April 2021, Vol. 35, Issue 2 The Scientist

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APRIL 2021 | VOL. 35, ISSUE 2 | WWW.THE-SCIENTIST.COM

ADVANCING AGAINST METASTASIS

CANCER CELLS CAN SPREAD EARLY AND LIE DORMANT FOR YEARS

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APRIL 2021

Contents

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THE SCIENTIST

THE-SCIENTIST.COM

VOLUME 35 ISSUE 2

Features

ON THE COVER: © ISTOCK.COM, SPECTRAL-DESIGN

24

32

Advancing Against Metastasis

Cancer’s Extra Genome

It’s now clear that treating primary tumors early doesn’t always stop cancer’s deadly spread, prompting a hunt for ways to target disseminated cells directly.

In the past decade, researchers have come to realize that tumors harbor bits of extrachromosomal DNA that can drive malignancy.

BY SHAWNA WILLIAMS

BY PAUL MISCHEL

04 . 2021 | T H E S C IE N T IST

3

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APRIL 2021

Department Contents 18

11

FROM THE EDITOR

Our Expanding Universe

As with the evolution of astronomy, new insights in biology beckon just beyond our conceptual and observational reach. BY BOB GRANT

14

CRITIC AT LARGE

Machine Learning and Cancer Diagnosis

While artificial intelligence could improve detection of tumors at their earliest stages, it also risks identifying malignancies that would never cause patients any harm. BY ADEWOLE S. ADAMSON

18

Monkeys can account for others’ behavior and circumstances; fluorescent tool illuminates cell-cell contacts

xx 40

NOTEBOOKS

40 THE LITERATURE Flexible synapse strength may underpin mammalian brain complexity; “rogue” protein could contribute to humans’ high cancer rates; obesity-linked gut bacteria may worsen graft-versus-host disease

43 SCIENTIST TO WATCH Bisrat Debeb: Studying Cancer’s Spread BY CATHERINE OFFORD

44 BIOBIZ

LAUREN BRENT; © MELANIE LEE; PHOTO COURTESY OF MD ANDERSON

Getting to the Source

Many major biopharmaceutical companies are developing or acquiring drugs that target the NLRP3 inflammasome, a large intracellular complex that researchers say can spark inflammation and stoke diseases of lifestyle and aging.

xx

BY RACHAEL MOELLER GORMAN

xx 43

49 READING FRAMES But We’re Animals Too, Aren’t We?

Since Darwin published his landmark work on natural selection, we’ve understood that we’re animals. But that doesn’t mean we really believe it. BY MELANIE CHALLENGER

52

FOUNDATIONS

Bile and Potatoes, 1921 BY JEF AKST

IN EVERY ISSUE

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CONTRIBUTORS SPEAKING OF SCIENCE

PUZZLE ON PAGE 12

S I E RR A L O I L I T T OR U A M I T OC E H BON S A I B C R E F L E X L O U D I S C L S A U S K Y L A B

P A A L E HO Z O I A C

A S C A L Q L U V E A I W NDR I A F S P R E Y A T I ON U U OB S T E R S O E S GA S J E T

04 . 2021 | T H E S C IE N T IST

5

APRIL 2021

ONLINE AT THE-SCIENTIST.COM:

Sex Differences in Immune Responses to Viral Infection

Free Fallin’: How Scientists Study Unrestrained Insects

Science with Borders: Researchers Navigate Red Tape

Stronger interferon production, greater T cell activation, and increased susceptibility to autoimmunity are just some of the ways that females seem to differ from males.

Researchers are pulling from video games, sports broadcasting, meteorology, and even missile guidance technology to better investigate how insects have mastered flight.

Scientists who work with foreign biological specimens face a patchwork of permits that threaten to block their projects, with potentially harmful consequences for the ecosystems they study.

AS ALWAYS, FIND BREAKING NEWS EVERY DAY ON OUR WEBSITE.

Coming next month • Hybrid animals such as mules and ligers may play a critical role in evolution. • Researchers are reconsidering an old and controversial idea that single cells can learn. • Recruiting patients for a clinical trial doesn’t have to mean getting them to leave their homes. • Pain researcher quits academia but not science—he takes his lab home with him. AND MUCH MORE

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APRIL 2021

Contributors Paul Mischel credits his deep interest in science and the natural world to growing up in a house that contained nearly 3,000 books. “My favorite times were spent in the library with my father reading books about science,” he says. When his father died from stomach cancer when Mischel was just 14 years old, he says it was life-altering. “I promised myself I wanted to do something about it,” he says. After receiving his medical degree from Cornell University Medical College and completing postdoctoral research training at the Howard Hughes Medical Institute at the University of California (UC), San Francisco, Mischel joined the faculty of UC Los Angeles as a physician-scientist in 1998. Perplexed that some of his brain cancer patients weren’t getting better, Mischel realized that he was missing something important. “I decided to take a very different vantage point,” he says. After moving to UC San Diego in 2012, Mischel’s research focus switched to understanding how extrachromosomal DNA—circles of genetic material found outside the chromosomes—affects cancer progression and drug resistance, a problem he writes about on page 32. Now at Stanford University, Mischel and his company Boundless Bio are using this research to develop new cancer treatments.

LUDWIG INSTITUTE FOR CANCER RESEARCH; KELLY DAVIDSON STUDIO; COURTESY OF ASHER JONES

Ade Adamson says he has always enjoyed figuring out how things work, a curiosity

that drew him to study the human body. After receiving his undergraduate degree from Morehouse College, Adamson completed his medical degree at Harvard Medical School in 2011. He later trained in dermatology at the University of Texas (UT) Southwestern. “I love the immune system. And your skin can be thought of as a large immune organ,” says Adamson. “The skin is a window to the health of the rest of your body.” While at Harvard, Adamson also earned a public policy degree. Now, as an assistant professor at UT Austin, Adamson’s research focuses on health services and policy for patients with melanoma, a type of skin cancer. Adamson is also the director of the university’s Melanoma and Pigmented Lesion Clinic, where he cares for patients. On page 14, Adamson writes about the potential and limits of artificial intelligence (AI) for diagnosis of this cancer. Asher Jones’s love of nature, and of animals in particular, drove the New Zealander

to study biology as an undergraduate at the University of Auckland. But when it came time to take a course that involved dissecting her beloved subjects, Jones put it off, semester after semester. Ultimately, it impeded her getting into an entomology class, so she gloved up and dug in. “I hated it,” she says. On the other hand, Jones enjoyed the insect course immensely and she ended up doing her master’s degree on native dung beetles with the professor who taught it. After a five-year break from science—some of which was spent in London working in television programming— Jones decided to pursue a PhD in entomology at Penn State. She had a tough time deciding what exactly to study, so she did a bit of everything, from interrogating interactions among plants, caterpillars, and parasitoid wasps to researching caterpillars’ gut bacteria and pathogenic viruses. “All the chapters of my dissertation are totally different,” she says. Jones came to realize that this broad curiosity about all things biology could be satisfied through science journalism. Last summer, she did a AAAS Mass Media Fellowship at Voice of America, and interned with The Scientist in early 2021. In this issue of the magazine, starting on page 40, Asher writes about a new model of information processing in the neocortex of mouse brains, an immune signaling protein that’s been linked to cancer in humans, and the role of obesity in the success of bone marrow transplants. 04.2021 | T H E S C IE N T IST

9

CITY OF VISIONARIES Arthur Riggs, Ph.D., is a legendary scientist who is now dedicated to a simple mission: creating a world without diabetes. Dr. Riggs pioneered the science behind recombinant antibodies and the development of the first synthetic human insulin, which have improved the lives of millions of cancer and diabetes patients worldwide. To celebrate his many achievements, we are renaming our renowned Diabetes & Metabolism Research Institute in his honor. Mh`^ma^k%p^ee\hgmbgn^hnk`khng][k^Zdbg`k^l^Zk\aZg]k^ohenmbhgZkrZiikhZ\abgma^jn^lmmh\hgjn^k]bZ[^m^l% delivering hope to patients everywhere.

© 2021 City of Hope

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Cancer Vaccines:

Raising a T Cell Army Scientists are developing vaccines to treat cancers, not just prevent them, and research into vaccine targets and delivery systems is extensive. Most of the vaccines under investigation prompt T cells to attack and kill cancer cells, but because tumors can evade the immune system, combination treatments may be necessary to elicit a sufficient immune response.

T Cell Priming T cells are primed by three inputs to recognize and act upon their targets.1 Once armed, effector T cells can respond to and kill a target cell.

1.

An antigen-presenting cell (APC) collects peptides from the targets (cancer cells or pathogens), binds them to the majorhistocompatibility complex (MHC), and presents them to naïve T cells. 2.

3.

T cell receptor engagement

ANTIGEN PRESENTING CELL (APC)

CD8+ T CELL

Co-stimulation The APC presents other ligands, such as CD28, to the T cell. Without co-stimulatory binding, the T cell will not be activated.

SELF-STIMULATION BY CYTOKINES

CD4+ T CELL CYTOKINES

Inflammatory stimulus from cytokines Cytokines, such as interferons or interleukins, support the stimulation, expansion, and differentiation of T cells. Without proper cytokine stimulation, the proliferated T cells could die, causing immune system tolerance to the stimulating antigen.

Cancer Vaccine Targets When designing a vaccine, scientists consider what antigens to target, but the ideal target is difficult to find. Scientists choose between host proteins expressed abnormally in cancer or cancerspecific proteins.1 THE IDEAL TARGET 1.

Exclusively expressed on cancer cells

2.

Necessary for cancer cell survival, so that cells cannot downregulate it

3.

Never expressed on the host cells

4.

Highly immunogenic

Tumor-Associated Antigens (TAAs)

Neoantigens

žŦȦŦŸîĔÒŦŜşłŦŰÒŰúŦŜúîęƥîÒĸŰęčúĸȹŠ”ȺȦÒşúĸłşĶÒĭ host proteins that are abnormally expressed in cancer cells.2

gúłÒĸŰęčúĸŦȦÒĭŦłĪĸłƑĸÒŦĸúłúŜęŰłŜúŦłşŰŸĶłşɁŦŜúîęƥî antigens (TSAs), are peptides from proteins created via cancer cell mutations.3, 4, 5, 6

Advantages Advantages

•

Well-researched

•

Some clinical trials have shown positive responses7

•

May work well with combination therapies

Disadvantages •

T cell tolerance to host proteins may make them poorly immunogenic

•

TAAs are not exclusive to cancer cells, so they may trigger autoimmune toxicity

•

Easy to identify with next generation sequencing of healthy and tumor tissue

•

New algorithms predict if an antigen would be a good MHC class I-binding epitope

•

Exclusive to cancer cells, so they do not induce T cell tolerance

Disadvantages •

Sequencing tumor DNA from multiple patients is expensive and slow

•

Slow to manufacture

•

No consensus among experts on how to identify neoantigens that elicit an immune response

•

Cancers with a low mutational burden have few candidate antigens

TYPES OF NEOANTIGENS TUMOR CELL

ACTIVATED CD8+ T CELL

Hotspot Mutations •

Many cancer patients express the same neoantigens

•

Can be used to create “off-the-shelf” vaccines

Oncogenic Viral Antigens •

Viral infection may account for up to 10% of cancers

•

Tumors driven by viruses express viral proteins

•

Make good vaccine targets because they can be highly immunogenic

•

Generally used to make conventional, preventative vaccines

Delivery of Cancer Treatment Vaccines Immune Cell Vaccines These vaccines use either patient or cell culture-derived immune cells. They are typically made of APCs, such as dendritic cells (DCs), that have incorporated the target antigen. Upon reintroduction to the patient, they stimulate T cell expansion and differentiation.1, 7, 8, 9 There are some approved immune cell vaccines, but many were cancelled in clinical trials due to a lack of positive outcomes.7 However, scientists may use them in combined therapies.

PATIENT-DERIVED IMMUNE CELL VACCINES Scientists harvest a patient’s immune cells, induce antigen uptake in the laboratory, and reintroduce them back into the body. This is an expensive and time-consuming process.10

WHOLE CELL VACCINE FROM TUMOR CELL LINES Researchers develop off-the-shelf vaccines from tumor cell lines to quickly create treatments. One example, Algenpantucel-L for pancreatic cancer, is made of lethally irradiated cells. Upon injection, patient DCs take in these cells and display tumor antigens to T cells in the lymph nodes. Clinical trial results with this vaccine were very positive, with a nearly doubled disease-free survival rate.11

TUMOR LYSATE-BASED VACCINES Researchers expose DCs isolated from patients to tumor lysates prior to reinjection. No sequencing or computations to determine immunogenicity are required. However, most antigens are TAAs, or non-immunogenic self antigens. Very few of the re-injected DCs display neoantigens and most ultimately fail to stimulate a T cell response. Scientists could use this approach as a priming vaccination while they design and manufacture personalized neoantigenbased vaccines.12

Recombinant viral vector vaccines

Peptide vaccines

RNA and DNA vaccines

Recombinant viruses expressing tumor antigens are off-the-shelf vaccines administered by direct injection. Recombinant viral vaccines have low toxicity, similar to conventional vaccines.13

These vaccines are collections łČDZǯɁDzǯŰŸĶłşɁŦŜúîęƥîŜúŜŰęôúŦ that are typically 15 amino acids long. Clinical trials using this technology have failed to produce results in Phase JJJȦíŸŰŰĔúƗĶÒƗíúĸúƥŰČşłĶ combined therapies.14

RNA and DNA vaccines offer detailed design and delivery parameters. They can incorporate genes for T cell induction, selfadjuvants, or self-replication to enhance immune responses.15, 16 Much of the rapid deployment of COVID-19 vaccines is owed to RNA cancer vaccine research..17

Combination Therapies Many of the cancer treatment vaccines in clinical trials have not performed well. However, optimism is high for the use of combination therapies to attack cancers on multiple fronts.1, 8, 9

Immunostimulators Co-treatment with molecules such as cytokines and adjuvants could enhance the immune response.

Conventional chemo- or radiotherapies Conventional therapies help APCs see cancers by causing ĭłîÒĭęƠúôęĸƦÒĶĶÒŰęłĸÒĸôîÒĸîúşîúĭĭôúÒŰĔȫ

Immune checkpoint therapy One of the biggest breakthroughs in cancer treatment ƐÒîîęĸúŦĔÒŦíúúĸŰĔúęôúĸŰęƥîÒŰęłĸłČęĶĶŸĸúîĔúîĪŜłęĸŰ inhibitors (ICIs). Normally, the immune system shuts itself down after clearing an invading pathogen. Some cancers have evolved the ability to overexpress and secrete immune checkpoint inhibitors that tell the T cells to stand down. Blocking ICIs with antibodies prevents them from interfering with the T cell response.18

References 1.

R.E. Hollingsworth, K. Jansen, “Turning the corner on therapeutic cancer

11.

2. 3.

12. 13.

controlled trial of a Poxviral-based PSA-targeted immunotherapy in

ity and sensitivity to immune checkpoint blockade,” Science, 351:1463-

metastatic castration-resistant prostate cancer,” J Clin Oncol, 28:1099-

T. N. Schumacher, R.D. Schreiber, “Neoantigens in cancer immunothera-

105, 2010. 14.

7.

CD8+ T cells in myeloid malignancies,” Haematologica, 96:432-40, 2011.

T. Schumacher et al., “A vaccine targeting mutant IDH1 induces antitumour immunity,” Nature, 512:324-27, 2014.

15. 16.

1403:755-61, 2016.

9. 10.

17.

E. Dolgin, “How COVID unlocked the power of RNA,” Nature, 589:18991, 2021.

P. Sharma, J.P. Allison, “The future of immune checkpoint therapy,” Science, 348:56-61, 2015.

F.B. Scorza, N. Pardi, “New kids on the block: RNA-based influenza virus vaccines,” Vaccines (Basel), 6:20, 2018.

Vaccines for Human Diseases, Methods in Molecular Biology, vol. 8.

T. Beissert et al., “A trans-amplifying RNA vaccine strategy for induction of potent protective immunity,” Mol. Ther, 28: 119-28, 2020.

S. Thomas, G.C. Prendergast, “Cancer vaccines: a brief overview,” in S. Thomas (ed.), Vaccine Design: Methods and Protocols: Volume 1:

K. Rezvani et al., “Repeated PR1 and WT1 peptide vaccination in Montanide-adjuvant fails to induce sustained high-avidity, epitope-specific

py,” Science, 348:69-74, 2015. 6.

P.W. Kantoff et al., “Overall survival analysis of a phase II randomized

N. McGranahan et al., “Clonal neoantigens elicit T cell immunoreactiv69, 2016.

5.

A. Harari et al., “Antitumour dendritic cell vaccination in a priming and boosting approach,” Nature Rev: Drug Discovery, 19:635–52, 2020.

H. Matsushita et al., “Cancer exome analysis reveals a T-cell-dependent mechanism of cancer immunoediting,” Nature, 482:400-404, 2012.

4.

Gastrointest Surg, 17:94–101, 2013.

O. Hofmann et al., “Genome-wide analysis of cancer/testis gene expression,” PNAS, 105:20422-27, 2008.

J.M Hardacre et al., “Addition of Algenpantucel-L immunotherapy to standard adjuvant therapy for pancreatic cancer: a phase 2 study,” J

vaccines,” npj Vaccines, 2019.

18.

R.A. Madan et al., “Ipilimumab and a poxviral vaccine targeting prostate-

E. Blass, P.A. Ott, “Advances in the development of personalized neoanti-

specific antigen in metastatic castration-resistant prostate cancer: a

gen-based therapeutic cancer vaccines,” Nature Rev: Clin Oncol, 2021.

phase 1 dose-escalation trial,” Lancet Oncol, 13:501-508, 2012.

E.J. Small et al., “Placebo-controlled phase III trial of immunologic therapy with Sipuleucel-T (APC8015) in patients with metastatic, asymptomatic hormone refractory prostate cancer,” J Clin Oncol, 24(19): 3089-94, 2006.

with single cell and spatial multiomics Fundamentally alter your understanding of cancer and accelerate translational research with flexible and innovative solutions for single cell sequencing and spatially-resolved transcriptional profiling from 10x Genomics. • Unravel the complexities of heterogeneous cancer samples to detect tumor clones and unique cellular states that drive malignancy • Resolve the tumor microenvironment and explore the influence of cancer on its resident tissue • Advance immunotherapies by characterizing the tumor immune response and the molecular mechanisms underlying therapeutic response and resistance

Chromium Single Cell Solutions Single Cell Gene Expression Single Cell Immune Profiling Single Cell Epigenomic Profiling Single Cell Protein Expression Targeted Gene Expression Visium Spatial Solutions Spatial Gene Expression Spatial Protein Expression Targeted Gene Expression

Learn more at 10xgenomics.com/cancer

10x Genomics builds solutions to interrogate biological systems at a resolution and scale that matches the complexity of biology. Our rapidly expanding suite of products, which include instruments, consumables, and software, have enabled customers to make fundamental discoveries across multiple research areas, including cancer, immunology, and neuroscience.

FROM THE EDITOR

Our Expanding Universe As with the evolution of astronomy, new insights in biology beckon just beyond our conceptual and observational reach. BY BOB GRANT

ANDRZEJ KRAUZE

I

n The Scientist’s April 2021 issue, scientist/author Paul Mischel of Stanford University posits an interesting parallel between scientific advancement in the field of astronomy and how biologists are exploring the living world. In his feature story, “Cancer’s Extra Genome,” beginning on page 32, Mischel explains how maps in both fields can drive and sometimes derail the discovery and characterization of scientific truths. In the case of astronomy, maps have included Ptolemy’s Earthcentric view of the universe and the corrected maps made 1,400 years later by astronomy’s patron saint, Renaissance-era thinker Nicolaus Copernicus. In the case of cancer, maps of the genome are missing a key element: extrachromosomal DNA (ecDNA). Mischel likens the conceptual leap represented by the adoption of a sun-centered model of the solar system to what is now needed in science’s conception of cancer dynamics. One amazing thing about the observations on which Copernicus based his model is that he made them using the same tools at Ptolemy’s disposal in ancient times—that is, the naked human eye. Copernicus died more than 50 years before Galileo Galilei trained a new instrument, the telescope, on the starry night sky, confirming Copernicus’s observations using the revolutionary tool. Since then, astronomy has grown and evolved by leaps and bounds, new technologies have been brought to bear, and humanity’s understanding of the cosmos has expanded in the manner of the universe itself. So too has our understanding of cancer grown as scientists through the ages have employed their unique perspectives or technological advances in studying the phenomenon. Cancer was once a mysterious illness that would cut down young and old alike, but over the decades scientist have chipped away at bygone ignorance, expanding the edges of our concept of the multifarious scourge. From Percivall Pott’s groundbreaking, experimentally validated link between chimney soot and squamous cell carcinoma in 1775 to the US Food and Drug Administration’s 2017 approval of transformative CAR T cell therapies for certain lymphomas and leukemias, cancer science has come ever closer to pinning down the disease. But unlike astronomy, cancer science is bent toward practical concerns— the ability to diagnose and treat patients. This raises the stakes of each stretch of the edges of this realm of scientific understanding, in some cases to a matter of life or death. And that is the promise that insights such as the role of ecDNA hold. Incorporating this fresh component into existing models of cancer dynamics could lead to new methods of vanquishing the disease, in many of its deathly expressions. Incorporating ecDNA into

long-established genetic maps of cancer, which focus on the more tractable chromosomal DNA, does not upend the entire model. It merely refocuses it in a way that just might shift the center of gravity of the otherwise accurately charted molecular players. Like Galileo’s telescope, advanced genomic technologies are confirming the biological influence of ecDNA and are allowing researchers to find more and more contexts in which the newly appreciated genetic components may be driving disease. This is the soul of scientific progress. It is not a straightforward process of advancing toward a goal of ultimate, indisputable truth. It is rather a slow and often clumsy stumbling toward a more useful truth—one that is destined to serve its purpose before yielding its place to an even more useful truth. What lies beyond the horizon of our observational powers is what fuels many scientists I’ve known, from cosmologists to immunologists. And that quest into the darkness of the unknown and even the not-yet-knowable, be it light-years away or ensconced in our very cells, is what propels humanity forward. J

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BY EMILY COX AND HENRY RATHVON

ACROSS

DOWN

1. Range of an orologist’s knowledge? 5. French inventor of a 17th-century calculator 8. On a shore; of a coast 9. Optic layer below the sclera 10. Energy-making organelles 12. Little work for a horticulturist? 14. Fish-eating raptor 16. Response facilitated by neural pathways (2 wds.) 19. Intervertebral shock absorber 20. Bottom-dwelling decapods 22. NASA orbiter launched in 1973 23. Burner in a chemistry class (2 wds.)

2. 3. 4. 5. 6. 7. 11. 13. 15.

Upper portion of either hipbone Cycles through, as crops Word before bladder, pump, or pocket Era of the “Cambrian explosion” Eight-armed ink dispenser Silvery member of the herring family 66-million-year-old impact crater Pillar from the time of Thutmose III Great Basin indigenous peoples whose name is an anagram of “pause it” 17. Alternative to general, for operations 18. Kind of shark not as caring as it sounds 21. Wetland source of sphagnum Answer key on page 5

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—Pilar Pinto, a lecturer and PhD candidate in arts and humanities at University of Cádiz in Spain, commenting in an Equal Times story on how academic publishing clashes with scientific culture (March 1)

We’ve been concerned throughout this pandemic about the level of prolonged stress, exacerbated by the grief, trauma and isolation that Americans are experiencing. This survey reveals a secondary crisis that is likely to have persistent, serious mental and physical health consequences for years to come. —Arthur C. Evans Jr., CEO of the American Psychological Association, in a press release about the organization’s recently published “Stress in America” survey, which compiled responses from more than 3,000 adults in the US in late February (March 11)

© JONNY HAWKINS

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Note: The answer grid will include every letter of the alphabet.

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AZBEE WINNER 2020 WEBSITE OF THE YEAR A huge and sincere thank you goes out to our readers, to the American Society of Business Publication Editors, and to everyone at The Scientistt and LabX X Media Group who has helped to make the-scientist.com great.

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Machine Learning and Cancer Diagnosis While artificial intelligence could improve detection of tumors at their earliest stages, it also risks identifying malignancies that would never cause patients any harm.

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rtificial intelligence has the potential to revolutionize healthcare. Machine learning (ML), a form of AI that uses algorithms to simulate aspects of human decision making, has gained a lot of attention in recent years. While the potential application of ML to healthcare is broad, many recent breakthroughs have been in the realm of imagedriven diagnostics. The diagnosis of cancer, particularly solid tumors, relies heavily on the visual interpretation of histologic slides by pathologists who use their experience in pattern recognition to render a diagnosis. This is a difficult and time-consuming skill for humans to master, but an ideal task for ML technology, which can use thousands to millions of images to train algorithms in a relatively short period of time. Given the ability to “learn” from large amounts of data, ML-powered systems hold promise for delivering faster and more-consistent cancer diagnoses.

1 4 T H E SC I EN T I ST | the-scientist.com

The technology has important limitations, however. The reliability of ML algorithms depends on the validity of the data on which they are trained. In the diagnosis of cancer, this means a reliance on data that are correctly labeled with the ground truth about what is and what is not cancer. Unfortunately, disagreement among pathologists exists concerning what constitutes cancer. Numerous studies have shown that diagnostic agreement among pathologists is often poor across various tumor types, including prostate, thyroid, melanoma, and breast, particularly in cancer’s early stages. The lack of a gold standard in cancer diagnosis is a wellknown problem that has important implications for the use of image-driven, ML-powered diagnostics. In fact, ML technology could further expose the problem. Researchers and physicians eager to make ML-powered cancer diagnosis a clinical reality are concerned that the technology’s reliance on pathologists as an external standard could lead to an increase in overdiagnosis—

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ML-powered systems hold promise for delivering faster and more-consistent cancer diagnoses. The technology has important limitations, however.

the diagnosis of disease that meets the pathologic definition of cancer but never would have caused morbidity or mortality in a patient’s lifetime. The hope for ML-driven cancer diagnostics is that one day doctors could intervene in the early stages of disease and avoid death related to cancer. However, this hope relies heavily on the presumption that pathologists can correctly identify early cancers that are destined to be lethal to patients. Mounting evidence of cancer overdiagnosis suggests that this may not be the case. In fact, AI may worsen the problem by increasing our ability to diagnose slow-growing, non-lethal cancers. One potential solution could be to train ML algorithms using histopathology images divided into three categories: total agreement on the presence of cancer, total agreement on the absence of cancer, and disagreement on diagnosis. The placement of images into these categories would be based on the assessment of a diverse panel of pathologists. The disagreement category could be a subject of study for researchers on

the natural history of cancer. It may also be useful for clinicians and patients who may want to consider less-aggressive treatment for the lesions in the gray area of pathologist disagreement. The use of ML technology in healthcare is still nascent, but like any other technology, it needs to be adequately vetted before it is widely adopted, given the potential for unintended harms such as overdiagnosis. Hopefully, prospective randomized trials can be performed in order to assess the effect of ML on cancer diagnosis. The focus for ML in cancer diagnosis should not just be on finding more cancers, but also on finding cancers that kill people. J Adewole S. Adamson is a dermatologist and assistant professor in the Department of Internal Medicine at University of Texas at Austin Dell Medical School. He is a health services and health policy researcher, and his primary clinical interest is in caring for patients at high risk for melanoma. Follow him on Twitter @AdeAdamson.

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JEFFREY WEIDNER, PHD Founder QualSci Consulting, LLC

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• Creating dose response assays that are robust and reproducible • Enhancing dose-response curves using the Thermo Scientific™ Multidrop™ Pico Digital Dispensers

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The development of drug resistance is an almost universal characteristic of cancers and is an outstanding challenge in the fields of tumor biology and clinical oncology. While there have been many studies focused on genomic contributions to resistance in cancer cells, recent studies have shown that genetically homogeneous cells can undergo adaptive cell state changes leading to the rapid emergence of drug-tolerant phenotypes. The heterogeneous nature of tumors, coupled with the functional and metabolic changes that accompany adaptive resistance development, suggests that multi-omic, single cell approaches have the potential to provide deep insights into the cell state changes that lead to adaptive resistance, and may provide hypotheses for new therapies that can prevent resistance development. IsoPlexis’ multi-omic (metabolomics + functional proteomics) energy state application provides a critical and uniquely capable tool for addressing this biology.

JAMES R. HEATH, PHD President and Professor Institute for Systems Biology, Seattle

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WATCH NOW! www.the-scientist.com/Synthetic-Biology -in-the-Age-of-a-Pandemic JAMES J. COLLINS, PHD Institute for Medical Engineering and Science Department of Biological Engineering Massachusetts Institute of Technology Broad Institute of MIT and Harvard Wyss Institute, Harvard University

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Reading the Monkey Mind

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eople who live with animals often feel these creatures have some perception of our intentions and feelings— for example, that a pet senses when we’re sad and tries to cheer us up. But finding out whether this is true or unwarranted anthropomorphising requires a mind-reading ability that has so far remained outside the grasp of psychology and neuroscience. In research with humans, investigators can simply ask study participants about their perceptions of others’ beliefs and motivations. With nonhuman animals, how does one tease out the ability to infer another creature’s point of view, known as “theory of

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mind,” from simply responding to cues that the animal has learned to associate with a particular outcome? Michael Platt, who has studied cognition in monkeys for decades, and his team, then at Duke University, recently harnessed a combination of brain monitoring and machine learning in a bid to get closer to answering this question. They devised a computer game in which two monkeys faced each other and used joysticks to drive circles on a computer screen, choosing either to go straight or to swerve. As in a real-life game of chicken, if both monkeys went straight, both lost— meaning that they didn’t receive a reward in the form of a sip of juice. If a monkey swerved while its opponent steered straight, it got a small juice reward, and

APRIL 2021

SHARING THOUGHTS: Rhesus macaques,

pictured here on Cayo Santiago near Puerto Rico, offer researchers a chance to study theory of mind in nonhuman animals.

if it steered straight while the other monkey veered, it won, receiving a larger portion of juice. If both monkeys veered, they received the small sip of juice plus a bonus reward that varied from trial to trial. The rhesus macaques (Macaca mulatta) in the experiment were restrained in their seats and played the game over and over, with a few variations. Sometimes, instead of facing off against a live opponent that they could see on the other side of a computer screen, they played alone against the computer, or

LAUREN BRENT

Notebook

against a computer with a decoy monkey who was present but wasn’t playing. The sizes of the rewards were varied, as was the clarity of the visual signal the monkeys saw on the screen indicating how their opponent was steering. The monkeys learned to play the game with sophistication, Platt says, adapting their strategies to those of their opponents in a way that is consistent with a principle in game theory called the Nash equilibrium, in which individuals settle on the best strategies available to them in light of their opponents’ strategy. Using machine learning, the researchers found that they could successfully predict the monkeys’ actions only when their model incorporated details of their opponents’ circumstances and behavior, such as the rewards available to the opponents and how they steered their on-screen circles, indicating that the monkeys were incorporating these cues into their decision making. While he’s observed complex behaviors from primates in the wild, Platt says, “I’m always surprised when we are able to elicit behavior in a task like this one, which is super complicated.” The animals’ behavior in the wild clearly doesn’t include playing video games, but Platt, who’s now at the University of Pennsylvania, says that the game likely draws on the same cognitive resources that monkeys use to make strategic social decisions such as when to offer or accept grooming.

LAUREN BRENT

The monkeys learned to play the game with sophistication, adapting their strategies to those of their opponents.

One factor that bore on the monkeys’ play was their preexisting social hierarchy, notes Wei Song Ong, a postdoc at UPenn who worked in Platt’s lab and is the study’s first author. The animals knew one another, she explains, as they’re housed together in individual enclosures that enable them to see and hear one another, and they establish hierarchies that researchers can deter-

mine by putting them in the same room, equally distant from a piece of fruit: the subordinate monkey will allow the dominant monkey to grab the food. In the game, “the action of the same monkey is different [depending on] whether or not he’s playing a dominant monkey or a subordinate monkey,” Ong explains. “But the subordinate monkeys all play very similarly, and the dominant monkeys play very similarly, which basically means that if you’re a mid-ranking monkey, you change your strategy depending on who you’re playing with.” Moreover, the subordinate monkeys were more attuned to their opponent’s strategies. “The subordinate is always trying to learn about the dominant, and the dominant mostly just does whatever they want,” showing a lesser propensity to learn about their opponents, Ong notes. As the monkeys played, electrodes monitored the activity of single neurons in two areas of their brains thought to correspond with brain regions used during social decision making in humans: the superior temporal sulcus (STS), which is thought to be analogous to humans’ temporoparietal junction, an area involved in thinking about the beliefs or thoughts of another person; and the anterior cingulate gyrus (ACCg), an area that’s active when an ani-

MIND’S EYE: Researchers studied how

macaques use other individuals’ behavior to inform their own choices.

mal sees a conspecific receive a reward and is associated with empathy in humans. The results revealed that the animals drew on both the ACCg and the STS during the game, although more so on the STS, suggesting that “cooperation is a strategic act, not an empathetic act,” Ong says (Nat Neurosci, 24:116–128, 2021). By monitoring the firing of single neurons, says Ziv Williams, a neuroscientist at Massachusetts General Hospital and Harvard Medical School who was a peer reviewer on the new study, Platt’s group was able to show “that there are certain dedicated neurons that really are very good at representing these predictions. And that’s something that has never really been shown before.” “This is really a nice study,” says Liesbeth Sterck, who studies social behavior and cognition in primates at Utrecht University in the Netherlands and was not involved in the work. There’s a gap, she says, between the type of research she does—observing primates’ behaviors in more natural settings—and neuroscience, which has focused on very sim04 . 2021 | T H E S C IE N T IST 1 9

ple actions in the lab. “This is the kind of paper that’s really bridging” that gap, she says, by homing in on the neuroscience behind more complex behavior. Daniel Povinelli, a biologist at the University of Louisiana at Lafayette who studies behavior in chimps, humans, and other animals, argues that the fact that the monkeys seem to make those predictions doesn’t show whether the animals have any understanding of one another’s mental states. “I can keep very complicated track of what another organism [does] in certain [kinds of ] situations, and I can make predictions, therefore, about what they’re going to do, without ever reasoning about their internal private motivations for doing so,” he says. Platt concedes that the study didn’t “precisely test” for theory of mind. But by showing that the monkeys take into account factors such as one another’s past behavior and incentives, he and his colleagues have established that the animals have the “building blocks of theory of mind,” he says. 20 T H E SC I EN T I ST | the-scientist.com

Whatever the role of theory of mind— or lack thereof—in enabling the monkeys to make those predictions, Platt argues that it isn’t an all-or-nothing proposition. Autism is sometimes associated with impaired theory of mind, but Platt argues that even among neurotypical humans, “there’s a lot of variation in the degree to which any of the components that together make up theory of mind are actually utilized and contribute to behavior. So very few people go through the fullest steps of reasoning, in terms of intentionality with regard to other people. . . . Certainly, my wife thinks that I don’t engage in a high degree of depth of theory of mind in my own behavior.” —Shawna Williams

Bright Encounters When cancers metastasize, diseased cells travel to distant parts of the body, where they can invade many different tissues

and cause tumors to grow in these new locations. Monte Winslow, an immunologist at Stanford University, investigates the mechanics of cancer metastasis with a blend of in vivo models and genomics. But he’s long faced a challenge in understanding just what causes cells to move—mainly, he says, because it’s difficult to study how tumor cells are physically interacting with one another or with surrounding cells. Existing methods using green fluorescent protein (GFP) or spatial transcriptomics only allow researchers to study interactions indirectly, says Winslow. They give researchers an idea of “who’s close to who, but not who’s touching who, and not who touched who yesterday,” he says. Struggling with this problem a few years ago, Rui Tang, a postdoc in Winslow’s lab, decided to work on an alternative, and spent the better part of a year devising a new technique. Specifically, he created a system that allowed GFP on one cell’s surface to be transferred to a recipient cell upon interaction. The team named the result GFP-based Touching Nexus

ANDRZEJ KRAUZE

NOTEBOOK

(G-baToN). One cell bears the label tethered to its surface, and when it interacts with another cell, that marker is passed on to engineered receptors for that marker on the recipient, not only indicating that the cells interacted, but also where contact was made (eLife, 9:e61080, 2020). Although the system wasn’t infallible, Winslow says, he was amazed during the development process at how well it seemed to work. It would highlight interactions between two cells of the same type, and also between completely different cell types, such as lung cancer cells interacting with cortical neurons in vitro. “Rui does this, and it works, and then he does it across other cells, and it works,” Winslow says. “Then the lady from the lab next door says, ‘Yeah, but it’s never gonna work in primary cells, it’s just gonna be cell lines’ ”— the thinking being that primary cells have a greater variety of protein receptors than cell lines do, and thus would work less consistently with the receptor-tagging sys-

One cell bears the label tethered to its surface, and when it interacts with another cell, that marker is passed on.

tem. “And then Rui has it work across all of these primary cells.” This broad applicability is key, Winslow says, if the technique is to be used for understanding the dynamic nature of how cancers metastasize. Tang also developed red and blue fluorescent tags to use in addition to the green one, so that researchers could tag different sender cells in different colors and thus observe interactions between multiple cells at once. Now, in what they refer to as their beta-testing phase, Winslow and Tang are providing vectors and instructions to other scientists to try in their own

labs. Vanderbilt University cell biologist Ian Macara, who was not involved in its development, has tried G-baToN to explore epithelial cell extrusion mechanisms to understand how human skin is able to reject a dying or irregular cell, such as a metastasizing tumor cell, without compromising the integrity of the barrier. In short, he says, the technology is “a powerful and adaptable system.” Tang’s vision for the approach also goes beyond merely recording the interactions. “It’s also [a] sort of cell-cell contact–based delivery system,” Tang says, referring to early signs that the system could also be used to transport DNA, proteins, and other macromolecules from one cell to another. Going forward, this system could be used to alter the function of the receiver cell, he adds. To Geoff Wahl, who runs the Gene Expression Laboratory at the Salk Institute and did not participate in the research, G-baToN holds promise to

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“identify and isolate normal cells that had come into contact with, and perhaps had been changed by, their interactions with cancer cells.” In his lab,

22 T H E SC I ENTI ST | the-scientist.com

Wahl studies breast and pancreatic cancers, the latter of which is notorious for going undetected until the disease advances and spreads. (See “Advancing

Against Metastasis” on page 24.) Wahl’s lab is currently collaborating with Winslow’s group to create the next iteration of G-baToN, which Wahl says they hope will enable them to track receiver cells over time.  Winslow says he’s excited about how the technique could allow researchers to ask new questions that couldn’t be addressed before. For now, to get a better sense of G-baToN’s full capabilities as well as its shortcomings, they invite other labs with different research interests to try it out. “If you’re doing the brain, if you’re doing immune cells, whatever it is,” Winslow says, “[we want to know if ] it can work or not.” —Lisa Winter

© NICHOLAS J. KRAMER, ELIFE, 9:E61080, 2020

LIGHTING UP: Cancer cells (green) in this microscopy image send GFP granules to neurons (purple), illuminating how cells interact with their neighbors.

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It’s now clear that treating primary tumors early doesn’t always stop cancer’s deadly spread, prompting a hunt for ways to target disseminated cells directly.

BY SHAWNA WILLIAMS

E

ight years ago, Adrienne Boire had a conversation with a patient that would set the course of her research. Back then, she was dividing her time between her postdoctoral research in a lab studying metastasis at Memorial Sloan Kettering Cancer Center and treating patients there as part of a clinical fellowship. The patient, like Boire, was in her 30s, and she had recently been diagnosed with a condition called leptomeningeal metastasis, in which cancer cells invade the spinal fluid, causing death within a few months. “She asked me deceptively simple questions,” Boire remembers, “like, ‘Why did this happen to me? Why did my cancer go into the spinal fluid? How did it get there?’ . . . And, obviously, ‘What can we do to stop this?’” Boire didn’t have good answers. “And then she said, ‘I really hope somebody will study this someday.’” Boire agreed with the patient, and she was upset when she left the woman’s room. She exited the hospital and crossed the street to enter the revolving door into the building that housed her research lab, “and then I kept walking and kind of revolved myself right back towards the hospital, because I realized that this woman is actually telling me what I’m supposed to be studying.” Back in the patient’s room, Boire took detailed notes on the woman’s questions. Then she returned to the lab and began to think about how to go about answering them. That was in 2013; Boire, who now heads her own lab at Memorial Sloan Kettering, has studied leptomeningeal metastasis ever since. Many unanswered questions remain, not just about leptomeningeal metastasis, but about metastasis in general—that is, the process by which cancer cells move out from the site where they initially arose and colonize new tissues. Once seen as a late stage of cancer, metastasis is now recognized as a complex process that can involve very early dissemination of cancer cells from primary tumors and is therefore unlikely to be averted simply by early screening and treatment. But as researchers work to understand cancer’s spread, they are uncovering clues about the factors that enable or block it and are working to develop treatments that specifically target disseminated cancer cells or the healthy tissues where they make their homes. Their quest is an urgent one: there are few treatment options for metastatic disease, which is responsible for the vast majority of the nearly 10 million cancer deaths globally each year. Boire says she’s hopeful that researchers will ultimately find a way to head off metastasis completely and “put me and my clinic out of business. . . . It would be really lovely to not have any more patients to treat.”

Sleeper cells The scientific conceptualization of metastasis was long dominated by a model posited in a 1990 paper by Johns Hopkins Kimmel Cancer Center’s Bert Vogelstein and his colleague Eric Fearon.1 In that model, an accumulation of genetic mutations that activate oncogenes and tamp down tumor suppressor genes first makes normal cells form benign tumors, then turns benign tumors into malignancies, and finally enables metastatic cells 26 T H E SC I EN T I ST | the-scientist.com

to leave the primary tumor and establish themselves elsewhere in the body. Yet hints emerged early on that there was more to the story, at least in some patients and cancer types. In the early 2000s, for example, Christoph Klein, a metastasis researcher now at University of Regensburg in Germany, his former thesis advisor Gert Riethmüller of the Ludwig Maximilians University in Munich, and their colleagues analyzed the genomes of individual cancer cells taken from the bone marrow of breast cancer patients who did not have metastases that could be detected via imaging. Disseminated cancer cells could be distinguished from normal cells in the bone marrow because they bore hallmarks of epithelial cells, breast cancer’s tissue type of origin. These markers pointed to the fact that the cells didn’t belong in the marrow and must have migrated there from elsewhere in the body. But rather than carrying more cancer-associated mutations than the primary tumors, the researchers found that these disseminated cells actually had fewer, says Klein.2 “We were quite frustrated to see that many of the expectations that we thought we would find did not fulfill.” Once a patient had detectable metastases somewhere in the body, however, the picture changed, with individual disseminated cells in the bone marrow harboring multiple genetic changes typical of primary and metastatic tumors. “That was very surprising, [that] all the information [about whether metastasis has occurred] is already in a single cell genome,” Klein says, whether or not those cells are near at the actual site of metastasis. Klein and Riethmüller suspected that cancerous, latent cells can lodge not only in the bone marrow, but in organs distant from the primary tumor, where they later seed metastatic growth. The idea aligned with circumstantial evidence they’d uncovered earlier, including documented cases in which patients have metastatic disease with no detectable primary tumor, as well as cases in which metastatic cancer was inadvertently transferred from donors to transplant recipients via apparently healthy organs. This suggested, Klein and Riethmüller argued in a 2001 review article, that metastatic cells were able to disseminate to distant sites very early in a primary tumor’s formation, before the patient became symptomatic.3 But the conditions cited in that review are fairly rare, so it was a surprise when the 2003 study found early dissemination in most of the patients studied, Klein says. A few years later, he, Riethmüller, and their colleagues found further support for this idea when they detected early dissemination of metastatic cells in breast cancer–prone transgenic mice, and determined that in breast cancer patients, the size of the primary tumor wasn’t correlated with the number of disseminated tumor cells found in the bone marrow.4 Other labs have also found evidence for the phenomenon of early dissemination followed by dormancy. Icahn School of Medicine at Mount Sinai metastasis researcher Julio Aguirre-Ghiso says that a series of mouse studies in his and other labs suggests “that in breast cancer patients and in melanoma, cells can leave very, very

early before cancers acquire a certain size or are detectable. And the cells can colonize organs and eventually give origin to metastasis.” This idea is now well-accepted among metastasis researchers, and it comes with a sobering implication: namely, that catching and treating a primary tumor early in its growth won’t necessarily head off metastasis. “I don’t think we can prevent [early] dissemination,” says Maria Soledad Sosa, a cancer researcher at Icahn School of Medicine. “That won’t be a viable therapy.” Indeed, the fact that today’s most effective cancer therapies, such as surgical removal and radiation, are largely aimed at localized, primary tumors rather than metastases may explain why most cancer fatalities are ascribed to metastatic disease, says Matt Vander Heiden, a cancer researcher at MIT’s Koch Institute for Integrative Cancer Research and a medical oncologist at the Dana-Farber Cancer Institute. “We’re not as good at treating metastatic cancer.” Many researchers have now turned their attention to dormant metastatic cells in hopes of better understanding these cells’ characteristics and what factors determine whether they remain quiescent or wake up and begin dividing. Ideally, this will lay the groundwork for new therapies that could kill the cells before they begin to proliferate or ensure they stay in a dormant state.

now being tested in a clinical trial among patients with prostate cancer at Mount Sinai to see if it can delay or prevent metastasis, Sosa says. Another tack for treating metastasis is to target not the disseminated cancer cells themselves, but the niche where they reside. “There’s a lot of thought and effort in the field into how to basically get the microenvironment to fight against the tumor,” says Ekrem Emrah Er, who studies metastasis at the University of Illinois at Chicago. For instance, healthy endothelium gives off cues that can drive disseminated breast cancer cells into quiescence and keep them there, explains Cyrus Ghajar, a metastasis researcher at the Fred Hutchinson Cancer Research Center in Seattle. “And when that homeostasis is lost,” through wounding, local inflammation, or other insult, Ghajar says, “that’s one of the triggers that can now catalyze the outgrowth of tumor cells in these distant organs.” In a 2019 study, Ghajar and colleagues found that by disrupting two molecular signals that enable communication between disseminated cancer cells and surrounding endothelial cells alongside blood vessels in mice, “you sensitize [cancer] cells to chemotherapy, and importantly, you do so without waking them up,” he explains. “By disrupting these interactions, you can deplete

Preventing metastasis

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The key to preventing dormant, disseminated cancer cells from growing into metastatic tumors, experts say, lies in signals the cells receive from their environments. Although a comprehensive picture of these factors has yet to emerge, and it’s likely that not all metastatic cancers involve a dormant phase, researchers have identified numerous signals involved in inducing dormancy or waking cells up. For example, while doing a postdoc in Aguirre-Ghiso’s lab, Sosa and her colleagues found that a transcription factor called NR2F1 helped induce dormancy in mouse models of head and neck, breast, and prostate cancers by, among other things, bringing about cell cycle arrest, causing epigenetic changes, and activating the retinoic acid pathway, which is involved in development. 5 Further work showed that a combination of azacitidine and retinoic acid, two cancer drugs approved by the US Food and Drug Administration (FDA), induced dormancy in cultured cancer cells. 6 The combo is

Oncee seenn ass a latee stagee off cancer, metastasiss iss now w recognizedd ass a complexx processs thatt cann involvee very earlyy disseminationn off cancerr cells from m primaryy tumors. VOL. 35 ISSUE 2 | THE SCIENTIST

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THE ROAD TO METASTASIS In some cancers, such as breast cancer and melanoma, tumor cells can leave the primary tumor site early in the tumor’s formation and colonize new tissues, where they may receive molecular signals from surrounding cells, known as the niche, that keep them dormant for long periods. Mutations in the cancer cells themselves or changes to the niche may later awake these dormant cells, enabling them to proliferate and form metastatic tumors.

BREAST

Tumor cell

Blood vessel Milk duct Epithelial cells

Q 1 DISSEMINATION: In some cancers, including breast cancer, cancer cells can move away from the site of the primary tumor very early in the progression of the disease, before doctors can even detect a primary tumor.

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Cancer cell

Q 2

DORMANCY: It’s thought that most of these cells die, but a few disseminated cancer cells survive the bloodstream. These cells may already have mutations needed to colonize a new niche, such as the lungs, or they may adapt once they arrive. The cells tend to stay close to blood vessels, where they receive signals from epithelial cells directing them to stay dormant.

LUNG

Q 3 PROLIFERATION: If something changes—either in the Epithelial cells Dormancy-promoting cues

surrounding healthy tissue, where stress or other factors can alter the dormancy signals that cancer cells receive, or in the cells themselves, which sometimes stop responding to the signals, or both—the cancer cells can begin to proliferate, forming metastatic tumors.

BONE MARROW: A SENTINEL The presence of disseminated tumor cells in the bone marrow—which can be sampled from patients relatively easily—can indicate that such cells are present elsewhere in the body as well, predicting future metastasis. The bone marrow can also play a direct role in metastases at other sites by producing dormancy cues, or, conversely, by awakening resident, dormant cancer cells, which then enter the bloodstream and travel to other tissues, where they proliferate.

BONE MARROW

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Moving on out In addition to dormancy, another property of metastatic cells that has captured researchers’ attention is their ability to adapt to and thrive in new environments within the body. This represents a shift in emphasis over the past 5 to 10 years from a narrower focus on cancer cells’ ability to escape the primary tumor site, says Purdue University’s Mike Wendt. “You can imagine if you’re a tumor cell, and you’re growing in a primary tumor—primary tumors are very hard and collagen-rich and acidic and hypoxic—and then you’re thrust into the bloodstream or the lymphatics, then you’re deposited shortly thereafter into a normal tissue. . . . These are very oxygen-rich . . . very soft tissue,” he says. It’s not so much dissemination, then, but the abilities of tumor cells to survive and later initiate a new tumor that “really seem to be the rate-limiting steps of metastasis.” One factor that likely enables this adaptation in at least some tumor cells, Wendt says, is a mechanism called fibroblast growth factor receptor (FGFR) signaling. He notes that several organs in the body produce ligands that interact with FGFR. His group’s hypothesis is that “if a tumor cell can upregulate

the receptor, that might put it in a better position to respond to some of these FGF ligands that we know are important in normal organ physiology . . . allowing those cells to adapt and eventually grow in the context of a new and different organ.” One piece of evidence for this comes from a 2016 study in which researchers led by Razelle Kurzrock of the Moores Cancer Center at UC San Diego Health found that aberrations— such as amplification, in which the copy number of the gene increases—in the gene for this protein were relatively common, occurring in 7 percent of the thousands of tumors of various types that they analyzed. 8 FGFR amplification has also been found in the metastatic tumors of patients whose primary tumors lacked such abnormalities,9 and metastatic cells can ramp up FGFR production even without such genetic changes, Wendt notes. “All of these pieces of evidence point to importance of FGFR signaling as a metastatic driver.” Multiple groups have worked to identify inhibitors of FGFR or other components of its pathway, and in 2019, the FDA approved one such drug, erdafitinib, for patients with metastatic urothelial cancer with genetic alterations in FGFR. Yibin Kang, a metastasis researcher at Princeton University, says that in recent years there’s been a change in emphasis in metastasis research from mainly trying to target what are known as oncogenic driver genes—genetic elements that are needed for cells to become cancerous and maintain malignant growth—to going after cancer fitness genes that enable cells to cope with stress. Such cancer fitness genes, he says, “are critically important for highly aggressive metastatic cancer.” In addition to their need to adapt to new environments and their unchecked growth if and when they escape dormancy, metastatic cells face stressors such as chemotherapy and attack by the immune system. In studies of one such cancer fitness gene, metadherin (MTDH), which codes for a signaling protein that’s involved in multiple pathways for functions such as apoptosis, Kang’s group has linked its amplified expression to poor prognosis in several human cancers.10 Furthermore, the team showed that knocking out MTDH in mice inhibited metastasis without any apparent ill effects on the animals.11 He’s now working on identifying inhibitors of the metadherin protein that could be used therapeutically.12 In addition to adapting to new niches they colonize, metastatic cells can in some cases mold the niches to suit their needs. Using a mouse model of leptomeningeal metastasis, Memorial Sloan Kettering’s Boire and her colleagues reported in 2017 that cancerous cells in spinal fluid overproduce a protein called complement component 3 (C3), that cerebrospinal fluid (CSF) from patients with the disease also has a relatively high level of the protein, and that C3 disrupts the barrier between the blood and the CSF, allowing substances that the cancer cells need to leak across.13 Boire explains that the oxygen- and nutrient-poor space that houses CSF is a tough place for cancer cells to make their home, and that C3 helps them change the environment to make it more hospitable. The brain is a site where metastases seem to

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the reservoir of disseminated disease, and . . . doing so prevents metastasis down the road.”7 Much of the research on this phenomenon has taken place in animals, and Lewis Chodosh, a researcher and physician at the University of Pennsylvania who studies cancer dormancy, cautions that it hasn’t been conclusively demonstrated that dormant disseminated cancer cells exist and cause  recurrent metastatic cancers in humans. “It’s very rare that you ever have the opportunity to look in a patient and find dormant cells,” he notes. “It’s very, very hard in a clinical situation to know to what extent does dormancy really exist.”

ANOTHER PROPERTY OF METASTATIC CELLS THAT HAS CAPTURED RESEARCHERS’ ATTENTION IS THEIR ABILITY TO ADAPT TO AND THRIVE IN NEW ENVIRONMENTS WITHIN THE BODY. require particularly drastic adaptations in order to thrive. (See “Metastasis in the Brain” below.) Researchers have yet to identify a commonality among metastatic cells that can be targeted therapeutically without dealbreaking side effects on other tissues. Although no master key that stops metastases from forming has yet been found, Boire says she thinks it’s possible that one exists, and she’s hopeful that the field is making its way toward finding it. She still has the notebook where she took notes during her conversation with the leptomeningeal metastasis patient, who died a few weeks later. While she hasn’t yet arrived at any definitive answers, Boire says that now “I have much more complete ways of asking the questions.” J

References 1. E.R. Fearon, B. Vogelstein, “A genetic model for colorectal tumorigenesis,” Cell, 61:759–67, 1990. 2. O. Schmidt-Kittler et al., “From latent disseminated cells to overt metastasis: Genetic analysis of systemic breast cancer progression,” PNAS, 100:7737–42, 2003. 3. G. Riethmüller, C.A. Klein, “Early cancer cell dissemination and late metastatic relapse: Clinical reflections and biological approaches to the dormancy problem in patients,” Semin Cancer Biol, 11:307–11, 2001. 4. Y. Hüsemann et al., “Systemic spread is an early step in breast cancer,” Cancer Cell, 13:58–68, 2008. 5. M.S. Sosa et al., “NR2F1 controls tumour cell dormancy via SOX9- and RAR`driven quiescence programmes,” Nat Commun, 6:6170, 2015. 6. M.S. Sosa, “Dormancy programs as emerging antimetastasis therapeutic alternatives,” Mol Cell Oncol, 3:1, 2016. 7. P. Carlson et al., “Targeting the perivascular niche sensitizes disseminated tumour cells to chemotherapy,” Nat Cell Biol, 21:238–50, 2019. 8. T. Helsten et al., “The FGFR landscape in cancer: Analysis of 4,853 tumors by next-generation sequencing,” Clin Cancer Res, 22:259–67, 2016. 9. L.R. Yates et al., “Genomic evolution of breast cancer metastasis and relapse,” Cancer Cell, 32:P169–84.E7, 2017. 10. G. Hu et al., “MTDH activation by 8q22 genomic gain promotes chemoresistance and metastasis of poor-prognosis breast cancer,” Cancer Cell, 15:9–20, 2009. 11. L. Wan et al., “Genetic ablation of metadherin inhibits autochthonous prostate cancer progression and metastasis,” Cancer Res, 74:5336–47, 2014. 12. Y. Kang, “Identification, validation, and therapeutic targeting of a driver gene in poor-prognosis breast cancer, Cancer Res, 80 (Suppl):Abstract nr IA13, 2020. 13. A. Boire et al., “Complement component 3 adapts the cerebrospinal fluid for leptomeningeal metastasis,” Cell, 168:1101–13.e13, 2017.

METASTASIS IN THE BRAIN While some systemic cancer treatments can prolong patient survival by delaying metastasis, this isn’t true of brain metastasis. In fact, by extending patients’ lives, such treatments may even increase the chance that cancer will have time to spread to the brain. Prior to the advent of Herceptin and other targeted therapies, brain metastases generally only occurred in very late-stage cancer patients who were already dying of systemic disease, explains National Cancer Institute (NCI) researcher Patricia Steeg. Despite years of experience in metastasis research, she hadn’t heard of cancer spreading to the brain until about a decade ago, when she attended a conference where some colleagues mentioned seeing the phenomenon in their patients. “What started happening was this explosion of patients that had brain [metastases] who were not at that advanced stage—they had a life to live. And they were often responding from the neck down to Herceptin and the eventual other” therapies for breast cancers that overproduce the HER2 receptor. Steeg says that while patients were living longer with Herceptin treatment—long enough for their cancers to metastasize to the brain—she suspects that another factor in the uptick in brain metastases was that Herceptin can’t penetrate brain tumors. In addition, notes Matt Vander Heiden of the Koch Institute for Integrative Cancer Research at MIT, brain metastases have more metabolic differences from the primary tumor than do metastases elsewhere in the body, which may explain their resistance to therapies that work on the primary tumor. (See “Bisrat Debeb: Studying Cancer’s Spread” on page 43.) Steeg has been studying brain metastasis in animal models ever since she first learned about it and says she now sees some hope for moreeffective treatment and prevention of the condition. She points, for instance, to an ongoing NCI-funded trial led by Priscilla Brastianos of Massachusetts General Cancer Center in which researchers are analyzing brain metastasis tissue and, on that basis, trying to match patients to an existing approved treatment. There’s reason to think this approach could be promising; last year, Brastianos and her colleagues reported that in more than one-third of breast cancer patients with brain metastases, the receptors displayed on the tumor cells in the breast differ from those displayed on the cells in the brain tumor, such that the cancers at the two sites would call for different targeted treatments (Neuro-Oncology, 22:1173–81, 2020). For example, a cancer might overexpress the estrogen receptor in the primary breast tumor but not in the brain metastasis. The findings offer a glimmer of hope for patients suffering from brain metastases, which are exceptionally deadly and come with debilitating symptoms such as cognitive changes, seizures, and loss of limb function, from both the cancer itself and the treatment, Steeg notes. “This is not how these patients care to live their lives. This is why we have to do something.” VOL. 35 ISSUE 04.2021 2 | T THE HE S SCIENTIST C IE N T IST 3A1

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GENOME

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In the past decade, researchers have come to realize that tumors harbor bits of extrachromosomal DNA that can drive malignancy. BY PAUL MISCHEL

CREDIT LINE

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n the spring of 2012, my colleagues and I began to notice something strange in tumor cells from patients with glioblastoma, a highly aggressive form of brain cancer, who were coming into our clinic at the University of California, Los Angeles. From genomic sequencing of their tumors, we knew they displayed amplification of a specific growthpromoting oncogene. Despite being treated with drugs designed to target this gene, the patients were not getting better, and when we interrogated the genomes of their cancers after the tumors were surgically removed following treatment, we saw that they had changed. The tumors had dramatically reduced the number of copies of the targeted epidermal growth factor receptor (EGFR) gene, presumably giving them an advantage to escape the drugs, and they had evolved these genetic differences at a rate that seemed to make no sense— within just one to two weeks. Normally, we think of cancers evolving over many cell divisions, as the cells carrying genetic changes that provide a fitness advantage—such as an ability to resist a particular treatment— will be more likely to survive and divide. Here, we were noticing a change in the copy number of the gene within just a few generations. There was no way that we could explain how the tumors were altering their DNA so quickly. Even stranger, we could take any cell from the tumor, and whether it had high or undetectable protein levels of EGFR, it would give rise to a new tumor when cultured in the lab or implanted into a mouse. Each of these new tumors would then display the full spectrum of cells found in the original tumor, varied in their EGFR copy number. This makes no sense according to what we know about classical genetics. We would have expected that tumors arising from a cell with low levels of EGFR 04 . 2021 | T H E S C IE N T IST 3 3

would give rise to a tumor with low EGFR levels, whereas a tumor arising from a cell with high levels of EGFR would give rise to a tumor with high EGFR levels. In 2012, in a project spearheaded by a graduate student in my lab named David Nathanson, who is now an associate professor at the University of California, Los Angeles, we set out to understand why. We wanted to see where copies of EGFR were located. Typically when we sequence cancers, we grind up a tumor and “read” all of the genes present, looking for mutations and copy number variations, and then we assign the location of these genetic alterations to the chromosomes where those genes are in the human reference genome. But when we looked at a cell getting ready to divide—the only time when it’s possible to tell where a specific piece of DNA is located—we were surprised to find that EGFR was not where we’d predicted. In

Extrachromosomal DNA was once thought to be a rare event in cancer, but our group showed that ecDNA is very common indeed. fact, it was not sitting on a chromosome at all. Rather, all of the amplified oncogenes were found on circles of extrachromosomal DNA (ecDNA). We could see these extra pieces of DNA near the chromosomes inside the nucleus of cancer cells.

Oncogene ecDNA

In contrast to chromosomal DNA, which is replicated and divvied up equally among daughter cells during cell division, the extrachromosomal DNA (ecDNA) found in some cancer cells is not always split evenly. Lacking centromeres, these circular bits of DNA are often unevenly parceled to daughter cells. Some daughter cells receive more ecDNA, which they then duplicate, so the copy number of oncogenes in those cells rises quickly. Moreover, because each cell division is essentially a “coin flip” with regard to ecDNA inheritance, variation among the cancerous cell population is preserved, providing an ample supply of the fuel needed for natural selection. These two features in combination could enable cancers containing ecDNA to evolve much more rapidly than cancers lacking ecDNA can.

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HOW ecDNAs MIGHT DRIVE CANCER EVOLUTION

When we removed the treatment with the EGFR inhibitor from cultured tumor cells, EGFR copy number quickly rebounded, but again, not on chromosomes. When we saw this, we realized that ecDNA might explain why some cancers can become resistant to treatment so quickly, allowing tumors to evolve at a rate that far exceeds anything that could be accounted for by classical genetics. We published our results in Science in 2014,1 but they were not immediately accepted by the community. Although we had only studied one tumor type, glioblastoma, we began to wonder whether this might be the tip of the iceberg. Without realizing it, this study led us, and now others, to a series of discoveries that have changed the way that researchers view cancer in general, revealing frightening ways that tumors can evolve. We have learned that ecDNA is central to

the behavior of some of the most aggressive forms of cancer, enabling remarkably elevated levels of oncogene transcription, creating new gene regulatory interactions, and providing a powerful mechanism for rapid change that can drive very high oncogene copy numbers or allow cancer cells to resist treatment. Along the path of discovery, we found that we were not the first to have seen these extrachromosomal particles. But with new tools in hand, and new questions in mind, we saw them in a very different way. And when we took the time to peer deeply into the nucleus of cancer cells, we saw ecDNAs in abundance. We eagerly pursued studies to understand their importance in cancer progression and drug resistance, and even founded a biotech company, Boundless Bio, to identify and develop new ways to treat patients whose cancers are driven by ecDNA.

Misleading maps Humans depend on maps to navigate complex landscapes and to understand the world around us. Without our maps, we’re lost. However, our maps also limit us. If something is missing from the map—or even worse, the map is wrong—we can be thrown off course. Consider Ptolemy’s and Copernicus’s maps of the solar system. Ptolemy made precise measurements of the planets moving across the heavens, but he got the orientation of his map wrong by placing Earth in the center. It took nearly 1,500 years for Copernicus to correct it. Researchers have been making maps of cancer for a long time. First we made organ-based maps, then cell type–based maps. These are still the dominant maps researchers and physicians use to navigate cancer, but the advent of powerful DNA sequencing technologies has ushered in a new era of cancer mapping at the level of the genome. We can take DNA from a patient’s tumor

and find genetic mutations that are to blame. This has become the basis for cancer diagnosis and treatment. But, as we learned in our seminal study several years ago, something was missing from genome-based maps: ecDNA. Since 1965, scientists have recognized that ecDNA elements exist in cells of certain cancers, in particular neuroblastoma. These elements were initially referred to as “minute chromatin bodies,” and then as “double minutes” because they were often paired. In the late 1970s and 1980s, a number of research teams showed that these double minutes could lead to gene amplification. In contrast with this earlier work, we found that ecDNAs are not always found in pairs. In fact, ecDNAs occur as paired bodies only 30 percent of the time.2 Therefore, for clarity, here we use the term “ecDNA” to describe both singlet ecDNA particles and double minute ecDNA. Until very recently, despite efforts to decipher ecDNA structure, the importance of these ecDNA particles remained unclear, and as

HOW ecDNAS MIGHT SUPPORT CANCEROUS GROWTH The circular nature of ecDNAs can enable gene interactions that may support the increased transcription of oncogenes, as genetic elements normally found in distant parts of the genome may come together to interact. While insulators in the chromosomal DNA sit at the stem of a loop structure and ensure that regulatory sequences such as enhancers work only on their nearby target genes, the circular shape of ecDNA generates new interactions with additional regulatory sequences that would not normally occur on chromosomal DNA.

Chromosomal DNA

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Enhancers

Oncogene

Additional regulatory sequences

Additional regulatory elements can support higher oncogene expression.

Insulators

Histone mark

Open chromatin arrangement can also support oncogene expression.

Additionally, ecDNAs tend to have a more-open chromatin structure than chromosomes that promotes increased gene expression. DNA is wound around histone cores into units of organization called nucleosomes. On chromosomes, some regions can become highly compacted, rendering the DNA inaccessible to the transcriptional machinery, but ecDNAs have an altered chromatin structure in which the nucleosomes do not compact, resulting in highly accessible DNA that is primed for transcription. Moreover, ecDNAs are loaded with active histone marks but have a paucity of repressive histone marks, promoting high levels of transcription. 36 T H E SC I EN T I ST | the-scientist.com

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Histone

the field developed new techniques to generate genome information–rich maps of cancer, most researchers stopped thinking about ecDNA, which was thought to occur rarely. Indeed, according to the Mitelman database of chromosomal aberrations, ecDNA only occurred in about 1.4 percent of cancers. Thus, after we published our 2014 study showing that ecDNA is a very common event in cancer, there was what I would call a colossal scratching of heads. I would give talks about the work and people would say, for example, “That’s funny, the cancer-causing gene that I am studying is on chromosome 8.” I would then ask how they knew that, and they would present me with a copy of a cancer map based on the human reference genome. I realized that we might be looking at a “Ptolemy” map.

Appreciating extrachromosomal DNA After our Science paper was published, I began working very closely with Vineet Bafna, a computer scientist at the University of California, San Diego, who had played a critical role in decoding the human genome. In a study spearheaded by a researcher from my lab named Kristen Turner (now at Boundless Bio), along with computer scientists Viraj Deshpande and Doruk Beyter from Bafna’s group, we integrated the old with the new—modern genomic tool kits, powerful next-generation sequencing, advanced computational methods, and visualization under the microscope of the locations of ecDNA. This work revealed the presence of ecDNA in almost every cancer type we looked at. We further found that ecDNA enabled cancers to change their genomes quickly, and even to move genes back onto chromosomes, but not in their original locations.3 In early 2017, I gave a seminar on these findings at Stanford University, where I met Howard Chang, a physician-scientist there who had developed techniques for analyzing the chromatin architecture and epigenetic structure of cancer cells. Chang had recently discovered major changes in the structure of chromatin in cancer that allow transcriptional machinery greater access to the DNA to drive transcription. After my talk, we discussed how ecDNA might generate a far more open chromatin pattern, enabling enhanced transcription of oncogenes, in line with what Chang was finding. Working closely with Chang, as well as with Bafna and the University of California, San Diego’s Bing Ren, my postdoctoral fellows Turner, Sihan “Sean” Wu, and Nam Nguyen set out to better understand the structure of ecDNA and its transcriptional consequences. We first demonstrated that ecDNA is circular. These ecDNAs differ from the more common small extrachromosomal circular DNA particles (eccDNAs) that are found in the genomes of eukaryotes from yeast to flies to humans. For one, eccDNAs average only about 100–1,000 base pairs in length and are thus relatively unlikely to contain intact genes or other functional elements, while ecDNAs are relatively large, averaging around 1.3 million base pairs. ecDNAs are chock-full of growth-promoting oncogenes, and they contain other genes and regulatory regions that may be involved in tumor formation and progression. Moreover, unlike other types of small circular DNAs such as eccDNAs and ribosomal DNAs that can be found in nor-

Researchers have been making maps of cancer for a long time, but we now know that we’ve been missing something from our maps.

mal cells at low levels, ecDNAs are unique to cancer, and they are highly amplified. And, because like all circular DNAs they lack the centromeres used to segregate homologous chromosomes, they are often not evenly divided between daughter cells upon cell division. This allows cancers with ecDNA to evolve rapidly, accumulating a great many copies of cancercausing genes or reducing the number of drug-targeted oncogenes, while maintaining the cell-to-cell variability that drives accelerated tumor evolution and drug resistance. (See illustration on page 34.) The three-dimensional structure of ecDNA also holds clues to its role in some cancers. By combining ultrastructural microscopy and whole-genome sequencing with computational reconstruction and long-range DNA optical mapping in which very long fragments of DNA, sometimes more than 150,000 base pairs, can be resolved, my collaborators and I showed that ecDNAs are circular and that the DNA is wound around histone cores into nucleosomes like chromosomal DNA is, but in a highly abnormal fashion. ecDNA chromatin displays a significantly lower degree of compaction compared with the same DNA segments that reintegrate into chromosomes, suggesting that altered DNA shape may actively contribute to the transcription of oncogenes found on ecDNA. (See illustration on opposite page.) In fact, singlecell imaging further revealed that ecDNA is among the most transcriptionally accessible chromatin in the cancer genome in actively cycling tumor cells. We also found that ecDNA is loaded with chromatin modifications that promote transcription and has a paucity of repressive chromatin marks, suggesting that it is poised for high levels of gene expression. Further, we found that ecDNA chromatin is well organized into loops that are normally an important part of gene regulation, but with a three-dimensional topology that is distinct from that of chromosomal DNA. As the DNA segment becomes circular, in a process that is still incompletely understood, distal DNA elements are brought into proximity, enabling ultra-long-range chromatin interaction that cannot be achieved by chromosomal DNA. This could potentially form new gene regulatory circuits, including new active contacts that drive oncogenic transcription. Consistent with these findings, we found that oncogenes residing on ecDNA are in 04 . 2021 | T H E S C IE N T IST 37

the top 1 percent of the most-transcribed genes in cancer cells that have them. We published these results in Nature in November 2019, and the paper was highlighted in a story by Carl Zimmer in The New York Times. Very shortly thereafter, two other groups— one led by Peter Scacheri of the Cleveland Clinic and Case Western Reserve University and Jeremy Rich of the University of California, San Diego,4 and another by Anton Henssen of Charité Hospital in Berlin and Richard Koche of Memorial Sloan Kettering Cancer Center5—added further evidence that ecDNAs may play a pivotal role in reorganizing the transcriptional control of cancer genomes by bringing regulatory elements encoded on ecDNA into contact with genes with which they would never interact in chromosomes. Recent work from Chang, in collaboration with myself, Bafna, and Henssen, has begun to suggest a very exciting new way that these circular pieces of DNA, instead of acting alone, often organize themselves into nuclear bodies called ecDNA hubs.6 These hubs are tethered by proteins and appear to provide a platform for cooperative transcription, in which ecDNAs work together to drive the expression of cancer-promoting genes. The question then became, how do we capitalize on this new understanding of ecDNA to improve patient outcomes? In 2018, Chang, Bafna, and I, with other scientists, cofounded Boundless Bio, where several staff scientists now seek the answer to that question.

Cancer Research UK recently designated ecDNA as one of the Cancer Grand Challenges that must be addressed. It is exciting to see mounting interest and an influx of talented investigators aiming to decipher the key aspects of ecDNA biology. We look forward to the development of new tools, new collaborations, and new treatments for patients. Joshua Lederberg wrote in his landmark 1952 paper in Physiological Reviews: “I propose plasmid as a generic term for any extrachromosomal hereditary determinant.”9 In bacteria, circular plasmids are a powerful mechanism for gaining selective advantage because they enable rapid evolution, including drug resistance. Similarly, yeast, weeds, and even parasites can evade drugs and environmental toxins by encoding resistance genes on circular extrachromosomal DNA. ecDNAs may do the same for cancer, providing a potent vehicle for rapid tumor evolution that maximizes critical oncogenic gene variants—or reduces them to evolve drug resistance. Just as explorers rely on maps of the Earth, and astronomers on maps of the galaxy, cancer biologists depend on maps to navigate the complexities of cancer. We now know that we’d long been missing a critical element. So here we are once again, as physiological cartographers, rolling up our sleeves and making new, topographically informed maps of cancer to help us navigate the multifarious disease and develop new and more effective treatments for patients. J

Translating ecDNA to clinical application

Paul Mischel is a professor and Vice Chair for Research for the Department of Pathology at Stanford University School of Medicine and an Institute Scholar in ChEM-H at Stanford University.

38 T H E SC I EN T I ST | the-scientist.com

References 1. D.A. Nathanson et al., “Targeted therapy resistance mediated by dynamic regulation of extrachromosomal mutant EGFR DNA,” Science, 343:72–76, 2014. 2. K.M. Turner et al., “Extrachromosomal oncogene amplification drives tumour evolution and genetic heterogeneity,” Nature, 543:122–25, 2017. 3. S. Wu et al., “Circular ecDNA promotes accessible chromatin and high oncogene expression,” Nature, 575:699–703, 2019. 4. A.R. Morton et al., “Functional enhancers shape extrachromosomal oncogene amplifications,” Cell, 179:1330–41.E13, 2019. 5. R.P. Koche et al., “Extrachromosomal circular DNA drives oncogenic genome remodeling in neuroblastoma,” Nat Genet, 52:29–34, 2020. 6. K.L. Hung et al., “EcDNA hubs drive cooperative intermolecular oncogene expression,” bioXriv, doi:10.1101/2020.11.19.390278, 2020. 7. O. Shoshani et al., “Chromothripsis drives the evolution of gene amplification in cancer,” Nature, doi:10.1038/s41586-020-03064-z, 2020. 8. H. Kim et al., “Extrachromosomal DNA is associated with oncogene amplification and poor outcome across multiple cancers,” Nat Genet, 52:891–97, 2020. 9. J. Lederberg, “Cell genetics and hereditary symbiosis,” Physiol Rev, 32:403– 430, 1952.

© ISTOCK.COM, MELETIOS VERRAS

Working closely with Chang, Bafna, and Roel Verhaak of the Jackson Laboratory (also a co-founder of Boundless Bio), we are trying to understand some of the clinical implications of ecDNA. Publicly available databases, including The Cancer Genome Atlas and the Pan-Cancer Analysis of Whole Genomes, contain a large number of whole-genome sequences of cancer samples, yielding a golden opportunity for discovery. We applied the AmpliconArchitect, a tool developed by Bafna that looks for the telltale signs of ecDNA in whole-genome sequencing data, including amplified regions that map to a circle, and then uses algorithms that deconvolute these circular structures. This enabled us to analyze the frequency and potential structural composition of ecDNA in more than 3,200 cancer samples of a wide range of histological types alongside matched whole blood and normal tissue. Our findings indicated that ecDNA is unique to cancer, and that at a minimum, 14 percent of human tumors, including some of the most malignant forms of cancer, harbor ecDNA.7 Further, we found that patients whose cancers have ecDNA have significantly shorter survival than cancer patients whose tumors are driven by lesions in chromosomal DNA. It remains to be seen how commonly ecDNAs play a role in the evolution of drug resistance, as we saw hints of in our initial study. Many other questions remain as well. Recent studies have shed light on how ecDNA may form,8 although we and others strongly suspect that there may be multiple routes to its development. The problem of ecDNA in cancer, and the challenge that it represents, has become clear. The National Cancer Institute and

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Developing effective vaccines using IsoPlexis’ highly

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cientists are still exploring and testing the requirements for successful

Multifaceted functional proteomics accelerates the future of medicine

vaccines. In addition to inducing antibody-based immunity, vaccines

elicit protection through T cells. Assessing polyfunctionality—the ability

With the IsoPlexis platform, the entire workflow is completely automated and

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hands-off. When running single-cell analysis, researchers load the samples onto

understanding T cell responses against infection and for developing effective

the chip and insert it into the IsoLight or IsoSpark, which automates the incubation,

vaccines. Highly polyfunctional “superhero” cells perform a broad range of

ELISA, data analysis, and more. The same IsoLight/IsoSpark system can also run

functions, leading to protective immunity through strong, persistent responses.

highly multiplexed serum analysis with only five minutes of hands-on sample

But protein assay technologies such as ELISpot and flow cytometry, which have

loading time. The automation for both single-cell and bulk serum analyses allows

been used for infectious disease research and vaccine development for decades,

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measure only a few cytokines per cell. As such, they cannot effectively predict

per replicate) experiments is particularly advantageous for small animal models—

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such as the mouse model used by Zhou et al.—and precious clinical samples.

IsoPlexis’ functional single-cell proteomics overcomes the limitations

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References

in creating effective therapies and vaccines for a broad range of diseases.

Functional single-cell proteomics sheds light on malaria vaccine durability Malaria still ranks among the most prevalent infectious diseases globally. Approximately 200 million people become infected yearly, with relatively high rates of morbidity and mortality. Challenges in designing durable vaccines include the lack of knowledge of key targets of immunity as well as achieving sustained, high efficacy, meaning very few effective malaria vaccines so far. In a Human Vaccines & Immunotherapeutics article, Zhou et al. from the Aaron Diamond AIDS Research Center characterized T cell responses to a sporozoitebased malaria vaccine in a mouse model with functional single-cell proteomics. Sanaria’s Plasmodium falciparum sporozoites (PfSPZ) malaria vaccine proved to be safe and effective in a Phase 1 clinical trial. The PfSPZ vaccine elicits a potent response from CD8+ T cells in the liver in non-human primates. Other studies have also emphasized the role of hepatic CD8+ T cells induced by the sporozoite-based malaria vaccine in a mouse model. But it has not been clear which characteristics of hepatic CD8+ T cells are associated with protective anti-malaria immunity. To identify the functional mechanisms, Zhou et al. used IsoPlexis’ single-cell proteomics to identify polyfunctionality in hepatic CD8+ T cells, induced by PfSPZ vaccine, and the potential biomarkers for evaluating vaccine efficacy. The cytokines/chemokines secreted by these polyfunctional CD8+ T cell XZGXJYX NSHQZIJ 2.5Ⱥ 7&39*8 .+3ȼ FSI .1&9MJXJ FXXTHNFYJ \NYM protective T cell responses against certain pathogens. The results suggest

1. J. Zhou et al., “CD8+ T-cell mediated anti-malaria protection induced by malaria vaccines; assessment of hepatic CD8+ T cells by SCBC assay,” Hum Vaccin

Immunother 

that successful induction of such polyfunctional hepatic CD8+ T cells may lead to the development of an effective human malaria vaccine.

isoplexis.c om

VOL. 35 ISSUE 2 | THE SCIENTIST

A

EDITOR’S CHOICE PAPERS

The Literature NEUROSCIENCE

Size Matters THE PAPER

S. Holler et al., “Structure and function of a neocortical synapse,” Nature, doi:10.1038/ s41586-020-03134-2, 2021.

4 0 T H E SC I EN T I ST | the-scientist.com

Neurotransmitter vesicle

Activated receptor

Unactivated receptor

BRAIN FLEX: By monitoring changes in voltage (graphs) in individual neurons in the mouse neocortex, researchers measured the strength of connections between a presynaptic neuron’s axon terminal (purple) and the postsynaptic neuron’s dendrite (beige). In contrast to the widely accepted notion that neocortical synapses can only release one neurotransmitter-filled vesicle per firing, the research team found evidence that multiple vesicles can be released at these synapses, indicating that their strength is more plastic than previously appreciated. The researchers also confirmed the widely held assumption that physically larger synapses are stronger.

Stephanie Rudolph, a neuroscientist at Albert Einstein College of Medicine who was not involved with the study, calls it a “technical tour de force” that confirms the relationship between synapse size and strength, a longstanding question in neuroscience. Some researchers have measured neuron connectivity simply by counting the number of synapses between a neuron pair. The finding that larger synapses are stronger will allow connection strength to be assigned to a synapse based on its size, providing “a much more accurate picture of the connection,” says Schuhknecht. This should inform maps of the brain’s connectome that scientists are developing for fruit flies and mice, with the goal of understanding how information flows through the brain. The strength of a synapse also depends on the number of neurotransmitter release sites in an axon terminal and the probability of a vesicle being released. Until

now, most neuroscientists’ understanding was that synapses in the neocortex could release only a single vesicle of neurotransmitter per action potential, says Schuhknecht. But the research team calculated that the number of release sites exceeded the number of synapses between each neuron pair in the mouse brain slices, indicating that each synapse may be capable of releasing multiple vesicles. This means that the strength of synapses in the neocortex—the largest region in human brains—is more flexible than has been recognized. “[This finding] profoundly alters the way we think about the predominant mode of synaptic transmission,” says Rudolph. “We can hypothesize that [multivesicular release] increases the ability of the brain to adapt to inside and outside challenges and allows a broader range of computational processing and information storage.” —Asher Jones

© MELANIE LEE

Brain cells use a language of neurotransmitters to pass messages to each other at junctions called synapses. A single neuron can have tens of thousands of synapses, allowing it to talk to thousands of other brain cells. These connections mediate information flow through the brain, and the plasticity of synapse strength is thought to underlie memory, learning, and other forms of cognition. Researchers have long suspected that synapses with greater surface areas are stronger, but have lacked experimental evidence for this, says Gregor Schuhknecht, a neuroscience postdoc at Harvard University. To answer this question, Schuhknecht, then a graduate student at the Institute of Neuroinformatics at the University of Zurich and ETH Zurich, and his colleagues identified synapses between neuron pairs in the neocortex region of mouse brain slices. When an electrical impulse known as an action potential triggers the release of neurotransmitterpacked vesicles from a neuron’s axon terminal, these chemicals f low across the synapse and are recognized by receptors in the receiving neuron’s dendrite, which in turn may trigger an action potential in this cell. The team recorded the change in voltage of the receiving neuron with a tiny electrode to measure synapse strength and used electron microscopy to calculate synapse size. Sure enough, they found that synapses with a larger postsynaptic density area—the part of the dendrite that houses neurotransmitter receptors—produced greater voltage changes.

FASEB BIOADVANCES, DOI:10.1096/FBA.2020-00092, 2020; LAM KHUAT

CANCER’S HELPER?: A tissue section from a prostate cancer patient who

TRANSPLANT TROUBLE: Gut bacteria partly explain why obese mice had

produces Siglec-XII (stained brown), which is much more highly expressed in malignant cells (lower) than normal cells (upper).

worse outcomes than lean individuals after bone marrow transplantation.

CANCER BIOLOGY

MICROBIOLOGY

Rogue Protein

Gut Bomb

THE PAPER

THE PAPER

S.S. Siddiqui et al., “Human-specific polymorphic pseudogenization of SIGLEC12 protects against advanced cancer progression,” FASEB BioAdvances, 3:69–82, 2021.

L.T. Khuat et al., “Obesity induces gut microbiota alterations and augments acute graft-versus-host disease after allogeneic stem cell transplantation,” Sci Trans Med, 12:eaay7713, 2020.

Among a group of cell surface proteins known as sialic-acid-binding immunoglobulin-like lectins (Siglecs), CD33-related Siglecs are found mainly on innate immune cells and are involved in cell signaling. One Siglec, however, appears to have “gone rogue” in humans, according to Ajit and Nissi Varki, a husband-and-wife team at the UC San Diego School of Medicine. Siglec-XII, encoded by the gene SIGLEC12, no longer binds sialic acid and seems to be involved in abnormal cell signaling in humans, the researchers report. The Varkis argue that the protein plays a role in cancer progression and could help explain why humans have much higher rates of carcinoma—cancers that arise from epithelial cells, where Siglec-XII is abundant—than do other great apes. Only about 30 percent of humans produce this rogue protein; most people have a mutation that inactivates SIGLEC12. The Varkis and their colleagues found Siglec-XII in about 80 percent of carcinoma samples but in just 35 percent of normal tissues. When they forced production of Siglec-XII in a human prostate cancer cell line, the result was higher expression of cancer progression–related genes than in prostate cancer cells that lacked the protein. And comparing cohorts of cancer patients, the team found that functional SIGLEC12 was associated with poor prognosis in late-stage colorectal cancer patients. “The study proposes very interesting hypotheses,” says Jun Wang, an immunologist at NYU Langone Health who was not involved in the research. But, he says, more evidence is needed to confirm Siglec-XII’s role in cancer progression because artificial overexpression of the protein in prostate cancer cells could differ from how the protein behaves in tumors. He notes that it would also be interesting to examine how Siglec-XII in immune cells contributes to cancer. “The cancer cell is just part of the puzzle. The whole picture is cancer and the immune system.” —Asher Jones

Bone marrow transplants are widely used to treat certain cancers and blood diseases, but these procedures run the risk of a serious complication called graft-versus-host disease (GVHD). This immune disorder occurs when donor T cells recognize the recipient’s body as foreign, triggering inflammatory immune responses that damage the patient’s organs and can cause death. Previous studies have shown that obesity can influence immune responses, but its effects on GVHD are poorly understood. To interrogate the effect of obesity on bone marrow transplant outcomes, UC Davis Health immunologist William Murphy and his team used a donor-recipient mouse strain combination in which transplantation usually causes GVHD in the form of scaly skin and hair loss. In lean recipients, the transplant caused skin symptoms and all of the mice survived, as expected. In contrast, most mice with dietinduced obesity died within weeks from severe damage to the gut epithelial lining associated with greatly elevated levels of inflammatory cytokines, an outcome that resembled acute GVHD. “That was a shock,” says Murphy. “Just by making [the mice] fat . . . we got markedly different outcomes.” In humans, the researchers found that obese bone marrow recipients had higher levels of a GVHD biomarker, more-severe gut damage, and lower survival than healthy-weight patients. Obese mice and humans both had lower gut bacterial diversity than their lean counterparts and lower levels of bacteria associated with good GVHD outcomes. When the team gave obese mice antibiotics before transplantation, survival improved by about 50 percent, suggesting that obesity-induced changes to gut bacteria affect GVHD severity. But “while the microbiome definitely is playing a role, it’s not the sole cause,” says Murphy. “It’s nice work,” says Anna Staffas, a cancer researcher at the University of Gothenburg in Sweden who was not involved with the study. The results, she adds, “need to be validated in larger cohorts to see if the same conclusions can be drawn for humans.” —Asher Jones 04.2021 | T H E S C IE N T IST 41

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Bisrat Debeb: Studying Cancer’s Spread Assistant Professor in the Department of Breast Medical Oncology, MD Anderson Cancer Center BY CATHERINE OFFORD

PHOTO COURTESY OF MD ANDERSON

B

israt Godefay Debeb used to get some odd looks when he asked people to hand over bits of sheep they were preparing for dinner. As an undergraduate in veterinary medicine at Addis Ababa University in Ethiopia, he was enthralled by his studies and would collect hearts and lungs that family members had removed from the animals at their home in Tigray to show younger students how the respiratory and circulatory systems functioned. Even when his family planned on cooking the organs, Debeb recalls, “I was like, ‘No, you cannot do this. I have to show this to my students.’” Debeb had always been interested in biology, but the realization that he wanted to turn this interest into a research career came toward the end of his degree. For his senior project, he analyzed raw cow’s milk at dairy farms and milk collection centers and detected bacterial contamination, some of which posed a health risk. The university awarded him a medal for academic excellence, and the findings were published in 2000, with Debeb as the first author (Berl Munch Tierarztl Wochenschr, 113:276–78). “That really introduced me to how to do research,” he says. After a stint spent lecturing at Addis Ababa, Debeb moved to the US on a USAID fellowship. He completed a master’s in epidemiology at Tuskegee University in Alabama in 2005 and went straight to Texas A&M University for a PhD. There, he started out working on lentiviruses, but soon switched to stem cell biology, and began characterizing extraembryonic endoderm cells, which compose the yolk sac of mammalian embryos (PLOS ONE, 4:e7216, 2009). Toward the end of his PhD, he attended a talk by Baylor College of Medicine’s Jeffrey Rosen, who “presented about cancer stem cells—a small population of cells that have an enhanced capacity to generate tumors,” Debeb recalls. “More importantly, they are the ones that mediate metastasis and also cause resis-

tance to standard therapy.” Intrigued, Debeb began looking for postdoc opportunities on the subject. Rosen recommended Wendy Woodward’s lab at the University of Texas MD Anderson Cancer Center. Debeb had “done really interesting work on stem cells,” says Woodward. “And he was such a well-spoken, eloquent, smart interviewee that he really stood out as a great candidate.” Joining Woodward’s lab after earning his PhD in 2008, Debeb dived into studying stem cells in aggressive breast cancer. In 2012, the team published findings suggesting that HDAC inhibitors, drugs then being investigated in clinical trials for breast cancer, can actually boost the population of cancer stem cells (STEM CELLS, 30:2366– 77). The work inspired a clinical trial to see how HDAC inhibitors might be combined with other drugs to produce more-favorable responses. Although the study stalled for

logistical reasons, Woodward says, “it was really interesting, and a striking achievement for a postdoctoral fellow.” Debeb had meanwhile become interested in another problem: a lack of animal models to replicate the spread of breast cancer to the brain. He began developing a handful of mouse models to do just that, recreating metastasis by using breast cancer cells injected into the tail vein or transplanted onto the mammary gland (J Natl Cancer Inst, 108:djw026, 2016). He identified the microRNA miR-141 as a key regulator and potential biomarker of brain metastasis along the way. “Bisrat has been sort of a pioneer in developing the brain metastasis model for inflammatory breast cancer,” Rosen says. “This was a major advance.” Since 2017, Debeb has been an assistant professor at MD Anderson, where he continues to investigate metastasis. Recently, he and collaborators identified depleted T cell populations, increased oxidative metabolism, and other cellular and metabolic changes associated with the spread of cancer to the brain (Acta Neuropathol, 141:303–21, 2021). He’s also continuing work on E-cadherin, a cell-surface protein upregulated by miR141 (Commun Biol, 4:72, 2021). The research is supported by a four-year grant from the American Cancer Society. From Houston, where he’s happily settled with his wife and three children, Debeb has also been establishing collaborations with researchers in Tigray. One recent project with Ayder Hospital aimed to improve screening and treatment for cancer patients—although the work came to a standstill when armed conflict erupted in the region last November. Worried for family members and for Tigray’s future, Debeb’s now thinking about how to contribute to recovery efforts. With violence and communication blackouts plaguing the region, the possibility feels distant, he says, “but I want to . . . help in that, in rebuilding Tigray.” J 04 . 2021 | T H E S C IE N T IST 4 3

BIO BUSINESS

Getting to the Source Many major biopharmaceutical companies are developing or acquiring drugs that target the NLRP3 inflammasome, a large intracellular complex that researchers say can spark inflammation and stoke diseases of lifestyle and aging. BY RACHAEL MOELLER GORMAN

4 4 T H E SC I EN T I ST | the-scientist.com

NLRP1 inflammasome when he was a PhD student in the late Jürg Tschopp’s lab at Lausanne. “When they started to inject him with the anti–IL-1 drug, within [a] few hours the skin rashes on his body were gone, the fever was gone—that was kind of a game-changer and kind of spectacular.” Martinon, now faculty at the university, later helped confirm that elevated NLRP3 inflammasome activity had indeed caused the British man’s symptoms. Fast forward almost two decades from the man’s treatment, and the field

SPARKING INFLAMMATION: Once activated,

the NLRP3 inflammasome (illustrated) triggers a suite of pathways that may play an important role in many different diseases.

of inflammasome research is booming. Studies in animals have suggested that the NLRP3 inflammasome’s impact can be felt far beyond rare genetic diseases such as Muckle-Wells. Microparticles that accumulate in tissue as a result of modern Western lifestyles and longer lives may activate the NLRP3 inflammasome, stoking inflammation and exacer-

ARTWORK REPRODUCED WITH PERMISSION FROM PROFESSOR KATE SCHRODER, THE UNIVERSITY OF QUEENSLAND

E

very morning of his life, no matter what he did, a young British man woke up with a fever, a headache, a rash on his trunk and limbs, joint pain, and ever-worsening deafness. His doctors had diagnosed him with a rare illness called Muckle-Wells syndrome, in which chronic inflammation rages out of control; no treatment had been able to cure his symptoms. Then, in 2001, researchers in California discovered the genetic explanation for his disease: a mutation in a gene called CIAS1 (since renamed NALP3 and then NLRP3), which was later found to cause the resulting protein to be constantly activated. Around the same time, a group of scientists at the University of Lausanne in Switzerland found that NALP1/NLRP1, a protein from the same family, forms a large intracellular complex called an inflammasome, which helps generate the IL-1` cytokine, triggering a cascade of inflammation. Taken together, the findings suggested that in Muckle-Wells, inflammasomes made with NLRP3 instead of NLRP1 were constantly forming and could be producing huge amounts of IL-1`, causing the man’s illness. So researchers Philip Hawkins of the Royal Free Hospital and University College Medical School (now UCL Medical School) and Michael McDermott of St. Bartholomew’s and the Royal London School of Medicine (he’s now at the University of Leeds) gave the man an existing arthritis drug called anakinra (Kineret), which blocks the IL-1 receptor. By the time the man received treatment in 2002, at 22 years old, “he had experienced fever, flu-like symptoms for his entire life, that was his normalcy, he didn’t know what it is to feel well,” recalls Fabio Martinon, who helped discover the

bating or even causing dozens of diseases of aging and lifestyle, from Alzheimer’s disease and cancer to type 2 diabetes and cardiovascular disease. These developments have attracted the interest of biotech companies, too. Unlike the injected IL-1 receptor blockers that the British man received, which can dampen broader immune responses needed to fight infection, NLRP3 inhibitors could be taken orally and only inhibit NLRP3-mediated inflammation, offering a more practical, more effective, and safer therapy. Since 2016, several small pharmaceutical companies have developed targeted NLRP3 inhibitors, and in the past year, large companies have scooped up those smaller companies in huge deals or have started developing inhibitors of their own. Torreya Capital’s Biopharmaceutical Sector Update Market Outlook ranked “Inflammasome Science” as one of the top five biopharma events of 2020. While no NLRP3-targeting drugs have hit the market just yet, “the science is unequivocal,” says Kate Schroder, an inflammasome researcher at the University of Queensland in Australia and one of the co-inventors on inflammasome inhibitor patents originally licensed to the Irelandbased company Inflazome (since acquired by Roche). “It’s really clear that this is an important pathway for human health, and if we can drug it, we will be able to improve a whole bunch of different diseases, some of which are diseases for which we don’t have any disease-modifying drug.”

The inflammasome’s early days In the years since the discovery of the NLRP1 inflammasome in 2001, researchers have learned that there are up to 15 types of inflammasomes (though only eight are well established), each responding to a unique danger signal. Most of these complexes, including the NLRP1, NLRC4, and AIM2 inflammasomes, can be activated by pathogens. NLRP3, by contrast, seems to be activated primarily in the presence of accumulated intracellular debris, including cholesterol crystals (which lead to atherosclerosis),

amyloid-`particles (which are associated with Alzheimer’s disease), and tobacco smoke particulates (which contribute to lung disease), among others. Although the precise mechanisms are still unclear, scientists have established the basics of how individual NLRP3 proteins come together to create an NLRP3 inflammasome. First, phagocytosing white blood cells such as macrophages engulf particles or crystals, which damage these cells’ plasma and lysosomal membranes. This causes potassium ions to move out of the macrophages, which in turn activates individual NLRP3 proteins in those cells. Once active, each NLRP3 associates with several others, forming a snowflake shape; then, so-called adaptor proteins assemble into long filaments that dangle from the NLRP3 wheel, resulting in a huge protein complex: the NLRP3 inflammasome can be the size of one of the cell’s organelles.

There’s obviously a whole portfolio of indications where inhibiting the inflammasome could make a lot of sense. —Guido Junge, Novartis Institutes for BioMedical Research

“They’re quite beautiful,” says Matt Cooper, a chemist at the University of Queensland. “It looks a bit like a Doric column with a starfish on the top.” On the surface of this complex, pairs of caspase-1 enzymes bind to each other, become active, and cleave precursor molecules for IL-1` and IL-18, transforming them into their mature inflammatory forms, which are then secreted from the cell. Caspase-1 also processes other compounds that trigger a form of inflammatory cell death known as pyroptosis. “NLRP3’s major role is to sense things like particles and crystals that need to be digested by the body, but which one cell can’t cope with,” says Schroder. “It makes sense for that cell to ring the alarm by

triggering IL-1 production, which recruits new cells to the site of the crystals or particles so you have a whole bunch of phagocytes to clean up the mess.” Disease can result, she suggests, when there are so many of these particles that they keep activating NLRP3 in new cells even after the previous cells die, relentlessly ramping up inflammation.

Drugging the inflammasome For years after the inflammasome’s initial discovery, the search for drugs that target it yielded no promising leads, Cooper says. “Big pharma all went and screened their libraries of drugs and said, can we find an inhibitor? But no one could.” Several years ago, that began to change. Luke O’Neill, an immunologist at Trinity College Dublin, was one of the people searching for an NLRP3 inhibitor, with colleagues at his company Opsona. He knew that a scientist named Chris Gabel had been working at Pfizer in the late 1990s on a molecule called CRID3, which appeared to block IL-1` production. O’Neill was intrigued and in 2012 teamed up with Cooper to study the compound. They were eventually able to show that CRID3, which the team renamed MCC950, was a highly selective and potent inhibitor of NLRP3. Together with Schroder’s group and others, the researchers filed patents and published a paper on the compound in 2015 in Nature Medicine. Soon, independent teams of researchers began showing that MCC950 improved symptoms and biomarkers of disease in animal models of Alzheimer’s, atherosclerosis, asthma, inflammatory bowel disease, stroke, and many other conditions. In a rodent model of Parkinson’s disease, for example, inhibiting NLRP3 with MCC950 protected against neurodegeneration and reversed the animals’ motor deficits. Meanwhile O’Neill and Cooper set out to raise venture capital to create the company Inflazome, based on the work their teams had done on MCC950 and derivatives. But it wasn’t easy. “Back then, people thought you were making it up. Like, it can’t possibly be true that one protein can 04 . 2021 | T H E S C IE N T IST 4 5

BIO BUSINESS

INFLAMMATION ON TRIAL Various companies, several of which have now been acquired, are investigating the effects of inhibiting NLRP3 inflammasomes.

Company

Drug(s)

Disease

Trials

Inflazome Dublin, Ireland (acquired by Roche in 2020)

Inzomelid, Somalix

Cryopyrin-associated periodic syndrome (CAPS) and others

Both drugs have completed Phase 1 trials. Inzomelid completed Phase 1b in people with CAPS.

NodThera Cambridge, UK; Seattle; and Boston

NT-0167 (two additional candidates moving to clinical trials soon)

Liver and lung fibrosis, neurodegenerative diseases

Completed Phase 1

IFM Tre Cambridge, Massachusetts (acquired by Novartis in 2019)

IFM-2427 (DFV890)

COVID-19 plus others

Completed Phase 1 and Phase 2 in COVID-19 patients with pneumonia (no results released yet)

Olatec Therapeutics New York City

Dapansutrile (OLT1177)

Heart failure, gout, COVID-19, melanoma

Completed Phase 1 and Phase 1b in patients with heart failure and Phase 2a in patients with acute gout flares. A Phase 2 trial for people with moderate COVID-19 symptoms is ongoing, and the company is planning a Phase 2 trial in late-stage melanoma, along with an approved immunotherapy.

mediate all these different diseases,” says Schroder, who met in 2015 and 2016 with potential investors to explain inflammasome biology. In the end, though, Fountain Healthcare Partners, headquartered in Ireland, and Novartis Venture Funds provided Inflazome with $17 million in series A funding, and Cooper moved to Dublin to be CEO, with O’Neill as chief scientific officer (CSO). Delving more into MCC950’s mechanism of action, Schroder’s lab, along with another group, recently determined that the compound inhibits NLRP3 by directly binding with a particular protein domain in a way that prevents NLRP3 from opening up into its active form. Although it worked well in animals, MCC950 raised blood levels of a liver enzyme in early human trials, so the team instead used it as a starting point to develop new compounds. By the time they sold Inflazome to Roche last fall, the researchers had devel4 6 T H E SC I EN T I ST | the-scientist.com

oped two drugs, Inzomelid (for diseases of the brain such as Alzheimer’s and Parkinson’s) and Somalix (for diseases in the rest of the body). Both have completed Phase 1 clinical trials for safety and tolerability, and Inzomelid also showed positive results in a small Phase 1b trial in patients with cryopyrin-associated periodic syndrome (CAPS), a group of diseases (including Muckle-Wells syndrome) in which mutations cause NLRP3 to be activated all the time. Last July, the US Food and Drug Administration (FDA) granted Inzomelid orphan drug status for the treatment of CAPS. A few months later, Roche bought the company and its portfolio of NLRP3 inhibitors for 380 million euros ($451 million), plus additional milestone payments. O’Neill and Cooper weren’t the only ones looking to commercialize drugs that target the NLRP3 inflammasome. New York–based Olatec Therapeutics,

for example, had been working since 2011 on a compound called dapansutrile that later turned out to be a selective NLRP3 inhibitor, and in 2016, several companies used the Nature Medicine paper to launch searches for their own inhibitors based on MCC950’s structure. Like Olatec, NodThera (where Chris Gabel is now VP of Biology), IFM Tre (recently bought by Novartis), and others have now moved compounds into Phase 1 trials; some companies already have Phase 2 trials underway. (See table “Inflammation on Trial” above.) “This is truly the new frontier,” says Charles Dinarello, a professor at the University of Colorado School of Medicine and co-CSO and chairman of the Scientific Advisory Board at Olatec; the company’s compound dapansutrile recently advanced through early-stage trials for heart failure and acute gout flares, and will also be studied in late-stage mela-

It looks a bit like a Doric column with a starfish on the top.

THE UNIVERSITY OF QUEENSLAND

ARTWORK REPRODUCED WITH PERMISSION FROM PROFESSOR KATE SCHRODER,

—Matt Cooper, University of Queensland

noma patients. “Now the big challenge is to go further, crossing into cancer with a safe-in-humans nontoxic drug.” Olatec and, separately, Novartis are even testing their respective drugs as possible treatments for COVID-19. (See “NLRP3 and COVID-19” below.)

Too good to be true? While inhibiting NLRP3 in humans seems promising to the researchers who spoke with The Scientist, more late-stage clinical data are needed to demonstrate that

NLRP3 inhibitors can halt the progression of various diseases of aging and lifestyle. “There is a small issue here: there are very few studies in humans on NLRP3 and the inflammasome. Most come from mice,” says inflammasome discoverer Martinon, who has no stake in any company. “Clearly there are differences in the regulation and the role of these proteins in [mice] and humans. So we will see what the clinical trials will tell us in humans.” Larger trials could also provide more information about possible toxicities.

Martinon notes too that scientists still don’t have a complete understanding of the precise molecular structure of the enormous inflammasome complex, making mechanistic studies challenging. “We still don’t really understand how it gets activated,” he says. Studies also suggest that NLRP3 inflammasomes play a role in adaptive immunity, an effect that needs to be further studied. Still, most inflammasome researchers are extremely hopeful. “There’s obviously a whole portfolio of indications where inhibiting the inflammasome could make a lot of sense,” says Guido Junge, who oversees inflammasome clinical activities at Novartis Institutes for BioMedical Research. Schroder agrees. “It may not be our compounds that get to the clinic first, or it may not be our compounds that get to the clinic at all,” she says. “But there are so many different compounds out there now that are being developed by so many different companies to inhibit NLRP3. As long as some of them get out into the clinic, I’ll be delighted because it’s all about helping the patients in the end.” J Rachael Moeller Gorman is a science writer based outside Boston.

NLRP3 AND COVID-19 One of the most dangerous phases of infection with SARS-CoV-2 is thought to be when the immune system launches an inflammatory overreaction called a cytokine storm, releasing a blast of cytokines into the bloodstream that can damage organs and may eventually kill the patient. Damaris Skouras, founder and CEO of New York–based Olatec Therapeutics, says that early in the pandemic, one of the first things the data revealed globally was the various comorbidities that increase risk of hospitalization and death: high BMI, diabetes, cardiovascular or pulmonary disease, and older age, among others. She says that research has already established that the ill effects associated with many of these conditions are modulated by dysregulated IL-1` inflammation, which is then exacerbated by SARS-CoV-2 infection. Olatec’s co–Chief Scientific Officer Charles Dinarello, for example, showed activation of NLRP3 and associated inflammation in blood samples from patients early in the course of infection with SARS-CoV-2. Two companies have launched Phase 2 trials with NLRP3 inhibitors to treat COVID-19 patients. In January, Novartis completed its randomized, controlled, open-label multicenter study using its compound DFV890 on 143 patients infected with SARS-CoV-2 and suffering from pneumonia and impaired respiratory function. A Novartis spokesperson says in an emailed statement that the company is analyzing the data now. Olatec has a Phase 2 randomized, double-blind, placebo-controlled trial on the safety and efficacy of dapansutrile for COVID-19 patients early in disease progression and not sick enough to be hospitalized. Launched last December, the trial has a target of 80 subjects and aims to test if dapansutrile will inhibit IL-1` and head off the cytokine storm. “We are addressing an unmet need for an ambulatory treatment for those most at risk to keep them out of the hospital, keep them safe, keep them at home,” says Skouras, “and most important, keep them from progression into severe COVID.”

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But We’re Animals Too, Aren’t We? Since Darwin published his landmark work on natural selection, we’ve understood that we’re animals. But that doesn’t mean we really believe it. BY MELANIE CHALLENGER

FROM HOW TO BE ANIMAL BY MELANIE CHALLENGER, PUBLISHED BY PENGUIN BOOKS. COPYRIGHT © 2021 BY MELANIE CHALLENGER

A

sk most scientists if we’re animals and you’ll get a funny look. Of course we’re animals! But very commonly, humans are set apart from animals using arbitrary delineations. Consider that in 2018, after many failed attempts, a lab in China created the first cloned primates, a pair of long-tailed macaques to be used in biomedical research. A few years earlier, the United Nations Declaration on Human Cloning stated that cloning people is “incompatible with human dignity.” That may be a good ruling. But what exactly is “dignity,” and why do humans have it while intelligent, aware primates such as macaques do not? A macaque may not think or talk about dignity, but does that mean it doesn’t possess it? The fact is, biology won’t leap to our assistance here because dignity is a human invention. This is typical of how philosophy and value assumptions creep into scientific practice or policy in ways that feel intuitive but are difficult to clarify. “Dignity” originates in Enlightenment dualist ideas separating cognition from physicality and instinct. Dualism has taken many forms through history, such as the idea that humans are made of two substances, the body and the soul. Once rationalism was foregrounded, a new binary emerged: the human mind and our physical bodies. In this worldview, the impulses and feelings of animal bodies (including our own) are viewed as less important than our mental experiences. This isn’t mind over matter. It is the idea that the mind is all that matters, and that it is some kind of separate and separable thing from the body. Some of this can be pinned on French philosopher René Descartes. He took the old religious idea of the soul and gave it a scientific makeover. Animals are just animals, he declared, mindless as clocks. Humans, on the other hand, are made of

two parts: a body (like any animal has) and a special mind of an altogether different substance, something that is unique to humans. This was hugely appealing. Not only was it a new way to escape the moral uncertainty of being animals, but it justified carving up the world into humans on one side and everything else on the other. This way, the moral confusion of being animal could be neatly sidestepped. Many of our laws and guidelines today create hard borders between humans and other animals on the basis of our supposedly superior mental properties. To maintain a strict boundary between our experiences and those of all other animals, we find ourselves prioritizing what we consider mind-based phenomena when making policies. So we demote experiences of fear and promote the idea that knowing fear—recognizing it, naming it—is what matters morally. We brush under the carpet the fact that these cognitive capacities, if present at all, change throughout our lives without altering our moral status. For example, babies experience fear but can’t conceptualize it. This doesn’t alter our belief in their moral subjecthood. In my book, How to Be Animal: A New History of What It Means to Be Human, I look at the history of science and psychology research to understand how our discomfort with some aspects of being animals—from moral confusion to mortality—can spur us to tell ourselves that we’re not really animals. I conclude that it’s time for a much-needed paradigm shift that no longer splits the world into unhelpful and unscientific binaries. The overwhelming bulk of scientific data from the past few decades points to how essential our animal, physical life is to our actions, feelings, and thoughts. Take the way we learn. Susan

Penguin Books, March 2021

Goldin-Meadow’s research into child development has shown that significant, measurable advantages in acquiring language and mathematics follow the use of physical movement and gesture. A growing body of work is also coloring in the complex and conscious worlds and experiences of an ever-expanding diversity of animals. It is getting much harder to separate out what is mental from what is sensual, physical, and experiential, and harder still to separate human from animal. We urgently need to get a handle on what it means to be an animal and move beyond outworn dualism. It’s not just that we’re animals: embodied, physical experiences are richer and more meaningful than we’ve recognized. If that means we need to rethink our relationship to other animals, so be it. J Melanie Challenger is a writer who focuses on philosophy of science and bioethics. Read an excerpt of How to Be Animal at the-scientist.com. 04 . 2021 | T H E S C IE N T IST 49

The Guide

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FOUNDATIONS

Bile and Potatoes, 1921 BY JEF AKST

I

52 T H E SC I EN T I ST | the-scientist.com

SEEKING PROTECTION: Although the Bacille Calmette-Guérin (BCG) vaccine has never been

routinely administered in the United States due to the country’s low incidence of tuberculosis, it is sometimes given to children who are regularly exposed to others with the disease. It was originally administered in an oral formulation, but researchers subsequently developed a version given as a shallow injection into the skin, shown here being administered by Reuben Erickson, the chief of the Division of Tuberculosis at Albany Hospital, in 1949.

for humans. On July 18, 1921, at the Charité Hospital in Paris, doctors gave an oral dose of BCG to an infant whose mother had died of tuberculosis only hours after giving birth. “No one knows exactly who made a decision and why . . . but they gave this newborn the BCG vaccine—experimental vaccine, at the time—and that was kind of the beginning,” Kupz says. Despite having been exposed not only by his mother, but by his grandmother, who had clinical tuberculosis, the child never developed any signs of the disease. As more and more babies received the vaccine, Calmette and Guérin gained confidence in its safety. Their initial report published in 1924 spurred the Pasteur Institute to begin mass production, and a subsequent publication tracking 114,000 vaccinations confirmed that BCG recipients suffered no adverse effects. At the same time, Calmette

and Guérin documented a drop in childhood deaths from tuberculosis among those who had been vaccinated. Other countries quickly adopted the vaccination for their own infants. Despite its widespread use, BCG has its shortcomings. It is not 100 percent protective, for example, and the protection it does provide in childhood often wanes in young adulthood. To this day, no one really knows why, largely because research on BCG dropped off as tuberculosis was all but eliminated from the West thanks to improved hygiene in addition to vaccination. As a result, “TB is still a large problem in the developing world,” says Kupz, who is now working to update the BCG vaccine. Killing nearly 1.5 million people in 2019, it stood as the leading global cause of death from an infectious pathogen, according to the World Health Organization. J

CORNELL CAPA/THE LIFE PICTURE COLLECTION VIA GETTY IMAGES

n the early 20th century, French bacteriologists Albert Calmette and Camille Guérin at the Pasteur Institute in Lille set out to develop a vaccine to protect against tuberculosis, a potentially severe lung infection that has been responsible for more human deaths than any other pathogen in history. It would take more than a decade of painstaking work before they had a TB vaccine ready to test in humans, but it would be worth the investment. Despite its shortcomings, that vaccine remains the primary tool for preventing the disease 100 years later. Inspired by English physician and scientist Edward Jenner, who developed the world’s first vaccine after discovering that inoculation with cowpox was protective against deadly smallpox infection, Calmette and Guérin turned to Mycobacterium bovis, a bacterium that infects cows and is closely related to the human pathogen M. tuberculosis. Because M. bovis itself can cause disease in humans, the duo began culturing the bacterium, and soon noticed that adding ox bile to glycerol-soaked potato slices used to grow the bacteria somehow lowered the microbes’ virulence. Starting in 1908, Calmette and Guérin passaged the bacteria to fresh potato slices every three weeks or so—more than 200 times—testing it on animals including guinea pigs, rabbits, cows, monkeys, and horses along the way to monitor its waning deadliness. Even in the throes of World War I, when Germans occupied Lille, the price of potatoes skyrocketed, and ox bile became difficult to get, they kept going. “It was all empirical,” says Andreas Kupz, a microbiologist at James Cook University in Australia. “At the time there was obviously no genetic modification. . . . All they did was to play around with it.” Eleven years later, they had bacteria in their cultures that no longer generated disease in a variety of animal models, from guinea pigs to cows. Two years after that, the experimental vaccine, dubbed Bacille Bilié Calmette-Guérin, later shortened to Bacille Calmette-Guérin (BCG), was deemed ready

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