Jammed Cells Expose the Physics of Cancer


The subtle mechanics of densely packed cells may help explain why some cancerous tumors stay put while others break off and spread through the body.

In 1995, while he was a graduate student at McGill University in Montreal, the biomedical scientist Peter Friedl saw something so startling it kept him awake for several nights. Coordinated groups of cancer cells he was growing in his adviser’s lab started moving through a network of fibers meant to mimic the spaces between cells in the human body.

For more than a century, scientists had known that individual cancer cells can metastasize, leaving a tumor and migrating through the bloodstream and lymph system to distant parts of the body. But no one had seen what Friedl had caught in his microscope: a phalanx of cancer cells moving as one. It was so new and strange that at first he had trouble getting it published. “It was rejected because the relevance [to metastasis] wasn’t clear,” he said. Friedl and his co-authors eventually published a short paper in the journal Cancer Research.

Two decades later, biologists have become increasingly convinced that mobile clusters of tumor cells, though rarer than individual circulating cells, are seeding many — perhaps most — of the deadly metastatic invasions that cause 90 percent of all cancer deaths. But it wasn’t until 2013 that Friedl, now at Radboud University in the Netherlands, really felt that he understood what he and his colleagues were seeing. Things finally fell into place for him when he read a paper by Jeffrey Fredberg, a professor of bioengineering and physiology at Harvard University, which proposed that cells could be “jammed” — packed together so tightly that they become a unit, like coffee beans stuck in a hopper.

Fredberg’s research focused on lung cells, but Friedl thought his own migrating cancer cells might also be jammed. “I realized we had exactly the same thing, in 3-D and in motion,” he said. “That got me very excited, because it was an available concept that we could directly put onto our finding.” He soon published one of the first papers applying the concept of jamming to experimental measurements of cancer cells.

Physicists have long provided doctors with tumor-fighting tools such as radiation and proton beams. But only recently has anyone seriously considered the notion that purely physical concepts might help us understand the basic biology of one of the world’s deadliest phenomena. In the past few years, physicists studying metastasis have generated surprisingly precise predictions of cell behavior. Though it’s early days, proponents are optimistic that phase transitions such as jamming will play an increasingly important role in the fight against cancer. “Certainly in the physics community there’s momentum,” Fredberg said. “If the physicists are on board with it, the biologists are going to have to. Cells obey the rules of physics — there’s no choice.”

The Jam Index

In the broadest sense, physical principles have been applied to cancer since long before physics existed as a discipline. The ancient Greek physician Hippocrates gave cancer its name when he referred to it as a “crab,” comparing the shape of a tumor and its surrounding veins to a carapace and legs.

But those solid tumors do not kill more than 8 million people annually. Once tumor cells strike out on their own and metastasize to new sites in the body, drugs and other therapies rarely do more than prolong a patient’s life for a few years.

Biologists often view cancer primarily as a genetic program gone wrong, with mutations and epigenetic changes producing cells that don’t behave the way they should: Genes associated with cell division and growth may be turned up, and genes for programmed cell death may be turned down. To a small but growing number of physicists, however, the shape-shifting and behavior changes in cancer cells evoke not an errant genetic program but a phase transition.

The phase transition — a change in a material’s internal organization between ordered and disordered states — is a bedrock concept in physics. Anyone who has watched ice melt or water boil has witnessed a phase transition. Physicists have also identified such transitions in magnets, crystals, flocking birds and even cells (and cellular components) placed in artificial environments.

But compared to a homogeneous material like water or a magnet — or even a collection of identical cells in a dish — cancer is a hot mess. Cancers vary widely depending on the individual and the organ they develop in. Even a single tumor comprises a mind-boggling jumble of cells with different shapes, sizes and protein compositions. Such complexities can make biologists wary of a general theoretical framework. But they don’t daunt physicists. “Biologists are more trained to look at complexity and differences,” said the physicist Krastan Blagoev, who directs a National Science Foundation program that funds work on theoretical physics in living systems. “Physicists try to look at what’s common and extract behaviors from the commonness.”

In a demonstration of this approach, the physicists Andrea Liu, now of the University of Pennsylvania, and Sidney Nagel of the University of Chicago published a brief commentary in Nature in 1998 about the process of jamming. They described familiar examples: traffic jams, piles of sand, and coffee beans stuck together in a grocery-store hopper. These are all individual items held together by an external force so that they resemble a solid. Liu and Nagel put forward the provocative suggestion that jamming could be a previously unrecognized phase transition, a notion that physicists, after more than a decade of debate, have now accepted.

Though not the first mention of jamming in the scientific literature, Liu and Nagel’s paper set off what Fredberg calls “a deluge” among physicists. (The paper has been cited more than 1,400 times.) Fredberg realized that cells in lung tissue, which he had spent much of his career studying, are closely packed in a similar way to coffee beans and sand. In 2009 he and colleagues published the first paper suggesting that jamming could hold cells in tissues in place, and that an unjamming transition could mobilize some of those cells, a possibility that could have implications for asthma and other diseases.

Lucy Reading-Ikkanda for Quanta Magazine

The paper appeared amid a growing recognition of the importance of mechanics, and not just genetics, in directing cell behavior, Fredberg said. “People had always thought that the mechanical implications were at the most downstream end of the causal cascade, and at the most upstream end are genetic and epigenetic factors,” he said. “Then people discovered that physical forces and mechanical events actually can be upstream of genetic events — that cells are very aware of their mechanical microenvironments.”

Lisa Manning, a physicist at Syracuse University, read Fredberg’s paper and decided to put his idea into action. She and colleagues used a two-dimensional model of cells that are connected along edges and at vertices, filling all space. The model yielded an order parameter — a measurable number that quantifies a material’s internal order — that they called the “shape index.” The shape index relates the perimeter of a two-dimensional slice of the cell and its total surface area. “We made what I would consider a ridiculously strict prediction: When that number is equal to 3.81 or below, the tissue is a solid, and when that number is above 3.81, that tissue is a fluid,” Manning said. “I asked Jeff Fredberg to go look at this, and he did, and it worked perfectly.”

Fredberg saw that lung cells with a shape index above 3.81 started to mobilize and squeeze past each other. Manning’s prediction “came out of pure theory, pure thought,” he said. “It’s really an astounding validation of a physical theory.” A program officer with the Physical Sciences in Oncology program at the National Cancer Institute learned about the results and encouraged Fredberg to do a similar analysis using cancer cells. The program has given him funding to look for signatures of jamming in breast-cancer cells.

Meanwhile, Josef Käs, a physicist at Leipzig University in Germany, wondered if jamming could help explain puzzling behavior in cancer cells. He knew from his own studies and those of others that breast and cervical tumors, while mostly stiff, also contain soft, mobile cells that stream into the surrounding environment. If an unjamming transition was fluidizing these cancer cells, Käs immediately envisioned a potential response: Perhaps an analysis of biopsies based on measurements of tumor cells’ state of jamming, rather than a nearly century-old visual inspection procedure, could determine whether a tumor is about to metastasize.

Käs is now using a laser-based tool to look for signatures of jamming in tumors, and he hopes to have results later this year. In a separate study that is just beginning, he is working with Manning and her colleagues at Syracuse to look for phase transitions not just in cancer cells themselves, but also in the matrix of fibers that surrounds tumors.

More speculatively, Käs thinks the idea could also yield new avenues for therapies that are gentler than the shock-and-awe approach clinicians typically use to subdue a tumor. “If you can jam a whole tumor, then you have a benign tumor — that I believe,” he said. “If you find something which basically jams cancer cells efficiently and buys you another 20 years, that might be better than very disruptive chemotherapies.” Yet Käs is quick to clarify that he is not sure how a clinician would induce jamming.

Castaway Cooperators

Beyond the clinic, jamming could help resolve a growing conceptual debate in cancer biology, proponents say. Oncologists have suspected for several decades that metastasis usually requires a transition between sticky epithelial cells, which make up the bulk of solid tumors, and thinner, more mobile mesenchymal cells that are often found circulating solo in cancer patients’ bloodstreams. As more and more studies deliver results showing activity similar to that of Friedl’s migrating cell clusters, however, researchers have begun to question whether go-it-alone mesenchymal cells, which Friedl calls “lonely riders,” could really be the main culprits behind the metastatic disease that kills millions.

Some believe jamming could help get oncology out of this conceptual jam. A phase transition between jammed and unjammed states could fluidize and mobilize tumor cells as a group, without requiring them to transform from one cell type to a drastically different one, Friedl said. This could allow metastasizing cells to cooperate with one another, potentially giving them an advantage in colonizing a new site.

The key to developing this idea is to allow for a range of intermediate cell states between two extremes. “In the past, theories for how cancer might behave mechanically have either been theories for solids or theories for fluids,” Manning said. “Now we need to take into account the fact that they’re right on the edge.”

Hints of intermediate states between epithelial and mesenchymal are also emerging from physics research not motivated by phase-transition concepts. Herbert Levine, a biophysicist at Rice University, and his late colleague Eshel Ben-Jacob of Tel Aviv University recently created a model of metastasis based on concepts borrowed from nonlinear dynamics. It predicts the existence of clusters of circulating cells that have traits of both epithelial and mesenchymal cells. Cancer biologists have never seen such transitional cell states, but some are now seeking them in lab studies. “We wouldn’t have thought about it” on our own, said Kenneth Pienta, a prostate cancer specialist at Johns Hopkins University. “We have been directly affected by theoretical physics.”

Biology’s Phase Transition

Models of cell jamming, while useful, remain imperfect. For example, Manning’s models have been confined to two dimensions until now, even though tumors are three-dimensional. Manning is currently working on a 3-D version of her model of cellular motility. So far it seems to predict a fluid-to-solid transition similar to that of the 2-D model, she said.

In addition, cells are not as simple as coffee beans. Cells in a tumor or tissue can change their own mechanical properties in often complex ways, using genetic programs and other feedback loops, and if jamming is to provide a solid conceptual foundation for aspects of cancer, it will need to account for this ability. “Cells are not passive,” said Valerie Weaver, the director of the Center for Bioengineering and Tissue Regeneration at the University of California, San Francisco. “Cells are responding.”

Weaver also said that the predictions made by jamming models resemble what biologists call extrusion, a process by which dead epithelial cells are squeezed out of crowded tissue — the disfunction of which has recently been implicated in certain types of cancer. Manning believes that cell jamming likely provides an overarching mechanical explanation for many of the cell behaviors involved in cancer, including extrusion.

Space-filling tissue models like the one Manning uses, which produce the jamming behavior, also have trouble accounting for all the details of how cells interact with their neighbors and with their environment, Levine said. He has taken a different approach, modeling some of the differences in the ways cells can react when they’re being crowded by other cells. “Jamming will take you some distance,” he said, adding, “I think we will get stuck if we just limit ourselves to thinking of these physics transitions.”

Manning acknowledges that jamming alone cannot describe everything going on in cancer, but at least in certain types of cancer, it may play an important role, she said. “The message we’re not trying to put out there is that mechanics is the only game in town,” she said. “In some instances we might do a better job than traditional biochemical markers [in determining whether a particular cancer is dangerous]; in some cases we might not. But for something like cancer we want to have all hands on deck.”

With this in mind, physicists have suggested other novel approaches to understanding cancer. A number of physicists, including Ricard Solé of Pompeu Fabra University in Barcelona, Jack Tuszynski of the University of Alberta, and Salvatore Torquato of Princeton University, have publishedtheory papers suggesting ways that phase transitions could help explain aspects of cancer, and how experimentalists could test such predictions.

Others, however, feel that phase transitions may not be the right tool. Robert Austin, a biological physicist at Princeton University, cautions that phase transitions can be surprisingly complex. Even for a seemingly elementary case such as freezing water, physicists have yet to compute exactly when a transition will occur, he notes — and cancer is far more complicated than water.

And from a practical point of view, all the theory papers in the world won’t make a difference if physicists cannot get biologists and clinicians interested in their ideas. Jamming is a hot topic in physics, but most biologists have not yet heard of it, Fredberg said. The two communities can talk to each other at physics-and-cancer workshops during meetings hosted by the American Physical Society, the American Association for Cancer Research or the National Cancer Institute. But language and culture gaps remain. “I can come up with some phase diagrams, but in the end you have to translate it into a language which is relevant to oncologists,” Käs said.

Those gaps will narrow if jamming and phase transition theory continue to successfully explain what researchers see in cells and tissues, Fredberg said. “If there’s really increasing evidence that the way cells move collectively revolves around jamming, it’s just a matter of time until that works its way into the biological literature.”

And that, Friedl said, will give biologists a powerful new conceptual tool. “The challenge, but also the fascination, comes from identifying how living biology hijacks the physical principle and brings it to life and reinvents it using molecular strategies of cells.”

The Dirty Little Secret of Cancer Research


For 50 years, scientists have ignored widespread cell contamination, compromising medical research. Why are they so slow to fix it?

trial-and-error
In the field of thyroid cancer, 58-year-old Kenneth Ain is a star. As director of the thyroid oncology program at the University of Kentucky at Lexington, Ain has one of the largest single-physician thyroid cancer practices in the country and more than 70 peer-reviewed publications to his name. Until recently, Ain was renowned for a highly prized repository of 18 immortal cancer cell lines, which he developed by harvesting tissue from his patients’ tumors after removal, carefully culturing them to everlasting life in vials. Laboratories around the world relied on the “Kentucky Ain Thyroid,” or KAT lines, both to gain insight into cellular changes in thyroid carcinoma and to screen drugs that might treat the disease, which strikes more than 60,000 Americans each year.
kenneth-ain
Kenneth Ain, a thyroid cancer physician and researcher at the University of Kentucky, found that many of his cell lines were contaminated.

In June 2007, all that changed. Ain attended the annual Endocrine Society meeting in Toronto, where Bryan Haugen, head of the endocrinology division at the University of Colorado School of Medicine, told Ain that several of his most popular cell lines were not actually thyroid cancer. One of Haugen’s researchers discovered that many thyroid cell lines their laboratory stocked and studied were either misidentified or contaminated by other cancer cells. Those included some of Ain’s. They were now hard at work unraveling the mystery.

There was a disaster brewing — it just wasn’t yet official.

Ain was shocked, and justifiably so. Research based on such false cell lines would undermine the understanding of different cancers and possible treatments, and clutter the scientific literature with bogus conclusions.

“At first I thought perhaps their samples were contaminated, not mine,” Ain recalls. “So I undertook systematic thawing and genotyping of all my lines.”

He found that 17 of the 18 most frequently shared KAT lines were imposters.

It was only a matter of time before Ain figured out what went wrong. “Early in my career,” says Ain, “the head of radiation oncology at UCLA, Guy Juillard, created a number of cancer cell lines. He generously shared a few of the thyroid lines with me. And it turns out that from the beginning those original lines were likely melanoma and colon cancer.” Both grow rapidly and can easily overtake slower-growing thyroid malignancies. Human error — a researcher working on two lines at the same time, a pipette used more than once, two scientists sharing the same incubator or lab space as they worked — had likely led to the original contamination and all the lines subsequently contaminated in Ain’s and other laboratories around the country.
But rampant contamination is not the shocker in this story. Ain retired all the lines; he never sent any of them out again. He also sent letters to 69 investigators in 14 countries who had received his lines. He heard back from just two.

Across different fields of cancer research, up to a third of all cell lines have been identified as imposters. Yet this fact is widely ignored, and the lines continue to be used under their false identities. As recently as 2013, one of Ain’s contaminated lines was used in a paper on thyroid cancer published in the journal Oncogene.
“There are about 10,000 citations every year on false lines — new publications that refer to or rely on papers based on imposter (human cancer) cell lines,” says geneticist Christopher Korch, former director of the University of Colorado’s DNA Sequencing Analysis & Core Facility. “It’s like a huge pyramid of toothpicks precariously and deceptively held together.”
Toxicologist Thomas Hartung of the Johns Hopkins Bloomberg School of Public Health in Baltimore agrees: “Clinical drug trials in general are very well designed. They fail too often because we’re simply betting on the wrong horses in the first place. It’s a disaster.”

Like Edgar Allan Poe’s famous purloined letter, the problem of rampant laboratory contamination is out in the open for all to see, widely acknowledged by the National Institutes of Health (NIH), the National Cancer Institute, major journals and innumerable bench scientists. Yet efforts of concerned scientists have failed to stanch the tide.
“I now give regular lectures about cell line contamination,” says Ain, “and every last person in the audience is shocked and horrified. But most scientists are not willing to test and verify their lines. The NIH doesn’t require it. Very few journals require it. And I can tell you that many scientists are reluctant to disembowel their curriculum vitae, even after they find out a cell line is false. What is an ethical researcher to do?”

Too Big To Fail
Cell lines are the workhorses of biology, routinely stocked and studied in every laboratory to understand cellular pathways, receptors, targets, hormones, and all aspects of normal and malignant physiology. “There is no cancer drug in current use that was not first tested in a cultured cell model,” says molecular geneticist Michael Gottesman of NIH. “Cell lines are an immortal, tissue-specific, physiologic test tube,” he says. “We need to know precisely which cell a culture represents.”

hela-cells
Containers of cultural HeLa cells are stored on shelves in an incubation cabinet. The cells are grown in the incubator in different concentrations in a culture medium. HeLa cells grow rapidly and have proven invaluable to all kinds of research. But their fast growth can be problematic.
When cell cultures were first crafted in 1907, they seemed to defy nature. Researchers coaxed cells to life in a broth of nutrients in hanging contraptions made of glass slides. By 1943, scientists had established the first cell line in mice.
The most studied and cherished cell line in all of biology is HeLa, cultured in 1951 from the strange, soft, purple cervical cancer of a young woman, Henrietta Lacks. The HeLa line proved extraordinarily robust. Viruses can multiply a million times in a few days in rapidly growing HeLa cells. HeLa allowed researchers to study polio, measles, papilloma virus (HPV), HIV and tuberculosis; it was used to create the first human-mouse cell hybrid, and even sent into space. It has played a role in more than 70,000 studies.

HeLa is also, unfortunately, the most common cell line contaminant, responsible for more than 20 percent of contaminated cell lines. This is not news: The first widespread HeLa contamination was identified in 1967, when geneticist Stanley Gartler of the University of Washington typed 18 different human cell lines. He found that every one was actually HeLa.
The HeLa discovery was just the beginning. Work on contaminated esophageal cancer lines has led to more than 100 scientific publications, 11 patents, three NIH research grants and ongoing clinical trials involving cancer patients. The cell lines were actually lung, colon and stomach cancers. A much-studied breast cancer line turned out to be melanoma and, according to Belgian biochemist Marc Lacroix, was assumed by scientists to represent a late-stage breast cancer with an unrivaled ability to metastasize. In truth, says Lacroix, unlike the melanoma it really was, “this particular metatastic behavior is rarely seen in breast cancer progression.”

Risk of Exposure
Exposing contaminated cell lines cost Walter Nelson-Rees his career. He was an expert in culturing human and animal cells at the University of California, Berkeley, and ran a cell line bank in Oakland. From 1975 to 1981, he published a series of articles in Science outing contaminated lines and naming the laboratories where they had originated. His angry colleagues called his publications a “hit list.” In an editorial, Nature’s editor-in-chief, John Maddox, railed against “self-appointed vigilantes.”

hela-cells-microscope
HeLa cells, with proteins labeled in blue and DNA in red, are responsible for over 20 percent of the cell line contaminations.
Nelson-Rees’ work made it clear that HeLa contamination was far from the only problem. Eventually, the NIH terminated his contract, and he became so isolated from his peers that he left science and became an art dealer.
In 2007, a 77-year-old retired cell biologist named Roland Nardone decided to spend the final chapter of his life ending the rampant contamination. Supported by 18 other cell biology experts, he drafted a widely distributed “white paper” calling for an end to contamination. Chastened, top officials with the federal Office of Research.

Integrity sent out an email to 45,000 scientists, exhorting them to test their lines to be sure they were authentic.

By that time, the cost of validating cell lines had dramatically fallen, and the power of the testing technology had dramatically increased. A nearly foolproof form of DNA fingerprinting called short tandem repeat (STR) — sequences of DNA that are highly variable from one individual to another and therefore useful for DNA profiling — was increasingly popular. Forensic pathologists often use the technique to fingerprint DNA from blood at a crime scene. It works exactly the same way in bench science. If two cell lines share the same STR fingerprinting result, they are indeed the same. According to chemist John Butler, a former group leader with the National Institute of Standards and Technology, the chance of two cell lines coming up with the same STR fingerprinting profile is 1 in 100 million. “Scientists need to follow the fingerprinting where it leads,” says Butler. “If it’s not what you expect, it is not a problem with the fingerprinting. It’s a problem with the sample.”
But the NIH did not require fingerprinting for its grants, and so nothing changed. In 2009, immunologist Linda Miller, then executive editor at Nature, penned an unsigned editorial blasting funding agencies for allowing cell line contamination to continue. Miller suggested a global database of STR-fingerprinted cell lines. But her salvo went nowhere.

Today, cell lines known for nearly 50 years to be imposters are still in wide use under their assumed names — wrong identities regularly invoked in peer-reviewed publications. How can this be?

A Sisyphean Task
It’s just too damn hard.

How do you dismantle the false lines of an entire field? And just how long does it take to clean up one ordinary laboratory? Years. Ask Rebecca Schweppe, who in 2006 was recruited by endocrinologist Haugen to join the team of thyroid cancer researchers at the University of Colorado School of Medicine. Her expertise was in melanoma, which shares some features with thyroid cancer, including much-studied mutations in a gene known as BRAF.

dna-fingerprinting
Reprinted by permission from Macmillan Publishers Ltd John masters/Nature Reviews Molecular Cell Biology 1, 233-236 (December 2000)

Schweppe had no idea that her work was going to set off an avalanche powerful enough to reshape the field of thyroid cancer. Soon after she arrived, she ran an experiment on six different thyroid cancer lines, but her results came back impossibly clean: Three lines gave one identical result, and the other three gave another identical result. “I thought maybe I had only two thyroid lines, not six — that a number of them were misnamed and were actually redundant.”

Schweppe then turned to geneticist Korch, of the university’s DNA sequencing center. Korch was obsessed with cell line contamination. A 70-year-old Swede with silvery hair, neatly trimmed beard and gentle features, Korch has helped unmask more than 78 imposters since 2000.

Korch fingerprinted Schweppe’s lines and found that indeed there were only two. Schweppe and Haugen, worried that the contamination had occurred in their lab, asked colleagues to send them a fresh batch of the same six lines. “They turned out to be identical to our two lines,” and therefore false, says Schweppe.

But nobody yet knew what the cells actually were, since fingerprinting does not tell a scientist the origin of a cell. Without an original tissue sample to compare the cells to, the provenance of Schweppe and Haugen’s mysterious cells could remain forever uncertain. One can only pore through online databases, comparing their STR bar codes to the thousands that have already been fingerprinted, hoping to find a match.
Schweppe started searching the American Tissue Culture Collection (ATCC) database of nearly 1,200 fingerprinted human cell lines, both normal and malignant, from tissues, organs and tumors. It was as nerve-wracking as trying to find someone who had vanished into a witness protection program.

exponential-errors
Alison Mackey/Discover after data from Christopher Korch.
“One Friday at midnight,” says Schweppe, “long after my husband and kids had gone to sleep, I was at the computer once again, searching the database, and found a match for one of our lines.” The ATCC called it breast cancer, but STR fingerprinting revealed the cells were actually melanoma cells. And they were an exact match for three of Schweppe’s false thyroid cancer cell lines.
So does it matter? Yes. Those false lines were used in the early trials of two drugs that were later tested in human patients with thyroid cancer. One, called bexarotene, had no significant benefit in 17 patients after a year, according to University of Colorado School of Medicine oncologist Joshua Klopper, the study’s lead author. “I’m not sure we would have moved ahead with the trial had we found out the truth earlier,” he says.

The other drug studied, vemurafenib, also failed; thyroid cancer patients are generally resistant to the drug. “If we had still been working with melanoma cell lines, thinking they were thyroid cancer,” Schweppe says, “we wouldn’t know why thyroid cancer patients were not responding in the clinic.”

Half Were False
Schweppe and her colleagues fingerprinted the remaining thyroid cancer lines. In fall 2008, they reported that 17 of 40 widely used lines were imposters. During the years they were compiling their results, Schweppe would warn other scientists when she knew they were researching on false lines. But until her team’s results appeared in a peer-reviewed journal, it was difficult to get the word out. She even served as a reviewer for papers using the false lines, but couldn’t say a word. “I hated it,” she recalls.

She and the lab then won part of a $1 million grant to characterize the remaining good lines and to establish new lines from thyroid tumors. “It’s very hard to create a new line,” says Schweppe. “We worked on it for two years and produced only two new cell lines.”

christoper-korch
Geneticist Christopher Korch, of the University of Colorado’s DNA sequencing center, has helped uncover 78 imposter cell lines since 2000.
When head and neck cancer surgeon Jeffrey Myers at the University of Texas MD Anderson Cancer Center read her paper, he realized he too had published on one of the false lines. It took Myers three years to clean up his own laboratory. “I sent every cell line we had to be fingerprinted. My head technician, Mei Zhao, spent three years of her career doing nothing but unfreezing cell line stocks and sending samples of DNA for fingerprinting.” Myers tossed out 4,000 vials, and contamination forced 50 lines into retirement. “We rebuilt and fingerprinted the entire stock with more than 70 new head and neck cell lines from their original source laboratories,” says Zhao.
Even then, with a completely clean lab highly focused on meticulous technique, a new contamination struck in 2013 — something Myers identified when several different cell lines showed up with exactly the same mutation. The cause of the contamination remains a mystery. Zhao thinks the most likely possibilities are a labeling error or cross-contamination. “I was shocked to learn that contamination can even occur under close watch. Scientists need to develop standard and reliable methods to solve these issues fundamentally,” says Zhao.

Myers sees contamination as an inevitable part of science, just as errors and complications are an inevitable part of clinical medicine. “Everybody thinks, ‘Our lab is not sloppy, so nothing will happen.’ But mistakes happen in every single lab. Our field needs to establish checklists, and post them in laboratories, and make sure everybody follows them.”
Today, MD Anderson urges all its scientists to fingerprint new cell lines as soon as they arrive in the lab and before publishing any data, and to complete an annual revalidation of all lines in their laboratories. Some universities and centers have their own sequencing facilities; others send cell lines to a contractor for $30 to $80 per sample. The results come back in a few days.
Sleight of Hand
In 2012, Korch was the lead author on a paper exposing 19 false endometrial and ovarian cell lines. Two of those endometrial false lines had been highly prized because they supposedly were drawn from normal tissue, which can be invaluable for studying the earliest steps to malignant transformation — potentially helping prevent cancers.

Once fingerprinting has revealed a line’s true provenance, such as HeLa, scientists sometimes claim their line does not behave or look like HeLa (or melanoma, or colon cancer, or whatever cell has invaded their line). “People treat their lines like pets,” says Gartler, who outed HeLa contamination in the ’60s. “They feel that merely by looking through a microscope they will know their cells by sight.”
But STR fingerprinting doesn’t lie. “This tool is extremely powerful and precise. It’s definitive,” he says. “If fingerprinting shows it’s HeLa, it is HeLa. Period.”

Experimental pathologist John Masters of University College London tells of a normal endothelium line that turned out to be bladder cancer, but researchers still refer to it as “endothelial-like” so they can use it in studies. (Endothelium cells line the interior of blood vessels and lymphatic vessels.) “They clearly know that these are not endothelial cells, but to get around it and not admit they are bladder cancer cells, they call them ‘endothelial-like.’ I don’t know how they reconcile the sleight of hand,” Masters says. “It is beyond my comprehension.”
Korch believes some of the waffling scientists truly are innocent: They actually don’t understand fingerprinting. They are also, he thinks, too enamored of testing cells’ behavior and function. As Korch points out, cells change appearance and function as they grow on plastic and are chemically manipulated while being studied. They also may mutate as successive generations are grown out over the years in different laboratories. Behavior is not proof of origin.

A Tarnished Reputation
In 2005, microbiologist Thomas Klonisch of the University of Manitoba created a highly prized “normal” uterine endometrial cell line, hTERT-EEC. It was a novel cell line to investigate the role of estrogen and progesterone in the endometrium, or uterine lining — including their role in endometriosis, miscarriage and cancer. “We hoped,” says Klonisch, “that researchers could use them to gain novel insights into important aspects of reproductive biology and the evolution of endometrial cancer.”

researcher-working
A researcher works with HeLa cells beneath a protective sterile hood, known as a laminar flow cabinet. Despite careful precautions such as these, cell contamination still regularly occurs in labs throughout the world.
Three years later, Korch’s colleague, Andy Bradford, a molecular biologist who studies gynecologic cancers, ordered three samples of the line. He sent them to Korch for fingerprinting as a matter of course. “I was very disappointed when they turned out to be breast cancer cells,” Bradford says. “Sometimes the truth sucks!” He asked Klonisch for another batch, hoping that perhaps his own lab had contaminated the first ones. But the new samples Klonisch sent were also breast cancer cells. After that, Korch and Bradford say, repeated emails to Klonisch went unanswered. During that time, research groups in at least three countries unwittingly studied and published papers on the line.
Klonisch struggles to explain why he did not immediately pay to fingerprint the line himself. One reason, he says, was the cost. At that time, the cost ranged from $67 to $475. “I offered to profile their samples as blind samples, with the identities sent to a neutral party ‘arbitrator,’ ” Korch says.

Klonisch felt he had a reasonable alternative. “We went through very extensive functional testing, and our data seemed to contradict that of Dr. Bradford. The uniqueness of our line meant that a lot was at stake. And our line did not seem to behave like breast cancer.”
How can scientists be so easily fooled by the behavior of a cell? “It’s the same concept as raising identical twins in different environments,” says Bradford. “They will look and behave differently, but their DNA remains the same.” If a breast cancer line had silently contaminated Klonisch’s cell culture early on, it would have been subject to the usual technique for immortalizing a normal cell (which involves applying enzymes, antibiotics and antibiotic-resistant genes). The only breast cancer cells that would proliferate would survive every one of those chemical manipulations. They would emerge functionally different than an untouched breast cancer cell because their environment had so radically changed.

In 2012, Korch, Bradford and colleagues published a paper detailing their false line and its breast cancer provenance. By May 2013, Klonisch did what a good scientist must: He offered corrections to the relevant journals, which have since published them. “My reputation was tarnished,” says Klonisch soberly, “and all my research in this field has been shut down. And we never intended any of this.”

Winds of Change
A global correction for cell line science has begun. In 2012, Korch, Masters and 16 other scientists formed the International Cell Line Authentication Committee (ICLAC). They agreed on STR fingerprinting as the global standard for authenticating cell lines. The committee also set up a public database (found at iclac.org) of all known false lines, which numbers more than 400.

Recently, the top four cell line repositories, in America, Germany and Japan, made plans to merge their online databases of cell lines validated through STR fingerprinting, with each fingerprint converted to a searchable genetic “bar code.” The consortium’s online tool (known as OSTRA, for Online STR Analysis) can beaccessed online.
As of this writing, at least 22 journals now require cell line authentication from authors. Norbert Fusenig, the Germany-based associate editor of the International Journal of Cancer, notes that the journal has had a steady increase in submissions since 2012, when it began to require authentication. “Our impact factor, a common measure of a journal’s success, has increased,” he says.

In April 2013, Nature published more stringent requirements, in which every author had to report the source of a study’s cell lines and whether the lines had been verified recently.
Immunologist Linda Miller, whose 2009 Nature editorial called for a global database of STR-fingerprinted lines, says, “Encouraging the community to authenticate, that’s the first step, but not the final step. Ultimately, it must become a requirement. Science, and the flow of money to scientists, depends on the public trust.”

Damage Control

Scientific integrity matters more than ever, and not just for cancer research. There is a rising tide of worry over the spike in fraudulent scientific papers.
• In the last 10 years, retractions of scientific papers have rocketed more than tenfold, while actual publications have increased by only 44 percent. — Nature, 2011

• The Office of Research Integrity, which pursues cases of scientific misconduct, received more than 400 allegations in 2012 — double the average from 20 years before.

• In October 2013, Science correspondent John Bohannon published an article reporting a sting operation. He
concocted a fraudulent scientific paper studded with anomalies and ethical approval problems, and sent it to more than 300 open-access peer-reviewed journals; more than half accepted the fake manuscript.

• Of 53 papers deemed “landmark” studies over the last decade, only six held up and were reproducible. — Commentary in March 2012 in Nature by oncologist Lee Ellis

• “To be successful, today’s scientists must often be self-promoting entrepreneurs whose work is driven not only by curiosity but by personal ambition, political concerns and quests for funding.” — Ferric C. Fang and Arturo Casadevall, editors-in-chief, Infection and Immunity — J.N.

The Liquid Biopsy: A Noninvasive Tumor Tracker.


To date, the “liquid biopsy,” a blood test that detects evidence of cancer in the circulation, has generated a lot of excitement in the lab but little in the clinic.

The only liquid biopsy currently approved by the US Food and Drug Administration (FDA) for clinical use is a prognostic survival tool with no potential to guide treatment decisions (CellSearch, Janssen Diagnostics).

But research published in the February 19 issue of Science Translational Medicine shows how liquid biopsies can provide a noninvasive, ongoing picture of a patient’s cancer, offering valuable insight into how best to fight it.

Work from 2 different groups shows how liquid biopsies are being used in the lab to identify tumors at a very early stage, monitor them for metastasis, and even pick up signs of early treatment resistance.

In the future, instead of extensive imaging and invasive tissue biopsies, liquid biopsies could be used to guide cancer treatment decisions and perhaps even screen for tumors that are not yet visible on imaging.

“I think early detection is the Holy Grail of cancer research,” said Luis Diaz Jr., MD, from Johns Hopkins University School of Medicine in Baltimore. Liquid biopsies will likely offer a screening method for most cancers one day, he told Medscape Medical News.

However, this exciting potential is probably furthest from being ready for the clinic, he acknowledged; other potential applications include genotyping, detection of minimal residual disease, and detection of treatment resistance.

In their research, Dr. Diaz and colleagues show that a liquid biopsy measuring the serum level of circulating tumor (ct)DNA could one day be a very useful tool in cancer decision-making, giving clues about what type of cancer a patient has and whether it has spread.

“Mutant DNA fragments are found at relatively high concentrations in the circulation of most patients with metastatic cancer and at lower but detectable concentrations in a substantial fraction of patients with localized cancers,” they write.

The team found this to be particularly true in cases of breast, colon, pancreas, and gastroesophageal tumors, where “detectable levels of ctDNA were present in 49% to 78% of patients with localized tumors and 86% to 100% of patients with metastatic tumors.”

They evaluated 136 metastatic tumors in 14 different tumor types, and found that “most patients with stage III ovarian and liver cancers and metastatic cancers of the pancreas, bladder, colon, stomach, breast, liver, esophagus, and head and neck, as well as neuroblastoma and melanoma, harbored detectable levels of ctDNA. In contrast, less than 50% of patients with medulloblastomas or metastatic cancers of the kidney, prostate, or thyroid, and less than 10% of patients with gliomas, harbored detectable ctDNA.”

In addition to offering clues about stage and spread, liquid biopsies can be used to monitor the effects of cancer treatment, give an early warning about possible recurrence, and offer clues to the reasons for treatment resistance.

A second team of researchers used liquid biopsies in colorectal cancer patients to show that early resistance to treatment with epidermal growth-factor receptor (EGFR) inhibitors could be identified by the presence of certain mutations in the blood.

In their research, Sandra Misale, a PhD student from the Department of Oncology at the University of Torino in Italy, and colleagues showed that this resistance can be overcome by concomitant treatment with mitogen-activated protein kinase (MEK) inhibitors.

“We reasoned that tissue biopsies would only offer a snapshot of the overall tumor mass and might therefore be ill suited to capture the multiclonal feature of the resistant disease,” the researchers note, explaining that liquid biopsies are “more likely to capture the overall genetic complexity of tumors in patients with advanced disease.”

In fact, Dr. Diaz’s team found the same mutations in treatment-resistant colorectal cancer patients, suggesting a future clinical application for liquid biopsies. “These data therefore strongly suggest that patients being considered for treatment with EGFR blockading agents should be tested for these additional mutations,” they advise. Patients harboring such mutations “are unlikely to benefit from these agents and would be better served by other therapeutic approaches.”

Tissue Biopsy Can Be Challenging

There is good reason to want to learn about cancer through the blood, said Terence Friedlander, MD, from the Helen Diller Family Comprehensive Cancer Center at the University of California, San Francisco. “For most tumors, a tissue biopsy is quite challenging, in that it’s costly, painful, and potentially risky for the patient,” he explained.

The research by both teams illustrates that there is “a lot of reason to be excited” about liquid biopsies, he told Medscape Medical News. “Together, both of these papers show that you can detect resistance as it’s happening in real time.”

Although the current FDA-approved liquid biopsy measures intact circulating tumor cells (CTC) to give a prognosis of overall survival, the potential predictive value of ctDNA is much more exciting, he said.

“Predictive markers are better because they help guide treatment decisions. In a sense, the ctDNA liquid biopsy allows us to understand specifically what kind of molecular changes are happening in the tumor in real time, which is a very big step beyond where CTCs are today, clinically.”

Alternative Type of Brachytherapy Proves Effective in Mice.


An injectable genetically engineered peptide polymer may one day offer an alternative to conventional brachytherapy, a commonly used radiation therapy technique, according to the results of a new study in mice. The treatment could eliminate some of the difficulties associated with brachytherapy, the researchers believe, and could be used to treat more cancer types than brachytherapy. The study results were published November 15 in Cancer Research.

Using mouse models of two different cancer types, the researchers showed that their alternative brachytherapy approach—directly injecting tumors with a biodegradable elastin-like polypeptide (ELP) that is “labeled” with radioactive iodine—effectively shrank tumors and, in many cases, eliminated them completely.

In conventional brachytherapy, radioactive seeds are implanted in tumors and later removed. This type of internal radiation therapy is used frequently to treat localized prostate cancer and, to a lesser extent, to treat breast cancer. The seeds must be implanted and removed surgically, however. Another disadvantage is that implanted seeds can travel to other, healthy tissues, explained the study’s lead investigator, Dr. Wenge Liu of Duke University, and his colleagues.

By contrast, ELPs are liquid at room temperature and can be injected into tumors. Once inside tumors, they assemble into small seeds, or depots. In the study, the researchers tested ELP formulations that varied in their amino acid composition, size, and concentration to determine which one developed the most stable depots and was retained longest in the tumor before breaking down.

The researchers identified the ELP that had demonstrated the best tumor retention, and they tested three variations in mouse models of prostate and head and neck cancers. After only a single administration, the most effective variation shrank tumors in all of the mice regardless of tumor type. It also completely eliminated tumors in two-thirds of the mice with head-and-neck tumors and all of the mice with prostate tumors.

A potential advantage of the ELP is that it eventually breaks down into nontoxic forms that are naturally excreted by the body, Dr. Liu and his colleagues wrote. They reported no clinical signs of side effects in the treated mice, and additional examinations showed that the radioactive iodine was concentrated at the tumor site, with very little accumulation in healthy tissues.

In addition to treating several types of localized tumors, an ELP has other potential uses, the researchers wrote, including shrinking (debulking) tumors considered to be inoperable so that they can be removed surgically.

The researchers are continuing to refine the approach, Dr. Liu said in an e-mail message, including investigating whether the radioactive iodine dose can be lowered without sacrificing efficacy and working to improve delivery of the ELP “to solid tumors located deep in the body, such as in the esophagus, bronchus, stomach, and colon or abdominal cavity.”

Source:NCI.