Phenotypic Variance among Cells Isolated from Spontaneous Mouse Mammary Tumors in Primary Suspension Culture1

A method is given for selecting epithelial cells directly from primary mammary tumors in Methocel suspension culture. The frequency of colony-forming units in primary tumors was ap proximately 10~4. Colonies grew by cell division; formation and growth of colonies was cell density dependent. Five Methocel isolates were established in monolayer culture and character ized. Two were epithelial, evidenced by functional occluding junctions. The other three were not typed in vitro, although they formed carcinomas in vivo. All were subtetraploid by passage 10. There were variations in ability of the five Methocel isolates to redone in suspension that appeared to be due to the evolution of anchorage-dependent variants during their growth in monolayer culture. These variants could be purified by limiting dilution plating on solid substrates. The five Methocel isolates and their derivative variants were used to determine correlations between transformation markers and tumorigenicity. Only three Methocel-derived sublines of nine tested, including two recloned in Methocel, were tumorigenic at all when inoculated in two sites of three syngeneic hosts, one athymic. The other six were nontumorigenic. The tumorigenic sublines were less tumorigenic than uncul tured cells of parent tumors or parent tumor cells grown in primary monolayer culture. Thus, anchorage-independent growth is not a reliable marker for the tumorigenic mammary phenotype. No correlation was found between two other "con tact-related" transformation markers, rapid growth rate and monolayer overgrowth, and tumorigenicity. The three transfor mation markers were expressed independently. Both tumori genic and nontumorigenic sublines expressed mammary tumor virus antigens M.W. 28,000 protein and M.W. 52,000 glycoprotein, although only a minor fraction of cells contained the M.W. 52,000 glycoprotein. These data emphasize the hetero geneity of phenotypes in mammary tumors as well as differ ences between fibroblasts and mammary epithelium in models of neoplastic transformation.