Identification and Characterization of the Ewing’s Sarcoma Stem Cell
One of the most important ideas driving cancer research over the past several years is the concept of the cancer stem cell. The cancer stem cell hypothesis runs counter to previous understanding of cancer cells – that any cell from a tumor is capable of limitless growth and the ability to spread throughout the body, forming new tumors at distant sites. Rather, this new model predicts that there is a specific population of cancer cells with these capabilities (Figure 1). These cells are also capable of self-renewal and differentiation, characteristics of a stem cell. These cells are proposed to be resistant to chemotherapy and therefore are thought to be the cells that are responsible for relapse, metastasis, and patient death.
Interestingly, in carcinomas (like breast cancer and colon cancer), cancer stem cells have mesenchymal characteristics.1 Mesenchymal cells are the kind of cells from which sarcomas are derived. This leaves open the question of whether all mesenchymal cancer cells might have stem cell characteristics, and if not, what a sarcoma stem cell might be like. Our laboratory has been investigating the possibility that Ewing's sarcoma contains a defined population of cancer stem cells, with the hope that we can answer these interesting scientific questions as well as developing treatments that target these cells will dramatically improve the prognosis of patients who present with metastatic disease – a group whose outcomes have not improved over the past three decades despite advances in chemotherapy treatments that have greatly benefitted patients with localized disease.
With the generous support of the Liddy Shriver Sarcoma Initiative, we have begun the process of identifying and characterizing Ewing sarcoma stem cells. Based on findings reported for breast cancer (and other carcinomas), we hypothesized that these cells would express high levels of an enzyme called aldehyde dehydrogenase (ALDH).2 ALDH plays an important role in retinoic acid metabolism in a variety of stem cell types,3 and there is a commercially available kit that makes determining how much ALDH activity an individual cell has, and sorting cells based on this activity, very straightforward. This kit, called the Aldefluor Assay, allowed us to demonstrate that there is a population of Ewing’s sarcoma cells, representing less than 5% of the total population, which contains very high levels of ALDH.
These cells have the characteristics that would be predicted of tumor stem cells. They demonstrate self-renewal activity when grown in the laboratory. When grown in culture for 2 weeks, cells with high levels of ALDH activity (designated ALDHhigh) regenerate a complex population, containing both ALDHhigh and ALDHlow cells (and, like the parent population, most of the cells are ALDHlow). In contrast, ALDHlow cells grown in culture do not result in an ALDHhigh subpopulation – all of the cells remain ALDHlow (Figure 2). The ALDHhigh cells grow much more rapidly than the ALDHlow cells do. Most importantly, the ALDHhigh cells show tumor initiating activity – grown in culture, these cells, but not the ALDHlow cells, form colonies in soft agar and grow in spheres (called "sarcospheres") when not allowed to stick to culture dishes. Furthermore, far fewer ALDHhigh cells than ALDHlow cells are needed to grow a tumor in mice. All of this data supports our hypothesis that there is an identifiable subpopulation of Ewing’s sarcoma stem cells, and that high levels of ALDH activity is an important characteristic of these cells. We expect to submit a manuscript describing these findings before the end of the year.
Identifying Ewing's sarcoma stem cells based on ALDH expression is only the first step. High ALDH activity is not limited to stem cells, so the cell population we have been studying probably contains a mixture of stem cells and other cells. Future work will be aimed at more completely purifying the Ewing's sarcoma stem cells so that we can focus our efforts as specifically as possible on these key cells.
Once the stem cell population is identified and purified to the extent possible, we will focus our attention on developing therapies that target this population specifically. As discussed above, these cells are resistant to standard chemotherapy drugs, and that is why so many patients relapse despite an early good response to treatment. If we were able to treat the stem cells in such a way that they became more sensitive to chemotherapy, or if we were able to develop treatments that the stem cells are sensitive to, despite their resistance to chemotherapy, this would dramatically improve the outcomes for patients with Ewing's sarcoma.
Finally, our work has implications for a deeper understanding of the origins of this enigmatic disease. One question that no one has been able to answer yet is what is the "cell of origin" of Ewing's sarcoma? Data from several laboratories has suggested that Ewing's sarcoma may arise from a neuronal precursor cell, from an epithelial cell, or perhaps from mesenchymal stem cells. We believe that a close examination of the gene expression pattern of a purified population of Ewing's sarcoma stem cells might shed important light on this question.
Summary and Conclusions
In summary, with the support of a grant from the Liddy Shriver Sarcoma Initiative, we have demonstrated that a subpopulation of Ewing's sarcoma cells characterized by high expression of aldehyde dehydrogenase has characteristics of stem cells: these cells are capable of self-renewal and of tumor initiating activity in immune deficient mice. We will continue to work to further purify the stem cell population so that we can gain a better understanding of the biology of this key cell type, as well as developing treatments that target them. It is our hope that therapeutic targeting of Ewing's sarcoma stem cells will result in dramatic improvements in the outcome of patients with Ewing's sarcoma, especially those with metastatic disease who rely on chemotherapy for a cure.
By David Loeb, MD, PhD
Assistant Professor of Oncology and Pediatrics
Director, Musculoskeletal Tumor Program
The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins
1. S. A. Mani et al., Cell 133, 704 (May 16, 2008).
2. S. Corti et al., Stem Cells 24, 975 (Apr, 2006).
3. J. P. Chute et al., Proc Natl Acad Sci U S A 103, 11707 (Aug 1, 2006).
V6N5 ESUN Copyright © 2009 Liddy Shriver Sarcoma Initiative.
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.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 accepted2 and there is good evidence supporting the existence of stem cells in a variety of other solid tumors, including brain tumors.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.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:
- 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
- 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.
Defining Cancer Stem Cells
The gold standard definition of a cancer stem cell would be the identification of an individual cell capable of giving rise to a serially-transplantable tumor upon injection into immune deficient mice. Clearly, this assay cannot serve as a basis for isolation of the cells, because the majority of the cells in such a tumor would not be stem cells. Thus, surrogate assays have been developed. The so-called "side population" assay is based on the ability of cells to efflux the fluorescent dye Hoechst 33342 by a mechanism that is inhibited by verapamil. The Aldefluor assay is based on the use of a fluorochrome that is a substrate for aldehyde dehydrogenase (ALDH). Cells with high ALDH expression will fluoresce brightly upon incubation with Aldefluor, and this fluorescence will be inhibited by the inclusion of DEAB, a specific inhibitor of ALDH.
Immunophenotyping can also be used to identify stem cells, although there is no single "stem cell marker." The immunophenotypes of cancer stem cells differ by tumor type: breast cancer stem cells are CD44+/CD24-/low/ESA+ but CML stem cells are CD34+/CD38-/Thy1-/IL3Ra+. Thus the immunophenotype of Ewing sarcoma stem cells will have to be determined after they are isolated by other means.
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.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.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.
By David Loeb, MD, PhD
Assistant Professor of Oncology and Pediatrics
Director, Musculoskeletal Tumor Program
Co-Director, Sarcoma Program
The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins
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.
V5N1 ESUN Copyright © 2008 Liddy Shriver Sarcoma Initiative.
The Liddy Shriver Sarcoma Initiative funded this $37,800 grant in February 2008. The study was made possible by a generous donations from the Arlo and Susan Ellison family; by generous donations made in memory of Christie Campbell, Jeremy Zimmer, Brad Rice, Peter Skelton, and Paul Onvlee, who fell victim to this disease; and by generous donations made in honor of Teri Marriage, Matthew Beaver, and Nick Gibboni, who are fighting the disease. Donations were also received in memory of Jeremy's grandfather, Robert Pickrell.
Copyright © 2012 Liddy Shriver Sarcoma Initiative.
The observation that not every cell in a tumor is clonogenic can be explained by two competing models: the stochastic model and the stem cell model. The stochastic model (A) predicts that any individual cell might be clonogenic, and stochastic events allow some, but not all, to give rise to colonies in in vitro assays of clonogenicity. The stem cell model (B) predicts that some, but not all, cells from a tumor are clonogenic, and that the clonogenic cells have characteristics that would allow their isolation and characterization. Thus, the prospective isolation of clonogenic cells based on characteristics such as immunophenotype would support the stem cell hypothesis. These models are discussed more extensively by Wang and Dick (Ref. 7).
The relative resistance of cancer stem cells to chemotherapy has significant implications for drug development. The above figure demonstrates the effect of cytotoxic chemotherapy on stem cell frequency within a large tumor. Treatments that target the bulk tumor population (such as most currently used chemotherapy) will yield a residual tumor population enriched for cancer stem cells (though these cells will remain a small minority of the total population (top panel). In contrast, therapies that are equally effective against cancer stem cells and their differentiated progeny will yield a residual tumor population that is not enriched for stem cells (bottom panel).
The stochastic model predicts that any tumor cell, given the chance, will be able to separate from the bulk tumor and form a new tumor. In contrast, the cancer stem cell model predicts that only a subset of cells (the blue cells in the figure) can generate a new tumor, while the other cells cannot.
The bulk population, at the top of the figure, is composed of a small subpopulation of (green) ALDHhigh cells and a larger population of (blue) ALDHlow cells. These cells can be separated from each other and grown in culture. The ALDHhigh cells, after 2 weeks, give rise to a population of cells consisting of a majority ALDHlow, with some ALDHhigh (bottom left). In contrast, the ALDHlow cells expand in culture, but do not give rise to any ALDHhigh cells (bottom right).