Linking EMT to ovarian cancer stem cells
Authors: Robert Strauss, ZongYi Li, Ying Liu, Pavel Sova, Charles Drescher, Nicole Urban, Andre Lieber
Journal: Cancer Research (2008)

2028  There is increasing evidence that tumor growth is driven by a small subset of tumor-initiating cells. These cancer stem cells have now been reported for brain tumor, breast, prostate, pancreatic, colon, ovarian carcinoma and melanoma.  Cancer stem cells have I) an extensive capacity for proliferation, II) the ability of self-renewal and for asymmetric cell division, III) multilineage differentiation potential and IV) the capacity to initiate heterogeneous tumors in NOD/SCID mice from a single cell. Additionally pluripotency is thought to be the basis for the ability of these cells to generate heterogeneous tumors in xenograft models and cell cultures in vitro.  Epithelial to mesenchymal transition (EMT) is an indispensable mechanism during embryogenesis/morphogenesis, where polarized epithelial cells acquire mesenchymal gene expression patterns and properties. It is thought that EMT also plays a major role during progression of tumor growth and the gain of metastatic competence in epithelial cancers.  We established a total of 42 primary ovarian cancer cell cultures derived from biopsies or ascites fluids from grade III or IV patients. Interestingly, only 1 of 20 tested cultures (ovc316m) was capable of forming tumors in NOD/SCID mice, even in numbers as low as 100 injected cells. We focused our study on this culture and found high numbers of ESA+/CD44+ hybrid-epithelial/mesenchymal (E/M) cells. In the transitional region between epithelial and mesenchymal cells these cells also expressed cancer stem cell markers Nanog, Oct4 and CD133.  In order to prevent EMT, cells isolated from an ovc316m xenograft were cultured under serum and growth factor free conditions for 3 passages. The resulting culture was >85% CD133+/ESA+/CD44+ and showed comparable purity to CD133+ cells isolated directly from xenografts by magnetic bead sorting. Application of growth factors EGF, insulin and 10% fetal bovine serum from passage 3 on resulted in a differentiation of ovc316m cells into epithelial and mesenchymal cells over passaging in vitro. This process appeared to be reversible, when the cell culture was medium starved again and hereby selected for cells that were able to form tumor spheres containing cells in an E/M hybrid stage only.  We show the existence of E/M-hybrid cells, which are able to differentiate into both epithelial and mesenchymal cells. We are able to enrich and culture E/M-hybrid cells under serum and growth factor free conditions in high numbers. Additionally we show that E/M-hybrid cells express cancer stem cell markers and are able to form tumors in NOD/SCID mice. Their ability to self-renew was demonstrated by heterogeneous, tumor-forming clonal cell cultures derived from ovc316m and the repetitive isolation of single cells that form heterogeneous cultures. Ongoing and future studies will include the ability of tumor formation of CD133+/ESA+/CD44+ and CD133-/ESAlow/CD44low cell fractions and genome wide gene expression analysis on mRNA and miRNA of these fractions.