Identification and Characterization of the Ewing’s Sarcoma Stem Cell

David Loeb, M.D., Ph.D.

Assistant Professor of Oncology and Pediatrics

Director, Musculoskeletal Tumor Program

Co-Director, Sarcoma Program

The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins

One of the most exciting concepts being explored in cancer research today is the idea of the cancer stem cell. The cancer stem cell hypothesis proposes that not all of the cells in a tumor are capable of dividing indefinitely. Instead, the hypothesis is that there is a small population of cells that are capable of indefinite proliferation, of self-renewal, and of differentiation (developing into more specialized cells) – the hallmarks of a stem cell. These so-called cancer stem cells are proposed to be resistant to chemotherapy, and therefore to be the cells that are responsible for disease relapse and for patient death.

The cancer stem cell hypothesis is supported by the important clinical finding that for most tumors, there is little correlation between response to therapy and long term survival. Certainly, patients who do not respond to treatment do not survive, but a large proportion of patients with cancer die of their disease despite responding well to chemotherapy. A good example of this phenomenon is the treatment of patients with metastatic Ewing’s sarcoma.

Most of these patients achieve a complete remission, but overall survival is less than 20%, and has stayed low despite decades of alterations in chemotherapy regimens that have resulted in significant improvements in the survival of patients with localized disease. Observations such as these are interpreted by proponents of the cancer stem cell hypothesis to mean that our therapies are missing the most important target: the cancer stem cell. By killing the daughter cells that make up the bulk of the tumor, we see dramatic responses, but we leave behind the cells that are capable of indefinite growth, and these cells eventually grow back, leading to relapse, metastasis, and ultimately to the death of the patient.

This is actually an old idea, first proposed in the 1960’s, but at that time scientists lacked the technology to adequately investigate the hypothesis, and the idea lay dormant for decades. In the 1990’s, a group of scientists in Toronto, led by Dr. John Dick, first conclusively demonstrated that leukemia is a stem cell-driven disease (Ref. 1). They isolated single leukemia cells from patients and showed that only a small subset could behave like stem cells, but the majority could not. Subsequently, numerous investigators have identified populations of cells within particular tumors that they propose represent cancer stem cells. The existence of breast cancer stem cells is reasonably well accepted (Ref. 2) and there is good evidence supporting the existence of stem cells in a variety of other solid tumors, including brain tumors (Ref. 3). Despite these findings, there is far from universal acceptance of the truth of the cancer stem cell hypothesis, and some tumor types (especially some forms of lymphoma) do not seem to contain a stem cell population. Thus, it is not at all clear that there are sarcoma stem cells; however, it is our hypothesis that stem cells exist in Ewing’s sarcoma, and one of our primary goals is to identify and characterize these cells.

We will begin to look for Ewing’s sarcoma stem cells with cell lines growing in the lab. There are a number of tests that can be done to identify cells that might be stem cells (Ref. 4). Although none of these tests alone can identify a stem cell, we believe that applying these tests sequentially will allow us to isolate a population of cells that is highly enriched for stem cells. The ultimate test of a cancer stem cell is to determine if the injection of a very small number of cells (or ideally just a single cell) into a mouse will allow a tumor to grow. We will test the population of cells we isolate by injecting them into mice to see if they can grow tumors that look and behave like Ewing’s sarcoma. A defining element of stem cells is the ability to self-renew, meaning that they create more of themselves. An important element of our work, then, is to not only show that the cells we identify can form a tumor, but to show that these tumors also contain stem cells. It is possible that the cell population we identify can cause a tumor when injected into a mouse, but if that tumor does not contain stem cells, then our original population did not undergo self-renewal in the mouse and therefore must not contain bona fide cancer stem cells. To check for this, we will do two things: 1) we will remove tumors from mice that have been injected with our target cell population and we will attempt to identify cells in the tumor that have the same characteristics that allowed us to originally identify these cells and 2) we will inject these cells into a new set of mice, to show that they can also give rise to a tumor. The ability to generate a tumor that can be serially transplanted from mouse to mouse proves that the original cell was capable of self-renewal and limitless proliferation, and therefore was a stem cell.

After we identify Ewing’s sarcoma stem cells in cell lines, we will have to prove that these cells are not found only in cells that have been adapted to growth in the laboratory. Our next step will therefore be to determine if Ewing’s sarcoma xenografts (human tumors growing in laboratory mice) also contain the same population of cells. If they do, we will then try to demonstrate that these cells can also be found in Ewing’s sarcomas taken directly from patients. This step-wise approach, we believe, will allow us to find Ewing’s sarcoma stem cells efficiently, and save the nonrenewable resource (patients’ tumors) for the most important, confirmatory experiments.

Demonstrating that there are Ewing’s sarcoma stem cells and identifying them in primary tumors is just the first step. We will next begin to explore the specific properties of these important cells so that we can begin to develop therapies that target them specifically. We will begin this process by characterizing their sensitivity to chemotherapy. As discussed above, cancer stem cells are proposed to survive standard chemotherapy treatments because they are particularly resistant to chemotherapy (Ref. 5). We will therefore determine how sensitive Ewing’s sarcoma stem cells are to various chemotherapy drugs compared to the bulk tumor population. We can do this in two ways. First, we can treat cells with chemotherapy drugs in the lab, and try to isolate stem cells from the cells that survive the chemotherapy. If stem cells are resistant, then the fraction of cells that are stem cells should be greater after chemotherapy treatment than it was before. Second, we can isolate stem cells and determine the concentration of drug needed to kill 50% of them (called the LD50). We predict that the LD50 of Ewing’s sarcoma stem cells should be substantially higher than the LD50 of the total tumor cell population. If both of these findings are true, we can use these experiments to identify chemotherapy drugs that might be useful in killing stem cells. Such drugs should not have an LD50 that differs from the bulk population, because they would kill stem- and non-stem cells equally well; therefore, treating the bulk population with these drugs should not enrich stem cells.

In the future, not only can we use this approach to screen drugs for their ability to kill stem cells, but we can study the stem cells in isolation to learn how they resist being killed. If we identify a specific mechanism of drug resistance, we can then target that mechanism with drugs that, when combined with traditional chemotherapy, might make those drugs more effective. Alternatively, if we identify a mechanism of drug resistance that affects some drugs and not others, we can tailor future clinical trials for patients with metastatic Ewing’s sarcoma to rely more heavily on drugs predicted to be effective against stem cells, and in this way hopefully improve the outcomes of those patients. Evidence that this approach can work was presented in a recent article describing melanoma stem cells (Ref. 6).

Ultimately, we believe that the future of Ewing’s sarcoma therapy lies not with developing new and better cytotoxic chemotherapy, but rather with developing targeted therapies. These therapies need to be targeted against the most important cells, the cells that are responsible for drug resistance, disease relapse, and patient death – the cancer stem cells. Only by first identifying these cells and developing a method for purifying them can we study them in the sort of detail necessary to learn enough about them to develop stem cell targeted therapies that will finally bring hope to patients with metastatic Ewing’s sarcoma.

References

1. Bonnet D, Dick JE: Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 3:730-7, 1997

2. Al-Hajj M, Wicha MS, Benito-Hernandez A, et al: Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A 100:3983-8, 2003

3. Singh SK, Clarke ID, Terasaki M, et al: Identification of a cancer stem cell in human brain tumors. Cancer Res 63:5821-8, 2003

4. Clarke MF, Dick JE, Dirks PB, et al: Cancer Stem Cells--Perspectives on Current Status and Future Directions: AACR Workshop on Cancer Stem Cells. Cancer Res 66:9339-44, 2006

5. Todaro M, Perez Alea M, Scopelliti A, et al: IL-4-mediated drug resistance in colon cancer stem cells. Cell Cycle 7, 2007

6. Schatton T, Murphy GF, Frank NY, et al: Identification of cells initiating human melanomas. Nature 451:345-9, 2008

7. Wang JC, Dick JE: Cancer stem cells: lessons from leukemia. Trends Cell Biol 15:494-501, 2005

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