April 2020 The Scientist

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APRIL 2020 | WWW.THE-SCIENTIST.COM

EXERCISE FOR CANCER MOLECULAR CLUES LINK PHYSICAL ACTIVITY TO IMPROVED PATIENT OUTCOMES

BACTERIAL ROBOTS VERSUS TUMORS TARGETING CANCER EVOLUTION NATURAL KILLER THERAPIES

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*Weller, MG, Analytical Chemistry Insights:11, 21-27 (2016). Antibodies shown: PD-L1 (A700-016) & Lamin A/C (A303-430A) ©2019 Bethyl Laboratories, Inc. All rights reserved.

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APRIL 2020

Contents

LUCY READING-IKKANDA; © SCIENCE PHOTO LIBRARY, STEVE GSCHMEISSNER; © STOCKSY.COM, IVAN GENER

THE SCIENTIST

THE-SCIENTIST.COM

Features

VOLUME 34 NUMBER 04

ON THE COVER: ADAPTED FROM © ISTOCK.COM, MSAN10; ©ISTOCK.COM, NTONIOKHR

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New therapeutic approaches in oncology take aim at the very thing that makes cancer so untreatable: its ability to evolve drug resistance.

Autonomous, living microrobots that seek and destroy cancer are not as futuristic as one might imagine, thanks to the fusion of robotics and synthetic biology.

Studies point to a role for physical exercise in fighting malignancies, improving treatment outcomes, and fostering overall health in patients.

BY CATHERINE OFFORD

BY SIMONE SCHUERLE AND TAL DANINO

BY BENTE KLARLUND PEDERSEN

Cancer's Game

Bacteria Take on Cancer

Exercise and Cancer

04.2020 | T H E S C IE N T IST

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Read The Scientist on your iPad!

PHENOTYPING THE TUMOR MICROENVIRONMENT WITH MULTIPLEX IMMUNOHISTOCHEMISTRY

PHENOTYPING THE TUMOR MICROENVIRONMENT WITH MULTIPLEX IMMUNOHISTOCHEMISTRY The tumor microenvironment (TME) is a complex and dynamic landscape. Cells within the TME interact to either stifle or promote cancer progression. Immune cells make up much of this environment, where they detect and destroy cancer cells. Cancer cells can evade the immune response by suppressing immune cell activity and recruiting additional cells that dampen the anti-cancer response.

Researchers analyze the TME to find novel cell phenotypes and disease mechanisms, develop targeted therapies, study drug efficacy, and plan patient treatments. Identifying biomarkers provides crucial information on tumor and immune cell status and their spatial arrangement. Traditional TME analysis methods, such as flow cytometr y, destroy tissue morphology to obtain single cell suspensions. New immunohistochemistr y (IHC) techniques stain tissue sections for multiple biomarkers concurrently and allow researchers to image an entire slide. These methods are non-destructive and can be combined with H&E staining to precisely locate cells of interest in the tissue section, identify immune cell infiltration, and detect cell interactions.

MA JOR PL AYERS IN THE TUMOR MICROENVIRONMENT

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ANTI-TUMOR T CELLS

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T cells play multiple roles in the tumor microenvironment depending on their phenotype. Memor y T cells are primed against tumor antigens and cytotoxic T cells directly destroy cancer cells by releasing lytic substances. T cells may become exhausted due to antigen over-stimulation or through immunosuppressive cell effects.

APCs initiate and regulate anti-cancer immunity, often by activating T cells. However, within the TME, some APCs may stimulate Tregs and help tumor cells avoid the immune response. • Dendritic cells • Macrophages • B cells

• Memor y T cells • Cytotoxic T cells • Proliferating T cells • Exhausted T cells

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TUMOR EVASION AND IMMUNE INFILTRATION Cancerous cells often have immune evasion mechanisms that block T cell effector functions. Immune cells may also promote cancer progression by acting against the anti-cancer immune response.

IMMUNOSUPPRESSIVE T CELLS 2

ANTIGEN-PRESENTING CELLS (APC S )

Regulator y T cells (Tregs), a subclass of T helper cells, in the TME suppress the anti-tumor immune response. Other subsets of T cells may also promote cancer progression.

• PD-L1-upregulated cancer cells • Tumor-associated macrophages (TAMs)

• Treg cells • Double positive T cells

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MYELOID-DERIVED SUPPRESSOR CELLS (MDSC S ) MDSCs expand in response to chronic infections and cancer, inhibit anti-cancer immune cells, and stimulate Tregs.

Ultivue products are for research use only; not

• Monocytic MDSCs

for use in diagnostic procedures.

• Polymorphonuclear MDSCs

Cytotoxic T cell

Exhausted

CD3+ CD8+

T cell

T helper cell CD3+ CD4+

CD3+ PD-1+

PHENOTYPING THE TME USING INSITUPLEX TECHNOLOGY The InSituPlex ® IHC technology allows researchers to stain tissues samples with multiple biomarkers for rapid hypothesis testing. With the ability to colocalize multiple markers in the same cell compartment, researchers can identify a multitude of cell phenotypes in the tumor microenvironment with a single 8-plex panel (left – 8 plex kit: CD3, CD4, CD8, CD68, FoxP3, PD-1, PD-L1, CK).

CD8 Regulatory T cell CD3+ CD8+ FoxP3+

Regulatory Immunoevading tumor cell CK+ PD-L1+

T cell CD3+ CD4+ FoxP3+

Immunosuppressive macrophage CD68+ PD-L1

INSITUPLEX IHC ASSAY WORKFLOW

AMPLIFY DNA-barcodes are simultaneously amplified

STAIN

Mixture of fluorescent probes

to maintain relative biomarker expression and to increase the number

Using the InSituPlex ® technology, antibody-DNA conjugates are

1st set

of potential hybridization sites for complementar y

simultaneously introduced into the tissue sample and bind to their respective antigens.

fluorescent probes.

DETECT AND DNA-EXCHANGE

Mixture of fluorescent probes 2nd set

Complementar y fluorescent probes hybridize with the DNA-barcodes and are detected. Probes are gently removed using DNAExchange, allowing for an additional complementar y fluorescent probe set to be introduced in order to detect additional biomarkers.

SAME-SLIDE IHC AND H&E STAINING Whole-slide H&E staining performed after InSituPlex® multiplex IHC provides both biomarker multiplexing and tissue morphology. This view of both brightfield and fluorescence gives a more complete view of the TME, including the localization of specific cell types, and facilitates phenotype identification.

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PHENOTYPING THE TUMOR MICROENVIRONMENT WITH MULTIPLEX IMMUNOHISTOCHEMISTRY

Ultivue provides researchers with multiplex biomarker assays for tissue phenotyping and digital pathology. Ultivue’s InSituPlex® technology enables scientists to unmask and analyze the true biological context of tissue samples. With the ability to run a same-slide fluorescent and H&E stain, Ultivue’s technology is enabling pathologists to connect traditional morphological analysis with multiplexed immunofluorescent data for comprehensive single-cell phenotyping.

APRIL 2020

Department Contents 11

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FROM THE EDITOR

52

Natural Killers Catch Up with CAR T

Avoiding illness can be as valuable as fighting it.

NK cell therapies offer a potentially cheaper and safer route to cancer treatment than their T cell–based predecessors.

BY BOB GRANT

11

CRITIC AT LARGE

Cannabis May Not Be a Cancer Pain Panacea

BY BIANCA NOGRADY

58

Studies are lacking, and in the highquality research that scientists have published, the results are not promising.

A new book revives a controversial hypothesis that proponents say may shake up scientists’ view of human evolution.

21 © ISTOCK.COM, ILYAST; © GEORGE RETSECK; © THE ORA LEE SMITH CANCER RESEARCH FOUNDATION

BY PETER RHYS-EVANS NOTEBOOK

Body-Snatching Fungi; Flu Shot for Tumors; Breaking Away; Blasting Through Biofilms

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READING FRAMES

Humanity’s Watery Beginnings

BY JASON BOLAND AND ELAINE BOLAND

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BIO BUSINESS

Pounds of Prevention

MODUS OPERANDI

Precision Membrane Puncture

A device for piercing individual holes in cell membranes allows vector-free DNA delivery while maintaining cell viability. BY RUTH WILLIAMS

46 THE LITERATURE

60 FOUNDATIONS Ideal Patients, 1896–Present BY AMY SCHLEUNES IN EVERY ISSUE

8 10 59

CONTRIBUTORS SPEAKING OF SCIENCE THE GUIDE

CORRECTION:

In the March profile of Joanne Chory, the size of the Arabidopsis genome was incorrect. It is not 135,000 base pairs but 135,000,000 base pairs. The Scientist regrets the error.

Stealth liposomes deliver CRISPR to rid mice of tumors; human connective tissue’s role in metastasis; Zika infects glioblastoma cells but not healthy brain tissue

51

48 PROFILE

PUZZLE ON PAGE 10

Cracking Down on Cancer

Through his studies on oncogenic viruses, University of California, Los Angeles, professor Owen Witte has helped develop lifesaving treatments. BY DIANA KWON

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SCIENTIST TO WATCH

Hadiyah-Nicole Green: Laser Focus BY EMILY MAKOWSKI

04.2020 | T H E S C IE N T IST

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APRIL 2020

Online Contents

THIS MONTH AT THE-SCIENTIST.COM: VIDEO

VIDEO

VIDEO

Aquatic Apes?

Witte's Work

Lasering Cancer

Watch Reading Frames author Peter RhysEvans and documentarian Sir David Attenborough discuss the new book The Waterside Ape and the impact it may have on our understanding of human evolution.

UCLA researcher and April Profilee Owen Witte discusses his studies of rare genetic cancers and other diseases.

See physicist Hadiyah-Nicole Green, this month’s Scientist to Watch, describe her research into using lasers to treat solid tumors.

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APRIL 2020

Contributors Simone Schuerle was the kind of girl who built cars for her Barbies instead of brushing their hair.

“That was maybe an early indication that I was more interested in engineering,” she says. Focusing at first on industrial engineering as an undergrad at the Karlsruhe Institute of Technology in Germany, Schuerle then gravitated toward the physics department after she happened to hear a lecture on microsystems technology. “I learned . . . how to fabricate very tiny systems, and I was just fascinated about the huge space and rich engineering opportunities at the nanoscale,” she says. Schuerle is now an assistant professor of biomedical systems engineering at ETH Zürich in Switzerland, where her research combines “the intelligence of bacteria,” which are especially adept at finding nutrient-rich, low-oxygen tumors, with magnetic fields to design microrobots to traverse the body and deliver drugs. Synthetic biologist Tal Danino still remembers his dad buying a 1989 Packard Bell computer with a 40-megabyte hard drive and a “turbo button” that launched the machine into a processing speed of 12 megahertz. “I broke that computer so many times,” he says, “and had to fix it both by replacing parts inside of it and learning how to do little programming things.” After studying math, chemistry, and physics as an undergrad at the University of California, Los Angeles, Danino pursued a PhD in bioengineering from the University of California, San Diego. There, he learned to engineer new kinds of systems, including genetic circuits to “make up an entirely new function in a bacteria,” he says. Danino’s own lab at Columbia University is engineering microbes to express different kinds of drugs, with the ultimate goal of treating many types of cancer. On page 30, Schuerle and Danino write about the latest advances in this field.

internal medicine, became interested in the immune system early on in her education, choosing to study the effects of intense exercise on immune function. She conducted some of the original studies that identified the first exercise-related myokine, a peptide that originates in the muscle and facilitates communication between the muscles, liver, bones, fat, and the brain. Researchers have since found hundreds of myokines, some of which they’ve shown to mobilize immune cells or to directly attack tumors. Pedersen is currently a specialist in infectious diseases and internal medicine at Rigshospitalet and directs the Centre of Inflammation and Metabolism and the Centre for Physical Activity Research, both at the University of Copenhagen, where she continues to study how exercise improves health and fights disease. Pedersen, who runs nearly every day, emphasizes that people don’t have to be athletic or engage in extreme sports in order to reap the benefits of exercise. The most important thing, she says, is “avoiding physical inactivity.” On page 38, Pedersen reviews the many ways in which exercise can help patients combat cancer.

When otolaryngologist Peter Rhys-Evans started to look closely at the anatomy of human skulls and those of other apes in the 1980s, he became intrigued. “I just couldn’t reconcile the differences,” he says. After reading Elaine Morgan’s 1985 book, The Aquatic Ape Hypothesis, however, “everything fell into place,” he recalls. That book, along with zoologist Desmond Morris’s 1967 classic The Naked Ape, explored the idea that some uniquely human characteristics, including bipedalism, scant body hair, and an unusual layer of subcutaneous fat, may have resulted from a period of hominin evolution when ancestors of Homo sapiens spent much of their time in and around water. But the hypothesis lacked concrete evidence. Rhys-Evans’s experience as an ear, nose, and throat doctor led him to consider exostoses, bony growths that occur in the ears of divers and surfers who spend much of their time in water. He posited that if these growths were found in the skulls of ancient hominins, it would provide strong support for a semi-aquatic phase of human evolution. Sure enough, archaeologists eventually published evidence of exostoses in the skulls of Neanderthals and other ancient hominin species, prompting Rhys-Evans to continue researching this possible alterative story of human evolution and write his new book, The Waterside Ape. “We don’t know all of the answers, but I just wanted to contribute to the debate,” he says. Read more about these controversial ideas on page 58. 8

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

LEN RUBENSTEIN; TAL DANINO, COLUMBIA UNIVERSITY; COURTESY OF MIKE DAINES

Bente Klarlund Pedersen, a researcher and physician who completed residencies in infectious disease and

FROM THE EDITOR

Pounds of Prevention Avoiding illness can be as valuable as fighting it. BY BOB GRANT

ANDRZEJ KRAUZE

A

t The Scientist, we report stories that deal mostly with the nuts and bolts of biology. Whether we’re writing about cutting-edge cancer research or the latest ecological insights, we tend to focus on new conceptual or technical ground that scientists are breaking in the lab and field, and how this knowledge changes, confirms, or enhances our understanding of living things. But when one considers the practical ripples that biology sends through societies—issues of public health and the shared goal of minimizing the impact of diseases on a global scale—human behavior and prevention become vitally important. As I write this piece, prevention of this type is on the lips of just about everyone, with infections of the newly discovered SARS-CoV-2 virus sweeping around the world, causing illness and death and prompting closures and cancellations aplenty. Public health officials are rightly championing preventive measures, such as frequent and thorough hand washing and limiting social interaction, as keys to slowing the spread of the novel pathogen. Prevention has been playing a growing role in other diseases, infectious and otherwise, long before this latest global pandemic. Cancer, the focus of this issue, is ubiquitous, and one would be hard pressed to find a person anywhere on Earth whose life wasn’t in some way touched by the complex and vexing malady. But its distribution is far from uniform. Cancer exists in patches. To be sure, some of these patches, or “clusters” as epidemiologists often refer to them, result from environmental carcinogens affecting populations that are, by dint of geography, occupation, or some other demographic feature, particularly exposed to them. Other clusters, though, owe more to lifestyle factors such as diet, smoking, and the uneven application of preventive measures, such as frequent medical screening and, more broadly, access to quality healthcare. In 2017, the US Centers for Disease Control and Prevention reported a disturbing trend across the US that strikes at the heart of prevention’s power. “While rural areas have lower incidence of cancer than urban areas, they have higher cancer death rates,” the agency found. “The differences in death rates between rural and urban areas are increasing over time.” Residents of rural areas tend to be poorer, with less access to medical services than their city-dwelling counterparts.

This disparity leads not only to a higher death rate in rural places from all cancer types, but also to a higher incidence of some cancer types, such as colorectal and cervical cancers, that can be more effectively treated or avoided altogether with measures for prevention and early detection, such as regular screening. As the oft-quoted Benjamin Franklin once said, “An ounce of prevention is worth a pound of cure.” This cancer-focused issue features a cover story in which we explore one facet of cancer prevention: exercise. On page 38, Danish researcher Bente Klarlund Pedersen explains that studies have shown frequent exercise to be useful in avoiding cancer as well as in helping cancer patients lessen the side effects of their cancers and treatments. Her research and that of others is seeking to enumerate the molecular and cellular mechanisms that underlie the benefits exercise seems to offer cancer patients. I sincerely hope that by the time you are reading this, the thoughtful preventive measures that are being taken now at the recommendation of scientists and public health officials have served to dampen the effect of SARS-CoV-2 and turn the tide of infection and death seen around the world. Only time will tell, but it’s comforting in times like these that researchers are studying the biology of prevention and seeking to apply rigorous science to help people and societies change their behavior, to the benefit of humanity’s health.

Editor-in-Chief [email protected] 04 . 2020 | T H E S C IE N T IST

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QUOTES

Speaking of Science 3

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BY EMILY COX AND HENRY RATHVON

ACROSS

DOWN

1. 5. 8. 9.

2. Brain membrane (2 wds.) 3. Like some gaseous elements 4. Developmental period 5. Bill who wrote Everything All at Once 6. Essential micronutrient 7. Founder of analytical psychology 11. Bunsen’s sort of implement (2 wds.) 12. Adjective for an alpha male 14. Companion of a canine 17. Side of a sapphire, say 18. About 97 percent of a modern penny 20. Asteroid, parrot genus, or dawn goddess

Titular season in a Rachel Carson book Tribe with the largest US reservation Body studied in limnology Outcome of eccrine and apocrine activity 10. Polysaccharide thickener in many foods (2 wds.) 13. NASA project between Mercury and Apollo 15. Organ or device for detecting impulses 16. Potentilla by another name 19. Physics Nobel winner of 1921 21. Quahog, for one 22. Early casualty of one-man flight? 23. Emu, rhea, or kiwi

10 T H E SC I EN TIST | the-scientist.com

Answer key on page 5

Examples of the stereotypes we observed included perceptions that African Americans were less knowledgeable about cancer research studies, less likely to participate due to altruism, or simply less likely to complete all facets of the research study. These and other examples of bias based on stereotypes of potential minority participants raise concerns that non-whites may be offered fewer opportunities to participate in cancer research studies. —University of Alabama at Birmingham researcher Soumya Niranjan in a press release announcing a Cancer paper she coauthored reporting the results of interviews with cancer center leaders, principal investigators of clinical trials, referring clinicians, and others, to determine if biases can explain the underrepresentation of racial and ethnic minorities in clinical trials (March 9)

We’re going to have to figure out what our American approach is to this and how we’re going to take more aggressive mitigation steps. We’re losing a pretty narrow window to step in. —Scott Gottlieb, former commissioner of the US Food and Drug Administration, addressing the country’s response to the mounting COVID-19 epidemic during his keynote speech at the Miami Breast Cancer Conference (March 7)

JONNY HAWKINS

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

1

CRITIC AT LARGE

Cannabis May Not Be a Cancer Pain Panacea Studies are lacking, and in the high-quality research that scientists have published, the results are not promising. BY JASON BOLAND AND ELAINE BOLAND

© ISTOCK.COM, ILYAST

C

annabis products, in the form of the plant’s flowers, isolated cannabinoids, or synthetic cannabinoid preparations, are increasingly used as medicine, especially with the intention of treating cancer-related ills. But there is a serious dearth of rigorous data to support many of these indications. There is no randomized controlled evidence for the effect of any cannabinoid (the active chemicals in medicinal cannabis) on cancer-related pain. Indeed, there is evidence that a product containing an equal ratio of cannabidiol (CBD) to tetrahydrocannabinol (THC), the psychoactive ingredient of marijuana, does not reduce cancer-related pain compared with placebo, and that the CBD:THC combo treatment could lead to more side effects. To take the next step in strengthening the evidence clinicians use to support their practice, we and our collaborators recently published a systematic literature review in BMJ Supportive & Palliative Care that analyzed data from five recently published high-quality studies that assessed the effect of cannabinoids for cancer-related pain. Uncontrolled studies with no placebo group or other comparator tend to overestimate effects of interventions, so we rigorously searched the literature specifically for randomized trials assessing the effects of cannabinoids versus a placebo on pain in patients with cancer. All published studies that we found used Sativex, a spray consisting of CBD:THC at a 1:1 ratio, compared with placebo in patients on prescription opioids for cancer pain. We could detect no difference between cannabinoids and placebo in reducing pain scores across the five studies. Moreover, cannabinoids carried a

higher risk of adverse events, especially somnolence and dizziness, when compared with placebo. No other cannabinoid or combination of cannabinoids has been studied in a randomized trial for cancer pain, so no data are available on these agents; as such, they could not be included in our systematic review. Therefore, the effect (and side effects) of other cannabinoids or combinations compared with placebo or other treatments is unknown. In addition, our systematic literature review was only looking for studies of cancer pain. This review did not assess the use of cannabinoids for nonpain indications, such as poor appetite, in patients with cancer.

pathic) pain. But these findings need confirming in larger follow-up trials. If cancer patients are considering taking medical cannabis, they should first consult their healthcare provider. However, there are many approved, conventional analgesics and other interventions that are commonly used for controlling cancer

We could detect no difference between cannabinoids and placebo in reducing pain scores across five high-quality studies. That’s not to say that cannabinoids don’t serve some medicinal purposes. The UK’s National Institute for Health and Care Excellence (NICE) guideline provides a list of cannabis-based products backed with evidence of their effectiveness; treatments include the orally active synthetic cannabinoid nabilone for intractable chemotherapy-induced nausea and vomiting in adults (which tends to persist with optimized conventional antiemetic treatment); CBD:THC spray for moderate or severe spasticity in adults with multiple sclerosis; and cannabidiol with a drug called clobazam for seizures associated with Lennox-Gastaut syndrome and Dravet syndrome. There have also been a few very small preliminary studies showing benefits of smoked or vaporized marijuana for easing noncancer (especially neuro-

pain that have undergone clinical trials and have been shown to be beneficial. Based on existing evidence, there is no role for cannabinoids in alleviating cancer-related pain, and NICE does not recommend cannabinoids for chronic pain relief. Further research to understand the effects/side-effects of different cannabinoids or combinations of cannabinoids is needed. g Jason Boland is senior clinical lecturer and honorary consultant in palliative medicine at Hull York Medical School in the UK. Follow him on Twitter @JasonWBoland. Elaine Boland is a consultant in palliative medicine and honorary senior lecturer at Hull University Teaching Hospitals NHS Trust in the UK. Follow her on Twitter @elaineboland17. 04 . 202 0 | T H E S C IE N T IST 1 1

NEWS AND ANALYSIS

Body-Snatching Fungi

A

fter the Chimney Tops 2 Wildfire charred 11,000 acres of the Great Smoky Mountains National Park along the North Carolina–Tennessee state line in November 2016, rangers closed affected trails to visitors. Mycologists Andy N. Miller and Karen Hughes and their teams were an exception. Toting hard hats and sample collection kits, these scientists jumped at the opportunity to track down their research subjects: pyrophilous (“fire-loving”) fungi, which produce mushrooms prolifically after forest fires and then disappear as the forest recovers.

12 T H E SC I EN TIST | the-scientist.com

The severely burned areas of the Smokies were almost completely lifeless two months after the blaze, when the group first ventured into the affected zone. “The level of destruction was incredible,” recounts Hughes, a researcher at the University of Tennessee, Knoxville, in an email to The Scientist. “Everything I touched left black carbon on my hands. It was incredibly quiet.” Miller, who is based at the University of Illinois Urbana-Champaign, also noted a surreal lack of activity. “There’s nothing running around, no birds singing,” he says. To him, the site smelled “like a house had burned up.” When the researchers returned to their collection sites a few months later, however, their mushrooms of interest had risen from the ashes. Miller noticed that when these fungi surface, they

APRIL 2020

SMOKY INDEED: A wildfire swept through the Great Smoky Mountains National Park in late 2016.

do so en masse: “They’re less than a millimeter in diameter, but there’s a lot of them, and once you train your eyes, they’re just all over the place.” The researchers were interested in documenting which species of pyrophilous fungi are present in the Smokies. They also wanted to test a theory about where the fungi go during the long periods between forest fires. Some fire-loving fungi are known to lie dormant in the dirt as spores or other heat-tolerant structures until post-fire soil conditions trigger growth and reproduction. Other species exist between burns in a vegetative state, aiding decomposition of

KAREN HUGHES

Notebook

dead organisms or interacting with tree roots. But many fire-loving fungi don’t fit into any of these categories. A new proposal, known as the body snatchers hypothesis, posits that some pyrophilous fungi hide out inside plants or lichens in between fires, nestling among host cells in a so-called endophytic or endolichenic state.

The level of destruction was incredible. Everything I touched left black carbon on my hands.

DANIEL RAUDABAUGH

—Karen Hughes University of Tennessee, Knoxville

To test the hypothesis, the researchers traveled to the burn site every few months for more than a year to sample the soil, as well as the mosses and lichens that sprang up while the forest recovered. They also gathered specimens from unburned areas of the park for comparison. In May 2018, members of Miller’s lab began analyzing the samples they’d collected. DNA sequencing results identified a total of 22 pyrophilous fungal species in the Smokies. Of these, three species were present only in the soil, while the remaining 19 were found inside plant samples from burned and unburned areas, either exclusively or in addition to being found in the soil. In line with the body snatchers hypothesis, “almost all of our pyrophilous fungi were found as endophytes,” Miller says. Mosses and lichens often live in difficult-to-reach places such as rock crevices and may be hardy enough to withstand minor flames, so fungi living inside these hosts could theoretically survive a low-intensity wildfire. But it’s still unclear how all of these organisms might persist through a moderate or severe burn, and how a fire-loving fungus would escape its host to recolonize a charred forest. Hughes has a hypothesis based on her observations at the burn site: “After the fire, I saw numerous tiny lichen fragments on the burned soil, as if they had been lofted into the air while trees were

burning and settled on the ground after the fire,” she says. These burned plant fragments may inoculate the soil with the fungi they harbor, giving the fire-loving fungi a way into the dirt. This is a feasible way for both a pyrophilous fungus and its host to rebound after a fire and maintain their relationship, according to Keith Clay, who studies plant-fungus interactions at Tulane University and was not involved with this study. “If [a moss fragment] lands in a good place, it can regenerate the whole plant,” says Clay. “If the endophyte is in that fragment, presumably it can just colonize these newly grown plants as well from the get-go.” Post-fire fungi may also acquire new hosts after a burn, Miller notes. One mushroom can produce millions of airborne spores that likely land on nearby mosses and lichens, germinate, and invade the tissues of these new hosts, he says.

BOUNCING BACK: Pyrophilous fungi such as Geopyxis carbonaria start producing mushrooms following fires.

To check whether their findings might apply to other forests, Miller and Hughes analyzed moss and lichen samples from other sites around the US. A handful of the fireloving fungi identified in the Smokies were also present as endophytes in Indiana and Alaska. That result was surprising because “there was really no evidence that a fire had occurred in the last few years in those areas,” Miller says. “What are they doing there if they’re not waiting for a fire to come along?” One possibility is that, while body snatching between fires, pyrophilous fungi use their plant hosts as nutrient sources, says Clay. He notes that many plants and fungi have mutualistic endophytic relationships, where the plant typically provides the fungus with “a home where they can live and sugars, carbon, from photosynthesis.” In return, the fun04.2020 | T H E S C IE N T IST 1 3

NOTEBOOK

—Annie Greene

Flu Shot for Tumors Nearly 5,000 years ago, Egyptian physician Imhotep observed a grotesque but revealing detail about tumors: some grew so large that they burst open—

and eventually disappeared. Seeing this happen, ancient texts suggest, he developed a radical cancer treatment: pierce patients’ tumors and then wait to see if they got smaller, cancer researcher Andrew Zloza of Rush University Medical Center in Chicago tells The Scientist. Sometimes they did. With no knowledge of the human immune system, Imhotep had hit on an essential connection between tumors and infections that wouldn’t appear again in the scientific literature until the turn of the 20th century, when bone surgeon and cancer researcher William Coley began injecting live bacteria and later bacterial toxins into individuals with sarcoma (Proc R Soc Med, 3(Surg Sect):1–48, 1910). Although Coley’s technique showed some success in treating patients’ cancer, it was quickly abandoned in favor of emerging chemotherapy and radiation therapy, Zloza says. (See “Fighting Cancer with Infection, 1891,” The Scientist, March 2016.) Now, as immunotherapy captures cancer researchers’ attention, Zloza and others have begun to recognize that Imhotep and Coley might have been onto a major

breakthrough in immunotherapy: they were using infections to kick-start cancer patients’ own immune systems to target and kill their tumors. Zloza and his colleagues recently added to the evidence for this approach with a study of tumorbearing mice treated with the seasonal flu vaccine: injecting the vaccine, which consists of inactivated flu viruses, directly into mice’s skin tumors dramatically slowed the growth of tumors and in some cases reduced their size, the researchers reported in January in PNAS (117:1119–28, 2020).

Imhotep developed a radical cancer treatment: pierce patients’ tumors and then wait to see if they got smaller. “Having this fairly bland vaccine have such a profound effect on tumor immunity is super surprising,” says Thomas Kupper, a dermatologist who studies skin tumor treatments at Dana-Farber Cancer Institute in Boston and was not involved in the study. If the results hold

ANDRZEJ KRAUZE

gus often produces alkaloids that benefit the host. Yet for the pyrophilous fungi examined in this study, Clay says, “what they offer the plant is not clear.” Sydney Glassman, a microbial ecologist at the University of California, Riverside, who was not involved with the study, notes that in vitro assays using carbon isotopes could help uncover these trade-offs by revealing “nutrient transfer between the plants and the fungus.” Miller and his team plan to examine the details of fungus-host interactions by recreating body snatching in the lab and conducting long-term field studies, he notes. After all, many forests where pyrophilous fungi live go for decades without fire, he says. “So how is that relationship maintained?”

up in human clinical trials, he says, it could offer an innovative way to target certain types of tumors that have been exceedingly tricky to treat, as well as cancer cells that are on the move to other parts of the body. Zloza and his team were focusing on just those sorts of difficult-to-treat tumors, known as cold tumors because they don’t have many immune cells infiltrating them. Compared to other tumors, in cold tumors there are fewer chances for the immune system to identify the presence of cancer cells that differ from normal cells in the body, and any immune cells that are present tend to suppress rather than activate the immune system. The researchers suspect that the injection of flu-associated proteins into the mouse skin tumors signaled to the mice’s innate immune system that foreign material had entered the body. The resulting immune response, the scien-

tists hypothesized, converts cold tumors to hot ones. Consistent with this idea, the team found that the treatment only worked when flu proteins were injected directly into the skin tumors. Injecting the live influenza virus into the tumors did not affect the cancerous cells, probably because the virus is unable to replicate and produce viral proteins in these cells, the researchers suggested in their paper. Injecting heat-inactivated virus or viral proteins outside of the tumor—into the mice’s muscles, for example—didn’t affect the tumors, either. When the team injected the flu vaccine into the tumors, dendritic cells, the foot soldiers of the innate immune system, swarmed the cancerous cells. Those dendritic cells began to pick up bits of the flu virus called pathogen-associated molecular patterns (PAMPs), which help trigger an immune response, and to engulf bits of the tumors. When the den-

dritic cells present viral and tumor antigens on their surfaces to attract T cells, they may trigger attacks on both the flu and the cancer cells. “We haven’t proved this yet, but we think what happens . . . is that you turn the tumor microenvironment into an immune hotbed, and so the antitumor response is aided in that way,” Zloza says. The team detected an increase in killer T cells carrying receptors for a specific tumor antigen, suggesting that those cells had indeed been primed to target tumor cells. The researchers also showed that when a mouse had two tumors, both the treated and untreated tumors, grew more slowly after the flu shot injection compared with tumors in untreated mice. “That would mean you elicited an adaptive immune response that’s specific to the tumor itself,” and that the anticancer T cells circulate through the body primed to kill, says immunologist David

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tumors, tumors that are already infiltrated with T cells,” Kupper says. There’s a lot of interest in turning cold tumors into hot tumors, and this work on using the flu vaccine is “a good first step towards that.” —Ashley Yeager

Breaking Away When Avri Ben-Ze’ev first came across the cell adhesion molecule L1 in 2005, the protein was known only for its role in brain development. It had been linked

to the migration and differentiation of nerve cells, and to helping axons find their way, but hadn’t been found in cells outside the brain. Oddly, though, Ben-Ze’ev, a cancer researcher at the Weizmann Institute of Science in Israel, and his colleagues were finding that the L1 cell adhesion molecule (L1CAM) was also a target in a signaling pathway associated with colorectal cancer. Even more unexpectedly, when the researchers tested the protein’s effects by loading up human cancer cells with a virus that made them express the

LEADING THE CHARGE: Cells at the invading

front of a colorectal tumor express L1 cell adhesion protein (red).

MESRUH TURKEKUL

Masopust of the University of Minnesota who was not involved in the study. “That would be important in the setting where the tumor has metastasized, which is often the case.” To see if something similar would happen with human tumors, Zloza’s team implanted human breast cancer cells into mice’s mammary fat pads. As happens in this form of human cancer, the fatty tissue tumors metastasized to the mice’s lungs. Just as the team had found when working with mouse tumors, a flu shot into the primary tumor in the fat pad led to reduced growth of both that tumor and any metastatic lung tumors that had started to form. However, there’s an important difference between mice and humans that intrigues both Zloza and Masopust. Lab mice have never been exposed to the flu, but humans have, and many get vaccinated every year against it. That means that there could be lingering T cells that are primed to respond to the inactivated virus if it’s injected into humans’ tumors. Masopust and his colleagues have been studying whether they can use this idea to tap the adaptive immune system, harnessing these virus-primed T cells to quickly target the viral proteins once the vaccine is injected into tumors and then spur the immune system to target the tumors too. Sure enough, in tumor-bearing mice that had T cells primed to remember infection with a particular RNA virus, injecting a peptide from that virus into the tumor did jump-start the mice’s immune system to target the tumor, Masopust and his colleagues reported last year (Nat Commun, 10:567, 2019). The result suggests that in humans, memory of a flu virus may actually add to the immune response incited by a viral injection into a tumor. Kupper notes that the research also shows that clinicians could use viral immunotherapy to boost the effectiveness of another form of cancer treatment, checkpoint inhibitors, which are known to be less effective in cold tumors. “Checkpoint inhibitor therapy, when it works, tends to work in patients with hot

L1CAM gene and injecting them into mice, the cells behaved far differently than unaltered controls. When normal cancer cells are injected, “it will form only a local tumor, but now that there is L1, it will metastasize to the liver,” Ben-Ze’ev says. Furthermore, when the researchers looked for L1CAM in colorectal tumor samples from patients, they found it—not in all the cells, but in those at the invasive edge, leading the charge of the cancer’s spread. “What was really surprising to me is that a gene . . . can so dramatically reprogram the properties of an already tumorigenic cell to form [a] metastasis,” Ben-Ze’ev recalls (J Cell Biol, 168:633–42, 2005). Research groups have since found that L1CAM is associated with metastasis in a variety of cancers, and antibodies against the protein have been tested as cancer therapies in mice with some success. Recently, Karuna Ganesh

of Memorial Sloan Kettering Cancer Center (MSKCC) in New York and her colleagues took a new approach to further investigate how L1CAM confers metastatic powers on cancer cells. Ganesh, who is a practicing oncologist as well as a researcher, explains that while tumors that remain localized are usually treatable, cancers that have metastasized are far more deadly and difficult to treat. Indeed, the vast majority of cancer deaths are from metastatic cancer. “Something is fundamentally different about the biology of these metastatic tumors that makes them so impossible to treat, and this is really a pressing clinical need, to find better ways of attacking metastatic disease,” Ganesh says. With that need in mind, in 2014 Ganesh joined the lab of MSKCC metastasis researcher Joan Massagué, where she worked to harness what was then a brand-new model for studying

Something is fundamentally different about the biology of these metastatic tumors that makes them so impossible to treat. —Karuna Ganesh Memorial Sloan Kettering Cancer Center

cancer: tumor organoids, tiny threedimensional models grown in a dish using tumor samples that were removed from patients. (See “Treatment in 3-D,” The Scientist, July/August 2019.) Looking in these models for the factors that distinguished metastasizing cells from tumor cells that stayed put, Ganesh and her colleagues came across L1CAM. Consistent with Ben-Ze’ev’s findings from a decade before, metastasis-initiating cells had lots of the protein, while other cancer cells didn’t.

NOTEBOOK

Ganesh and her colleagues decided to use the organoids, along with experiments in mice, to probe for factors that caused relatively mild-mannered tumor cells to switch on L1CAM and other genes needed to initiate metastasis. What they found, she says, is that the tumor’s physical environment plays a role in tilting cancer cells toward striking out to colonize new territory. The colon is made of epithelial tissue, meaning that its cells grow in sheets, Ganesh explains. Colon cells “like to be attached to their neighbors, and when they let go of their neighbors, they are designed to die.” A key finding of the new study, she says, is that the metastasis-inducing cancer cells respond to physical detachment from surrounding cells not by undergoing apoptosis, but by gaining new abilities—a phenomenon that the team also detected in cells that regenerate tissue after wounding. Specifically, the team identified L1CAM expression in a specialized population of cells in mice with colitis that appeared to use the protein to facilitate healing of damage to the colon caused by inflammation. By contrast, levels of L1CAM RNA were negligible in the colon epithelia of control mice without colitis (Nat Cancer, 1:28–45, 2020). Both the regenerative cells and the metastasis-initiating cells switch on L1CAM as they change to a mode in which they can survive without their neighbors. In the case of the metastasizing cells, Massagué says, the team’s recent study indicates that the protein allows them to seek out and adhere to a membrane that surrounds capillaries at sites of future metastasis. Once it’s latched on, L1CAM signals the cells to begin proliferating. But Massagué adds that it’s just one of the proteins enabling metastasis; he plans to investigate the actions of others as well, in hopes of finding additional therapeutic targets. While L1CAM has long been recognized as a metastasis marker, “actually effectively targeting and linking it to the changes in the epithelial status of the tumor, I think, are very interesting,” says David Menter, a gastrointestinal cancer 18 T H E SC I EN TIST | the-scientist.com

researcher at MD Anderson Cancer Center who was not involved in the study. “It provides new avenues for us to think about targeting metastasis.” Another recent paper by Massagué and colleagues similarly identifies likenesses between metastasizing and regenerating cells in the lung (Nat Med,  26:259–69, 2020). For Judith Agudo, a cancer immunologist at the Dana-Farber Cancer Institute who was not involved in the work, a key takeaway of these studies is that “they bring this new angle in which metastasis takes advantage of pathways that take place in the body during wound healing or regeneration, which can explain a lot [about] how metastasis works. And that new view was really interesting.” For his part, Ben-Ze’ev feels that the new study is sound, but says some of its findings echo insights about L1CAM and colon cancer progression and metastasis that were previously reported by his group and others—a viewpoint he lays out in a review on the site F1000. In Ganesh’s view, the study highlights the importance of using models for drug discovery that include metastasiscapable cells. “Many of the models that people used for drug discovery actually don’t contain this metastasis stem cell state,” she says, but rather consist of mice with benign tumors. “If you want to treat metastasis with drugs that treat metastatic cancer, which is ultimately what all cancer drugs are, then we really need to have model systems that capture this metastasis stem cell state.” —Shawna Williams

Blasting Through Biofilms When Kevin Braeckmans and Tom Coenye first teamed up in 2009 to devise new ways to treat badly infected wounds, it seemed like a natural pairing. Braeckmans was a drug delivery expert with

no microbiology experience, while Coenye was a microbiologist lacking drug delivery expertise. These two researchers, both at Ghent University in Belgium, aspired to outfox biofilms—cooperative clusters of bacteria that infect 90 percent of chronic wounds and stymie many antibiotics due to their sticky, tightly packed nature.

Laser-triggered nanobubbles were often as effective at aiding antibiotic delivery as breaking up biofilms using sound or stirring. The standard therapy for biofilmafflicted wounds is to scrape away infected tissue before the infection becomes lethal. But after four years of working on an alternative to this painful and sometimes ineffective approach, Braeckmans and Coenye were stuck. They had learned how the electrical charges and molecular sizes of antibiotic compounds affected the drugs’ movements through biofilms. And with that information, they had managed to get antibiotics deep into biofilms by packaging them into appropriately sized and shaped nanoparticles, which help ferry small compounds a bit like a taxi shuttles passengers, says Braeckmans. It still wasn’t enough. “We could get the drug molecules into the taxi [and] the taxi could drive inside the biofilm. That was not a problem,” Braeckmans recalls. “But we could not get the passengers out again.” So in 2014, the scientists started to test a different approach. Instead of incorporating drugs into nanoparticles, they tried percolating gold nanoparticles into biofilms and zapping them with pulses of yellow-green laser light. These zaps rapidly heat the particles and the water enveloping them, creating vapor nanobubbles. When these bubbles implode, they create high-energy shock waves that tear the biofilm’s structure and clear passageways for antibiotics.

ZAPPING THROUGH BARRIERS: Researchers

COURTESY OF KEVIN BRAECKMANS

in Belgium used a setup involving lasers to aid the delivery of antibiotics into bacterial biofilms.

To test the efficacy of the method, Braeckmans, Coenye, and colleagues grew Pseudomonas aeruginosa or Staphylococcus aureus, two bacterial species commonly found in chronic wounds, into hardy biofilms in lab dishes. They then tested how well a panel of common antibiotics and antiseptics killed bacteria in biofilms that were laserzapped, compared with those that were left untouched or broken up with sound or manual stirring—processes known to break up biofilms, but which run the risk of heating up healthy tissue or sending fragments of destroyed biofilms into the bloodstream, respectively. Previous work by Braeckmans showed that the transfer of energy from laser zaps to vapor nanobubbles is highly efficient, and thus doesn’t generate excess heat. And while the nanobubbles generate high-energy shock waves when they collapse, these waves aren’t powerful enough to fragment a biofilm. Laser-triggered nanobubbles, the team found, were often as effective at aiding antibiotic delivery as breaking

up biofilms using sound or stirring. One antibiotic became 100 percent effective against a biofilm zapped with just five rounds of laser fire, the team notes in a paper published in the December issue of Biofilm (1:100004, 2019). “I think this is a really cool idea,” says Kendra Rumbaugh, a microbiologist at Texas Tech University Health Sciences Center in Lubbock who was not involved in the study. “They’ve got some important next steps and have to show that it works in animals, but I think they’re on the right track.” Paul Stoodley, a microbiologist at Ohio State University in Columbus, thinks the findings are encouraging and warrant further investigation. But he cautions that the approach would likely only be applicable to skin-deep infections, and not to biofilms that attack bone or cause persistent urinary tract infections, for example. “If you’re using lasers, you need line of sight,” says Stoodley, who was not involved in the study. “If you’ve got a biofilm inside the body, laser light is going to be [challenging].”

The findings come with other caveats. Certain antimicrobials worked just as well on biofilms that hadn’t been zapped as on those that had, suggesting that those drugs didn’t need the lasertriggered implosions in the first place. And the laser bursts sometimes enhanced a drug’s effect on biofilms of one bacterial species but not another. Nevertheless, Braeckmans and Coenye’s approach might work against a wider range of biofilm-forming bacteria than other biofilm-busting strategies under development. Some scientists are testing small molecules that fool biofilm-bound bacteria into resuming life as single cells, for example, while others are searching for enzymes that chop up the sugary matrix that makes biofilms so impenetrable. These strategies will likely only work for biofilms containing certain bacterial species, says Rumbaugh, while the laser-triggered nanobubbles developed by Braeckmans and Coenye could in theory blast through biofilms regardless of their composition. The Belgian group plans to develop the technique in future studies and will test the safety and efficacy of the approach in more-complex skin and animal models. Coenye says a natural place to start would be with a laboratory mimic of skin in which structural molecules such as collagen sit atop a mixture of blood and bacterial growth medium. Researchers could infect this skin model with biofilm-forming bacteria to test whether laser nanobubbles combined with antibiotics help clear the infections. “This approach is, I think, potentially widely applicable,” Coenye says. “One of the goals I have, before I retire, is to be able to say that I’ve contributed to bringing one new drug or one new approach to treat biofilm-related infections to patients.” —Jonathan Wosen 04 . 202 0 | T H E S C IE N T IST 1 9

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the Secrets of the Tumor Microenvironment for Cancer Therapeutics

Cancer cells survive by manipulating direct interactions with other cells, but they thrive by changing the environment they live in. This tumor microenvironment (TME) favors cancer cell survival and protects against antagonists, making it a major obstacle for many potential therapeutic approaches. A greater understanding of the TME will enable scientists to manipulate it to create an environment more amenable to anticancer strategies.

References 1. M. Rajabi, S.A. Mousa, “The role of angiogenesis in cancer treatment,” Biomedicines, 5(2):34, 2017. 2. L. Yadav et al., “Tumour angiogenesis and angiogenic inhibitors: a review,” J Clin Diagn Res, 9(6): XE01-XE05, 2015. 3. G. Lupo et al., “Anti-angiogenic therapy in cancer: downsides and new pivots for precision medicine,” Front Pharmacol, 7:519, 2016. 4. S. Spranger, T.F. Gajewski, “Mechanisms of tumor cell-intrinsic immune evasion,” Annu Rev Cancer Biol, 2:1,213-28, 2018. 5. J. Cheng et al., “Understanding the mechanisms of resistance to CAR T-cell therapy in malignancies,” Front Oncol, 9:1237, 2019.

6. P. Darvin et al., “Immune checkpoint inhibitors: recent progress and potential biomarkers,” Exp Mol Med, 50:165, 2018 7. K.E. Allison et al., “Metabolic reprogramming in the tumour microenvironment: a hallmark shared by cancer cells and T lymphocytes,” Immunology, 152(2):175-84, 2017. 8. V. Gouirand et al., “Influence of the tumor microenvironment on cancer cells metabolic reprogramming,” Front Oncol, 8:117, 2018. 9. L. Sun et al., “Metabolic reprogramming for cancer cells and their microenvironment: beyond the Warburg Effect,” Biochim Biophys Acta Rev Cancer, 1870(1):51-66, 2018. 10. A. Luengo et al., “Targeting metabolism for cancer therapy,” Cell Chem Biol, 24(9):1161-80, 2017.

11. B. Wegiel et al., “Metabolic switch in the tumor microenvironment determines immune responses to anti-cancer therapy,” Front Oncol, 8:284, 2018. 12. V.M. Ngwa et al., “Microenvironmental metabolism regulates antitumor immunity,” Cancer Res, 79(16):4003-8, 2019. 13. V.M. Youngblood et al., “The Ephrin-A1/EPHA2 signaling axis regulates glutamine metabolism in HER2-positive breast cancer,” Cancer Res, 76:1825-36, 2016. 14. E. Shiuan, J. Chen, “Eph receptor tyrosine kinases in tumor immunity,” Cancer Res, 76:6452-7, 2016. 15. S.R. Pillai et al., “Causes, consequences, and therapy of tumors acidosis,” Cancer Metastasis Rev, 38:205-22, 2019.

Angiogenesis Paves the Road to Tumor Growth

Like any tissue, a tumor needs nutrients and oxygen from the blood to survive. Cancer cells stimulate new vessel formation through a variety of signaling pathways. This tumor-directed angiogenesis is haphazard and unchecked, making it difficult to control. Key pathways involved include those of hypoxia-inducible transcription factors (HIFs), pro-inflammatory molecules (prostaglandins and cyclooxygenases), and growth factors. Neovascularization is essential for tumor expansion and metastasis1,2.

Putting the Brakes on Angiogenesis

Roadblocks to Vessel Formation

Turning Angiogenesis Against Tumors

Researchers have used molecular antagonists (including custom antibodies) to downregulate proangiogenic signaling, either by blocking receptors or targeting ligands to prevent binding1. Over a dozen such inhibitors have US FDA approval, and they are currently employed concurrently with other anticancer therapeutic approaches1.

Proteins such as angiostatin can target the physical processes of neovascularization. These compounds block vessels from assembling by preventing endothelial cell migration, preserving extracellular matrix integrity, or inducing endothelial cell apoptosis2.

While furthering tumor growth, blood vessels also carry therapeutic agents to their intended targets. Researchers are now exploring the possibility of stimulating angiogenesis in order to maximize anti-cancer agent delivery. As stabilizers of vascular integrity and maturation, pericytes appear essential to this approach3.

Immune Evasion Lets Cancer Cells

Hide in Plain Sight

The body launches an immune response to kill cancer cells upon detection, both before and after tumor formation. Cancer cells evade this response by deactivating effector immune cells such as cytotoxic T cells and macrophages. Evasion mechanisms include blocking immune cell activation signaling, downregulating surface antigens to prevent detection, and secreting cytokines and chemokines to prevent immune cell recruitment and infiltration into the TME4.

Metabolic Reprogramming Gives Cancer Cells Advantages

A hallmark of cancer cells is altered metabolic programming. Cancer cells are directed by signals from each other and from stromal sources to prefer anaerobic to catabolic energy-producing processes7,8. This gives them multiple energy production options, including lactate, acetate, ketone bodies, and ammonia metabolism pathways7,9. They take up and use more glucose than healthy cells7. The resulting rapid ATP generation enables elevated rates of proliferation and allows cancer cells to outcompete other cells for resources7,9. Finally, cancer cell metabolic pathways also secrete immuno-suppressive metabolites such as prostaglandin E₂ and kynurenine9.

Starving Cancer into Submission The metabolic differences between cancer and healthy cells present opportunities for researchers to target the former without overly impacting the latter. However, directly blocking glucose metabolism involves navigating a fine line between efficacy and hypoglycemia; this has led scientists to target other pathways, such as fatty acid synthesis and NAD+/NADH homeostasis10. In addition, the elevated metabolic and proliferation rates of cancer cells make them susceptible to oxidative stress-induced apoptosis and DNA synthesis inhibitors10,11.

Fueling Immune Responses Activated T cells rely on aerobic glycolysis to function, so they directly compete with cancer cells for resources12. As such, inhibiting cancer cell energy production can restore immune function. In order to avoid side effects on immune cells, scientists are looking for metabolic targets, pathways, and transporters selectively used only by cancer cells. One example of this is EphA2, which promotes glutamine metabolism in breast cancer cells but is not expressed by T cells13,14. Alternatively, modulating immune cells ex vivo to make them more metabolically fit could improve functional efficacy12.

Neutralizing Acidosis The acidic TME induces genomic instability, leading to more oncogenic mutations and creating more aggressive tumors. This also stimulates invasion and metastasis. Attempts to directly alter TME pH using buffer solutions have proven difficult to translate to the clinic. Scientists have therefore focused their efforts on inhibiting metabolic pathways that create acidic byproducts. However, targeting compounds that create alkaline products or absorb acidic ones have demonstrated some feasibility in clinical testing15.

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Precision Membrane Puncture A device for piercing individual holes in cell membranes allows vector-free DNA delivery while maintaining cell viability. BY RUTH WILLIAMS

V

iral vectors are efficient at transporting desired pieces of DNA into cells, and are used for, among other things, transfecting chimeric antigen receptor (CAR) genes into patient lymphocytes for CAR T cell therapy. But for some gene therapies, vectors come with “a litany of frustrations,” says Masaru Rao, a mechanical engineer at the University of California, Riverside. Some viral vectors are limited in the size of DNA they can carry, and they integrate that DNA randomly into the genome, risking damaging mutations, Rao explains. The presence of viral particles in the body can, in certain types of gene therapies, induce an innate immune response in patients, he adds. Furthermore, the production of viral vectors, which depends on culturing cell lines, can be difficult to scale up. “A non-viral transfection method is critical for the field,” says biomedical engineer Abraham Lee of the University of California, Irvine. He, Rao, and others are now working to develop mechanical alternatives for gene delivery. Most of the approaches developed so far, however, including electroporation, cell squeezing, and acoustic shearing, indiscriminately disrupt the cells’ membranes to allow the entry of genetic material, says Rao. “The number and size of holes is not well controlled,” he says. As a result, some cells are ripped apart, while others may remain intact but do not take up the DNA.

 4  1

 2

 3

SPIKED CELLS: Cells in suspension are pipetted into a reservoir with an array of cell-size wells each

containing a single spike. Microfluidic channels running through the wells generate a suction force that draws cells into individual wells  1 , where the spikes pierce the cell membranes  2 . The flow is reversed to release the cells  3 , which are then mixed with the DNA of interest  4 . The DNA diffuses into the cells via the temporary pores, which then close up on their own.

There is often a trade-off between transfection efficiency and cell viability, he explains. To avoid this problem, Rao and colleagues created a device that generates a single transient pore in each cell, allowing DNA to enter but minimizing the rate of cell death. Using microfluidic manipulations, cells in suspension are guided into individual cell-size wells that are arranged in an array at the bottom of the cell reservoir. Each well houses a single spike, which pierces the cell as it slips into the well. The fluid flow is then reversed to release the perforated cells, which are collected and incubated with the desired DNA before the membrane heals itself. The team has optimized flow rates to maximize cell viability and tested the device

with various human cell types. The researchers achieved transfection efficiencies of greater than 80 percent for a T cell line as well as T cells isolated from blood. An electroporation protocol optimized for the same T cell line, by contrast, yielded an average efficiency of around 20 percent. The device currently pierces 10,000 cells at a time, but could be scaled up to house a larger array and could be automated for high throughput, says Rao. A typical CAR T therapeutic dose is several million to several hundred million cells. “With their fabrication technique, I believe they could [scale up],” says Lee, who was not involved in the project. “This is an elegant technology and . . . a great addition to the field.” (Nano Lett, 20:860–67, 2020) g

© GEORGE RETSECK

AT A GLANCE NON-VIRAL TRANSFECTION TECHNIQUE

HOW DNA GETS IN

CELL VIABILITY

TRANSFECTION EFFICIENCY

NUMBER OF CELLS TRANSFECTED AT ONCE

Electroporation

A current is passed through a suspension of cells, disrupting the cell membranes.

Varies greatly depending on cell type and electrical current

Varies, but in Rao’s report, using a protocol optimized for a human T cell line, 20 percent

5 million to 10 million

Deterministic mechanoporation

Cells are sucked into wells and pierced by a micro-scale spike.

Close to 100 percent

For the same human T cell line, 88 percent

Currently 10,000, but could be scaled up

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Cancer’s Game New therapeutic approaches in oncology take aim at the very thing that makes cancer so untreatable: its ability to evolve drug resistance. BY CATHERINE OFFORD

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n the early 2000s, back when biologist Olivia Rossanese worked as an investigator at GlaxoSmithKline, fighting cancer was an exercise in brute force. Researchers at the company had set their sights on developing inhibitors of B-Raf, a protein kinase involved in cell signaling that becomes dysfunctional in many cancers, and “what we were thinking was that we needed to hit this . . . protein so hard,” says Rossanese. “You had to inhibit it 99.999 percent to shut down the signaling pathway.” In 2008, Rossanese and her GSK colleagues discovered just the sort of compound they were after: a small mole-

22 T H E SC I EN TIST | the-scientist.com

cule, dabrafenib, that potently inhibited B-Raf and showed striking effects in melanoma patients with certain mutations in the BRAF gene. With dabrafenib, says Rossanese, “we see really amazing responses, and melanomas go away.” The drug was approved by the US Food and Drug Administration (FDA) in 2013. But in a majority of patients, the effect doesn’t last long. The cancer usually comes back within just six months or so—and when it does, it’s resistant to dabrafenib. GSK’s data showed that less than half of metastatic melanoma patients treated with dabrafenib alone survived more than two years.

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04 . 202 0 | T H E S C IE N T IST 2 3

It’s a familiar refrain. The vast majority of cancer deaths in the US come about not because of a lack of treatment, but because the treatments themselves stop working in the patients receiving them. Accordingly, the emergence of drug resistance is now widely regarded by oncologists as the biggest challenge in cancer therapy. Insensitivity to drugs may arise due to changes in gene expression that allow a cancer cell to rewire its metabolism to circumvent the targeted pathway. Resistance also arises through genetic mutations, which, provided they offer a survival or growth advantage, can come to dominate a population of replicating cancer cells much as they would a population of organisms undergoing adaptive evolution in a new environment. (See “Resist or Desist,” The Scientist, April 2017.) Growing appreciation of cancer’s capacity to evolve drug resistance is revealing fatal weaknesses in the drug-

it-until-it-dies mentality that dominates cancer treatment and drug discovery efforts, says Mel Greaves, director of the Centre for Evolution and Cancer at the Institute of Cancer Research (ICR) in the UK. During traditional drug screening, for example, oncologists “take a drug, put it in a test tube with a cell culture or cell line of cancer, and ask if it kills the cells,” says Greaves. At the ICR, which launched its Centre for Cancer Drug Discovery last summer, “we’re saying, that’s just wrong.” Instead, several groups of cancer biologists are looking for therapies and treatment strategies that target cancer evolution itself, says Rossanese, now head of biology at the new center, which claims to have the “world’s first ‘Darwinian’ drug discovery program” specifically designed to tackle drug resistance. This can take the form of manipulating the course of cancer evolution to clinicians’ advantage, or putting the brakes

on the processes that drive it in order to limit a tumor’s capacity to adapt. In taking this approach, researchers “just assume resistance from the start,” Rossanese says. “If you do that, and you change your mindset that way, then how would you design drugs? How would you design trials?”

Cornering cancer The emergence of resistance to potent inhibitors such as dabrafenib isn’t surprising, says Rossanese. By the time of diagnosis, a typical tumor might already comprise more than 1 billion cells, each of which has the entire human genome at its disposal. During the tumor’s development up to that point, the accumulation of mutations in replicating cells will have led to heterogeneity, the substrate for evolution, among different subpopulations of cancer cells. When a clinician administers high doses of a drug that blocks an important

PLAYING TRICKS ON TUMORS Treating cancers with high doses of tumor-targeting drugs often triggers the evolution of drug resistance, which leads to tumor progression. Researchers are consequently exploring alternative treatment strategies that manipulate tumor evolution to a patient’s advantage—by exploiting drug resistance instead of trying to avoid it. EVOLUTIONARY DEAD END

Clinicians administer a drug and thus select for cells with resistance-conferring mutations. Then, having narrowed the population down to just those resistant cells, they administer a second drug designed to target a weakness, what researchers refer to as a “collateral sensitivity,” in those same cells.

Strong selection for resistant cells

First drug added

Resistant cells killed thanks to collateral sensitivity

Second drug added LUCY READING-IKKANDA

Heterogeneous tumour

Just assume resistance from the start. If you do that, and you change your mindset that way, then how would you design drugs? —Olivia Rossanese, Centre for Cancer Drug Discovery, Institute of Cancer Research

cellular pathway, “the pressure on the cells to come up with a resistance mechanism is quite strong,” Rossanese says. Any mutation conferring an advantage in that scenario, even if it’s present in just a few cells, offers an escape route, and can quickly sweep through the population to produce a drug-resistant cancer that thwarts further treatment. One way to try to block cancer’s evolutionary escape routes is to use drug combinations that target multiple oncogenic pathways at once. For example, the combination of dabrafenib and trametinib—a drug that targets another

central protein in cellular signaling, MEK—was approved in 2014 for certain types of melanoma and later for other cancers after showing improvement in survival rates compared with dabrafenib treatment alone. However, many cancers eventually go on to evolve multidrug resistance. There’s also the issue of toxicity: generally, the more drugs a clinician administers, the higher a patient’s risk of side effects. An alternative strategy is to set a sort of evolutionary trap by administering a combination of drugs in a particular order. The aim is to select for resis-

tance to the first therapy before hitting surviving cancer cells with a second therapy designed to target a vulnerability created by the very mutations that conferred resistance to drug 1. Known as evolutionary herding, the method exploits the fact that any biological adaptation often involves trade-offs; being better at surviving in one environment may mean being worse at surviving in another. As part of an announcement last year about the ICR’s new drug discovery center, computational biologist Andrea Sottoriva likened the approach to sending cancer “down dead ends and to its own destruction.” To turn the idea of evolutionary herding into practical cancer therapies, oncologists are using computational and experimental techniques to predict which combinations of drugs, and in what order, are most likely to work. In one 2016 study, MIT researchers exploited the evolution of drug resis-

HARNESSING COMPETITION

Researchers administer low levels of a drug, enough to kill most, but not all, of the vulnerable cells in the tumor population while favoring the survival of drug-resistant lineages. Once the tumor has shrunk, clinicians stop administering the drug. The drug-sensitive cells, which tend to have a competitive edge over cells that have invested in a costly drug-resistance mechanism, can now begin to grow back. Competition between drug-sensitive and drug-resistant cells for resources in the tumor microenvironment keeps the tumor size in check.

Low levels of drug added

Low levels of drug added

Drug withdrawn

Heterogeneous tumour

Drug withdrawn

Regrowth of non-resistant cells, control of resistant subpopulation

Weak selection for resistant cells

Regrowth of non-resistant cells, control of resistant subpopulation

Reselection of resistant cells: tumour size under control

tance in a murine model of an aggressive type of acute lymphoblastic leukemia (ALL) called Philadelphia chromosome–positive ALL. 1 This cancer is characterized by the fusion of two genes, BCR and ABL1, and is often treated with high doses of small-molecule drugs that inhibit the resulting BCRABL1 oncoprotein. Blasting ALL cells in vitro with dasatinib or bosutinib, two common BCR-ABL1 inhibitors, resulted in the emergence of resistance to both drugs, regardless of which compound the cells were exposed to, the researchers found. But drug screening revealed that these resistant cells showed increased vulnerability, or “collateral sensitivity,” to a selection of other drugs. Sequencing assays revealed a single base mutation, a guanine-to-cytosine substitution, in ABL1 that appeared to be responsible both for protection against dasatinib and bosutinib and for sensitivity to at least four other small-molecule drugs— a weakness that, the researchers write in their paper, should be “therapeutically exploitable.” More recently, Sottoriva and colleagues applied a similar approach to

manipulate the evolution of non-small cell lung cancer (NSCLC) cells in vitro.2 The researchers first bombarded their cell lines with trametinib, which, as expected, caused major cell death followed by the growth of drug-resistant cells a few weeks later. Sequencing revealed that these trametinib-resistant cell lines had all lost functional copies of the gene coding for CDKN2A, a protein that helps regulate cyclin-dependent kinases (CDK) and, consequently, cell division. Because the loss of CDKN2A is known to lead to increased production of certain CDKs, the team predicted that the cells would be particularly sensitive to CDK inhibitors. Sure enough, such drugs proved twice as lethal in the trametinib-evolved lines as in control NSCLC cells. Efforts to target collateral sensitivity bring their own challenges, however. For starters, collateral sensitivity may be relatively rare, or at least difficult to identify. In their study, Sottoriva and colleagues described a second set of NSCLC lines, this time treated with gefitinib, which targets another protein involved in cell signaling called epidermal growth factor receptor (EGFR).

The cells duly gained gefitinib resistance, but they didn’t show collateral sensitivity to any of the nearly 500 other drugs the team hit them with, including several that the researchers identified as good candidates based on genetic sequencing of resistant cell lines. Even when researchers can identify collateral sensitivity, work by several groups suggests that it often arises unpredictably and may be temporary. Tumors continue to diversify as cells replicate and accumulate mutations, so cancers may eventually evolve resistance to both the original treatment and the therapy designed to take advantage of resulting collateral sensitivities. Charles Swanton, a clinician scientist at the Francis Crick Institute and University College London, notes that in lung cancer, for example, tumor sequencing data suggest that evolution becomes “less constrained, not more constrained” as cancer progresses. “In terms of forcing tumors down culde-sacs, I think perhaps in very early stages of disease that might be a fruitful approach,” he says. “But I think in later stages of disease, the tumor is too diverse for that to be possible.”

TRACKING CHANGES To effectively manipulate a tumor’s evolution, researchers need a way of monitoring the various subpopulations of cancer cells within that tumor. Standard tissue biopsies are impractical for many cancer types and tend to provide poor measures of tumor heterogeneity: one study of renal carcinoma patients found that a single biopsy identified on average just a little more than half of the mutations in each tumor (NEJM, 366:883–92, 2012). Many researchers have consequently switched their attention to liquid biopsies, which pick up cancer-related biomarkers circulating in the blood and may prove to be cheaper, less-invasive, and more-effective ways of monitoring within-tumor changes over time. A proof-of-concept study by a team at Institut Curie in France a couple of years ago used whole-exome sequencing analyses of circulating DNA to detect the rise of tumor subpopulations resistant to chemotherapy in 19 patients with neuroblastoma tumors (Clin Cancer Res, 24:939–49, 2018), while a team at Asahikawa Medical University in Japan did the same with non-small cell lung cancer patients receiving tyrosine kinase inhibitors (BMC Cancer, 18:1136, 2018). One analysis published last year found that, in nearly 80 percent of cases, this approach identified clinically relevant, resistance-inducing mutations that had been missed by tissue biopsies, raising researchers’ hopes that the technique could be effective for monitoring tumor evolution (Nat Med, 25:1415–21, 2019). Other types of liquid biopsies include analyses of circulating tumor cells and of genetic material (usually RNAs) in tumor-derived extracellular vesicles. These approaches are being evaluated in multiple clinical trials as a way to monitor patient responses to cancer therapies, but it will be a while before they’re ready for use in long-term, evolution-focused treatments, says Robert Gatenby of the Moffitt Cancer Center. Researchers don’t know, for example, if the proportions of various types of circulating DNA directly mirror the proportions of each tumor subpopulation, or whether they “represent disproportionately the populations that are losing—or the populations that are winning—the evolutionary battles,” he says. “We’re hoping [these techniques] will help us, but there’s a lot of work that has to be done.”

Hunger games About 15 years ago, while perusing internet news, Robert Gatenby came across an article about the diamondback moth, and the damage that this pest species had been wreaking on cabbages and other cruciferous plants around the world for much of the last century. Ecologists had realized, the article explained, that by smothering their crops in chemicals, farmers were merely encouraging this fast-reproducing species to evolve insecticide resistance, while killing off any competing insects in the ecosystem that might

toward resistance instead of growth and replication, for example. This idea has been bolstered by years of clinical data. For example, one small 2015 study of patients with colorectal cancer who were receiving EGFRtargeting drugs found that, soon after the study started, several patients’ tumors were taken over by cells with mutations in KRAS, a well-known oncogene that helps cancer cells bypass normal metabolism and confers drug resistance. Yet when clinicians stopped administering the drugs, that same KRAS-mutated subpopulation took a hit, with liquid biop-

LUCY READING-IKKANDA

A cancer cell with a resistance mechanism would have an advantage over other cancer cells competing for space and resources— but only when the relevant drug was present. have helped keep moth populations in check. To Gatenby, a radiologist and codirector of the Center of Excellence for Evolutionary Therapy at the Moffitt Cancer Center, the parallels to cancer were obvious. High doses of cancer therapy are “the same as high-dose insecticide,” he says. “You’re selecting for resistance, and you’re taking away competitors.” He says the story made him wonder whether oncologists might harness competition within the cancer ecosystem—that is, among the various clonal subpopulations making up a tumor— to stave off the evolution of resistance. A cancer cell with a resistance mechanism would have an advantage over other cancer cells competing for space and resources, but only when the relevant drug was present, Gatenby reasoned in theoretical papers during the 2000s and 2010s. In the drug’s absence, it would have no such edge—indeed, the resistance mechanism might even come with a cost, if the cell directs resources

sies showing declining numbers of these cells with respect to other subpopulations. The drug-resistant cells appeared to be drug-dependent. To turn the idea into a therapeutic strategy, Gatenby proposed cycling between providing a treatment and withholding it, thus growing and shrinking different cancer cell subpopulations in a way that would limit the overall size of the tumor and block drug-resistant cells from taking over completely. A team at Novartis tested this strategy in the early 2010s using patient-derived melanoma xenografts in immunocompromised mice. The researchers found that while a continuous high-dose treatment of melanoma tumors with the B-Raf inhibitor vemurafenib led to lethal drug-resistant disease in mice within a few months, intermittent dosing on a four-weekson, two-weeks-off schedule staved off resistance for the full 200 days of the study, and drastically slowed overall tumor growth. 3 04 . 202 0 | T H E S C IE N T IST 27

More recently, Gatenby’s group has used the method in patients with metastatic castration-resistant prostate cancer (mCRPC). Patients treated with the hormone therapy abiraterone usually progress to a drug-resistant and thus more lethal form of the disease after about 16 months. But instead of dosing continuously, the Moffitt trial’s clinicians monitored 11 patients’ blood levels of PSA, a biomarker for prostate cancer, and administered abiraterone only until PSA had dropped to 50 percent of its pretreatment level. Then, they suspended treatment, waited a few weeks or months until a patient’s PSA had risen back to pretreatment levels, and started over. Interim findings published in 2017 indicated that, in patients exposed to intermittent dosing, the average time to progression to the more aggressive form of the cancer was at least 27 months.4 Last year, after expanding the

noma, and, as of later this year, metastatic pediatric sarcoma. Theoretical work by the Moffitt researchers suggests that the approach could be more effective if clinicians use multiple drugs to control the proportions of cell subpopulations—taking advantage, for example, of any collateral sensitivities. Simulations the team published last year indicate that repeatedly alternating between abiraterone and the chemotherapy drug docetaxel could have significantly increased timeto-progression in the mCRPC trial, although effective implementation of such a regime would require precise techniques for monitoring the proportion of various tumor subpopulations. 6 (See “Tracking Changes” on page 30.) One implication of strategies that exploit competition among tumor subpopulations is that they shift the goalposts for treatment, notes Benjamin Roche, codirector of the Center for

We don’t try to remove the cancer, we try to keep it at a low level that doesn’t kill you or impact your life.

group to 15 men, the team published an update: six had progressed, but the rest had not, boosting the median time to progression to at least 30 months. 5 With the trial now wrapping up after having accrued more than 20 patients in total, “those patients, it looks like, are going to have about a twentymonth increase in their median time to progression,” says Gatenby. Plus, “they’re getting less than half the dose of drugs that they would have received otherwise”—a factor that could help reduce costs and potential side effects. The team is now testing this approach in patients with castration-sensitive prostate cancer, thyroid cancer, mela-

Ecological and Evolutionary Cancer Research in Montpellier, France. By trying to maintain “competition between resistant and sensitive cancer cells . . . we are completely changing our priorities about the fight against cancer,” says Roche, who has collaborated with Gatenby on papers in the past. “We don’t try to remove the cancer, we try to keep it at a low level that doesn’t kill you . . . or impact your life.” This mentality has taken a while to percolate through the oncology community, says Gatenby. A decade ago, the idea that clinicians might chose to manage, rather than obliterate, a cancer “was a difficult sell.” Understandably, he

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—Benjamin Roche, Center for Ecological and Evolutionary Cancer Research

says, “physicians as a group did not like it. [At conferences] the usual comment was, ‘This is bullshit. Just give me better drugs.’” Now, with clinical trials underway, Gatenby says, there’s more interest in these sorts of approaches for cancers that are all but untreatable. Greaves says he agrees that controlling, rather than curing, aggressive cancers may be a sensible goal. But there’s still “a bit of a gap between the modeling and the clinical practice,” he notes. Current theory can’t produce clear treatment recommendations for individual patients, he continues. “We aren’t there yet.”

Blocking evolution Efforts to herd or otherwise manipulate cancer evolution assume that the emergence of drug resistance is inevitable, and is thus best directed in order to achieve clinical benefit. But some researchers are interested in how a cancer gets to be so adaptable in the first place, and whether that process itself might be blocked or slowed down. The key, Rossanese explains, is the heterogeneity of the cancer cell population. “When you increase heterogeneity, you’re giving evolution a bigger substrate to act upon,” she explains. “So what if we could reduce some of the ways cancers generate heterogeneity?” Although research shows that some cancer-related mutations arise as a result of treatment itself, most DNA errors are generated spontaneously as cells in the tumor multiply. Data on when and where these mutations usually arise are pouring in from research projects, such as the Pan-Cancer Analysis of Whole Genomes (PCAWG), that sequence and analyze tumor DNA. For example, researchers working on the TRAcking Cancer Evolution through therapy (TRACERx) project, which Swanton leads, have sequenced tumors from hundreds of NSCLC patients in the UK to explore how mutation patterns change as cancer progresses. (Swanton also cofounded a company, Achilles Therapeutics, to develop personalized

T cell therapies targeting antigens identified by mutations in patient sequencing data.) Among other things, such data can help identify particular types of mutations in a tumor that are associated with elevated genetic diversity overall, and thus identify potential targets for evolution-stalling therapies. One such target generating interest among oncologists is the apolipoprotein B mRNA-editing catalytic polypeptidelike (APOBEC) protein family, a group of enzymes that modify nucleic acids by changing cytosine bases to uracil and are thought to be involved in the innate immune response. A 2019 study using TRACERx and other datasets containing genomic information on various lung and thoracic cancers found a strong correlation between APOBEC-driven mutagenesis and overall tumor heterogeneity, suggesting that APOBEC activity may be a significant contributor to the diversification of cancer cell subpopulations.7 Other research has linked the proteins to tumor diversity and disease progression in head and neck, breast, and bladder cancer, among others. “We know it’s an active process that’s driving heterogeneity in cancer,” says Rossanese. APOBEC3B in particular appears to be upregulated in at least half of all known cancers, and ICR researchers are currently in the early stages of developing small-molecule inhibitors of the enzyme, Rossanese tells The Scientist. “The idea is to test the hypothesis that reducing mutational load and heterogeneity will in fact delay drug resistance,” she explains. Researchers in New Zealand, meanwhile, are targeting the protein using oligonucleotides. In work published last fall, a team at Massey University described an oligonucleotide drug that selectively inhibited APOBEC3B in vitro. 8 Like other evolution-based approaches to therapy, the strategy has limitations. At diagnosis, most cancer patients have already accumulated substantial withintumor heterogeneity. While better cancer screening could help tackle that problem from a public health perspective through

earlier diagnosis, therapies targeting APOBEC or as-yet-undiscovered evolutiondriving proteins might have little effect on late-stage disease, notes Gatenby. “I think once the horse is out of the barn, it’s going to be very hard to suppress evolution,” he says, adding that cancer cells may well find new escape routes that researchers can’t predict. According to the axiom attributed to evolutionary biologist Leslie Orgel, “Evolution is cleverer than you are,” Gatenby says. “I take that to heart.” Rossanese isn’t dissuaded. She notes that evolution-stalling therapies would probably be used in conjunction with more-traditional approaches. Even for a patient who already has high tumor heterogeneity, “a lot of times, a primary therapy is going to take out 99 percent of the cancer cells, and that 1 percent that’s left is going to have to adapt its new condition,” she says. “We’re trying to hobble those remaining cells as much as possible.” g

References 1. B. Zhao et al., “Exploiting temporal collateral sensitivity in tumor clonal evolution,” Cell, 165:234–46, 2016. 2. Acar et al., “Exploiting evolutionary herding to control drug resistance in cancer,” bioRxiv, doi:10.1101/566950, 2019. 3. M. Das Thakur et al., “Modelling vemurafenib resistance in melanoma reveals a strategy to forestall drug resistance,” Nature, 494:251–55, 2013. 4. J. Zhang et al., “Integrating evolutionary dynamics into treatment of metastatic castrate-resistant prostate cancer,” Nat Commun, 8:1816, 2017. 5. J. Zhang et al., “Integrating evolutionary dynamics into treatment of metastatic castrate-resistant prostate cancer (mCRPC): Updated analysis of the adaptive abiraterone (abi) study (NCT02415621),” J Clin Oncol, 37:5041, 2019. 6. J.B. West et al, “Multidrug cancer therapy in metastatic castrate-resistant prostate cancer: An evolution-based strategy,” Clin Cancer Res, 25:4413–21, 2019. 7. N. Roper et al., “APOBEC mutagenesis and copy-number alterations are drivers of proteogenomic tumor evolution and heterogeneity in metastatic thoracic tumors,” Cell Rep, 26:2651–66, 2019. 8. F.M. Barzak et al., “Selective inhibition of APOBEC3 enzymes by single-stranded DNAs containing 2′ -deoxyzebularine,” Org Biomol Chem, 17:9435–41, 2019.

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Bacteria (pink) cozy up to dividing colorectal cancer cells (blue) in this false-color scanning electron micrograph.

© SCIENCE PHOTO LIBRARY, STEVE GSCHMEISSNER

CANCER INTRUSION:

Bacteria Take On Cancer Autonomous, living microrobots that seek and destroy cancer are not as futuristic as one might imagine, thanks to the fusion of robotics and synthetic biology. BY SIMONE SCHUERLE AND TAL DANINO

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n the 1966 movie Fantastic Voyage, a team of scientists is shrunk to fit into a tiny submarine so that they can navigate their colleague’s vasculature and rid him of a deadly blood clot in his brain. This classic film is one of many such imaginative biological journeys that have made it to the big screen over the past several decades. At the same time, scientists have been working to make a similar vision a reality: tiny robots roaming the human body to detect and treat disease. Although systems with nanomotors and onboard computation for autonomous navigation remain fodder for fiction, researchers have designed and built a multitude of micro- and nanoscale systems for diagnostic and therapeutic applications, especially in the context of cancer, that could be considered early prototypes of nanorobots. Since 1995, more than 50 nanopharmaceuticals,

basically some sort of nanoscale device incorporating a drug, have been approved by the US Food and Drug Administration. If a drug of this class possesses one or more robotic characteristics, such as sensing, onboard computation, navigation, or a way to power itself, scientists may call it a nanorobot. It could be a nanovehicle that carries a drug, navigates to or preferentially aggregates at a tumor site, and opens up to release a drug only upon a certain trigger. The first approved nanopharmaceutical was DOXIL, a liposomal nanoshell carrying the chemotherapeutic drug doxorubicin, which nonselectively kills cells and is commonly used to treat a range of cancers. The intravenously administered nanoshells preferentially accumulate in tumors, thanks to a leaky vasculature and inadequate drainage by the lymphatic system. There, the nanoparticles slowly 04 . 202 0 | T H E S C IE N T IST 3 1

release the drug over time. In that sense, basic forms of nanorobots are already in clinical use. Scientists can manipulate the shape, size, and composition of nanoparticles to improve tumor targeting, and newer systems employ strategies that specifically recognize cancer cells. Still, precise navigation to tumor sites remains a holy grail of nanorobot research and development. A 2016 meta-analysis assessing the efficiency of nanodelivery vehicles tested in animal studies in the previous 10 years revealed that a median of fewer than 1 percent of the injected nanovehicles actually reached the tumor site, and that this could be only marginally improved with active targeting mechanisms, such as surface decoration with specific antibodies or peptides for tumor-specific receptor binding.1 How can we make these nanobots better at steering themselves to tumor sites? Wireless energy transmission remains a huge challenge, and batteries are not yet efficient at the nanometer scale. Researchers have used external forces such as ultrasound or magnetic fields to promote the homing of nanomedicines to tumor tissues, but the fluid dynamics of the circulatory system work against nanoshuttles, whose surface-to-volume ratio is 1 billion times that of objects on the scale of meters. This causes surface and drag forces to become more dominant: to the nanoparticle, it might feel like moving through honey when navigating the aqueous environment of the vasculature. But as it so often does, nature might just have a solution: bacteria. The microscopic organisms swim autonomously through fluids, driven by molecular motors that spin their cilia or flagella in a corkscrew-like fashion—a very effective propulsion mechanism at this scale that has inspired many nanoroboticists that try to mimic this functionality. Researchers have fabricated helical, magnetic swimmers that can be spun forward by a rotating magnetic field, for example.2 But bacteria, especially in the context of treating cancer, are more than just role models for efficient swimming; some are actually themselves therapeutic. In addition, microbes can sense biochemical cues and adjust their trajectories accordingly, similar to the envisioned on-board computation. The idea of using bacteria to treat cancer is not new. One of the earliest reports on bacteria as a cancer therapy comes from the immunotherapy pioneer William Coley, who in the late 19th century recognized that some cancer patients also suffering from skin infections were more likely to get better. He began injecting bacterial toxins, heat-inactivated microbes, or even live cultures of Streptococcus bacteria into his patients with inoperable bone and soft-tissue cancers, often leading to remissions. It was a bold approach, given the risk of uncontrollable infections from these bacterial formulations before the widespread availability of antibiotics. Largely because of that danger, and the promise of the nascent concepts of radiation and chemotherapy, the clinical use of bacteria as therapeutic agents for cancer went undeveloped. Today, this revolutionary idea has been experiencing a renaissance. Thanks to the convergence of fields from biology and chemistry to materials science, engineering, and computer sciences, new avenues for the development of bacterial therapies for cancer are opening up. The toolkits made available thanks to 32 T H E SC I EN TIST | the-scientist.com

reduced costs of both sequencing and synthesis of DNA, along with synthetic-biology approaches for custom genetic design of bacterial-like behaviors, are paving the way for the emerging fields of micro- and nanorobotics.

Bacteria with anti-cancer payloads Bacillus Calmette-Guérin (BCG), an attenuated bacterium typically used as a vaccine strain for tuberculosis, has been repurposed for the last several decades to locally treat bladder cancer. The concept behind this approach, similar to that postulated by Coley, is that the administration of bacteria stimulates the patient’s immune system to fight off the cancer. Even better, though unbeknownst to Coley, many bacteria (though, for unknown reasons, not BCG) also have the potential to selectively grow within solid tumors, in the bladder and elsewhere; reduced immune surveillance in the tumor’s hypoxic and acidic environment provides anaerobic bacteria with a safe haven to grow and thrive. While inside tumors, some bacteria produce toxins and compete with cancer cells for nutrients. Ultimately, the accumulation of bacteria within the tumor induces immune-cell infiltration, which can then lead to anti-cancer responses. Still, despite having tested many naturally occurring and laboratory-made bacterial strains in animal models of cancer, and having conducted human trials testing bacteria to treat cancer, researchers have observed little efficacy beyond the benefits that continue to be seen in bladder cancer patients.

Precise navigation to tumor sites remains a holy grail of nanorobot research and development. As a result, the field has shifted to genetically engineering bacteria to serve as ferries for recombinant payloads. The selective targeting and subsequent growth of bacteria in tumors, along with local delivery of therapeutics facilitated by the microbes themselves, could minimize the collateral damage to healthy cells that is common with systemic cancer therapies. Several groups have engineered bacteria to produce a wide variety of cargo, including anticancer toxins, cytokines, and apoptosis-inducing factors. The production of potentially toxic therapeutic cargo necessitates further control over the bacteria, in case they land in locations they shouldn’t. Thus, researchers are now moving toward engineering next-generation bacterial systems to sense a physiological cue and respond by producing a therapeutic at the local disease site. To aid in this goal, over the last two decades the field of synthetic biology has developed a repertoire of genetic circuits to control microbial behaviors. These circuits consist of pos-

WIKIMEDIA COMMONS, NIAID

Salmonella typhimurium

itive and negative feedback motifs to modulate dynamic cellular functions, acting as toggle switches, oscillators, counters, biosensors, and recorders—tools that researchers have used to design cancer-fighting microbes. One example of genetic control over cancer-fighting bacteria is the synchronized lysis circuit developed in 2016 by Jeff Hasty’s group at the University of California, San Diego, in collaboration with Sangeeta Bhatia’s laboratory at MIT, where both of us did our postgraduate training. (T.D. was a coauthor on this 2016 study.) In this circuit, bacteria localize to tumors and grow to a critical density, then synchronously rupture to release therapeutic compounds that the microbes had been producing. This approach, which takes advantage of natural bacterial quorum sensing, improves upon several features of previously developed bacterial therapies, most of which constitutively produce drugs, meaning they might make and release the therapeutics in unintended areas of the body. Because bacteria only reach critical density within tumors, they will only self-destruct and release their therapeutic payload there. This leads to pruning of the microbial population, preventing uncontrolled growth of bacteria in the tumor or elsewhere. In a colorectal liver metastasis mouse model, this system resulted in a twofold increase in survival when paired with chemotherapy, as compared with chemotherapy or bacteria alone.3 Several groups have further developed this approach. In 2019, for example, one of us (T.D.), along with Columbia University microbiologist and immunologist Nicholas Arpaia and colleagues, created bacteria that produced molecules known to block immune checkpoints, such as CD47 or PD-L1, which ordinarily put the brakes on immune cells and thereby decrease anti-tumor activity.4,5 As a result of blocking these pathways in tumors, bacteria were able to prime T

cells and to facilitate the clearance of cancer in a lymphoma mouse model. Most surprisingly, untreated tumors within treated animals also shrank, suggesting that local priming could trigger distant and durable antitumor immunity. The approach of using bacteria as a cancer therapy is starting to attract the attention of the biotech industry. One company, BioMed Valley Discoveries, has been testing injections of the spores of Clostridium novyi-NT, an obligate anaerobe that can only grow in hypoxic conditions and is genetically attenuated so that a lethal toxin is not produced, in several clinical trials. In rats, dogs, and the first human patient, the treatment showed “precise, robust, and reproducible antitumor responses,” according to a 2014 report.6 Another company, Synlogic, is developing intratumorally injected bacteria designed to produce a STING (STImulator of INterferon Genes) agonist and act as an innate immune activator. The bacteria are sensed and engulfed by antigen-presenting cells that have infiltrated the tumor, and within those immune cells they activate the STING pathway, resulting in interferon release and tumor-specific T cell responses. A Phase 1 clinical trial is underway to evaluate this therapy for the treatment of refractory solid tumors, and trials for use in combination with a checkpoint inhibitor are planned. The results of these and other trials will serve to guide further innovations in safety and efficacy for engineered bacterial cancer therapies. For instance, these studies will shed light not only on therapeutic efficacy, but on bacterial colonization levels and distribution in patient tumors, shedding or off-target colonization, and stability of genetic modifications over time— factors that have only been studied at a detailed level in mouse models. Once a proof-of-principle is established in humans, 04 . 202 0 | T H E S C IE N T IST 3 3

BUILDING BACTERIA TO FIGHT CANCER Synthetic biologists are applying new strategies in genetic engineering to encode traits and smart circuits in bacteria for more effective in vivo monitoring and drug delivery. At the same time, engineers are developing instruments for external control and guidance of bacteria with the aim of enhancing their ability to find and access tumors. Here are a few examples.

BACTERIAL BOMBS

Cancer therapeutic

Jeff Hasty of the University of California, San Diego, in collaboration with Sangeeta Bhatia of MIT (and T.D. in Bhatia’s lab), engineered an attenuated Salmonella enterica bacterial strain to synchronously release cancer therapeutics when the population reaches a critical density, allowing periodic drug delivery in mouse tumors (Nature, 536:81–85 , 2016). The effect is based on quorum lysis, meaning when a critical bacteria cell density is sensed by the population, they lyse and release the drug, while surviving bacteria keep proliferating until the critical threshold is reached again to repeat the cycle.

Tumor

ENCODED NANOSTRUCTURES FOR IMAGING Mikhail Shapiro of the University of California, Berkeley, and colleagues encoded gas-filled nanostructures in microorganisms, including bacteria and archaea (Nat Nanotechnol, 9:311–16, 2014). These structures, when produced by the microbes, serve as contrast agents for ultrasound imaging, allowing researchers to visualize where they go in the body—critical for cancer diagnostics as well as to monitor treatment status by allowing researchers to visualize bacterial accumulation in tumors over time. The group recently demonstrated multiplexing of this approach by encoding a distinct reporter in each of two bacteria, E. coli and Salmonella, to localize and distinguish the microbe in the guts and tumors of mice (Nature, 553:86–90, 2018).

ULTRASOUND

Gas-filled nanostructures

MAGNETICALLY ASSISTED NAVIGATION

MAGNETIC FIELD Magnetic nanoparticles

Nanoliposomse

SHINING LIGHT ON TUMORS

LIGHT Nitric oxide

Semiconductor nanomaterials

© MESA SCHUMACHER

Sylvain Martel of Polytechnique Montréal and colleagues attached drug-containing nanoliposomes onto a magnetotatic bacterial strain called MC-1 that was injected in close proximity to tumors in mice. These bacteria naturally biomineralize magnetic nanoparticles inside their membranes, allowing the researchers to use magnetic fields to guide the bacteria to—and into— tumors (Nat Nanotechnol, 11:941–47, 2016), where they can deliver therapeutics or serve as imaging contrast agents.

Di-Wei Zheng and colleagues at Wuhan University in China used light to enhance the metabolic activities of E. coli by attaching to the bacteria’s surfaces semiconductor nanomaterials that under light irradiation produce photoelectrons. These triggered a reaction with the bacteria’s endogenous nitrate molecules, increasing the formation and secretion of a cytotoxic form of nitric oxide by 37-fold. In a mouse model, the treatment led to an 80 percent reduction in tumor growth (Nat Commun, 9:1680, 2018).

New genetic toolkits are paving the way for the emerging fields of micro- and nanorobotics.

there will be a big push to determine the optimal bacterial strain, payload, circuitry, and appropriate clinical settings in which to use these types of therapies.

Conquering tumors While researchers are succeeding in engineering bacteria to carry or produce anticancer compounds, fewer than 1 percent of those microbes will reach tumors on their own. Since most tumors are not accessible by direct injection, clinicians need to be able to effectively navigate bacterial therapies to tumor sites, where the microbes should reliably and controllably release the toxic drugs they encode. This is where synthetic biology has been influenced by the principles of microrobotics. For example, E. coli bacteria can be engineered with genes from marine microorganisms to sense and make use of light energy. In 2018, the University of Edinburgh’s Jochen Arlt and coworkers showed that such photosynthetic strains of motile E. coli could be guided through spatially

patterned light fields.7 In response to patterns of light exposure, the bacteria moved to certain locations; tracking their position informed the next light input to guide them forward along a predefined path—a process that’s known as closed loop control, a fundamental part of robotics. In the same year, Xian-Zheng Zhang and colleagues at Wuhan University in China used light to locally trigger a 37-fold increase in bacterial cytotoxin production by attaching to the bacteria’s membranes nanomaterials that, upon light exposure, release photoelectrons that promote the toxin’s synthesis. In a mouse model of breast cancer, these anaerobic bacteria were found to accumulate in the hypoxic microenvironment of the tumors, and the subsequent light-boosted cytotoxin production resulted in around 80 percent inhibition of tumor growth.8 This is an example of how the integration of synthetic material into live bacteria can allow remote control of certain actions or functionality, another feature borrowed from classic robotics. While optically triggered navigation and control has enormous potential, light’s limited ability to penetrate tissue hampers the approach. A more widely used form of external energy is ultrasound. It has long had applications in medical diagnostics and monitoring. More recently, gas-filled microbubbles, due to their strong and distinct acoustic response, are used to enhance contrast on ultrasound images of tissues, and special forms of highpowered, focused ultrasound have been applied in therapy to boost the transport of drug-filled nanobubbles by using the acoustic

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E. coli

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pressure waves as external energy to push them deep into tumor tissues. This approach achieved especially promising results in glioblastoma, because the blood-brain barrier is particularly hard to overcome for drugs. A couple of years ago, researchers used ultrasound to track therapeutic bacteria in vivo. Mikhail Shapiro and colleagues at Caltech genetically engineered bacteria to express what they termed acoustic reporter genes (ARG), which encode the components of hollow structures called gas vesicles that scatter ultrasound waves, generating an echo that enabled them to detect the bacteria’s location deep inside living mice.9 Other common sources of external energy that can be safely and remotely applied in the human body are magnetic fields. While magnetic resonance imaging systems have been used clinically for decades, the development of systems for magnetic guidance and control are still fairly new. So far, researchers have applied the approach to guide magnetic catheters for high-precision surgery. The most renowned example is the NIOBE system from St. Louis–based Stereotaxis for the treatment of cardiac arrhythmias. A magnetic catheter tip is precisely steered along abnormal heart tissue, where electrical pulses heat or cool the device to ablate misfiring cells. The use of similar magnetic instrumentation to guide bacteria in the context of cancer therapy has been proposed by groups that work with magnetotatic bacteria—marine microbes that naturally synthesize strings of iron oxide nanoparticles wrapped in a lipid shell. This trait has evolved to help them navigate in the water by sensing the Earth’s magnetic field, with these strings working as compass needles inside their unicellular bodies. This was first discovered in the 1970s by Richard Blakemore of Woods Hole Oceanographic Institution in Massachusetts. Roughly 40 years later, Sylvain Martel of Polytechnique Montréal’s NanoRobotics Laboratory and colleagues coupled these magnetotactic bacteria to DOXIL, the liposome-wrapped chemotherapeutic that earned the title of the first approved nanomedicine. Martel’s group, too, took advantage of the fact that anaerobic bacteria tend to home to tumors for their low-oxygen environment, and coupled that natural homing mechanism with an external directing magnetic field, demonstrating increased accumulation and penetration of the therapy in mouse tumors.10 In another recent study, one of us (S.S.), with researchers at MIT and ETH Zurich, showed in tissue models on a chip that applying rotating magnetic fields could drive swarms of such magnetotactic bacteria to act as little propellers, creating strong flows to push companion nanomedicines out of blood vessels and deeper into tissues.11 While the use of such magnetotactic species inside the human body might occur decades in the future, encoding magnetosensation in other, more clinically translatable or already-tested bacterial strains might be an achievable goal in the near term. Several of the proteins involved in the complex biomineralization process that forms the magnetic compounds in magnetotactic bacteria have been identified, 12 and in a preprint published earlier this year, researchers reported engineering E. coli to form magnetite particles and controlling them by external magnetic fields.13

Another route to making non-magnetic bacteria controllable by magnetic fields is to simply attach magnetic materials to them. Researchers have taken one or even multiple bacterial strains and bound them to magnetic micro- or nanoparticles. When exposed to an external magnetic field, these magnetic particles will orient with the field, and so will the bacteria, which will then travel in that direction. In 2017, Metin Sitti and colleagues at the Max Planck Institute for Intelligent Systems in Stuttgart, Germany, attached E. coli bacteria to microparticles made of layers of the chemotherapeutic doxorubicin and tiny magnetic nanoparticles. Using cancer cells in a dish, the researchers showed that they could remotely control these drug-carrying bacterial bots with magnets to improve tumor cell targeting compared with just adding drugloaded microparticles to the cells.14 No matter how, genetically engineered bacteria empowered by external energy sources providing triggers, control, and guidance are a fascinating new direction in this field. Fueled by the convergence of synthetic biology, mechanical engineering, and robotics, these new approaches might just bring us one step closer to the fantastic vision of tiny robots that seek and destroy many cancer types. g Simone Schuerle is an assistant professor at ETH Zurich and a member of the university’s Institute for Translational Medicine. Tal Danino is an assistant professor at Columbia University and a member of the Herbert Irving Comprehensive Cancer Center and the Data Science Institute.

References 1. S. Wilhelm et al., “Analysis of nanoparticle delivery to tumours,” Nat Rev Mat, 1:16014, 2016. 2. L. Zhang et al., “Artificial bacterial flagella: Fabrication and magnetic control,” Appl Phys Lett, 94:064107, 2009. 3. M.O. Din et al., “Synchronized cycles of bacterial lysis for in vivo delivery,” Nature, 536:81–85, 2016. 4. S. Chowdhury et al., “Programmable bacteria induce durable tumor regression and systemic antitumor immunity,” Nat Med, 25:1057–63, 2019. 5. C.R. Gurbatri et al., “Engineered probiotics for local tumor delivery of checkpoint blockade nanobodies,” Sci Trans Med, 12:eaax0876, 2020. 6. N.J. Roberts et al., “Intratumoral injection of Clostridium novyi-NT spores induces antitumor responses,“ Sci Trans Med, 6:249ra111, 2014. 7. J. Arlt et al., “Painting with light-powered bacteria,” Nat Commun,9:768, 2018. 8. D.-W. Zheng et al., “Optically-controlled bacterial metabolite for cancer therapy,” Nat Commun, 9:1680, 2018. 9. R.W. Bourdeau et al., “Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts,” Nature, 553:86–90, 2018. 10. O. Felfoul et al., “Magneto-aerotactic bacteria deliver drug-containing nanoliposomes to tumour hypoxic regions,” Nat Nanotechnol, 11:941–47, 2016. 11. S. Schuerle et al., “Synthetic and living micropropellers for convection-enhanced nanoparticle transport,” Sci Adv, 5:eaav4803, 2019. 12. A. Peigneux et al., “Learning from magnetotactic bacteria: A review on the synthesis of biomimetic nanoparticles mediated by magnetosome-associated proteins,” J Struct Biol, 196:75–84, 2016. 13. M. Aubry et al., “Engineering E. coli for magnetic control and the spatial localization of functions,” doi:10.1101/2020.01.06.895623, 2020. 14. B.-W. Park et al., “Multifunctional bacteria-driven microswimmers for targeted active drug delivery,” ACS Nano, 11:8910–23, 2017.

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Exercise Title Here in Serif and Cancer Deckline vent et volum dolut odis aut molupta consecta venimus consequ iassim sintur, sitiatat qui auta necum doluptium qui omnimilit velectias doluptat.

BY AUTHORStudies HERE point to a role for physical exercise in fighting malignancies, improving treatment outcomes, and fostering overall health in patients.

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40 T H E SC I EN TIST | the-scientist.com

people could reduce their cancer risk with moderate to vigorous leisure-time exercise training. The phenomenon held across several different cancers, including breast, colon, rectum, esophagus, lung, liver, kidney, bladder, and head and neck. 1 And the combined results of approximately 700 unique exercise intervention trials, involving more than 50,000 cancer patients in total, leave little doubt that patients benefit from physical activity, showing improvements such as reduced toxicity of anticancer treatment, decreased disease progression, and enhanced survival. The same studies showed that exercise training improves mood, decreases loss of muscle mass, and helps cancer patients return to work earlier after successful treatment.2 Some studies show that 150 minutes per week of moderate exercise nearly double the chance of survival compared with breast cancer patients who don’t exercise during treatment.3 Hundreds of animal studies, conducted over decades, suggest that the link is likely causal: in mice and rats, exercise leads to a reduction in the incidence, growth rate, and metastatic potential of cancer across a large variety of models of different human and murine tumor types. But how exercise

helps fight cancer is a bit of a black box. Exercise may improve the efficacy of anticancer treatment by boosting the immune system and thereby attenuating the toxicity of chemotherapy and immunotherapy. Cancer patients are also likely to benefit from the overall healthpromoting properties of physical activity, such as improved metabolism and enhanced cardiovascular function. Uncovering the mechanisms whereby exercise induces anticancer effects is crucial to fighting the disease. Exerciserelated factors that have a direct or indirect anticancer effect could serve as valuable biomarkers for monitoring the amount, intensity, and type of exercise required to best aid cancer treatment. Such research could also potentially highlight novel therapeutic targets.

Each workout matters Regardless of the nature of the training, the primary setting of exercise’s effect on cancer is the bloodstream. Long-term training has been associated with a reduction in the blood levels of systemic risk factors, such as OUTRUNNING CANCER: Tumors on the lungs

of sedentary mice (left) and animals that ran on wheels (right) after injection with melanoma cells.

L. PEDERSEN ET AL., CELL METAB, 2016

M

athilde was diagnosed with breast cancer at the age of 44. Doctors treated her with surgery, chemotherapy, and radiation, and Mathilde’s physician informed her that, among many other side effects of her cancer treatment, she could expect to lose muscle mass. To fight muscle wasting, Mathilde began the intensive physical training program offered to cancer patients at the Rigshospitalet University Hospital of Copenhagen. The program consists of 3.5-hour sessions of combined resistance and aerobic training, four times a week for six weeks. Although the chemotherapy made her tired, Mathilde (a friend of mine, not pictured, who requested I use her first name only) did not miss a single training session. “In a way it felt counterintuitive to do intensive, hard training, while I was tired and nauseous, but I was convinced that the training was good for my physical and mental health and general wellbeing,” Mathilde told me in Danish. She followed the chemo- and radiotherapy strictly according to the prescribed schedule. She was not hospitalized, acquired no infections, and did not develop lymphedema, a failure of the lymphatic system that commonly occurs following breast cancer surgery and leads to swelling of the limbs. Physical exercise is increasingly being integrated into the care of cancer patients such as Mathilde, and for good reason. Evidence is accumulating that exercise improves the wellbeing of these patients by combating the physical and mental deterioration that often occur during anticancer treatments. Most remarkably, we are beginning to understand that exercise can directly or indirectly fight the cancer itself. An increasing amount of epidemiological literature strongly indicates that exercise training may lower the risk of cancer, control disease progression, amplify the effects of anticancer therapy, and improve physical function and psychosocial outcomes. For example, a 2016 study of more than 1.4 million individuals in the US and Europe found that

sex hormones, insulin, and inflammatory molecules.4 However, this effect is only seen if exercise training is accompanied by weight loss, and researchers have not yet established causal direct links between regular exercise training and the reductions in the basal levels of these risk factors. 5 Alternatively, the anticancer effect of exercise could also be the result of something that occurs within individual sessions of exercise, during which muscles are known to release spikes of various hormones and other factors into the blood. To learn more about the effects of individual bouts of exercise versus longterm training regimens, Christine Dethlefsen, a graduate student in my laboratory, incubated breast cancer cells with serum obtained from cancer survivors at rest before and after a six-month training intervention that began after patients completed primary surgery, chemotherapy, and radiotherapy. For comparison, she incubated other cells with serum obtained from blood drawn from these patients immediately after a two-hour acute exercise session during their weeks-long course of chemotherapy. Her study revealed that serum obtained following an exercise session reduced the viability of the cultured breast cancer cells, while serum drawn at rest following six months of training had no effect.6 These data suggest that cancer-fighting effects are driven by repeated acute exercise, and each bout matters. In Dethlefsen’s study, incubation with serum obtained after a single bout of exercise (consisting of 30 minutes of warm-up, 60 minutes of resistance training, and a 30-minute high-intensity interval spinning session) reduced breast cancer cell viability by only 10 to 15 percent compared with control cells incubated with serum obtained at rest. But a reduction in tumor cell viability by 10 to 15 percent several times a week may add up to clinically significant inhibition of tumor growth.7 Indeed, in a separate study, my colleagues and I found that daily, voluntary wheel running in mice inhib-

its tumor progression across a range of tumor models and anatomical locations, typically by more than 50 percent.8

Exercise’s molecular messengers One prime candidate for helping to explain the link between exercise and anticancer effects is a group of peptides known as myokines, which are produced and released by muscle cells. Several myokines are released only during exercise, and some researchers have proposed that these exercise-dependent myokines contribute to the myriad beneficial effects of physical activity for all individuals, not just cancer patients, perhaps by mediating crosstalk between the muscles and other parts of the body, including the liver, bones, fat, and brain. The best-characterized myokine is interleukin-6, levels of which increase exponentially during exercise in humans. At least in mice, interleukin-6 is involved in directing natural killer (NK) cells to tumor sites. But there are approximately 20 known exercise-induced myokines, and the list continues to grow. Preliminary studies show that myokines can reduce cancer growth in cell culture and in mice. For example, when treated with irisin, a myokine best known for its ability to convert white fat into brown fat, cultured breast cancer cells were more likely to lose viability and undergo apoptosis than were control cells.9 A study I led found that oncostatin M, another myokine that is upregulated in murine muscles after exercise, also inhibits breast cancer proliferation in vitro.10 And a team led by Toshikazu Yoshikawa of Kyoto Prefectural University determined that in a mouse model of colon cancer, a myokine known as secreted protein acidic and rich in cysteine (SPARC) reduced tumorigenesis in the colon of exercising mice.11 Overall, skeletal muscle cells may be secreting several hundred myokine types, but of these, only about 5 percent have been investigated for their biological effects. And researchers have tested fewer for whether they regulate cancer cell growth. Not all of the molecular messengers released in response to exercise come from

Several myokines are released only during exercise, and some researchers have proposed that these exercise-dependent myokines contribute to the myriad beneficial effects of physical activity for all individuals, not just cancer patients.

04 . 2020 | T H E S C IE N T IST 41

EXERCISE’S ANTICANCER MECHANISMS Researchers are beginning to understand that not only can exercise improve cancer patients’ overall wellbeing during treatment, but it may also fight the cancer itself. Experiments on cultured cells and in mice hint at some of the mechanisms that may be involved in these direct and indirect effects.

 1 Exercising muscles release multiple compounds known as myokines. Several of these, including interleukin-6, have been shown to affect cancer cell proliferation in culture and tumor growth in mice.

Interleukin-6

 4

In mice, interleukin-6 appears to direct natural killer cells to home in on tumors.

Norepinephrine

Cancer cells

Epinephrine

Natural killer cell Interleukin-6

In lab-grown cells and in mice, epinephrine, norepinephrine, and some myokines hinder tumor growth and metastasis.

© SCOTT LEIGHTON

 5

 2

Exercise stimulates an increase in levels of the stress hormones epinephrine and norepinephrine, which can both act directly on tumors and stimulate immune cells to enter the bloodstream.

Epinephrine

Natural killer cell

Norepinephrine

 3 Epinephrine also stimulates natural killer cells to enter circulation.

Adrenal gland

Tumor

NK cells appear to be instrumental to the exercise-mediated control of tumor growth in mice.

the muscles. Notably, exercise induces acute increases in epinephrine and norepinephrine, stress hormones released from the adrenal gland that are involved in recruiting NK cells in humans. Murine studies show that NK cells can signal directly to cancer cells. In Dethlefsen’s study, when breast cancer cells incubated with serum obtained after a bout of exercise were then injected into mice, they showed reduced tumor formation. The exercise-induced suppression of breast cancer cell viability and tumor formation were, however, completely blunted when we blockaded β-adrenergic signaling, the pathway through which epinephrine and norepinephrine work.12 These findings suggested that epinephrine and norepinephrine are responsible for the cancer-inhibiting effects we observed. Epinephrine and norepinephrine, which activate NK cells, have also been shown to act on cancer cells through the Hippo signaling pathway, which is known for regulating cell proliferation and apoptosis. Exercise-induced spikes in these stress hormones activate this pathway, which somehow inhibits the formation of new malignant tumors associated with metastatic processes.

Calling the immune system In addition to acting directly on tumors, the myokines released during and after exercise are known to mobilize immune cells, par-

ticularly NK cells, which appear to be instrumental to the exercise-mediated control of tumor growth in mice. The late molecular biologist Pernille Højman of the Centre for Physical Activity Research at Rigshospitalet was a leader in discerning this mechanism. In the study described above that compared tumor growth in active and sedentary mice, on which I was also an author, Højman looked more closely at the tumors and found that the running mice had twice as many cytotoxic T cells and five times more NK cells than those animals housed without a wheel. Højman repeated the experiment on mice that had been engineered to lack cytotoxic T cells. Again, she found that mice with access to running wheels had smaller tumors. When she performed the same test on mice that had intact T cells but lacked NK cells, the tumors of all the mice grew to the same size. This suggested that the NK cells, and not the T cells, were the link between exercise and tumor growth suppression.8 Work by other groups had demonstrated that epinephrine has the potential to mobilize NK cells, and Højman and the rest of our team wondered if epinephrine had a role in mediating the anticancer effects of exercise. We

EXERCISE AND DEPRESSION Depression is a severe adverse effect of cancer and cancer therapy. The risk of depression can be as high as 50 percent for some cancer diagnoses, although this number varies a great deal depending on cancer type and stage (J Natl Cancer Inst Monogr, 32:57–71, 2004). In addition to its effects on a patient’s quality of life, depression can hinder compliance with treatment, and it’s a risk factor for mortality in cancer patients (Lancet, 356:1326–27, 2000). In recent years, healthcare providers have increasingly integrated physical exercise into the care of cancer patients with the aim of controlling disease and lessening treatment-related side effects, while researchers have amassed evidence supporting the assertion that such training can lower symptoms of depression in these patients (Acta Oncol, 58:579–87, 2019). The biological mechanisms behind this beneficial effect remain to be determined, although some clues have emerged. For example, a study in mice found that exercise-dependent changes in metabolism result in reduced accumulation of some neurotoxic products (Cell, 159:33-45, 2014). In cancer patients, systemic levels of kynurenine, a neurotoxic metabolite associated with fatigue and depression, are upregulated (Cancer, 121:2129-36, 2015). In mice, exercise enhances the expression of the enzyme kynurenine aminotransferase, which converts kynurenine into neuroprotective kynurenic acid, thereby reducing depression-like symptoms. Findings such as these, together with exercise’s well-documented effects in alleviating depression among patients without cancer, suggest that incorporating exercise into cancer treatment may benefit mental as well as physical health.

AND STAY OUT: Exercise activates natural killer

cells (purple) and helps them home to tumors.

Bente Klarlund Pedersen is a professor of integrative medicine at the University of Copenhagen and a specialist in infectious diseases and internal medicine at the university’s Rigshospitalet hospital. She directs both the Centre of Inflammation and Metabolism and the Centre for Physical Activity Research.

© ISTOCK.COM, SELVANEGRA

References

injected mice that had malignant melanoma with either epinephrine or saline and found that the hormone indeed reduced the growth of tumors, but to a lesser degree than what was observed in the mice that had access to a wheel. Something else had to be involved. To find out what, our team tested the effects of interleukin-6, which we suspected was the additional exercise factor involved in tumor homing of immune cells. When we exposed inactive mice to both epinephrine and interleukin-6, the rodents’ immune systems attacked the tumors as effectively as if the animals had been running.8

While much remains to be learned about how physical exercise influences cancer, evidence shows that exercise training is safe and feasible for patients with the disease and contributes to their physical and psychosocial health. (See “Exercise and Depression” on page 44.) Being active may even delay disease progression and improve survival. A growing number of patients, including Mathilde, are undergoing exercise training to fight physical deterioration during cancer treatment. As they do so, scientists are working hard to understand the pathways by which physical activity results in anticancer activity. g

1. S.C. Moore et al., “Association of leisure-time physical activity with risk of 26 types of cancer in 1.44 million adults,” JAMA Intern Med, 176:816–25, 2016. 2. J.F. Christensen et al., “Exercise training in cancer control and treatment,” Compr Physiol, 9:165–205, 2018. 3. M.D. Holmes et al., “Physical activity and survival after breast cancer diagnosis,” JAMA, 293:2479–86, 2005. 4. A. McTiernan, “Mechanisms linking physical activity with cancer,” Nat Rev Cancer, 8:205–11, 2008. 5. C. Dethlefsen et al., “Every exercise bout matters: linking systemic exercise responses to breast cancer control,” Breast Cancer Res Treat, 162:399–408, 2017. 6. C. Dethlefsen et al., “Exercise regulates breast cancer cell viability: systemic training adaptations versus acute exercise responses,” Breast Cancer Res Treat, 159:469–79, 2016. 7. P. Højman et al., “Molecular mechanisms linking exercise to cancer prevention and treatment,” Cell Metab, 27:10–21, 2018. 8. L. Pedersen et al., “Voluntary running suppresses tumor growth through epinephrine- and IL-6-dependent NK cell mobilization and redistribution,” Cell Metab, 23:554–62, 2016. 9. N.P. Gannon et al., “Effects of the exerciseinducible myokine irisin on malignant and nonmalignant breast epithelial cell behavior in vitro,” Int J Cancer, 136:E197–202, 2015. 10. P. Højman et al., “Exercise-induced musclederived cytokines inhibit mammary cancer cell growth,” Am J Physiol Endocrinol Metab, 301:E504–10, 2011. 11. W. Aoi et al., “A novel myokine, secreted protein acidic and rich in cysteine (SPARC), suppresses colon tumorigenesis via regular exercise,” Gut, 62:882–89, 2013. 12. C. Dethlefsen et al., “Exercise-induced catecholamines activate the Hippo tumor suppressor pathway to reduce risks of breast cancer development,” Cancer Res, 77:4894–904, 2017.

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EDITOR’S CHOICE PAPERS

The Literature gRNA

TECHNIQUES PEG coating

THE PAPER

46 T H E SC I EN TIST | the-scientist.com

E7 gene

Plasmid with gene for Cas9

L. Jubair et al., “Systemic delivery of CRISPR/ Cas9 targeting HPV oncogenes is effective at eliminating established tumors,” Mol Ther, 27:2091–99, 2019. When the human papillomavirus enters a cervix, it doesn’t lyse cells or cause inflammation. While some strains can cause genital warts, in most cases the body clears the virus without much fuss. But “in an unfortunate number of people, the virus gets stuck,” says Nigel McMillan, a cancer researcher at Griffith University in Queensland, Australia. Even 15 or 20 years after infection with certain human papillomavirus (HPV) strains, cervical and other cancers can develop as a result. Looking for a new way to treat these cancers, McMillan focused on two oncogenes, E6 and E7, that HPV delivers to host cells. If E6 and E7 are turned off, cancer cells will not survive—a phenomenon known as oncogene addiction. In the early 2000s, McMillan and others used short interfering RNAs (siRNAs) to reduce levels of the mRNA products of these two oncogenes. This treatment killed cancer cells in vitro, but there was no effective and commercially available way to get the siRNA to tumors in a live animal. So in 2009, McMillan and his colleagues began working with something called stealth liposomes. Unlike regular liposomes, which are spherical phospholipid containers that researchers can use to deliver drugs into cells but which are often targeted by the immune system to be removed from the body, these liposomes are coated with a polyethylene glycol (PEG) layer that’s nontoxic and nonimmunogenic. In a mouse model that had been injected with cancer cells, tumors shrank considerably when the animals were treated with siRNA-loaded stealth liposomes. But the tumors never completely disappeared.

Liposome

gRNA Cas9

Tumor cells

CUT OFF: Special stealth liposomes with a polyethylene glycol (PEG) coating that serves to hide the liposomes from the immune system are injected into mice with tumors  1 . There, the PEG covering spontaneously falls off  2 , allowing the liposomes to merge with cells and deliver CRISPR-Cas9 gene editing machinery  3 . The system is designed to slice the powerful oncogene E7  4 , triggering apoptosis  5 and wiping out the tumors.

In 2013, CRISPR-Cas9 gene editing burst onto the scientific scene, and by 2016 McMillan decided to try deploying it against the HPV oncogenes. With CRISPR, “we were actually attacking the very gene, the absolute primary cause of this cancer,” rather than its products, as siRNAs did, says McMillan. His team made guide RNAs targeting the E7 gene and put them into PEGylated liposomes along with the other components needed for CRISPR-Cas9 editing. They then injected the liposomes into the bloodstreams of mice with tumors. The PEG coating falls off within 24 hours of injection, allowing the liposome to merge with tumor cells and release the CRISPR-Cas9 system, shutting down E7. McMillan and graduate student Luqman Jubair gave some of the mice three injections, which caused the tumors’ growth to slow, but still, it didn’t stop. In a separate group of mice given seven injections, the tumors disappeared altogether. “It was like, ‘Holy moly! This is amazing,’” says McMillan.

“We kept being amazed each time we did a measurement.” McMillan says the study is the first example he knows of wiping out cancer in vivo using CRISPR. Edward Stadtmauer, a clinical oncologist and researcher at the University of Pennsylvania who was not involved in this study but recently demonstrated the safe use of CRISPR-edited cells in cancer patients, writes in an email that the work is “certainly innovative” and demonstrates “really interesting delivery of CRISPR technology to tumors in a mouse model.” McMillan hopes to launch a clinical trial of liposomes delivered via a patch placed on the cervix, rather than intravenously, in the next couple of years, working with Kevin Morris, a gene therapy researcher at City of Hope Hospital in California who wasn’t involved in the current study. “It’s the whole package,” Morris says of McMillan’s study. “He’s shown here that you can obliterate the cancer itself.” —Rachael Moeller Gorman

© KELLY FINAN

CRISPR’d Cancer

WELCOME MAT: Cow trophoblasts (green) invade a layer of human stromal

SEEK AND DESTROY: The Zika virus was unable to enter patient-derived

CANCER BIOLOGY

DRUG DEVELOPMENT

Another Break in the Wall

Zika, Cancer Warrior?

THE PAPER

THE PAPER

Kshitiz et al., “Evolution of placental invasion and cancer metastasis are causally linked,” Nat Ecol Evol, 3:1743–53, 2019.

Z. Zhu et al., “Zika virus targets glioblastoma stem cells through a SOX2-integrin αvβ5 axis,” Cell Stem Cell, 26:187–204.E10, 2020.

The study was conceived like a cliché joke: an evolutionary biologist, a cell-signaling specialist, and a cancer researcher walk into a happy hour at Yale University. The conversation turned to mammalian tumors, and how it was common to see horses in Austria and cows in India with prominent tumors that rarely killed the animals. It turns out that horses and cows have something else in common. “Interestingly, in the same animals, the pregnancy is very different from human pregnancy,” says the University of Connecticut Health Center’s Kshitiz, the cancer researcher, who uses a single name. Placental cells in ungulates don’t burrow into the uterine lining early in pregnancy as they do in apes and many other mammals. The researchers were not the first to draw a connection between cancer severity—specifically, whether the tumors metastasize to other locations in the body and become more deadly—and placental invasion. But they wondered if the key to that link was the action not of the invading cells, but of the tissue that was under attack. Together with colleagues, the trio cultured layers of human or bovine endometrial stroma cells and tested their resistance to invasion by placental cells from both species and by a melanoma cancer cell line. “The difference between cow and human was just like day and night,” Kshitiz says. “The cow cells will resist invasion; the human cells will . . . not only not resist, but may even assist invasion.” “I think the most striking feature of this paper is that they show that it’s not really the way in which the cancer cells behave that’s different, but it’s really the fibroblasts—the stromal cells— how they respond to the cancer cells,” says Karuna Ganesh, an oncologist and cancer researcher at Memorial Sloan Kettering Cancer Center. The finding suggests, she says, that healthy fibroblast tissue could be targeted by therapeutics with the aim of halting metastasis. That’s a possibility Kshitiz’s lab is already exploring in follow-up work. —Shawna Williams

In 2017, University of California, San Diego, regenerative medicine researcher Zhe Zhu and colleagues found that the Zika virus, which sparked a widespread epidemic in the Americas a few years ago and is known to cause microcephaly in fetuses by destroying neural stem cells, preferentially targets and kills glioblastoma stem cells (J Exp Med, 214:2843–57). The researchers argued that a modified form of Zika could potentially be used as an oncolytic virus therapy against glioblastoma, an aggressive type of brain cancer. Following up on that work, Zhu and collaborators set out to find “the unique property of this virus” that allows Zika to selectively target brain cancer stem cells while sparing adjacent tissues, says Zhu. Previous research has shown that a number of viruses use integrins, a group of transmembrane cell adhesion receptors, as gateways for entering host cells. The team wondered whether the same was true of Zika. Using antibodies, the researchers blocked different integrins in brain cancer stem cells and found that the integrin αvβ5, which is produced at higher levels in brain cancer tissue than in normal brain tissue, seemed to be responsible for helping the virus enter the cells. Silencing the same integrin in a brain cancer mouse model, human brain organoids, and glioblastoma surgical samples also blocked Zika infection. Zhu and his collaborators believe that understanding Zika’s entry mechanism can lead them closer to treatments for glioblastoma. “We hope that we can eventually use this virus in clinical trials and help to improve the outcome of the patient,” he says. Hongjun Song, a neural stem cell researcher at the University of Pennsylvania who was not involved in the study, agrees that a modified version of the Zika virus is a potential therapeutic tool for treating glioblastoma. The approach is still a long way from clinical trials, Song says, “but this is a really interesting mechanistic study to go towards that direction.” —Amy Schleunes

KSHITIZ ET AL., NAT ECOL EVOL; PINAR MESCI

cells (black), which may also be susceptible to metastasizing cancer cells.

glioblastoma stem cells (green) growing on a human brain organoid if the cells lacked a particular integrin in their membranes.

04 . 2020 | T H E S C IE N T IST 47

PROFILE

Cracking Down on Cancer Through his studies on oncogenic viruses, University of California, Los Angeles, professor Owen Witte has helped develop lifesaving treatments. BY DIANA KWON

48 T H E SC I EN TIST | the-scientist.com

FROM SUBURBIA TO STANFORD Born in Brooklyn in May 1949, Witte started his life as a city boy, then spent much of his childhood and adolescence in Levittowns along the East Coast of the US. Levittowns were large housing developments that sprang up after World War II to house veterans and their families. Abraham Levitt and his two sons, William and Alfred, founded the company that constructed these developments, which became the prototypical American suburbs—sprawling neighborhoods with rows upon rows of nearly identical homes, situated on rectangular land plots with driveways and neatly trimmed lawns.

That’s the great beauty of science. . . . Doing work because the question is interesting and not necessarily being able to anticipate when it might actually be useful down the road. — Owen Witte, UCLA

When Witte was in third grade, Levitt & Sons hired his father, and the family moved to one of the cookie-cutter suburbs in Pennsylvania. Witte credits his early interest in science to a fifth-grade teacher in the Pennsylvania Levittown. “She taught science not by reciting facts from a textbook, but with hands-on experiments,” Witte says, noting that one such experiment involved learning about the circulatory system by dissecting a cow’s heart. “That allowed us to learn what real science is about.” Witte continued to do hands-on science as he got older, working at the Department of Health conducting bacterial counts in lakes, swimming pools, and restaurants. But at the time, he wasn’t dead set on a career in science. In high school he was drawn to the culinary arts, and later, in 1967, he went off to study at Cornell University in Ithaca, New York, with an undecided major. While he was there, he considered a career in hotel management, but was drawn back to science after taking classes in the university’s College of Agriculture, where he was exposed to various scientific disciplines, including food science and microbiology. Witte developed an interest in research while working in the lab of Lawrence Slobin, a professor in the department of microbiology, studying how antibodies stop viruses from replicating within a host. It was 1971, right in the middle of the Vietnam War, when Witte was finishing up his final undergraduate

REED HUTCHINSON/UCLA

S

itting at a lab bench at MIT in the late 1970s, Owen Witte finally had to admit he was stuck. He had identified a cancer-causing protein encoded in the genome of the Abelson murine leukemia virus, which infects mice. Prior work had led Witte to hypothesize that this protein should be a kinase, an enzyme that attaches phosphoryl groups to amino acids. But after double- and triple-checking his methods and repeating the experiments several times, he failed to find evidence that this was the case. Witte’s idea that the Abelson murine leukemia virus (A-MuLV) protein should be a protein kinase stemmed primarily from work by molecular biologist Raymond Erikson, then at the University of Colorado, and his colleagues. They had shown that the oncogenic viral gene src was associated with protein kinases, and Witte expected to find something similar with A-MuLV. He followed the standard protocol for characterizing protein kinases by identifying their phosphorylated amino acid products, which involved adding the A-MuLV protein to an acidic solution, heating it, then waiting 24 hours for the enzyme to complete its action. But in the end, Witte could see only free phosphates—compounds that are transferred during a kinase reaction. Exasperated, he tried something different. He analyzed his solutions at several time points during the reaction rather than only at the end. The new protocol worked. Finally, after several months of trying, he’d identified the elusive amino acid, which appeared within a few hours (Nature, 283:826–31, 1980). To Witte’s surprise, the amino acid was neither phosphoserine nor phosphothreonine, which, at the time, were thought to be the primary phosphorylated amino acids produced by protein kinases in mammals. Instead, it turned out to be phosphotyrosine, which had never before been observed as the product of a protein kinase reaction. The A-MuLV protein was a newly discovered type of enzyme: a tyrosine kinase. Another team, led by Tony Hunter at the Salk Institute in California, nearly simultaneously reported tyrosine kinase activity associated with another virus that causes cancer in chickens. Witte, after setting up his own lab, demonstrated that a homolog of the A-MuLV tyrosine kinase, ABL1, was highly active in human leukemias—a discovery that contributed to the development of a lifesaving leukemia therapy called Gleevec (imatinib). The discovery of tyrosine kinases also revealed an entire class of enzymes that could be medical targets. It is “probably the single greatest discovery that I could ever imagine being associated with,” Witte says.

semester, and like many other male college students graduating at the time, he needed to make a choice: join the military or continue his education. Full-time students could get deferments from being drafted as long as they maintained a good academic record and demonstrated their ability to graduate. “I think that weighed heavily in my decision to end up applying to medical school,” Witte says. Witte’s undergraduate research had focused on the way antibodies neutralize a bacteria-infecting virus called bacteriophage f2 (J Immunol, 108:927–36, 1972). This work was in immunology, a discipline that fell under the umbrella of medicine at the time, so Witte decided to apply to MD and MD/ PhD programs. Accepted by Stanford University, he packed his bags and headed to the West Coast. Before that, he says, “I had never been west of the Mississippi River.”

DECIPHERING CANCER’S ORIGINS

CAREER TITLES/AWARDS Professor, Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles Presidential Chair in Developmental Immunology, University of California, Los Angeles Director, Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research Association of American Medical Colleges Award for Distinguished Research in Biomedical Sciences (2016) American Association for Cancer Research G.H.A. Clowes Memorial Award in Cancer Research (2015) Rowley Prize, International Chronic Myeloid Leukemia Foundation (2014)

Greatest Hits • Discovered that the Abelson (ABL) tyrosine kinase is highly active in human leukemias, a finding that led to the development of imatinib (Gleevec), a therapeutic for chronic myeloid leukemia and acute lymphocytic leukemia • Identified Bruton’s tyrosine kinase (BTK), an enzyme required for B-lymphocyte development and function, which enabled the development of drugs to treat certain leukemias and lymphomas • Helped identify surface antigens and kinases to target for prostate cancer therapies currently in development

When Witte arrived at Stanford in the early 1970s, he initially planned to work with Henry Kaplan. Kaplan was a radiologist famous for groundbreaking work that transformed Hodgkin’s disease, a cancer that originates in the lymphatic system, from a fatal illness to a treatable one. But after being introduced to one of Kaplan’s former mentees, Irving Weissman—a young professor who would go on to become a leading scientist in the fields of immunology and cancer biology—Witte saw a better fit. He joined Weissman’s lab while maintaining a relationship with Kaplan as a co–thesis advisor. “I kind of got the best of both worlds,” Witte says. “The younger professor energy in the lab and the more senior professor with a lot of experience and great [connections].” In Weissman’s lab, Witte studied retroviruses that cause cancers in mice. That research led to several important insights into the viruses’ morphology and the role of their proteins in processes such as replication and budding, where a virus exits a cell in an envelope derived from the host’s membrane. Some of this work contributed to crucial advances years later, when scientists studying human immunodeficiency virus (HIV) identified proteins that could inhibit proteases; the finding later served as the foundation for antiretroviral medications for HIV. “That’s the great beauty of science,” Witte says. “Doing work because the question is interesting and not necessarily being able to anticipate when it might actually be useful down the road.” After earning his medical degree, Witte moved to Boston to become a medical intern. But it wasn’t long before he 04 . 202 0 | T H E S C IE N T IST 49

PROFILE decided to abandon a career as a doctor and head back to the lab. “I just did not like it,” he says. Not only was the medical internship an imperfect fit, a family illness made this a particularly trying time in his life. Witte decided to return to Stanford, but Weissman suggested that before he packed his bags and crossed the country again, he reach out to David Baltimore, who was then a professor of microbiology at MIT. “I kind of laughed because David Baltimore had won the Nobel Prize about two years prior, and I was sure that the lineup of people trying to get into his lab was quite long,” Witte recalls.

I’m 70 years old and I’m still really jazzed up about this new finding. — Owen Witte, UCLA

Fortunately for Witte, Weissman knew Baltimore and called to put in a recommendation for his former mentee. “Irv said such wonderful things about him that I thought I would be missing out on a terrific person if I didn’t take him,” Baltimore recalls. “And of course, it worked out terrifically well.” At the time, a focus of Baltimore’s lab was the Abelson murine leukemia virus, a retrovirus that causes cancer in mice. As a postdoc, Witte worked diligently to characterize a protein encoded within the virus’s genome, which eventually led to the discovery that it was a tyrosine kinase. “My graduate work and my postdoc were the most enjoyable times during my [scientific career], because all I had to do was be in the lab and do the work that I love,” Witte says.

EXPLORING NEW FRONTIERS Witte left Boston in 1980 and headed west again to set up his own lab at the University of California, Los Angeles. Within the first few days of arriving, Witte met Jami McLaughlin, a research assistant in the department. The two started dating shortly after and got married in May 1984. A few months later, McLaughlin joined Witte’s lab, where, alongside Witte’s graduate students, she helped clone and sequence BCR-ABL, an oncogene that causes human leukemias. “It’s really been a partnership over many years, with her doing some of the really critical experiments that led to these important results,” Witte says. His team later identified Bruton’s tyrosine kinase (BTK), a protein produced by a mutated gene in X-linked agammaglobulinemia, a human immune deficiency marked by a loss of B cells (Science, 261:358–61, 1993). Further investigations of BTK’s expression and kinase activity eventually led to the develop of ibrutinib (Imbruvica), a drug used to treat B cell–associated leukemias and lymphomas. 5 0 T H E SC I EN TIST | the-scientist.com

For almost three decades, Witte’s research centered on leukemias and lymphomas. Then, in the mid-1990s, he started hearing from relatives who had been diagnosed with prostate cancer. “They would call me asking, ‘What should we do?’” he recalls. After digging through the literature, Witte discovered that there had been little change in the treatment of prostate cancer for several decades. The paucity of advances in this field led him to a radical decision: to shift his focus from blood cancers to prostate cancer. “That wasn’t easy at that stage of my career,” Witte says. “I had a very well-established reputation in one area, and no reputation whatsoever in this new area.” But just as he had persevered with his groundbreaking experiments in the 1970s, he pushed himself to work through any roadblocks that popped up as he started down a new research path. This work has led to, among other things, pinpointing the population of stem cells that gives rise to human prostate cancers (Science, 329:568–71, 2010). Another realm where Witte’s perseverance paid off was in his participation in industry, particularly with companies developing cancer drugs. “One of the important things about Owen was that he has been unafraid to get involved in companies,” Baltimore says. “Many researchers are comfortable working both with industry and as academics, but that was not true 20 years ago when Owen was first doing that.” It allowed him to have an even greater scientific impact, Baltimore explains. Witte has also had an impact on his students with his scientific curiosity and warmth. Witte was incredibly knowledgeable and was an insightful mentor with high expectations for his students, says Charles Sawyers, now a physician-scientist at Memorial Sloan Kettering Cancer Center. “I found [his lab] to be the most intellectually stimulating community of people,” he says. “I was like a sponge, soaking up everything, because I was so new to the area.” Prostate cancer researcher John Lee of Fred Hutchinson Cancer Research Center recalls Witte’s softer side. During his graduate work in Witte’s lab, Lee’s mother was diagnosed with metastatic bladder cancer and passed away a year after starting treatment. “Owen was very supportive during that period,” Lee says. “But the other really important thing that he told me was that [when it comes to cancer research], there’s a lot of work yet to be done.” Witte’s lab continues to contribute important discoveries to cancer research. One of the most recent was demonstrating that epithelial cancers starting in different organs, such as the prostate and lung, develop into highly aggressive, lethal late-stage tumors by a nearly identical mechanism (Science, 362:91–95, 2018). The discovery suggests that targeting a set of “master” gene regulators could potentially help treat a wide range of cancers, Witte says. “I’m 70 years old and I’m still really jazzed up about this new finding.” g

SCIENTIST TO WATCH

Hadiyah-Nicole Green: Laser Focus Associate Professor, Morehouse School of Medicine, Age: 39 BY EMILY MAKOWSKI

© THE ORA LEE SMITH CANCER RESEARCH FOUNDATION

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hen Hadiyah-Nicole Green was in kindergarten, she started helping one of her older brothers with his fourth-grade homework. She and her siblings lived in Saint Louis, Missouri, with their aunt and uncle, who raised them after their mother and grandparents died. “As a child, there were no scientists in my life. I didn’t dream of being a scientist, let alone a physicist. I didn’t have that example,” Green tells The Scientist. “But I loved learning . . . and that gave me the foundation that I needed.” For college, Green chose to attend Alabama A&M University, where a graduate student persuaded her to study physics. In the summers, she interned at the University of Rochester and then at NASA, where she helped calibrate lasers for the International Space Station. After graduating in 2003 with a perfect 4.0 GPA, she planned to work on improving fiber optics. But a series of family tragedies changed her career trajectory. First, the aunt who’d raised her announced she had female reproductive cancer and would forgo treatment. “She said that she would rather die than experience the side effects of chemotherapy or radiation,” Green explains. “I was her primary caregiver the last three months of her life, and I watched her go from this powerful matriarch in our family to being someone who couldn’t walk, speak, or stand on her own.” Her aunt died in 2005, and three months later, her uncle was diagnosed with esophageal cancer. Later, he was also diagnosed with prostate cancer. The long-term side effects of treatment contributed to his death in 2013. Her aunt’s death and uncle’s illness led Green to decide to use her knowledge of lasers to develop a cancer treatment that wouldn’t have side effects. She proposed the idea to Sergey Mirov, a physicist at the University of Alabama at Birmingham who agreed to accept her as a graduate

student. In Mirov’s lab, Green worked on a cancer therapy in which gold nanoparticles are injected into tumors. When lasers are directed at the nanoparticles, the particles start to vibrate and warm up, destroying the tumor cells with heat. Because the treatment is delivered locally, it does not affect the surrounding healthy cells. In 2011, Green and her colleagues showed that these nanoparticles could be attached to tumor-specific antibodies in cell culture (J Nanotechnol, 2011:631753), work that earned her a PhD in physics a year later. She is the 76th African American woman to receive a physics PhD from an American university, she says; the African American Women in Physics website keeps a database of the recipients. After graduation, Green joined Tuskegee University as an assistant professor and continued studying lasers and cancer, showing that mice with a form of skin cancer had nearly 100 percent tumor regression when treated using her gold nanoparticle method (Int J Nanomed, 9:5093–102, 2014). Using lasers to destroy tumor cells “is an extremely clever approach, very innovative, yet straightforward,” says James Lillard, an immunobiologist and associate dean for research at Morehouse School of Medicine. He recruited Green in 2016, and she launched the Ora Lee Smith Cancer Research Foundation, a nonprofit named for her aunt, to raise money to test her laser technique in human

clinical trials. That same year, she received a $1.1 million grant from the Veterans Affairs Historically Black Colleges and Universities Research Scientist Training Program. Landing that grant “is huge for somebody just beginning their career,” says Adeboye Adejare, a neurodegeneration researcher at the University of the Sciences in Philadelphia who mentored Green and helped her apply for the award. The award is a testament to the promise of Green’s laser treatment, which “obviously has a lot of applications,” Lillard says. “I imagine it having an impact initially in head and neck cancers, colorectal cancers, and anal cancers that often can be difficult to treat.” g

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BIO BUSINESS

Natural Killers Catch Up with CAR T NK cell therapies offer a potentially cheaper and safer route to cancer treatment than their T cell–based predecessors. BY BIANCA NOGRADY

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

and they don’t cause graft-versus-host disease, so you could potentially manufacture multiple doses of these cells . . . to treat multiple patients,” says Katy Rezvani, an immunotherapist at the University of Texas MD Anderson Cancer Center. This combination of efficacy, safety, and relative ease of supply is “the holy grail of cell therapy.” It is still early days for NK immunotherapies, which now face many of the same challenges that have limited CAR T cell therapies’ broader application, particularly in targeting harder-to-treat can-

cers such as solid tumors. NK cells also have their own disadvantages compared to their adaptive immune cousins: they don’t last as long in the body, for example, and they don’t proliferate as easily. But the excitement surrounding experimental NK-based cancer treatments is nevertheless translating into serious commercial interest. Biotechnology company Nkarta last year raised $114 million to take its NK cell therapy into clinical trials, and Celgene has paid a total of $83 million since 2017 in a partnership with Dragonfly Therapeutics for its NK cell programs.

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hen the first anticancer therapies based on engineered T cells hit the market a few years ago, they offered the possibility of what would have once been perceived as a medical miracle: a one-shot cure for certain blood cancers. Chimeric antigen receptor (CAR) T cell therapies, as they are known, involve harnessing the patient’s own immune cells, genetically modifying them with cancerspecific receptors for maximum potency against cancerous cells, then reinjecting them into the patient. But for all that cancerfighting ability, CAR T cells come at a cost: potentially severe side effects, massive price tags, and slow manufacture. Now a new cell therapy for cancer is edging into the spotlight. Natural killer (NK) cells have potential as a cellular anticancer therapy that could be significantly safer, cheaper, and faster, researchers say. While T cells are part of the adaptive immune system—they are primed to recognize a specific threat by the immune proteins (antigens) on a foreign cell surface—NK cells are part of the innate immune response, meaning that they respond to anything that appears to be non-self. This broad action makes them suitable for use not only as engineered cell therapies, but as unmodified cells administered on their own. Both the unmodified and the engineered forms of NK cell treatment are showing promise in early clinical trials in patients with cancer. And so far, they haven’t shown any of the significant toxicities—such as graft-versus-host disease, in which the transplanted cells attack the host as foreign, or cytokine release syndrome, in which immune cells pour out dangerous amounts of inflammatory signaling molecules—that plague CAR T cell therapies. “You have these cells that have an innate capability to recognize tumor cells,

Rezvani and her colleagues’ own research, meanwhile, has led to a partnership with the Japanese pharmaceutical company Takeda to take their NK cell work into multicenter clinical trials.

Natural killers NK cells were first described in the 1970s, when Swedish and British researchers independently discovered a new class of immune cells that didn’t match the features of T cells or B cells, but still laid waste to cancerous cells. Ever since that discovery, scientists have tried to harness the cells’ powers to fight cancer. But it took the more recent development of immune checkpoint inhibitors, which showed that it was possible to unleash and enhance the immune response against cancer, to throw open the door for cell-based immunotherapies. As it happened, CAR T cell therapy was the first such therapy out of the gate—albeit with a few obstacles. Most notably, T cell treatment currently has to be autologous— only a patient’s own T cells can be used. That’s because of the way in which T cells interact with the human leukocyte antigen (HLA) complex, a group of cell surface proteins that identifies a cell as being part of the self: any change to the HLA complex on the surface of a cell signals to a T cell that the cell is foreign. While this sensitivity makes T cells effective immune defenders, it has potentially deadly consequences for T cell–based therapy, as any mismatch between introduced and host cells can lead to cytokine release syndrome or graft-versus-host disease. NK cells, by contrast, are much less choosy about the HLA complex, says Soyoung Oh, a cancer immunologist at the biotechnology company Genentech. Stressed cells, such as those that are malignant or infected with a virus, may have reduced expression of HLA proteins, or, in some cases, none at all. They can also produce stress-related proteins on their surface. Either of these changes triggers NK cells to release two types of proteins that perforate a target cell’s membrane, damage its vital organelles, and induce cell suicide. This broad activity means that, unlike T cells, NK cells

don’t need any antigen-specific priming to provide a therapeutic anticancer effect. But typically, the mere presence of any HLA complex on a cell surface can be enough to signal NK cells to stand down. Consequently, using another person’s NK cells is less likely to trigger the dangerous immune reaction that an unfamiliar T cell might, says Oh, allowing researchers to envisage massproducing NK cell therapies as an offthe-shelf product that doesn’t need to be immunologically matched to a patient.

Unleashing the immune attack Biotech companies are exploring various sources of NK cells. Nkarta, for example, harvests cells straight from the peripheral blood of a donor using a technique called leukapheresis, in which immune cells are separated out from red blood cells. Other companies are looking to umbilical cord blood, which has a more concentrated supply of NK cells and their progenitors than peripheral blood.

So far, NK cell therapies haven’t shown any of the significant toxicities that plague CAR T cell therapies.

Still others are investigating the use of stem cells, which can in principle be differentiated into hematopoietic stem cells (HSCs) that then generate NK cells. Dan Kaufman, a hematologist at the University of California, San Diego, has been using both embryonic stem cells and induced pluripotent stem cells— stem cells derived from normal cells such as skin cells—to generate NK cells. (Kaufman is also collaborating with California-based Fate Therapeutics on its NK cell immunotherapy program.) Netherlands-based biotechnology company Glycostem, meanwhile, manufactures its NK cells from hematopoietic stem cells harvested from cord blood. In 2015, Glycostem released results from a Phase 1 clinical trial in which 10 patients

with acute myeloid leukemia who had relapsed after chemotherapy were infused with varying doses of the company’s unmodified cord blood–derived NK cells. The study recorded no instances of graft-versus-host disease or cytokine release syndrome, and patients showed significantly better survival compared with historical controls. Two patients even showed evidence of eradication of minimal residual disease—a low but persistent level of leukemic cells after treatment that usually heralds relapse. “For these patients, . . . they have no therapeutic option actually, they just have to wait for the relapse,” says Didier Haguenauer, chief medical officer at Glycostem. After the success of that single-dose study, the company is now launching a clinical trial testing a three-dose course of treatment. “We expect repeat administration to improve the efficacy of the treatment.”

Boosting the killer instinct Results from trials such as Glycostem’s suggest that simply boosting a patient’s population of unaltered NK cells could be enough to help their immune system overcome a cancer. But many researchers are also looking into mechanisms that might enhance or better engage NK cells’ anticancer actions. As is the case for T cells, NK cells are regulated and inhibited by immune checkpoints. They are “a natural brake, so that you potentially don’t lead to autoimmune diseases or immunopathologies from excessive activation,” says Nicholas Huntington, who directs the Cancer Immunotherapy Laboratory at Monash University in Melbourne and is the cofounder and CSO of oNKoInnate, an Australia-based biotech developing NK cell therapies. Overriding those checkpoints, which Huntington helped identify for NK cells, could be useful when it comes to targeting cancer. “If we genetically delete these checkpoints, then the natural killer cells remain active and hyperfunctional, and they can eradicate cancer much quicker, can eliminate metastases much quicker 04 . 202 0 | T H E S C IE N T IST 53

BIO BUSINESS

MAKING A KILLER THERAPY

Natural killer (NK) cells can be extracted directly from umbilical cord blood or from the peripheral blood of a donor  1 , or generated using stem cells from these or other sources  2 . Some biotechs are investigating the use of unmodified NK cells as cancer therapies  3 ; others are genetically engineering cells to carry chimeric antigen receptors (CARs) and other modifications  4 to make them more effective at targeting and killing cancer cells while sparing healthy tissue.

 1

EXTRACTED NK CELLS

 3

BLOOD

DONOR

PATIENT

than normal natural killer cells,” he says. Huntington and his colleagues are currently researching these molecular switches, and mechanisms that might turn them on or off. Other groups are interested in boosting NK cell activation. One receptor in particular, CD16, appears to be the equivalent of a gas pedal for NK cells. “It’s expressed on the NK cell and then once you engage it, it basically will [trigger] killing by the NK cell,” says Genentech’s Oh. Plans to target this receptor with drugs were the subject of a $96 million deal between Genentech and biopharmaceutical company Affimed in 2018. Oh notes that Genentech is addition5 4 T H E SC I EN TIST | the-scientist.com

 4 NK CELLS CULTURED AND EXPANDED

ally investigating whether the cancertargeting precision of CD16-activated cells might be enhanced through CARstyle engineering involving other protein receptors. Rezvani and colleagues are also combining NK cells with CAR technology. They have been engineering cord blood–derived NK cells with a CAR that targets the CD19 antigen—a well-studied molecule characteristic of certain B cell lymphomas. “We wanted to target CD19-positive malignancies, because that’s where the best results have been published with CAR T cells,” Rezvani says. To overcome NK cells’ relatively short half-life and poor proliferation

« CAR

in the body, the team also introduced a gene coding for a cell signaling protein, interleukin-15. This molecule is known to encourage NK cells to increase in number and hang around longer than normal. Lastly, they engineered a safety mechanism into the NK cell genome: a so-called suicide switch gene. Already used in some CAR T cell therapies, the switch can be activated with a drug in the event that the cell therapy shows signs of serious toxicity in patients. The researchers administered these triple-engineered NK cells to 11 patients with relapsed or treatment-resistant lymphomas or leukemias that were positive for the CD19 antigen. According to results published a few months ago, seven

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STEM CELL TECHNIQUES

 2

patients showed complete remission of disease and one showed partial remission. Most importantly, there were no signs of toxic effects such as graft-versushost disease or cytokine release syndrome. “The study has been so safe that the [US Food & Drug Administration] has allowed us to move into an outpatient setting,” Rezvani says. The team is now initiating an international multicenter study in partnership with Tokyobased Takeda.

NK unknowns Despite recent progress toward NK cell therapies, there are still many unanswered questions about how the immune cells function. For example, why does the normal complement of NK cells present in the body fail to automatically eliminate all cancer cells in the first place? One possibility is that cancer cells find ways to evade NK cells’ detec-

Unlike T cells, NK cells don’t need any antigen-specific priming to provide a therapeutic anticancer effect.

tion. “There have been some studies that [show that] tumor cells can lose [the] ligands or proteins on the surface that NK cells normally recognize,” says Kaufman. In addition to helping the cancer hide from NK cells, this loss of recognizable ligands could potentially play a role in the development of resistance to cell therapies, he adds. “Typically with a one-time dosing of NK cells, the disease does come back eventually; it might be months or years later. Whether that’s due to resistance to the natural killer cells or other mechanisms, I don’t think we really know.” It’s also still not clear whether NK cells will have better luck than T cells

getting into solid tumors—a harderto-reach environment than blood cancers. Huntington says he thinks it’s possible, particularly if researchers can find the right antigen to target. “I think that is certainly feasible,” agrees Kaufman, “but it will take a little bit more development.” Even if researchers do overcome these challenges, Oh says it’s unlikely that NK cell immunotherapy will entirely supplant CAR T therapy. Instead, the technologies might be effective in combination, particularly as there’s emerging evidence that “NK cells could produce other factors to recruit other immune cells that may actually then further potentiate the anti-tumor response,” she says. “I could envision where there may be benefits to using both together.” g Bianca Nogrady is a freelance science writer based in Sydney, Australia.

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CATHRYN HAIGH, PhD Chief, Prion Cell Biology Unit Laboratory of Persistent Viral Diseases National Institute of Allergy     and Infectious Diseases Division of Intramural Research Rocky Mountain Laboratories National Institutes of Health

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Humanity’s Watery Beginnings A new book revives a controversial hypothesis that proponents say may shake up scientists’ view of human evolution. BY PETER RHYS-EVANS

F

or the past 150 years, scientists and laypeople alike have accepted a “savanna” scenario of human evolution. The theory, primarily based on fossil evidence, suggests that because our ancestral ape family members were living in the trees of East African forests, and because we humans live on terra firma, our primate ancestors simply came down from the trees onto the grasslands and stood upright to see farther over the vegetation, increasing their efficiency as hunter-gatherers. In the late 19th century, anthropologists only had a few Neanderthal fossils to study, and science had very little knowledge of genetics and evolutionary changes. So this savanna theory of human evolution became ingrained in anthropological dogma and has remained the established explanation of early hominin evolution following the genetic split from our primate cousins 6 million to 7 million years ago. But in 1960, a different twist on human evolution emerged. That year, marine biologist Sir Alister Hardy wrote an article in New Scientist suggesting a possible aquatic phase in our evolution, noting Homo sapiens’s differences from other primates and similarities to other aquatic and semi-aquatic mammals. In 1967, zoologist Desmond Morris published The Naked Ape, which explored different theories about why modern humans lost their fur. Morris mentioned Hardy’s “aquatic ape” hypothesis as an “ingenious” theory that sufficiently explained “why we are so nimble in the water today and why our closest living relatives, the chimpanzees, are so helpless and quickly drown.” Morris concluded, however, that “despite its most appealing indirect evidence, the aquatic theory lacks solid support.” Even if eventually the aquatic ape hypothesis turns out to be true, he continued, it need not completely rewrite the story of human evolution,

5 8 T H E SC I EN TIST | the-scientist.com

but rather add to our species’ evolutionary arc a “salutary christening ceremony.” In 1992, I published a paper describing a curious ear condition colloquially known as “surfer’s ear,” which I and other ear, nose, and throat doctors frequently see in clinics. Exostoses are small bones that grow in the outer ear canal, but only in humans who swim and dive on a regular, almost daily basis. In modern humans, there is undisputed evidence of aural exostoses in people who swim and dive, with the size and extent being directly dependent on the frequency and length of exposure to water, as well as its temperature. I predicted that if these exostoses were found in early hominin skulls, it would provide vital fossil evidence for frequent swimming and diving by our ancestors. Researchers have now found these features in 1 million– to 2 million–year-old hominin skulls. In a recent study on nearly two dozen Neanderthal skulls, about 47 percent had exostoses. There are many other references to contemporary, historical, and archaeological coastal and river communities with a significantly increased incidence of aural exostoses. In my latest book, The Waterside Ape, I propose that the presence of exostoses in the skulls of ancient human ancestors is a prime support for an aquatic phase of our evolution, which may explain our unique human phenotype. Other Homo sapiens–specific features that may be tied to a semi-aquatic stage of human evolution include erect posture, loss of body hair, deposition of subcutaneous fat, a completely different heat-regulation system from other primates, and kidneys that function much like those of aquatic mammals. This combination of characteristics, which do not exist in any other terrestrial mammal, would have gradually arisen over several million years. The finding of the bipedal hominin named “Lucy,” dating to 3.5 million years ago, suggested that walking on two legs was

CRC Press, July 2019

the initial major evolutionary adaptation to a semi-aquatic habitat. By the time the Neanderthals appeared some 400,000 to 300,000 years ago, their semi-aquatic lifestyle—swimming, diving, and perhaps hunting for food on land and in the water—may have been firmly part of day-to-day life. In my opinion, the accumulated fossil, anatomical, and physiological evidence about early hominin evolution points to our human ancestors learning to survive as semi-aquatic creatures in a changing East African environment. After transitioning to bipedalism, ancient hominins had both forelimbs free from aiding in walking, which may have allowed for increasing manual dexterity and skills. Perhaps a marine diet with lipoproteins that are essential for brain development fueled the unique intellectual advances and ecological dominance of Homo sapiens. g Peter Rhys-Evans works in private practice as an otolaryngologist in London at several hospitals including the Harley Street Clinic. He is the founder and chairman of Oracle Cancer Trust, the largest head and neck cancer charity in the UK. Read an excerpt from The Waterside Ape at the-scientist.com. Follow Rhys-Evans on Twitter @TheWatersideApe.

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POOR PROGNOSIS:

Anna Reginalda Bolan, the head of the Field Museum’s roentgenology division between 1926 and 1932, examines an X-ray of the Egyptian Harwa mummy, which was donated to the museum in 1904 and is currently on display. According to the Field Museum’s 1932 annual report, X-rays of all Egyptian and Peruvian mummies were completed that year, and the Division of Roentgenology was closed.

tion experts quickly realized its potential for revealing ancient mortuary practices. Case in point: a 2011 CT scan at the Field Museum revealed wax figurines of the sons of the ancient Egyptian god Horus bound to individual organ packets stuffed inside the mummy. Because each of the sons designated certain organs in Egyptian culture, Brown was able to identify the intestines, stomach, liver, and lungs. He then used these findings to help identify unknown organ packets in other mummies that didn’t have wax figurines. “That was pretty awesome,” he says, because “apart from flagrant guessing, we had no previous methodological basis” for determining organ identity. Mimi Leveque, a conservator at the Peabody Essex Museum in Salem, Massachusetts, and self-described “mummy doctor” who has collaborated with Brown, recalls CT scanning an Egyptian mummy known as Padihershef at Massachusetts General

Hospital in 2013 and seeing it imaged “layer by layer by layer so you could see the face, you could see the bones . . . of the face, you could see inside the head . . . you could still see the brain tissue.” CT scanning has also helped Leveque design custom housings to support the deteriorated bones of North America’s oldest mummy, a roughly 4,000-year-old specimen from Egypt at the Michael C. Carlos Museum in Atlanta. Despite the long history of mummy scanning, Brown says that many questions remain about ancient mortuary practice that can’t be answered with individual scans. He points to archives being developed at the Penn Museum and the IMPACT mummy database, which compile scans of mummies and offer access to researchers who wish to study them, as steps toward improving our understanding of both the mummification process and its artifacts. g

© FIELD MUSEUM

hey don’t move, they don’t complain, and they’re impervious to X-ray damage. In other words, mummies are “a perfect subject for medical radiography,” according to conservator JP Brown of the Field Museum of Natural History in Chicago. Scientists figured this out early on: just months after Wilhelm Roentgen’s discovery of X-rays in the fall of 1895, a physicist, Walter Koenig, captured the first radiographic images of mummified remains at the Physical Society of Frankfurt-am-Main. Up until that point, studying mummies had mostly meant unwrapping them, a process that Brown notes is “necessarily destructive.” A few decades later, the Field Museum became a pioneer of mummy imaging. Edward Jerman of the Victor X-Ray Corpo-ration of Chicago volunteered his services and radiographed 32 ancient Egyptian and Peruvian mummies in the museum’s collec-tion with what curator Berthold Lauer called “such gratifying and convincing results” that museum president Stanley Field opened a Division of Roentgenology in 1926. In 1931, the museum published a radiographic study by paleopathologist Roy Moodie that captured many of its mummies in vivid skeletal detail, including child mummies from Egypt and Peru, and a skull with an outgrowth that Moodie diagnosed as a cranial tumor. The study also turned up “imitation mummies” made of assorted feathers, bones, and scraps of skin—believed to have been either created to help guide disintegrated bodies on their journey to the afterlife, or assembled by embalmers as a sly attempt to earn extra money. Although X-rays allow a noninvasive glimpse into unopened mummies, they create distortion by magnifying objects closer to the X-ray source, and they obscure the appearance of soft tissues and textiles. When CT scanning, which produces high-resolution, cross-sectional images of the body, emerged in the 1970s, mummy preserva-

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