Researchers Look to Single Cells for Cancer Insights


When asked about the biggest challenges to better understanding cancer, one word practically leaps from the mouths of many researchers: heterogeneity.

A tumor, the researchers stress, is not a uniform mass of identical cells with identical behaviors. Cells can act quite differently in one part of a tumor than in another. Genes critical to cell proliferation, for instance, may be active in one area but not another, or a subpopulation of cancer cells may be dormant, practically hiding from any drug that may try to enter their lair.

This heterogeneity has been blamed, for example, for the limited success of targeted therapies and of efforts to identify better diagnostic and prognostic markers of disease.

Researchers are now discovering what many have long suspected: much of what makes tumors heterogeneous overall is the substantial heterogeneity among individual cancer cells.

Until recently, the meticulous scrutiny of individual cells has been nearly impossible, particularly because of the relative scarcity in each cell of the key components that need to be measured, such as DNA and RNA. But thanks to technological advances that can help overcome some of those limitations, a growing number of investigators are beginning to delve deeper into the biology of the single cell.

The studies conducted to date “show us how much diversity there is among cancer cells in a given tumor,” said Dr. Garry Nolan, an immunologist at Stanford University whose lab is focused on mapping communication networks in individual cancer cells.

Even with improved technology, however, conducting studies at the single-cell level is difficult and can be time-consuming and expensive. But with growing interest—and $90 million over 5 years from the NIH Common Fund initiative (see the sidebar)—there is cautious optimism that over the next decade single-cell research may begin to pay dividends for patients with cancer and other diseases.

Moving beyond the Average

Most research on the molecular biology of tumors requires the use of mixtures of tens or hundreds of thousands of cells. Those samples “have immune cells, endothelial cells, and other infiltrating cells that make up the milieu of what a tumor actually is,” explained Dr. Dan Gallahan of NCI’s Division of Cancer Biology. “That really makes it difficult to get a grasp on what defines or, more importantly, how to treat a tumor.”

Results from studies that involve a bulk population of cells, Dr. Gallahan continued, essentially represent an average measurement.

Studying single cells is a way to “defy the average,” Dr. Marc Unger, chief scientific officer of Fluidigm Corporation, said earlier this year at a stem cell conference in Japan. (Fludigm, which develops tools for single-cell analysis, and the Broad Institute recently announced plans to establish a single-cell genomics research center.)

Single-cell analysis may be able to provide important clinical insights, said Dr. Nicholas Navin of the University of Texas MD Anderson Cancer Center, who has used next-generation sequencing to study variations in the number of genes (copy number variation) in single cancer cells.

Single-cell analysis might, for example, help identify “pre-existing [cell populations] that are resistant to chemotherapy or rare subpopulations that are capable of invasion and metastasis,” he said. “We may also be able to quantify the extent of heterogeneity in a patient’s tumor using single-cell data and use this index to predict how a patient will respond to treatment,” Dr. Navin continued.

Results from several recent studies have highlighted the challenges posed by tumor heterogeneity.

For example, researchers at BGI (formerly Beijing Genomics Institute) sequenced the protein-coding regions of DNA (the exome) of 20 cancer cells and 5 normal cells from a man with metastatic kidney cancer. The researchers found a tremendous amount of genetic diversity across the cancer cells, with very few sharing any common genetic mutations.

Much of the work in the analysis of single cells is still quite preliminary, and any potential clinical impact is still some years away, researchers agree.

“The problem with the single-cell data is that we don’t really know yet what they mean,” Dr. Sangeeta Bhatia of the Massachusetts Institute of Technology commented recently in Nature Biotechnology.

And studies involving bulk populations of cells will not be going away any time soon, noted Dr. Betsy Wilder, director of the NIH Office of Strategic Coordination, which oversees the NIH Common Fund.

“Single-cell analysis isn’t warranted for every question that’s out there,” Dr .Wilder stressed. “Studies using populations of cells will continue to be done, because it makes a lot of sense to do them.”

Technology, a Driving Force

Beyond just an interest in learning more about single cells—what Dr. Gallahan called “the operational units in biology”—technology has been the driving force behind the growth of this field.

Dr. Stephen Quake, also of Stanford, has pioneered the use of microfluidics, which typically uses small chips with microscopic channels and valves—often called lab-on-a-chip devices—that allow researchers to single out and study individual cells. Dr. Quake, who co-founded Fluidigm, and others are increasingly using these devices for gene-expression profiling and for sequencing RNA and DNA of individual cells.

Dr. Nolan’s research involves a hybrid approach that combines two technologies: a souped-up method of flow cytometry, which has been used for several decades to sort cells and to perform limited analyses of single cells, and mass spectrometry, which is often used to identify and quantify proteins in biological samples.

Dr. Nolan’s lab is using this “mass cytometry” approach—developed by Dr. Scott Tanner of the University of Toronto—to characterize the response of individual cells to different stimuli, such as cytokines, growth factors, and a variety of drugs. Much of the group’s work has focused on analyzing normal blood-forming cells.

They published an influential study last year in Science that revealed some of the subtle biochemical changes that occur during cell differentiation. The study also described how dasatinib (Sprycel), a drug used to treat chronic myelogenous leukemia, affects certain intracellular activities. The research, Dr. Nolan said, is a prelude to studying individual cells from patients with blood cancers. The approach, he believes, may prove particularly useful for identifying new drugs and for testing them in the lab.

A tumor is not a uniform mass of identical cells with identical behaviors. Cells can act quite differently in one part of a tumor than in another.

The Microscale Life Sciences Center (MLSC), an NIH Center of Excellence in Genomic Science that is housed at Arizona State University, develops and applies the latest technology to single-cell research.

The center—a collaboration of investigators from Arizona State, the University of Washington, Brandeis University, and the Fred Hutchinson Cancer Research Center—includes researchers from numerous disciplines, including microfluidics, computer science, physics, engineering, and biochemistry, explained principal investigator Dr. Deirdre Meldrum.

“All of these disciplines are needed to develop the new technologies we’re working on,” said Dr. Meldrum, an electrical engineer by training.

In its initial work, the MLSC has measured metabolic processes in single living cells, including cellular respiration—the process by which cells acquire energy—as it relates to an individual cell’s ability to resist or succumb to cell death. The workhorse of this effort is a platform called the Cellarium, developed by Dr. Meldrum’s team. Individual cells are isolated in controlled chambers, Dr. Meldrum explained, “where we perturb them and measure how they change over time.”

Investigators at the MLSC and elsewhere have also developed technologies to image single cells. MLSC scientists are using a device developed by VisionGate, called the Cell-CT, “that enables accurate measurement of cellular features in true 3D,” Dr. Meldrum said.

MLSC researchers have studied abnormal esophageal cells from people with Barrett esophagus, a condition that increases the risk of esophageal adenocarcinoma. In particular, they’ve looked at how these cells respond to very low oxygen levels, or hypoxia.

Acid reflux, which can cause Barrett esophagus, can damage the esophagus “and lead to transient hypoxia in the epithelial lining of the esophagus,” explained Dr. Thomas Paulson, an investigator at Fred Hutchinson. In effect, he continued, the Cellarium system provides a snapshot of how this hypoxic environment selects for variants of cells that are able to survive and grow in it, providing insights into the factors that influence the evolution of cells from normal to cancerous.

Although Dr. Paulson’s work at MLSC is focused on Barrett esophagus, he believes the approach represents an excellent model system for studying cancer risk in general.

“I think our understanding of what constitutes risk is probably going to change as we understand the types of changes that occur at the single-cell level” that can transform a healthy cell into a cancerous cell, he said.

Deeper Dives Ahead

There’s a general acknowledgement in the field that single-cell analysis still has important limitations. Technological improvements are needed that can allow for the same type of molecular and structural “deep dives” that can be achieved by studying batches of cells. And powerful computer programs will be needed to help interpret the data from single-cell studies.

In addition, the research will eventually have to move beyond the confines of the mostly artificial environments in which single cells are now being tested, Dr. Gallahan noted. “As the technology gets better, we should be able to do more of this work in an in vivo setting.”

Although much more work is needed, the potential for what can be learned from studying single cells is quite large, Dr. Nolan believes.

“The fact that we’ve been able to make good decisions and learn as much as we have, even at the level of resolution [of cell populations], means that there’s something of even greater value to mine when you get to the level of the single cell,” he said.

Transforming the Field of Single-Cell Research

This month, the National Institutes of Health will announce grant recipients for the NIH Common Fund’s single-cell analysis program.

The program, which includes three funding opportunities, “is largely a technology building program,” explained Dr. Wilder. The NIH Common Fund launched this program now because “there’s a sense that the technologies exist that can enable us to do the sort of analysis required to look at single cells in their native environment,” such as in a piece of excised tissue.

Although the focus is on technology, an important goal of the initiative is to support research that will “identify a few general principles of how single cells behave in a complex environment,” added Dr. Ravi Basavappa, the program director for the single-cell analysis program.

From the planning discussions, it was clear that the program should not limit the types of technology under consideration, Dr. Wilder commented. “Our analysis indicated that there are a lot of possibilities, so we left it up to the imaginations of the investigators to determine what technologies would be most transformative for the field as a whole.”

Source: NCI

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Tumor microenvironment helps skin cancer cells resist drug treatment .


Neighboring non-cancer cells may contribute to drug resistance

One of cancer’s most frightening characteristics is its ability to return after treatment. In the case of many forms of cancer, including the skin cancer known as melanoma, tailored drugs can eradicate cancer cells in the lab, but often produce only partial, temporary responses in patients. One of the burning questions in the field of cancer research has been and remains: how does cancer evade drug treatment?

New research by a team from Dana-Farber Cancer Institute, the Broad Institute and Massachusetts General Hospital suggests that some of the answers to this question do not lie in cancer cells themselves. To find the answers, scientists are looking beyond tumor cells, studying the interplay between cancer cells and their healthy counterparts. The research team has found that normal cells that reside within the tumor, part of the tumor microenvironment, may supply factors that help cancer cells grow and survive despite the presence of anti-cancer drugs. These findings appear online this week in a paper published in Nature.

“Historically, researchers would go to great lengths to pluck out tumor cells from a sample and discard the rest of the tissue,” said senior author Todd Golub, MD, the Charles A. Dana Investigator in Human Cancer Genetics at Dana-Farber Cancer Institute, pediatric oncologist at Dana-Farber/Children’s Hospital Cancer Center, and director of the Broad’s Cancer Program. Golub is also a professor at Harvard Medical School and an investigator at Howard Hughes Medical Institute. “But what we’re finding now is that those non-tumor cells that make up the microenvironment may be an important source of drug resistance.”

To investigate how the tumor microenvironment may contribute to drug resistance, the researchers designed experiments in which cancer cells were grown in the same wells (miniscule test tubes no larger than a pencil eraser) along with normal cells. These co-cultured cells were then treated with anti-cancer drugs. When grown alone, such cancer cells died in the presence of many of these targeted agents, but when grown together with normal cells, cancer cells developed resistance to more than half of the 23 agents tested.

These observations reflect what clinicians often see in patients with cancers such as melanoma. In the case of melanoma, targeted therapies have been developed against a specific, common mutation in a gene known as BRAF. While some patients’ tumors show an overwhelming response to BRAF inhibitors and seem to disappear, other patients’ tumors only respond by slightly decreasing in size. The failure to shrink tumors at the outset suggests that those tumors possess some level of innate resistance — the ability to evade drugs from the beginning of treatment.

“Even though recent advanced in targeted therapy have caused tremendous excitement in melanoma, the fact remains that drug resistance eventually develops in nearly all metastatic melanomas treated with RAF inhibitors, and in some cases is present at the outset of treatment,” said Levi A. Garraway, MD, PhD, an associate professor at Dana-Farber and Harvard Medical School and a senior associate member of the Broad Institute. “There are many different types of mechanisms that tumors may hijack to circumvent the effects of therapy…no single experimental approach can capture all of these potential mechanisms. Thus, the application of complementary approaches can offer considerable synergy in terms of discovering the full spectrum of clinically relevant resistance mechanisms.”

Scientists have uncovered resistance mechanisms that cancer cells develop over time – genetic changes in specific genes that may give cancer the ability to overcome the effects of a drug with time – but these acquired resistance mechanisms do not explain the innate resistance seen in many tumors.

“We can take cancer cells out of a melanoma patient, put them on a dish, and most times they will turn out to be extremely sensitive to the targeted agents, but that’s not what we see in patients,” said Ravid Straussman, MD, PhD, a postdoctoral fellow at the Broad Institute and first author of the Nature paper. “Why do we get just a partial response in most patients? We set out to dissect this question, and the next logical step was to think beyond cancer cells.”

After completing systematic, high-throughput screens of more than 40 cancer cell lines, the researchers chose to focus on melanoma, looking at whether factors normal cells secrete help cancer cells resist treatment. They measured more than 500 secreted factors and found that the factor most closely linked to BRAF inhibitor drug resistance was hepatocyte growth factor (HGF). HGF interacts with the MET receptor, abnormal activation of which has been tied to tumor growth in previous studies but never to drug resistance in melanoma.

In addition to studying cells in the lab, the research team sought to replicate their findings in samples from cancer patients. Keith Flaherty, MD, director of developmental therapeutics at Massachusetts General Hospital Cancer Center and an associate professor at Harvard Medical School, and his lab provided 34 patient samples for study. The team measured levels of HGF in these samples and saw a relationship between how much HGF was present and the amount of tumor shrinkage patients experienced. For example, tumors in patients with high levels of HGF shrank less than those in patients with low HGF levels.

“To try to explore in patient samples what factors in the microenvironment are not only present but functionally important in drug resistance would have been largely impossible. Coming up with candidates in the lab and then exploring relevance in humans in a targeted way is the only tractable approach,” said Flaherty. “By taking this high-throughput screening, hypothesis-generating approach, we could then follow up by looking at patient samples. In a case like melanoma, where you already have a targeted therapy available, it puts you on good footing to narrow in on specific factors that may be at play in drug resistance.”

Several HGF/MET inhibitors are in clinical development or are FDA-approved for other indications, making clinical trials combining these inhibitors with BRAF inhibitors feasible in the future. In addition, researchers could follow the same approach taken by the team to screen other drugs currently in development, identifying mechanisms of resistance and ways to counter them even before treatment begins.

“Drug resistance should no longer surprise us,” said Golub. “We’re thinking about how to do this – how to systematically dissect resistance – much earlier in the drug development process so that by the time a new drug enters the clinic, we have a good sense of what the likely mechanisms of resistance will be and have a strategy to combat them.”

Source: Dana Faber cancer Institute.