The metastatic properties of tumor cell clones isolated from individual lesions of B16 melanoma metastatic to lung have been examined at different stages in the evolution of metastasis. Clonal analysis of metastatic lesions produced by B16 melanoma populations containing clones with identifiable, stable drug-resistance markers revealed that the majority (>80%) of experimental metastases produced by intravenous injection oftumor cells are of unicellular origin. During the early stages of their growth (<25 days after initial tumor cell arrest), the majority of metastatic lesions contain cells with indistinguishable metastatic phenotypes (intralesional clonal homogeneity) although different clonally homogeneous lesions from the same host contain tumor cells with different metastatic phenotypes (interlesional clonal heterogeneity). Progressive growth of metastatic lesions is accompanied by emergence, within originally clonally homogeneous lesions, of variant tumor cells with altered metastatic properties (intralesional clonal heterogeneity). By 40-45 days after initial arrest of injected tumor cells in the lung, 90% of the metastatic lesions are populated by cells with heterogeneous metastatic phenotypes.Studies in several laboratories have shown that malignant tumors contain subpopulations of cells that differ widely in their metastatic abilities (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). This cellular heterogeneity is believed to result from the formation of variant subpopulations of tumor cells with altered metastatic properties during progressive tumor growth (15). The factors that influence the genesis and regulation of cellular diversity within malignant tumors are poorly understood. Recent studies (16)(17)(18)(19) suggest that the rate at which tumor cell variants with altered metastatic properties are generated in vitro is influenced by the extent of subpopulation diversity within the overall cell population. We have shown that the rate of formation of metastatic variants is significantly higher in populations containing a limited number of tumor cell subpopulations than in highly heterogeneous, polyclonal populations containing multiple cellular subpopulations (16,17). We have also shown that the majority oflung metastatic lesions produced by the murine B16 melanoma arise from the single cells (17). Formation of such lesions thus represents a situation in which subpopulation diversity is restricted. It was therefore considered of interest to determine whether this situation would stimulate rapid formation of new tumor cell variants to generate phenotypically diverse subpopulations oftumor cells within individual metastases. Cells. The origin and properties of the B16 melanoma B16-F10 subline have been described (14,20). Drug-resistant (Dr) variants were selected from the B16-FlO subline by treatment with the chemical mutagen N-methyl-N'-nitro-N-nitrosoguanidine as described (16,17). Variants were selected for resistance to trifluorothymidine (2 ,ug/ml; TFT%), diaminopurine (47 p.M; DAPr), vin...

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