Close-up view of tumors reveals new cancer biology

RNA sequencing reveals a previously unknown dimension of how malignant cells work – which could lead to novel treatments

When Brad Bernstein first looked at a cancer tumor cell by cell in 2014, what he found dismayed him: He realized that in any given tumor, there is not one type of cancer cell at work but several types. . "I was a little sad when I saw it," says Bernstein, a pathologist at the Massachusetts Institute of Technology and Harvard University's Broad Institute. "Some of the most difficult tumors are really heterogeneous mixtures [of cells]."

But for many physicians and surgeons working to find a cure for cancer, this observation about the nature of tumor cells was more a glass half-empty than half-empty. They already knew that some tumors are an incredibly difficult problem to treat, Bernstein says, "and now you've shed light on why it's an incredibly difficult problem."

To zoom in and view the tumor one cell at a time, Bernstein and his colleagues use a technique called single-cell RNA sequencing (scRNA seq). It shows, in theory, what each individual cell in the sample is doing and what genes are active within that cell. This technique is revealing a new dimension to cancer tumors – that they are mosaics of different cell types, more like loosely structured organelles than undisciplined masses of replicating cells as previously thought. And these tumor cells can fluidly change their developmental path, presenting unique challenges—and opportunities—for treatment.

Six years after Bernstein and his team got the initial up-close view, scRNA-seq is recognized as one of the most powerful new approaches in cell biology. The two types of RNA are intermediary molecules that help translate genes into proteins, so their presence in the cell indicates that the respective gene is active. It is now possible to isolate and identify the sequence of nearly every RNA in a given cell so rapidly that thousands of cells can be analyzed at once in a tissue sample.

For example, the technique can show researchers how the body's development unfolds in step-by-step detail as cells differentiate from an early stem cell state (in which they are capable of becoming any type of tissue) and into a receives her final "fate". special tissue. When used to analyze tumors, scRNA seq has revealed that cancer is not simply a breakdown of the developmental process, a glitch that produces rogue, uncontrolled cell proliferation.

Instead, says Bernstein, "we're learning that brain tumors are like deranged recapitulations of normal development." Single-cell RNA sequencing "opens your eyes to what's really in a tumor," he says, "and the answer is: a lot more than tumor cells." A tumor is not a mass of uniform cancer cells, but a closely bound mixture of several types of cells – including lots of non-malignant so-called healthy cells. Yet these nonmalignant cells, Bernstein says, "are not innocent bystanders." They seem to help support the tumor in some way.

While this condition makes the challenge of treating cancer more difficult, it may ultimately make treatment more effective. If researchers know that different types of malignant cells are present, they can tailor a specific cocktail of drugs to attack them. Such detailed information at the single-cell level is "clearly revolutionizing our understanding of cancer," says Mario Suva of Massachusetts General Hospital. Whereas previous work focused on certain functional properties of single cancer cells or the genomics of entire tumors, Suva says, researchers are now looking at everything at the cellular level.

For example, previous studies seem to suggest that one of four different types of cancer cells may be present in any given brain tumor, creating four different classes of tumors – each amenable to a different type of treatment. Is required. Bernstein's single-cell analysis of a particularly lethal type of brain tumor called glioblastoma, however, showed that all four types of cells were typically present in every tumor he and his colleagues looked at. But they occurred in different proportions, so large-scale studies tended to look at only the dominant type. "It was surprising to us," says Bernstein. Instead of four distinct classes of tumors, single-cell analysis revealed that there is a continuum of tumor types, each containing a mixture of those four malignant cell varieties (usually with a variety of healthy cells as well).

"We probably need a diagnostic test that can take a tumor and see which major cell groups are present," says Bernstein. The researchers would then hit them with so-called combination therapies, which apply several different pharmaceutical agents at once. Such mixtures are difficult to develop and test, he admits, but in the long run, they should be more effective at eliminating all dangerous cells.

The different types of cells in a tumor arise by differentiation from a single type of cancer "stem cell" – just as normal cells differentiate into normal tissues in a developing embryo. "The tumors mostly follow a similar hierarchy, starting in a stem-like position," says Mariella Philbin, a pediatric neuro-oncologist at the Dana-Farber Cancer Institute and Boston Children's Hospital who collaborated with Suva's team. But cancer Cells don't quite make it to the normal fate of other cells, Philbin explains. "Some get stuck and just grow."

Cancer cell differentiation also appears to be more fluid than that of healthy cells. "It's not like a typical hierarchy, where you differentiate, and then you commit [to a fortune]," says Bernstein. Cancer cells can break away for a bit and then come back, he said. Such reversibility and plasticity pose real challenges for therapies that target a single cell type: the interchangeability of these states gives cancer cells a survival strategy. "The tumors can become something else and survive our drug treatments," says Philbin. "It's too easy for them."

Another problem is that some tumors may be mostly non-malignant. In a study of squamous epidermal cancer of the head and neck three years ago, Bernstein and his colleagues learned that a group of patients' tumors had a significantly higher number of normal fibroblasts—connective-tissue cells—that are derived from the so-called epithelium. Was cells. "Some tumors may have just 5 to 10 percent tumor cells, and the rest are nonmalignant cells sitting in the tumor ecosystem," he says. But such tumors appear to be capable of using these cells for malicious purposes. In some cases, healthy epithelial cells transform into stage I mesenchymal cells, break free from the tumor and become mobile in the body. That is, these tumors restart an earlier growth process to bring about aggressive metastasis, spreading the cancer and making it much harder to treat. Bernstein notes, "None of this stuff comes out of bulk analysis."

A recent report by Moran Amit and colleagues at the University of Texas MD Anderson Cancer Center describes the troubling ability of cancer cells to hijack healthy cells. By looking at the RNA profiles of the cells in the squamous tumor cells in the head and neck, they found that cancer cells can reprogram normal neurons so that they promote tumor growth.

The new picture of cancer provided by scRNA seq may open up entirely new treatment possibilities. Suva has used the technique to study the immune system of tumors – specifically, the T cells they contain, which are the main agents of our normal immune response. Boosting the immune system has already been found to give it the ability to attack cancer cells. But doing so has only been effective for certain types of cancer—namely, leukemias. Suwa hopes that studying the immune status of tumors may point to new options for such cancer immunotherapy. Looking cell-by-cell, he says, "is a powerful tool for discovering new biology."

The insights this technology has already provided may suggest even more dramatic cures. The unusual plasticity of cancer cells – which can transition back and forth between different states more easily than normal cells – means that instead of just trying to kill them, they must be slowly reduced to a non-infectious state. It may be possible to "cure" by directing back. , This process is called differentiation therapy, and Philbin is hunting for small-molecule drugs that can do it. He and his colleagues are exploring approaches for neuroblastoma, an aggressive form of cancer that affects the nervous system of children. (The new technology will be used after treatment with conventional chemotherapy and radiotherapy.) And a team in Germany and Italy tried to treat a particularly challenging form of leukemia called APML (acute promyelocytic leukemia) without chemotherapy. . Instead the researchers employed only two differentiation agents: all-trans retinoic acid and arsenic trioxide. They found an almost 98 percent survival rate for patients after 50 months. "It's differentiation therapy at its best," says Philbin. She and her colleagues have also found that differentiation can put cancer cells into a chronic state that ultimately leads to cell death.

"Right now I'm treating brain tumors where we have nothing: They're resistant to chemo- and radiotherapy," says Philbin. "So we can't seem to kill those cells. But maybe we can isolate them." If so, she says, "I would be the happiest woman on the planet."

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