation of bubbles. The gel will start rapidly polymerizing after the addition of APS and TEMED, therefore these two reagents should be the very last to be added to the mix, and the gel should be poured immediately after their addition. Maintaining the mix on ice will retard the polymerization (see Notes 8 and 9). 3. Allow the gel to completely polymerize for 30–60 min at room temperature (see Note 10). Be sure to always use freshly prepared or thawed APS (avoid re-freezing of leftovers, discard them). Loss of APS activity is manifested in abnormally long polymerization times.

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4. Prepare the upper stacking gel as follows: in a 15 mL conical tube, mix 4.1 mL of distilled H2O, 1.0 mL of 30% acrylamide/ bis-acrylamide mix, 750 μL of 0.5 M Tris–HCl pH 6.8, and 60 μL of 10% SDS. Mix gently avoiding foam and bubbles. When ready to pour, add 60 μL of 10% APS and 6 μL of TEMED. Mix and add it to the glass plates (see Note 11). 5. Immediately after pouring the stacking gel, insert comb being careful not to form any bubbles at the base of the wells. Allow the stacking gel to polymerize for 30–60 min at room temperature. 6. Carefully remove the comb and the bottom spacer. We do not recommend that you remove the comb straight out of the dry gel. Rather, we recommend that first you assemble the whole electrophoresis apparatus, including inserting the gel inside it, fill the liquid reservoir with running buffer, and then remove the comb. We use a standard protein gel electrophoresis apparatus from Bio-Rad. Dilute the 10
SDS-PAGE running buffer to 1
with distilled water and fill the assembled apparatus with it until you cover the gel. Only then we recommend that you slowly and carefully remove the comb (see Note 12). Using a glass or plastic Pasteur pipette, rinse the wells with running buffer to remove excess acrylamide. 7. After denaturing the protein samples as indicated in Subheading 3.2, steps 4 and 5, load them into the wells, being careful not to over flood the wells (if this happens, you risk having a protein sample over flooding into an adjacent lane). Remember also to load the protein ladder. 8. Place the lid on the electrophoresis apparatus, connect to a power supply, and run the gel at 160 V for 60 min (do not set a limit for Amperes). Monitor the run by following the bromophenol blue dye (from the sample loading buffer) front. Stop the run when the dye front reaches about two-thirds of the length of the frontal glass plate. 3.4 Transfer of Proteins to Nitrocellulose Membranes

1. Disassemble the electrophoresis apparatus and remove the gel assembly. Very gently and carefully separate the glass plates from the gel inserting a fine spatula in between the glass plates, and slowly twisting the spatula until the plates start to separate from the gel. Use a razor blade to carefully remove the stacking gel without damaging the separating gel. Rinse the gel with transfer buffer, keep it submerged in transfer buffer, never let the gel dry. 2. Cut a piece of nitrocellulose membrane of the approximate size of the gel (a little bit larger so you can handle it by the edges without touching the gel) and immerse in cold transfer buffer for 2–5 min. Always handle the membrane with gloves or with tweezers. Never touch the membrane with bare hands, this may

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leave fingerprints oils on the membrane and this in turn will prevent even wetting of the membrane. This usually results in areas in the membrane where transfer of proteins is impaired. 3. Pre-wet several pieces of filter paper (cut in a size similar to the gel) in cold transfer buffer by submerging one side of the paper first and then slowly lowering it into the buffer. 4. Assemble the gel electro-transfer cassette following the manufacturers´ instructions. Avoid bubbles being trapped between the gel and the membrane, as protein transfer will not occur adequately at these sites. 5. Insert the transfer cassette into the electro-transfer unit. It is usual for electro-transfer units to have a special compartment for an ice block or any other cooling device. As the transfer process generates heat and the transfer buffer can get warm (or even hot), it is recommended that the ice block/cooling devices are used. The transfer can be done in a cold room, or the whole transfer apparatus can be inserted in a tray and surrounded with ice during the transfer process. 6. Transfer for 60 min at 100 V (do not set a limit for Amperes). 7. After the transfer is completed, disassemble the unit. Wash the membrane in TBST to remove residual SDS and potential gel fragments. 8. Check the efficiency of transfer by staining the membrane in Ponceau-S solution for 5 min at room temperature. Record an image of the Ponceau-stained membrane using a document scanner or camera. Even transfer (no air bubbles) of equal amount of proteins per lane should be seen in the Ponceaustained membrane. Never allow the membrane to get dry. Keep it moist during the documentation process by wrapping it in plastic wrap after soaking in transfer buffer. See Notes 13–15 for transfer troubleshooting tips. 3.5 Immunoblotting, Image Development, and Capture

1. Remove the Ponceau-S staining from the membrane by incubating in TBST containing 5% milk. You will notice the milk solution turning red. Discard and rinse the membrane with TBST (no milk). Repeat until no trace of the red Ponceau-S stain remains in the membrane. At this point you should only see the rainbow-colored protein markers. Repeat a final rinse with TBST (see Note 16). 2. Block membranes in TBST with BSA (see Note 16). You can block for 2 h at room temperature or overnight at 4  C. Use constant rotation to ensure that the membrane is constantly bathed by the solution. 3. Incubate membranes with the primary antibody. Like the blocking step, primary antibody incubation can be done either 2 h at room temperature, or overnight at 4  C. Use constant motion during this step as well (see Note 17).

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4. Wash three times in TBST. 5. Incubate with secondary antibody for 1 h at room temperature while agitating. 6. For development of the membrane chemiluminescence, we use the Supersignal West Pico Plus™ Chemilumiscent Kit (Thermo Scientific Cat. No. 34580), following the procedures exactly as described by the manufacturer. We add 1 mL of substrate solution per membrane. Do not leave it for more than 3 min (see Note 18). 7. Capture the chemiluminescent signal using a ChemiDoc imaging system or its equivalent (we use ChemiDoc XRS+), using the accompanying image software for image capturing and quantification of signal intensity. 3.6 Interpretation of Results

We usually run in parallel immunoblots using the phosphor-specific antibodies as well as antibodies recognizing total Rb protein. You need to assess phosphorylation of your protein of interest in particular residues, relative to the total amount of that specific protein. In our case, we document protein Rb phosphorylation as the ratio of phosphorylated Rb to total Rb [9, 14]. When using an antibody against the total protein, usually phospho-proteins can be appreciated in western blots as a doublet consisting of two bands migrating close to each other [9, 14]. In that doublet, the upper band usually corresponds to the phosphorylated form of the protein (as phosphorylation increases the protein’s molecular weight), while the lower band corresponds to the unphosphorylated form. Alternatively, if a doublet is not apparent, the phosphorylated form may appear as a single band but of higher molecular weight relative to the unphosphorylated form. You should still be able to see this doublet (or the higher weight band) in the sample treated with BIP buffer alone, since this is your negative control reaction. However, in the sample treated with BIP, the doublet should disappear (only the lower band should remain), or the higher molecular weight form should be displaced to a lower molecular weight position in the gel. When using the antibody that recognizes only the phosphorylated form, BIP treatment should eliminate immunoreactivity of the slower migrating higher molecular weight bands, confirming that these bands correspond to phosphorylated versions of the protein. This change is indicative of the removal of the phosphate groups from the protein, which translates into a faster electrophoretic mobility. This change should be reversed when the sample is treated with both the BIP and the phosphatase inhibitor cocktail, so that the band pattern in this sample is comparable to untreated samples or to samples treated with BIP buffer alone without enzyme.

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Notes 1. Protease inhibitors in the cell lysis buffer, combined with maintaining the protein lysate on ice at all times, minimize protein degradation during extraction and handling of the lysate. We use a broad specificity protease inhibitor cocktail from SigmaAldrich (Cat. No. P8340-5ML). Other alternatives are acceptable, follow manufacturer’s instructions regarding working concentration and handling. Be sure that the inhibitor cocktail does not contain phosphatase inhibitors, as these will inhibit BIP activity. 2. We use the premixed Bio-Rad acrylamide solution. Store at 4  C. Please be aware that polyacrylamide is toxic. Carefully read the accompanying Materials Safety Data Sheet for specific instructions on how to handle and dispose polyacrylamide solutions. 3. Make small volume aliquots and store at 20  C. Avoid repeated freezing and thawing cycles. Do not use leftovers for future experiments, a fresh aliquot should be thawed for each experiment. 4. Sodium azide is used as a preservative to prevent bacterial growth in the solution. This is highly recommended if you plan to reuse the primary antibody solution and store it for long term use. This solution can be stored and reused for several months, but you need to be attentive for signs of bacterial and fungi contamination such as a strong odor or cloudiness in the solution. In such case, discard and prepare a fresh solution. Use of contaminated antibody solution usually yields high background in western blots, meaning that it is time to replace the solution with a fresh one. 5. Avoid over-trypsinization as it may kill cells. Fresh trypsin solution should detach cells in under 5 min. If you find that 5 min under trypsin-EDTA are not enough to detach cells from the plate, it is better to get a fresh trypsin-EDTA batch than prolonging the trypsinization time. Avoid trypsin when studying membrane proteins. 6. We perform a standard protein quantification assay using Bradford Assay, following its instructions, and using BSA as a quantification standard. Other substitute assays can be used. After quantification, protein lysates that will not be used immediately can be stored at 20  C for up to 3 months (protein integrity cannot be ensured beyond that). 7. This treatment should eliminate immunoreactivity if the antibody is indeed recognizing a phosphorylated form of the protein of interest. This will be observed after western blot

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evaluation. Remember also run in the SDS-PAGE the controls treated with phosphatase buffer alone, as well as a control with phosphatase in the presence of phosphatase inhibitors. 8. It is recommended that you add a thin layer of isopropanol on top of the separating gel solution immediately after pouring it. Isopropanol is not miscible in water thus it will form a distinct layer. Adding isopropanol eliminates any bubbles on the surface of the separating gel solution and will produce a smooth surface. 9. For Rb, which has a molecular weight of approximately 110 kDa, we use a 10% polyacrylamide separating gel. The % of polyacrylamide you will choose depends on the molecular weight range in which you want to have good resolution. Take this into consideration if you wish to adapt this protocol to other phosphor-proteins. 10. After 45 min, you can verify if the gel has polymerized by gently tilting the casting apparatus sideways. Only the isopropanol layer should move while the underlying separating gel should be static if it has polymerized. 11. To save time, you can start preparing the stacking gel while the separating gel is polymerizing. However, do not add the TEMED and the APS until immediately before adding the stacking gel to the casting apparatus. The stacking gel without APS and TEMED can be kept on ice until pouring. The % of acrylamide of the stacking gel is usually smaller (4–6%) than that of the separating gel. 12. If you slowly remove the comb having the gel submerged in running buffer, you will notice that as you remove the comb the empty well space is immediately filled with buffer. This will avoid the collapse of the well that is experienced if you remove the comb out of the dry gel, as a vacuum is formed inside the well as the comb is retrieved. 13. Do not exceed the transfer time as this leads to gel shrinkage and distortion of the membrane. In case of poor transfer efficiency, opt for making thinner gels, rather that prolonging transfer time. 14. If there are unstained “white spots” on the membrane seen after Ponceau-S staining, this may have been caused by air bubbles trapped between the gel and the membrane. Make sure to remove all the bubbles when preparing the transfer cassette. An air bubble does not necessarily ruin an experiment, it depends on its size and on the molecular weight range in which it formed. If your protein of interest is not in this range, you may choose to proceed with the subsequent steps.

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15. Ponceau-S staining may also help you to spot degraded proteins, which are appreciated as a diffuse smear in the lower half on the membrane. In this case, ensure that you are taking all the precautions necessary to deal with protein degradation, such as not using protein samples that have been stored for prolonged times (or repeatedly frozen-thawed), ensuring that cell lysis was done on ice and the samples were kept cold or refrigerated all the times, and that you added protease inhibitors to the RIPA buffer. 16. When doing a western blot using antibodies against phosphorylated residues, it is important that you thoroughly remove any traces on milk from the membrane. Casein (milk protein) is heavily phosphorylated and any traces of milk in the membrane can lead to high background due to nonspecific antibody binding. For the same reason, the blocking solution must not contain milk. The blocking step is usually one of the steps in which you can make adjustments in case you experience high background levels. 17. Longer incubation periods are recommended if you are having trouble obtaining strong signals. However, be aware that longer incubation times also increase the likelihood of obtaining a strong background. The length of the incubation time (with primary antibody) is one of the factors that affect signal strength. Dilution of antibody also usually affects background noise. If you are obtaining too much background, in addition to extending blocking time, you can try diluting the primary antibody. Conversely, try concentrating the primary antibody if you obtain weak signals. 18. This step can be performed for 30 s to 3 min. If you are experiencing weak signals, in addition to using more concentrated primary antibody and/or increasing incubation time, you can try developing the membrane for longer (but do not exceed 3 min, as this may blacken the membrane). Some antibodies give a very strong signal and in such cases 30 s to 1 min is sufficient.

Acknowledgments Our work is supported by the U54 Moffitt Cancer Center-Ponce Health Sciences University Partnership (NIH-NCI #2U54CA163071-06), the PHSU-MCC Partnership Pre-doc to Post-doc Transition program (NIH-NCI #2U54CA163071-06 and 2U54CA163068-06), the NIGMS-RISE Program support (R25GM082406), the NIMHD- NIAID funded Puerto Rico Clinical & Translational Research Consortium (#U54MD007587), the Molecular Genomics (MAGIC) Core (MBCL-RCMI Grant

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RR003050 MD007579) and its staff, the PHSU RCMI Program (Award Number #5G12MD007579-33 from The National Institute on Minority Health and Health Disparities), the University of Puerto Rico at Ponce RISE Program (#R25GM082406), and the Post Hurricane Marı´a Aid for Researchers Grant Continuity Track Program. References 1. Downward J (2003) Targeting RAS signalling pathways in cancer therapy. Nat Rev Cancer 3:11–22 2. Biankin AV, Waddell N, Kassahn KS et al (2012) Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes. Nature 491:399–405 3. Hodis E, Watson IR, Kryukov GV et al (2012) A landscape of driver mutations in melanoma. Cell 150:251–263 4. Eblen ST (2018) Extracellular-regulated kinases: signaling from Ras to ERK substrates to control biological outcomes. Adv Cancer Res 138:99–142 5. Rubin SM (2013) Deciphering the retinoblastoma protein phosphorylation code. Trends Biochem Sci 38(1):12–19 6. Hatakeyama M, Weinberg RA (1995) The role of RB in cell cycle control. Prog Cell Cycle Res 1:9–19 7. Burkhart DL, Sage J (2008) Cellular mechanisms of tumour suppression by the retinoblastoma gene. Nat Rev Cancer 8(9):671–682 8. MacDonald JI, Dick FA (2012) Posttranslational modifications of the retinoblastoma tumor suppressor protein as determinants of function. Genes Cancer 3(11–12):619–633 9. Pe´rez-Morales J, Mejı´as-Morales D, RiveraRivera S et al (2018) Hyper-phosphorylation of Rb S249 together with CDK5R2/p39 overexpression are associated with impaired cell

adhesion and epithelial-to-mesenchymal transition: implications as a potential lung cancer grading and staging biomarker. PLoS One 13 (11):e0207483 10. Sigma-Aldrich. Procedures for Dephosphorylation|Sigma-Aldrich. https:// www.sigmaaldrich.com/life-science/met abolomics/enzyme-explorer/analyticalenzymes/alkaline-phosphatase/dephosphory lation.html#protein. Published 2018. Accessed 11 June 2018 11. PhosphoSolutions. Protocols – Phosphatase Treatments|PhosphoSolutions. https://www. phosphosolutions.com/protocols-phospha tase-treatments/#cellproteins. Accessed 11 June 2018 12. Brumbaugh K, Johnson W, Liao W-C et al (2011) Overview of the generation, validation, and application of Phosphosite-specific antibodies. Methods Mol Biol 717:3–43. https://doi. org/10.1007/978-1-61779-024-9_1 13. Bordeaux J, Welsh A, Agarwal S et al (2010) Antibody validation. BioTechniques 48 (3):197–209 14. Santiago-Cardona PG, Pe´rez-Morales J, Gonza´lez-Flores J (2018) Detection of retinoblastoma protein phosphorylation by Immunoblot analysis. Methods Mol Biol 1726:49–64. https://doi.org/10.1007/978-1-4939-75655_6

Chapter 8 Detection of Non-Small Lung Cell Carcinoma-Associated Genetic Alterations Using a NanoString Gene Expression Platform Approach Johan Staaf, Mats Jo¨nsson, and Anna F. Karlsson Abstract In non-small cell lung cancer (NSCLC), mutation detection and fusion gene status are treatment predictive and, hence, key factors in clinical management. Lately, alternate splicing variants of MET have gained focus as NSCLC tumors harboring a MET exon 14 skipping event have proven sensitive toward targeted therapy. Reliable methods for detection of genetic alterations in NSCLC have proven to be of increased importance. This chapter provides with hands-on experience of the NanoString gene expression platform for detection of genetic alterations in NSCLC. Key words NanoString, Gene expression, Fusion gene, Non-small cell lung carcinoma

1

Introduction Lung cancer accounts for 1.3 million deaths annually with an incidence rate of 1.6 million per year [1]. The majority of lung cancer patients are being diagnosed with advanced disease limiting the curative treatment options (mainly surgery). Lung cancer is broadly divided into small cell carcinoma (SCLC) and non-small cell carcinoma (NSCLC). NSCLC accounts for 80-85% of all lung cancers and is further subdivided into adenocarcinoma (AC), squamous cell carcinoma (SqCC), large cell carcinoma (LCC), and large cell neuroendocrine carcinoma (LCNEC) which represent the four main histological subtypes [2]. Histological prediction is made on the basis of morphology, growth pattern, and cell of origin. Histology is an important treatment predictive factor as, e.g., SqCC tumors have proved less sensitive toward specific cytotoxic agents like pemetrexed [3, 4]. Histological assessment of tumor material has traditionally been performed using morphological evaluation of hematoxylin and eosin (H&E) staining combined with immunohistochemical (IHC) analysis of protein markers associated with the

Pedro G. Santiago-Cardona (ed.), Lung Cancer: Methods and Protocols, Methods in Molecular Biology, vol. 2279, https://doi.org/10.1007/978-1-0716-1278-1_8, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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histological subtypes [2]. These methods are well established and relatively cheap but require skilled trained personnel and pathologists. Occasionally, the methods require a lot of material as multiple stains need to be performed in order to conclusively assess tumor histology. This is a major issue in a disease such as lung cancer where the majority of patients are diagnosed on the basis of small biopsies and cytology specimens obtained from e.g., bronchoscopy. For patients diagnosed with advanced disease, treatment options vary depending on histology, stage, tumor localization, and more recently molecular alterations. In total, about 50% of NSCLC cases have been associated with activating mutations in specific proto-oncogenes (typically different tyrosine kinases) including EGFR , KRAS , MET, BRAF, and HER2, as well as gene fusions involving ALK, RET, ROS1, and NTRK and FGFR families. Lately, a splicing variant involving translational skipping of exon 14 in MET has gained focus as tumors harboring this alteration have proven sensitive toward targeted therapy (i.e., crizotinib) [5]. Activating mutations and rearrangements in these genes represent highly desirable therapeutic targets due to: (1) the concept of oncogene addiction, i.e., genetic alterations that governs the oncogenic potential of malignant cells that are crucial for tumor survival, and (2) that these alterations are mutually exclusive to other driver events [6–11]. Based on the successful development of targeted therapy, e.g., tyrosine kinase inhibitors (TKIs), against activating alterations that prolong the progression free survival of patients, clinical management of lung cancer today requires methods for accurately assessing mutation, and gene fusion status for deciding first line therapy. In a clinical context, for NSCLC management there are several demands on such methods: (1) quick turn-around-time, (2) the ability to perform analysis on scarce amounts of tissue, (3) no technical restrictions on using degraded DNA or RNA (which often is the case when using archival tumor tissue such as formalin-fixed paraffin-embedded, FFPE, tissue), and (4) result reproducibility and stability. For gene fusion detection, the current standard procedure is still often IHC in combination with fluorescence in situ hybridization (FISH), which is labor intensive and highly dependent on skilled personnel for correct interpretation. To minimize turnaround time, simultaneous analysis of mutation status and gene fusion status would be preferable. Combined DNA and RNA methods are available such as the Foundation One [12] or the MSK-IMPACT [13] assays, but these methods are often non-accessible in a clinical setting where treatment predictive molecular tests are performed on local pathology departments. Although targeted assays for simultaneous mutation detection and gene fusion status are available through the Illumina (AmpliSeq Focus Panel) [14] and the Ion Torrent (Oncomine assays through ThermoFisher Scientific) [15] platforms (two commonly used

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sequencing platforms in local pathology departments), these combined assays often struggle with low sample processing success rates for the RNA-based fusion gene detection module. Fusion gene detection and splice variants such as MET exon 14 skipping events can be detected using a DNA-based approach but requires deep sequencing and have proven less sensitive compared with RNA-based methods [16]. One platform for simultaneous gene expression profiling, gene fusion detection, and detection of splice variants applicable to small amounts of FFPE-derived RNA is the NanoString technology [17]. This multiplexed gene expression platform has several advantages in a clinical context, as stated here before, including quick turnaround time, the ability to perform analysis on small amounts of degraded RNA and high reproducibility [18]. The NanoString platform is highly flexible regarding which genes to analyze, as the technique is based on custom designed probes that span the exon-exon junction of the fusion gene, a second probe that recognizes the target and a protector probe that utilizes the toehold exchange technology to create a thermodynamic balance to prevent off-target hybridization and ensure signal only when there is a perfect match between probes and target sequence (Fig. 1a) [19, 20]. NanoString is a hybridization-based technique where total RNA is hybridized to the custom designed probes, labeled, and counted. Elevated probe counts indicate highly expressed genes for which the probe in focus corresponds to. Specifically designed fusion probes indicate which specific fusion partner is involved (Fig. 1b). Designing probes so that they correspond to the 50 and 30 part of oncogenes known to be involved in fusion events gives the benefit of detecting fusions with novel partners (for which no fusion specific probe exists). Counts are used through a 30 /50 imbalance ratio where elevated 30 expression in a specific gene indicates fusion [21] (Fig. 1c). By this design, the user retrieves fusion gene status as well as gaining knowledge on gene expression profiles of the genes defined in one single, multiplexed assay. The flexibility of the platform also allows a user to combine different analysis modules. For instance, we have shown using the nCounter Elements chemistry that an assay can be created that provides both fusion gene status of therapeutically targetable genes and histological subtype prediction in a single experiment [22]. This chapter provides hands-on experience and pointers on detection of NSCLC associated genetic alterations such as fusion genes, splice variants, and gene expression profiling using the NanoString technology, including wet-lab protocols and data analysis.

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Fig. 1 NanoString probe design and example of fusion gene detection of the ALK gene. (a, b) NanoString probes are designed to span the exons of the ALK gene as well as to target specific fusions. Both cartoons in (c) represent the same results visualized in two different manners. The exon spanning probes are visualized in a 30 /50 fashion. Hence, calculating the ratio between the counts representing the 30 spanning probes and the 50 spanning probes of the ALK gene indicates fusion events involving ALK. The left pane visualizes the calculated ratio of the 30 and 50 probes of the ALK gene on the x-axis and fusion specific probe counts on the y-axis. Fusion negative samples appear in the lower left quadrant while fusion specific samples appear in the upper right quadrant. A fusion event involving the ALK gene with a novel partner would appear in the lower right quadrant. The right pane illustrates probe counts representing the 30 and 50 exons as well as fusion specific probes. Elevated bars representing the 30 probes and low bars representing the 50 of the ALK gene indicate fusion of the ALK gene. Elevated bar corresponding a fusion specific probe reveals that the specific fusion is EML4-ALK. ((a) is re-printed from [21] with permission from Elsevier through Copyright Clearance Center. (b) is re-printed from [17] with permission from NanoString Technologies)

2

Materials RNA and DNA from FFPE tissue can be extracted in many different ways and there are multiple vendors on the market that offer extraction kits. The extraction guidelines below have proven suitable for small amounts of fixated tumor material. Revising the protocol may be of importance (see Note 1). Extracted RNA should be stored at 80 ˚C and extracted DNA should be stored at 20 ˚C until further use in downstream applications.

2.1 Tumor Cell Content Assessment

Previous to the procedure, you need to have the following prepared beforehand. 1. You need to already have performed an H&E stain of the block of interest and to have assessed tumor cell rich areas (see Note 2). 2. You also need to have sectioned the tissue (see Note 3) and have the tissue sections stored in RNase/DNase-free safe-lock 1.5 mL tubes.

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2.2 DNA/RNA Extraction

1. We use the AllPrep DNA/RNA FFPE kit (QIAGEN, Hilden, Germany). After evaluation of multiple methods and kits, we selected the AllPrep DNA/RNA FFPE Kit for this procedure.

2.3 NanoString Fusion Gene Detection

1. nCounter Elements TagSet 72-plex (NanoString Technologies, Inc., Seattle, WA, USA). 2. Custom designed probes synthesized by IDT (Integrated DNA Technologies, Coralville, IA, USA) and provided through NanoString (see Note 4). 3. NanoString SPRINT instrument (NanoString Technologies). 4. TE-Tween® buffer. We buy a premixed, ready-to-use 10 mM Tris–HCl, pH 8.0, 1 mM EDTA (or 10 mM Tris–EDTA pH 8.8 solution), and to this we add Tween® 20 to a final concentration of 0.1%. 5. Thermal cycler with possibility to adjust the lid temperature. 6. Cell line pool or any other RNA reference material of choice. 7. Download and install R from https://cran.r-project.org/. 8. Install nSolver Analysis Software provided by NanoString. Installation packages are available through the NanoString website. (http://www.nanostring.com/products/nSolver requires registration).

2.4 NanoString Gene Expression

1. nCounter XT CodeSet Gene Expression Assays (NanoString Technologies, Inc., Seattle, WA, USA). 2. Custom designed probes synthesized by IDT (Integrated DNA Technologies, Coralville, IA, USA) and provided through NanoString (see Note 4). 3. NanoString SPRINT instrument (NanoString Technologies). 4. Thermal cycler with possibility to adjust the lid temperature. 5. Cell line pool or any other RNA reference material of choice. 6. Download and install R from https://cran.r-project.org/. 7. Install nSolver Analysis Software provided by NanoString. Installation packages are available through the NanoString website. (http://www.nanostring.com/products/nSolver requires registration).

2.5 General Laboratory Equipment, Supplies and Reagents

1. Micro-pipettes of different volumes, tips. 2. Benchtop microcentrifuge. 3. Ice bucket with ice. 4. Thermal cycle strip tubes. 5. DNase/RNase free H2O.

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Methods Fusion gene detection requires the use of the NanoString nCounter Elements chemistry. By using the Elements chemistry, fusion gene status in combination with gene expression status of many different genes can be retrieved simultaneously. This allows creation of a single assay that can assess both gene fusions and expression of genes associated with NSCLC histological subtypes [22]. The nCounter XT chemistry is suitable for mRNA gene expression profiling only.

3.1 RNA/DNA Extraction

After evaluation of multiple methods and kits the AllPrep DNA/RNA FFPE Kit was selected, and we have optimized this procedure using DNA/RNA extracted with this kit. We recommend that you strictly follow the kits instructions for the extraction step. Using this protocol, you will retrieve DNA as well as RNA from the same FFPE sections (see Note 1) (Fig. 2).

3.2 Fusion Gene Detection Using the NanoString Technology

This section describes sample hybridization and further processing on the NanoString instruments (see Note 5). Although this section is a step-by-step process of the NanoString hybridization process and data generation, it is highly recommended to always follow the manufacturer’s instructions primarily by using the supplied protocol by NanoString when purchasing the NanoString products. It is recommended to process 11 RNA samples and one reference RNA in a single experiment. The recommended RNA input varies from 100 ng to 500 ng. In our experience, 500 ng RNA input is to be preferred (see Note 6). We strongly recommend that you include an RNA reference (see Note 7).

3.2.1 Hybridization Using the nCounter Elements Chemistry (Combined Gene Expression and Fusion Gene Detection)

1. Program a thermal cycler to 24 h at 67  C followed by a 4  C indefinite step. The lid temperature should be set to 72  C. The decrease from 67  C to 4  C should be performed ramping down 1  C per second to increase hybridization efficiency. 2. Remove aliquots of TagSet, Probe A, Probe B, and Protector Probe from the freezer and thaw at room temperature. Invert several times to mix well and spin down reagents. 3. Start the thermal cycler and pause the program in order to keep the thermal cycler at 67  C. 4. Using strip tubes, dilute your reference RNA (100 ng) and sample RNA (500 ng) in 3μL DNase/RNase free H2O. Keep on ice. 5. Always create separate 30 working pools for Probe As, Probe Bs, and Protector Probes.

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Fig. 2 DNA and RNA extraction. Workflow of routine sample processing for nucleic acid extraction using the Qiagen AllPrep DNA/RNA FFPE kit

6. Make sure you have ready the TE-Tween® buffer, prepare it as described in Subheading 2.3. Due to the low concentration of the final probe pools, NanoString suggests using the TE-Tween® buffer when preparing probe pool dilutions. 7. Mix 4μL Probe A with 29μL of the TE-Tween® buffer. Mix well and quick spin to bring contents to the bottom of the tube. The final concentration of each Probe A in the working pool will be 0.6 nM. The final concentration of each Probe A in the 30μL hybridization assay will be 20 pM. 8. Mix 4μL Probe B with 29 of the TE-Tween® buffer. Mix well and spin to bring contents to the bottom of the tube. The final concentration of each Probe B in the working pool will be 3 nM. The final concentration of each Probe B in the 30μL hybridization assay will be 100 pM.

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9. Mix 4μL Protector Probe with 29μL of the TE-Tween® buffer. Mix well and spin to bring contents to the bottom of the tube. The final concentration of each protector probe in the working pool will be 1.2 nM. The final concentration of each protector probe in the 30μL hybridization assay will be 40 pM. 10. Add 70μL of hybridization buffer to the TagSet tube. 11. Add 7μL each of the 30 Working Probe A Pool and 30 Working Protector Probe Pool. Mix well by flicking the tube and briefly spin down. 12. Add 7μL of the 30 Working Probe B Pool to complete the master mix. Mix well by flicking the tube and briefly spin down again. 13. Keep the master mix on ice. 14. On ice, add 12μL of master mix to each of the 12 tubes containing the 3μL of RNA prepared in step 4 using a fresh pipette tip for each tube. Slowly pipette the master mix to avoid mechanically disrupting the TagSets. 15. Mix the reagents by inverting the strip tubes several times. Briefly spin down the hybridization reactions. 16. Transfer the strip tube containing RNA and master mix to the preheated thermal cycler to initiate hybridization. 17. Proceed to Subheading 3.2.3. 3.2.2 Hybridization Using the nCounter XT Chemistry (Gene Expression Only)

1. Program a thermal cycler to 24 h at 65  C followed by a 4  C indefinite step. Lid temperature should be set to 70  C. The decrease from 65  C to 4  C should be performed ramping down 1  C/s to increase hybridization efficiency. 2. Remove aliquots of both Reporter CodeSet and Capture ProbeSet reagent from the freezer and thaw at room temperature. Invert several times to mix well and spin down reagent (see Note 8). 3. Start the thermal cycler and pause the program in order to keep the thermal cycler at 65  C. 4. Using strip tubes, dilute your reference RNA (100 ng) and sample RNA (500 ng) in 5μL DNase/RNase free H2O. Keep on ice. 5. Create a master mix by adding 70μL of hybridization buffer to the tube containing the Reporter CodeSet. Do not remove the Reporter CodeSet from this tube. Do not add the Capture ProbeSet to the master mix. Invert repeatedly to mix and spin down master mix. 6. Add 8μL of master mix to each of the 5μL pre-aliquoted RNA samples from step 4. Use a fresh tip for each pipetting step to accurately measure the correct volume.

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7. Invert the Capture ProbeSet tube to mix and spin down the contents. Add 2μL of Capture ProbeSet to each tube immediately before placing at 65  C. Cap tubes and mix the reagents by inverting the tubes several times and flicking with a finger to ensure complete mixing. Briefly spin down and immediately place the tubes in the preheated 65  C thermal cycler (see Note 9). 8. Incubate reactions for 16 h (minimum) to 24 h. Maximum hybridization time should not exceed 48 h. Ramp reactions down to 4  C and process the following day. Do not leave the reactions at 4  C for more than 24 h or increased background may result (see Note 10). 9. Proceed to Subheading 3.2.3. 3.2.3 Operating the NanoString SPRINT Instrument

1. Make sure to have the run prepared with sample names and correct RLF file. 2. Bring out the cartridge for at least 30 min in room temperature. 3. Remove the strip from the thermal cycler and keep on ice or ice cooler until ready to load the samples on to the cartridge. 4. Spin down carefully the strip and add extra H2O, from 15μL to 30μL. Normally there is a volume loss during the hybridization process. Adding 17μL of H2O to each sample is normally sufficient but this can vary. 5. Transfer the samples with a pipette starting with sample nr:1 in well 1 on the cartridge. 6. Seal the wells/ports with provided transparent sealer. 7. Remove green seal. 8. Initiate run. 9. After the run is finished, remove the used cartridge, and proceed to Subheading 3.2.4.

3.2.4 NanoString Data Processing

The development of the analysis tools described below has been done based on a published method [21] and is applicable to data produced using the nCounter Elements chemistry as well as the nCounter XT chemistry (see Notes 11 and 12). 1. Output data files from the NanoString instrument (RCC files) and load them into the nSolver Analysis software (see Notes 12 and 13). 2. Load the exported csv file from the nSolver Analysis Software to R using the command: data