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 effusions did not form colonies under these conditions. The number of tumor colonies was proportional to the number of cells plated between concentrations of 104 to 106 cells/dish.

Morphological and histochemical criteria showed that the colonies consisted of cells with the same characteristics 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 provided 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 macrophages 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 cis-platinum) against the tumor stem cells from various ovarian cancer patients.

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Supported by Grants CA 21839 and CA 17094 from the National Cancer Institute.

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