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+
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 cancer16, 21
, colon cancer19, 20
, hepatocellular carcinoma18
, head and neck squamous cell carcinoma22
and ovarian cancer23, 24
. Based on these reports and current criteria including in vivo
tumor formation with limited cells, and sphere formation, we suggest that ALDH+
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 cells34
. 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+
cells are from original CD133dim
cells (which may not be detectable by current flow cyometry technique), or CD133−
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 types35
. Further genetic and functional studies are needed to dissect 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 distinct36
. 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 hour in the in vitro
serum-free culture, and 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, 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 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+
ovarian cancer cells are equally tumorigenic. It has been reported that CD44+
can be used to identify ovarian cancer stem cells12, 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 can’t 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−
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 utilized commercially available established ovarian cancer cell lines, or the unsorted tumors, or “the cells” isolated from the in vitro
formed spheres or the mouse xenografts12–15, 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, based on our multiple color flow cytometry analysis, we have excluded all the possible non-epithelial 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 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 niche38, 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
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 ovarian cancer patients. Our data indicate that the expression levels of multiple stem cell markers gradually diminish following prolonged culture in vitro. Fresh tumor cells are needed to investigate cancer stem cell biology.