98. Enrichment and Characterization of Ovarian Cancer Stem Cells
Authors: Zong Yi Li, Robert Strauss, Pavel Sova, Shaoheng Ni, Mujun Yu, Dmitry Shayakhmetov, Charles Drescher, Nicole Urban, Andre Lieber
Journal: Molecular Therapy (2006)

Cancer stem cells, e.g. cells with a capacity to self-renew and undergo pluripotential differentiation, have been identified in malignancies of hematopoietic origin and in solid breast and brain tumors. We have established primary cultures from 40 ovarian cancer biopsies. After in vitro culture, tumor cells were expanded in immunodeficient mice. Cells from an explanted xenograft were then cloned. Of these clones, 40% contained subsets of cells with different morphology, size, and expression of surface markers, indicating that the cells from which these clones originated were pluripotent. When injected into mice, clonal cultures formed tumors with different histological components that stained with surface markers for human cells. Among the histological features were poorly differentiated malignant cells and structures that resemble the ovarian follicles. The ability of a clonal culture to give rise to different phenotypes further indicates the presence of pluripotent stem cells. Tumors also had clearly developed ECM and vasculature derived from mouse tissue. We then attempted to separate different cell fractions from clonal cultures with polymorphic morphology using FACS for marker expression. Immediately after sorting, different fractions were separated into three parts: i) cells were set aside for RNA isolation and expression array studies, ii) cells were subjected to clonogeneic assays and the percentage of polymorphic colonies that developed was used as a parameter to assess whether a given subfraction contains pluripotent cells. iii) different cell numbers of each fraction were injected into mice and tumor formation and histology was monitored. Among the markers used for sorting were tumor-associated antigens (CEA, CA125, HE-4), epithelial cell markers (CD44, CD133. |[alpha]|v|[beta]|3, ESA, AE1/AE3), embryonic stem cell markers (laminin receptor), and hematopietic stem cell markers (SLAM receptors, aldehyde dehydrogenase). We also sorted cells based on their ability to exclude the fluorescent dye Hoechst 33342. [It is thought that hematopoietic stem cells reside in a Hoechst 33342 negative side population (SP) based on a high activity of ATP-binding cassette drug transporters such as ABCG2.] We found that the fraction of ovarian cancer SP cells contained the highest percentage of pluripotent cells among all fractions analyzed. Other fractions that were enriched for pluripotent stem cells included the fractions of small cells, of cells that were relatively resistant to trypsin, and of cells that were ALDH-/low. Notably, markers that have been described for breast or brain cancer stem cells, such as CD133 and CD44, did not discriminate cell fractions containing pluripotent cells. Furthermore, putative ovarian cancer stem cells were negative for CD31 and CD45 but expressed high levels of the B-group adenovirus receptor CD46. We are currently analyzing expression microarray data from different sorted cell fractions for new markers for ovarian cancer stem cells and the results will be presented.