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- Material and Methods
- Supporting Information
Identification of cancer stem cells is crucial for advancing cancer biology and therapy. Several markers including CD24, CD44, CD117, CD133, the G subfamily of ATP-binding cassette transporters (ABCG), epithelial specific antigen (ESA) and aldehyde dehydrogenase (ALDH) are used to identify and investigate human epithelial cancer stem cells in the literature. We have now systemically analyzed and compared the expression of these markers in fresh ovarian epithelial carcinomas. Although the expression levels of these markers were unexpectedly variable and partially overlapping in fresh ovarian cancer cells from different donors, we reliably detected important levels of CD133 and ALDH in the majority of fresh ovarian cancer. Furthermore, most of these stem cell markers including CD133 and ALDH were gradually lost following in vitro passage of primary tumor cells. However, the expression of ALDH and CD133, but not CD24, CD44 and CD117, could be partially rescued by the in vitro serum-free and sphere cultures and by the in vivo passage in the immune-deficient xenografts. ALDH+ and CD133+ cells formed three-dimensional spheres more efficiently than their negative counterparts. These sphere-forming cells expressed high levels of stem cell core gene transcripts and could be expanded and form additional spheres in long-term culture. ALDH+, CD133+ and ALDH+CD133+ cells from fresh tumors developed larger tumors more rapidly than their negative counterparts. This property was preserved in the xenografted tumors. Altogether, the data suggest that ALDH+ and CD133+ cells are enriched with ovarian cancer-initiating (stem) cells and that ALDH and CD133 may be widely used as reliable markers to investigate ovarian cancer stem cell biology.
Ovarian carcinoma is a deadly disease characterized by late diagnosis, early metastasis and resistance to therapy. Although the majority of patients initially respond to platinum-based chemotherapy, most will subsequently succumb to chemoresistant, recurrent disease. Long-term treatment success is limited by the development of chemoresistant disease. This may be partially due to the immunosuppressive networks formed in the human ovarian cancer microenvironment. In the last several years, our research team has focused on the human ovarian carcinoma microenvironment and has demonstrated that immune cells in human ovarian carcinoma have been reprogrammed by active tumor-mediated processes to defeat tumor immunity and in turn temper the clinical efficacy of chemotherapy.1–4
It is also possible that existing therapies primarily target the bulk of the ovarian carcinoma cell population rather than the cancer-initiating cells (or stem cells or stem-like cells).5–11 Although multiple markers including CD44, CD117,12 CD13313, 14 and Hoechst positive “side population”15 are used to identify ovarian cancer stem cells, the concept of ovarian cancer stem cells remains controversial, and the nature of these ovarian cancer stem cells has not been well defined in fresh ovarian cancer and primary tumor cells. Aldehyde dehydrogenase (ALDH) has been used to investigate multiple human cancer stem cells.16–22 Recent reports showed that ALDH+ ovarian cancer cells possess stem cell properties.23, 24 However, it is not well understood if ovarian cancer stem cells universally express ALDH, CD133 and other markers and if ALDH expression is phenotypically and functionally associated to many other reported markers for ovarian cancer stem cells. In our study, we revisited this issue and examined fresh ovarian cancer tissues, primary ovarian cancer cells and xenografted ovarian cancer cells in our laboratory and focused our studies on identifying and comparing potential ovarian cancer stem cells and the regulatory effects of in vitro and in vivo environments on the property of ovarian cancer stem cells.
- Top of page
- Material and Methods
- Supporting Information
In this study, we have examined and compared the expression of multiple cancer stem cell markers in fresh ovarian cancer and established primary ovarian cancer cell lines and investigated the stem cell properties of potential ovarian cancer stem cells in vitro and in vivo.
We have shown that although the levels of ALDH and CD133 expression are variable, expression is detectable in the majority of fresh ovarian tumors. Consistent with the cancer stem cell concept, ALDH+ and CD133+ cells are able to efficiently form spheres and heterogeneous tumors in vivo with limited numbers of cells. ALDH is thought to be a marker for defining stem cells in multiple human epithelial cancers including breast cancer,16, 21 colon cancer,19, 20 hepatocellular carcinoma,18 head and neck squamous cell carcinoma22 and ovarian cancer.23, 24 On the basis of these reports and current criteria including in vivo tumor formation with limited cells and sphere formation, we suggest that ALDH+ and CD133+ cells may be enriched with cancer stem cells in the majority of human ovarian cancer.
Although ALDH and CD133 can be used to identify ovarian cancer stem cells in fresh ovarian tumors, the expression of CD133, ALDH and other markers is gradually reduced following prolonged in vitro cell passages. In support of our observation, it has been demonstrated that tumor cells grown under standard serum-containing cell culture conditions result in the loss of tumor stem cells.34 The loss of stem cell markers in vitro culture system suggests that the stem cell phenotype or/and properties may possibly need to be supported in vivo in the tumor microenvironment and that the in vitro conventional culture conditions may not be appropriate for maintaining the cancer stem cell phenotype. In support of this possibility, the expression of cancer stem cell markers CD133 and ALDH is partially recovered in the in vivo formed xenograft tumors and in the in vitro serum-free culture. Our results also indicate that the loss of stem cell markers may be reversible. However, it is unknown whether the recovered CD133+ and ALDH+ cells are from original CD133dim and ALDHdim cells (which may not be detectable by current flow cytometry technique) or from CD133− and ALDH− cells. It has been suggested that the capacities for self-renewal and tumor initiation may not be restricted to a uniform population of stem-like cells, but can be shared by a lineage of self-renewing cell types.35 Further genetic and functional studies are needed to analyze if CD133 and ALDH are functionally and genetically relevant for controlling cancer stem cell properties and if fresh and induced (or rescued) CD133 and ALDH expressing cells are genetically and functionally distinct.36 Interestingly, once the cells are exposed to conventional culture conditions, the expression of CD133 and ALDH rescued (or induced) by the serum-free culture conditions or the in vivo tumor passages gets lost again. Nonetheless, given that the expression of CD133 and ALDH appears within 12 hr in the in vitro serum-free culture and that the appearance of their expression is dependent on the environmental conditions, we speculate that genetic mutations may not be the major cause of driving CD133 and ALDH induction in our experimental conditions. This does not contradict with the notion that the combination of multiple genetic changes or/instability is of fundamental importance in tumorigenesis. Furthermore, fresh and induced CD133- and ALDH-expressing cells express high levels of stem cell core genes and efficiently form spheres and in vivo tumors. Altogether, the data support the conclusion that CD133- and ALDH-expressing cells are enriched with cancer stem cells and that these cells are important tools for studying ovarian cancer stem cell biology.
In addition to ALDH and CD133, other markers may be used in ovarian cancer stem cell research. ESA is expressed in fresh epithelial ovarian tumor cells. The expression of CD24 and CD44 is highly expressed in many fresh ovarian tumor cells we examined. ABCG2+ and ABCG2− ovarian cancer cells are equally tumorigenic. It has been reported that CD44+ and CD117+ can be used to identify ovarian cancer stem cells.12, 37 Our data show that the expression of CD117 is not detectable in more than 50% of fresh ovarian tumors and in 100% primary ovarian cancer cells established in 10% FCS conventional culture. Furthermore, the loss of CD44 and CD117 expression cannot be rescued by in vivo xenograft tumor passage and in vitro sphere culture. Based on these results, our data suggest that CD133 and ALDH can more accurately identify ovarian cancer stem cells and can be broadly used for ovarian cancer stem cell research in the majority of ovarian cancer. However, given the high heterogeneity of ovarian cancer types, it is important to note that CD44, CD117 and other markers could be used to investigate cancer stem cells in certain ovarian cancer types. Additionally, we have shown that CD133− and ALDH− cells could be tumorigenic in vivo. It also indicates that CD133 and ALDH are not exclusive markers for ovarian cancer stem cells.
The majority of published reports on human ovarian cancer stem cells used commercially available established ovarian cancer cell lines (or the unsorted tumors) or “the cells” isolated from the in vitro formed spheres or the mouse xenografts.12–15, 37 Macrophages, fibroblasts and many other cells express some stem cell markers including CD44 and CD24. As these cells are substantial populations in the tumor mass and may promote tumorigenesis, it is important to absolutely avoid their contamination in the ovarian cancer stem cell compartments. To this end, on the basis of our multicolor flow cytometry analysis, we have excluded all the possible nonepithelial cell fractions, including the immune cells (e.g., macrophages, T cells, and B cells), fibroblasts, vascular endothelial cells and CD34+ progenitors and hematopoietic cells in our studies. We systemically compared multiple stem cell markers and directly sorted these cells from fresh human ovarian tumors and investigated their stemness properties, including self-renewal and in vivo tumorigenesis. Given that long-term culture may alter cancer stem cell properties, we have minimized the potential impact of long-term in vitro culture and the experimental manipulation on the properties of ovarian cancer stem cells. One of the key issues in cancer stem cell studies is the regulation of cancer stem cell self-renewal and expansion. These properties are not autonomous to stem cells, and recent evidence points to a level of external control from the microenvironment that defines the stem cell niche.38, 39 This may explain why tumor stem cell markers are lost in the in vitro culture and are partially rescued in the in vivo model. Recent studies demonstrate that IL-640, 41 and IL-842 promote cancer stem cell-mediated tumorigenesis in vivo. We suggest that cancer stem cells may renew and expand in the tumor environment in vivo. The next step is to further define the tumor environmental factors and molecular signaling pathway crucial for regulating ovarian cancer stem cell properties.
We conclude that ALDH and CD133 are useful and reliable markers for investigating human ovarian cancer stem cells in the majority of patients with ovarian cancer. Our data indicate that the expression levels of multiple stem cell markers gradually diminish following prolonged culture in vitro. Therefore, fresh tumor cells are needed to investigate cancer stem cell biology.