title: Malignant progression of B16 melanoma cells induced in vitro by growth factors produced by highly malignant cells.
abstract: 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.
authors.affiliation: Department of Experimental Pathology, New York Medical College, New York.
authors.forename: C W
authors.initials: CW
authors.lastname: Stackpole
authors.forename: L
authors.initials: L
authors.lastname: Groszek
authors.forename: S S
authors.initials: SS
authors.lastname: Kalbag
mesh_terms: D000818:Animals; D002455:Cell Division; D016895:Culture Media, Serum-Free; D005260:Female; D006133:Growth Substances / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D008546:Melanoma, Experimental / Q000473:pathology* / Q000556:secondary; D051379:Mice; D008810:Mice, Inbred C57BL; D010641:Phenotype; D014407:Tumor Cells, Cultured
chemical_list: D016895:Culture Media, Serum-Free; D006133:Growth Substances