Hope crushed can be a terrible thing. Oncologist Alessandro D. Santin, M.D., sees that despair all too often. Santin, professor of obstetrics, gynecology, and reproductive sciences, typically treats ovarian cancer patients when their tumors have grown dangerously large and spread to other parts of their bodies. Most of these women undergo surgery first, then chemotherapy. After they have endured these complicated and painful treatments, though, often the most he can offer is a small measure of good news.

Nearly four out of five patients have no detectable disease left in their body; however, cancer will return in nearly 90 percent of the apparently “cured” women—the second time with a vengeance—and chemotherapy will no longer work. “There is very little we can do for them at that time,” Santin says.

Sitting in his School of Medicine office almost swallowed by the stacks of papers and journals that surround him, Santin speaks of the deceptively malevolent power of ovarian cancer cells to re-erupt after treatment. He has been studying the source of that power and thinks emerging insights reveal new possibilities for stopping it from killing women. “I’m targeting the cells that are resistant to treatment and therefore responsible for the cancer’s recurrence and the death of my patients,” he says. He has come to believe a very specific type of killer lies deep within tumors: cells that are not ordinary tumor cells.

“We are trying to identify a subset of cells that looks different, acts different, and reacts differently among cancer cells. These are the cells you try to kill with anything,” but cannot, at least so far.

Santin’s research is among the most advanced efforts to translate the complicated biology of what some observers term “cancer stem cells” into novel treatments for one of the most aggressive and lethal forms of cancer. According to a controversial theory that is gaining wider acceptance among oncologists and cancer researchers, certain cells have a unique set of developmental differentiation and self-renewal powers—akin to stem cells that form tissues during embryonic development—needed to initiate tumors. According to this theory, tumors need these “cancer seeds” in order to spread through the body. Santin and a growing number of cancer biologists are also convinced that among the mass of tumor cells in an individual cancer, only a subset of tumor cells have the genetic endowment to resist treatment.

The idea that a separate type of tumor cell lies at the root of cancer completely reworks existing dogma about how cancers form, gain structure, and spread. The notion of a separate subset of cells within tumors, says Gil G. Mor, M.D., Ph.D., professor of obstetrics, gynecology, and reproductive sciences, who shares an office suite with Santin and also studies the role of ovarian cancer stem cells, marks “a big change in the concept we learned in biology as medical students. We were taught that tumors are a mass of identical cells.”

“This,” says Haifan Lin, Ph.D., professor of cell biology and of genetics, and director of the Yale Stem Cell Center, “is a paradigm-shifting idea.” He explains, “A small number of cells are much more important within a tumor than others. These are seeding cells—cancer cells that are different from other cancer cells.” But Lin added, “cancer stem cells may not be responsible for all types of cancers.”

Thomas J. Lynch Jr., M.D. ’86, the Richard Sackler and Jonathan Sackler Professor of Medicine (Medical Oncology); director of Yale Cancer Center; and a specialist in lung cancer, considers that notion controversial but says, “There is no doubt that a population of cells is responsible for propagating metastases. There is no doubt we have to target them to halt the process.” Lynch questions their designation as cancer stem cells, but he is convinced that “for therapy to be successful, you have to target that population.”

While oncologists almost universally acknowledge that only genetically aberrant hematopoietic stem cells can generate blood cancers, including most leukemias, there is no definitive way to distinguish cancer stem cells in solid-tumor cancers. Some say that’s because the cells are not there. But Santin and Mor say that they have characterized traits of these cells, including unique surface proteins, in certain cancers that distinguish them from other cells within solid tumors. That characterization may provide targets for the development of new anticancer agents. Several efforts are now under way at Yale and elsewhere to find therapies that attack those so-called stem cells.

If the researchers succeed, a revolution in the way oncologists view—and more importantly—treat cancer may be in the offing.

Cancer’s seeds

Recent decades have brought enormous advances in knowledge of cancer’s often baffling biology. But despite huge investments in treatment that have drawn on those insights, including the introduction more than 30 years ago of commonly used platinum-based chemotherapeutic agents, the odds and length of survival for most patients newly diagnosed with advanced solid-tumor cancers have barely increased. Eventually, in most cases of ovarian cancer—as well as lung, colorectal, breast, pancreatic, brain, and other solid organ-tissue cancers—the malignancy recurs after treatment, and that very often presages death.

Mainstream cancer biology points to tumor cells that escape even the most powerful targeted treatments as the culprits in cancer recurrence. Most oncologists believe that cure rates would rise if it were possible to improve the newer agents’ targeting accuracy or increase the potency of chemotherapy agents without putting the patient’s life at risk. But the cancer stem cell theory puts treatment resistance and cancer recurrence in a very different light. It isn’t that a few cells hide out only to proliferate anew; it’s that certain cells have a genetic endowment that resists chemotherapy and enables them to regenerate tumors and spread the cancer.

“There are definitely cancer cell types that can initiate tumors and some that do not,” says Don Nguyen, Ph.D., assistant professor of pathology, who studies how lung cancer metastasizes, “and the cells that form tumors may be unique or more frequent depending on the cancer type.”

That opinion contrasts with traditional models of cancer biology. According to the most widely held view, malignant cells have mutations that make them genetically unstable. They accumulate further mutations and evolve into tumors that grow rapidly, encourage blood vessel formation, discourage immune responses, metastasize, and resist treatment.

Mor finds this model of malignancy “oversimplified.” The cancer stem cell model posits that rather than being made up of identical cells, the tumor is composed of genetically and functionally different cell types. Furthermore, within the tumor there is a hierarchy. “Only these mother cells can give origin to cancer cells and all the cells in the tumor, including the blood vessels to nourish the tumor,” Mor says. “We have isolated the cancer stem cells and we can recreate the complexity of the human tumors in the mouse. That only happens with the cancer stem cells.” He hopes to test therapeutic agents designed to treat recurrent forms of the disease in ovarian cancer patients. These advanced tumors appear, he says, “to be a different monster altogether” from the primary untreated cancer; and require “a completely different approach” to target resistant cancer stem cells as well as the other non-stem cells that make up a tumor.

Lin agrees. He expects that new drugs specifically engineered to seek out and kill cancer’s seeds will stop at least some aggressive chemotherapy-resistant cancers. “The key is to find what is specific and different in cancer stem cells. That becomes their Achilles’ heel,” he says.

But the most basic questions about the biology of cancer stem cells await answers because the existence of uniquely potent tumor-initiating cells with stem cell-like characteristics remains unproven.

Where cancer comes from

The cancer stem cell theory arose out of a paradox observed in certain cancers in laboratory studies. Tumor cells—even large numbers of them—may fail to generate new tumors when transplanted into immunodeficient laboratory mice. At the same time, other tumor cells—sometimes just single cells—can generate new tumors physiologically identical to the parent cancer, and also spontaneously generate metastases.

In 1994 cancer biologists at the University of Toronto found that very specific human acute myeloid leukemia (AML) cells were needed to transmit the disease into immunocompromised mice. “In leukemia you can take out cancer stem cells and transplant them and create the cancer again,” says Lin. “Lots of leukemia cells cannot do that. Only leukemia stem cells can.” Evidence for the role of stem cells in other hematological malignancies followed. To cure AML patients, treatments require total destruction and replacement of the patients’ bone marrow to eliminate the cancer-causing stem cell component. Nine years after the Toronto discovery, however, the research focus began to shift from cancers of the blood to solid-tumor cancers. In 2003, University of Michigan researchers separated populations of what they said were rare stem cell-like cells from other cancer cells within specimens from human breast cancer tumors.

Since then scientists say they have characterized populations of cells resembling stem cells in many types of solid tumors through elevated levels of proteins typically found only on the surface of stem cell membranes. “There is a semi-consensus in certain cancers, probably not all of them, that stem cells exist,” says Joseph Schlessinger, Ph.D., chair and the William H. Prusoff Professor of Pharmacology.

The presence of such stem cells may explain a cancer’s ability to survive treatment. “A lot of drugs can kill regular cancer cells but not cancer stem cells,” says Lin. “They can evade every insult we throw at them.” Cancer stem cells, he believes, possess DNA repair mechanisms that allow them to resist chemotherapy. They also divide much more slowly than most cancer cells, making them less vulnerable to chemotherapeutic agents that attack fast-dividing cells. (Speed of division underlies the death of hair, nail, and certain blood cells during chemotherapy as well as the development of unbearably itchy skin and other chemotherapy-related side effects.) Moreover, studies indicate that when tumor cells die during treatment, they release signaling proteins that may stimulate surviving stem cells to reproduce and differentiate into a new tumor. In effect, treatment may actually increase the proportion of the highly aggressive and resistant cancer stem cells within tumors, perhaps explaining why fast-growing deadly malignancies often follow therapy.

What matters then is not just how many tumor cells a cancer therapy eliminates, but also its capacity to kill the cancer at its source.

Attacking cancer at the root

Lin studies genes that he calls the master regulators—they control other genes in the genome and decide whether a cell becomes a neuron or a heart, kidney, or skin cell. Genetic switches normally turn off stem cells once they have completed these tasks—except for small numbers involved in renewing and repairing blood, skin, bone, certain cells in the brain, and membranes in the lung and gastrointestinal tract. Lin believes that the stem cell genes may mutate through environmental insults like exposure to sun, tobacco smoke, and carcinogenic chemicals, or through genetic machinery gone awry; become overactive; and reawaken their self-renewing powers. This time, though, they lead to cancerous overgrowth. The parent cells that gave life transform into killers.

In the lab Lin’s team can manipulate genes in a normal stem cell to turn it into a cancer cell. In humans, he says, no one knows exactly how a stem cell becomes cancerous. Despite limited insights into how cancer stem cells develop, he contends that it is possible to develop drugs against them.

However, some cancer investigators question the notion of targeting cells that are so poorly understood. Scott Kern, M.D., associate professor of oncology and pathology at Johns Hopkins University School of Medicine, argues that alternate theories could explain the distinct class of treatment-resistant, tumorigenic cells under study. Says Kern, who studies cancer genetics, “We all know that the cancer stem cell theory explains at least some leukemias and teratocarcinomas [germ cell tumors, mainly cancers of the testes]. Nobody has debated that, to my knowledge. It is the extension of the idea to the common solid tumors ... that we find worthy of debate.” Existing theories, Kern argues, could explain the stem cell-like characteristics scientists claim to see in certain cell populations within tumors. He thinks that methods being used to identify cancer stem cells in a tumor represent “fuzzy math” that weights statistical results of studies to conform to a stem cell theory. And what some call a distinct class of treatment-resistant tumor-initiating cells could in his view just as likely result from cancer-causing genetic and environmental factors and physiological processes that encourage tumor growth. “Instead of chasing stem cell-ness,” he says, “you’re chasing an unknown.”

Definitive ways to distinguish cancer stem cells from normal ones have yet to emerge. That has not stopped cancer researchers from trying to identify biological markers by studying the differences between tumors that respond to therapy and those that resist. Patients with those markers that suggest a large population of cancer stem cells may benefit from more aggressive treatment. “The key,” says lung cancer biologist Nguyen, “is to identify these patients and get treatment to them as quickly as possible.”

For some cancers, skin cancer among them, the role of stem cells is a bit different. Douglas E. Brash, Ph.D., clinical professor and senior research scientist in therapeutic radiology, and professor of dermatology and of genetics, has found large clones of mutant progenitor cells, or skin stem cells, on the order of 30 per square centimeter in sun-exposed adult skin. These cells possess a mutation in the p53 tumor suppressor gene, which Brash showed came from sun exposure. When later exposed to beach-trip levels of ultraviolet radiation, the mutated stem cells produce more of themselves. In skin and other solid tissues, these progenitors are not professional stem cells: when they divide, they choose randomly between acting like a stem cell or acting like a differentiating cell. Brash theorizes that these mutated cells lead to a form of cancer known as squamous cell carcinoma. He is trying to create a mouse model in which the mutated skin stem cells can be observed while they are progressing to cancer. Shrinking a tumor 99 percent with a hammer may not be as effective as altering the stem cell’s choices with a screwdriver.

Santin does not claim certainty that a subpopulation of tumor-initiating cells exist in all or even most cancers, but he does believe that they exist in therapy-resistant ovarian cancer.

Much of Santin’s workday is spent either with patients or down the hall from his office in the laboratory where he and his colleagues are studying ways to kill ovarian tumor specimens enriched in chemotherapy-resistant cancer cells. During the past few years his laboratory team has published findings that Clostridium perfringens enterotoxin (CPE), a type of bacterial poison found in the intestine and responsible for foodborne illness and diarrhea in humans, may attack and kill only cancer cells possessing a specific protein called claudin-4 on their surface. Santin showed that the same protein can also be found on the surface of chemotherapy-resistant tumor cells.

When exposed to the bacterial toxin, the previously resistant cells die within minutes. “It’s hard to believe how effective the toxin can be against these biologically aggressive tumor cells,” he declares. Santin is now working with W. Mark Saltzman, Ph.D., the Goizueta Foundation Professor of Biomedical Engineering and professor of cellular and molecular physiology and of chemical engineering, and chair of the Department of Biomedical Engineering, to engineer a small section of that bacterial toxin into a nontoxic form suitable for use as a drug and as a diagnostic tool. The researchers are infusing a fragment of CPE linked to a fluorescent dye that lights up when it binds to a cancer cell. Santin hopes that within the next two or three years he can test the modified enterotoxin as a diagnostic method for chemotherapy-resistant ovarian cancer and then also be able to use it to treat patients with resistant ovarian cancer.

His departmental colleague Mor is looking for unique variants of the stem cell surface marker protein known as cd44 that may allow him to differentiate normal stem cells from the stem cells of ovarian cancer. Mor has already identified markers he believes may be present only in therapy-resistant ovarian cancer cells and has begun using the markers to look for compounds that might kill the cancer cells. “In recurrent disease,” he says, “you need to treat the cancer with a completely different approach. We have been successful in killing the fast-dividing cells. In the next 10 years, the challenge will be getting at the roots of cancer.” Even those who have raised doubts about the existence of cancer stem cells can agree with Mor about that.

Marc Wortman, Ph.D., is a freelance writer in New Haven.