Generation of phenotypic diversity in the B16 mouse melanoma relative to spontaneous metastasis

Serial s.c. transplantation of the B16 melanoma in syngeneic mice for nearly 30 monthly generations effected gradual changes in the incidence of spontaneous pulmonary metastasis. Cell lines derived from s.c. tumors and from secondary tumor growths in the lungs were comparably variable in metastatic predilection and also differed in ability to produce tumor colonies in the lungs following i.v. injection of cultured cells. Some lines metastasized to the lungs from s.c. tumors and also colonized the lungs; others were metastatic but noncolonizing, colonizing and nonmetastatic, or nonmetastatic and noncolonizing ("null"). Metastatic and colonizing activities of all cell lines except the most potent colonizers were unstable in culture and during s.c. growth. Over 150 clones and subclones were obtained from B16 melanoma cell lines, and four distinct categories were defined on the basis of dissemination-related phenotypic characteristics: slow-growing null (Ns); rapid-growing null; metastatic; and colonizing. No single cell belonged to more than one category at the same time, but interconversions occurred rapidly and consistently during growth in vitro and in vivo. Progenitor Ns cells generated metastatic cells in culture and in tumors and became rapid-growing null cells and colonizers solely within tumors. Metastatic activity was transient, with cells reverting back to an Ns phenotype in culture and s.c. or converting to rapid-growing null cells and colonizers in vivo. Only potent colonizers were stable, an apparent end result of phenotypic diversification, but formation or proliferation of these cells within tumors was somehow regulated. Comparable heterogeneity was generated within lung metastases, except that reversion of metastatic cells to Ns cells and regeneration of metastatic activity were not demonstrated; the result was a progressive loss of metastatic cells within developing metastases.