The Ewing's Sarcoma Stem Cell: |
An ESUN Article
Introduction
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.
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| 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. |
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.
Results
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.
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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). |
Future Directions
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.
References
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).
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