Malignant hemangioendotheliomas produced by subcutaneous inoculation of Balb/3T3 cells attached to glass beads

The Balb/3T3 mouse embryo cell line has been frequently used in cancer research as representative of nontumorigenic cells with the characteristic in vitro properties of postconfluence inhibition of cell division, low saturation density, and anchorage dependence. On the reasoning that anchorage dependence might also apply in vivo, each of nine mice were subcutaneously inoculated with an average of 15,400 Balb/3T3 cells attached to two glass beads 3 millimeters in diameter. After 8 weeks, all the mice had developed large bloody tumors that microscopically proved to be hemangioendotheliomas. Ther inoculation of Balb/3T3 cells alone or beads alone produced no tumors. Transplants of each tumor into normal mice grew to kill the animal within 6 weeks. Tumor cells from collagenase-disaggregated tumor tissue had a plating efficiency of 21.2 percent compared to that of normal adult subcutaneous fibroblasts of less than 0.1 percent. The tumor cells in vitro closely resembled Balb/3T3 cells in appearance and were tumorigenic at a dose of 10-4 cells. A second, repeat experiment produced the same type of tumors grossly and microscopically in 17 of 25 mice between 99 and 211 days after inoculation of the Balb/3T3 cells attached to glass beads. These findings require a reassessment of the postulate that low saturation density, postconfluence of cell division, and anchorage dependence are characteristic in vitro properties only of nonneoplastic cells.