Direct Cloning of Human Ovarian Carcinoma Cells in Agar1

We have recently developed an in vitro assay for human tumor stem cells that permits cloning of human ovarian adenocarcinoma cells in soft agar. Tumor colonies grew from both effusions and biopsies from 85% of 31 ovarian cancer patients. The cloning efficiency did not vary with the histology of the tumor. Growth was induced with medium conditioned by the adherent spleen cells of mineral oil-primed BALB/c mice. Up to 2000 colonies appeared after 10 to 14 days in culture, yielding a plating efficiency of 0.001 to 1%. Cells from nonmalignant effu sions did not form colonies under these conditions. The number of tumor colonies was proportional to the number of cells plated between concentrations of 10" to 106 cells/ dish. Morphological and histochemical criteria showed that the colonies consisted of cells with the same characteris tics as those of the original tumor. Results of cytogenic studies were also consistent with a malignant origin for the tumor colonies with marked hyperdiploidy in colonies from four patients and hypodiploidy in a fifth patient. [3H]Thymidine and hydroxyurea suicide indices pro vided evidence that in most cases a high proportion of ovarian tumor colony-forming cells were actively in transit through the cell cycle. Removal of phagocytic macro phages with carbonyl iron markedly reduced the plating efficiency, and 2-mercaptoethanol could only partially substitute for macrophages. The assay appears useful for screening differential cytotoxic effects of specific anticancer drugs (such as cisplatinum) against the tumor stem cells from various ovar ian cancer patients.