Role of anchorage in the expression of tumorigenicity of untransformed mouse cell lines

Cultured mouse cells were tested for tumorigenicity in nude mice with both a conventional assay (injection of cell suspensions) and a new test involving implantation of cells grown on gelatin sponges. Sublines of Balb/3T3 cells, obtained from different sources, varied in their tumorigenic potential with either assay. One subline (A) formed distinctive precancerous nodules only in the sponge assay; these nodules often became progressive after a latent period of 3-4 months. However, suspensions of cells of this subline also caused tumors after a similar latent period, but no nodular phase preceded tumor formation. Another subline of Balb/3T3 (M) has failed to form tumors in either assay. The Balb/3T3 sublines did not differ in vitro properties, such as low saturation density, failure to grow in methylcellulose, and monolayer morphology. A second experimental approach involved tests on nude BALB/c mouse-embryo fibroblasts at various passage levels. The cells were passaged from primary culture, through crisis, to heteroploid, established cell lines. Tumorigenicity was demonstrable earlier in the sponge assay, at which time in vitro parameters putatively associated with malignant behavior were unchanged. Possible relationships with the in vivo phenomenon of solid-surface sarcomagenesis are discussed.