Malignant Progression of B16 Melanoma Cells Induced in Vitro by Growth Factors Produced by Highly Malignant Cells

Four mouse B16 melanoma subclones (G3.15, G3.5, G3.12 and G3.26) exhibit progressively greater growth capacity in vitro and in vivo. Previously, non-metastatic G3.15 cells were sequentially converted, in monolayer cultures, to the moderately-metastatic G3.5 cells, and then to a highly-metastatic G3.5* phenotype. Both conversions were induced by hypoxia followed by confluence, and also occurred in tumors. G3.5* cells were comparable with, yet distinguishable from, G3.12 cells in being growth-autonomous in culture. In this study, the presumption that rapidly-growing G3.26 cells represented the ultimate progression step in this clonal system was examined. Both G3.12 and G3.5* cells converted in vitro to the G3.26 phenotype during growth in serum-free medium conditioned by G3.26 cell growth. By selective filtration of conditioned medium and characterization of the stability of growth- and conversion-promoting activities, three distinct activities were found to promote a two-step G3.12 to G3.26 phenotype conversion: (1) a < 10 kDa filtrate stimulated slight attachment and proliferation of G3.12 cells, effects that were reversible, partly attributable to accumulated lactate, and fully mimicked by medium acidification to pH 6.5; (2) medium acidification, together with a heat- and acid-stable but partially trypsin-sensitive > 10 kDa activity, induced G3.12-->G3.5* conversion that resulted in acquisition of growth autonomy; and (3) a heat-, acid- and trypsin-sensitive > 10 kDa activity induced G3.5*-->G3.26 conversion, characterized by anchorage-independent growth in soft agar, and potent lung colonization following intravenous injection. Phenotype analysis of G3.12 tumors and lung metastases revealed that G3.5*-like cells were regularly present in tumors and metastases, whereas G3.26-like cells occurred almost exclusively in large lung metastases. While G3.12 cells might convert to G3.5* cells in order to disseminate, G3.26 cells are apparently not involved in metastatic spread but probably account for the rapid growth of established metastases.