Modulation of clonal progression in B16F1 melanoma cells.

We have examined the effects of the microenvironment on the frequency and generation of metastatic variant cells in both parental B16F1 melanoma cells and nascent clones. The metastatic abilities of cultured B16F1 cells were tested after a period of growth in the presence or absence of a second cell population separated from each other by a transwell membrane (0.45 micron pore size). The first population is defined as the 'responder' cells and the second as the 'stimulator' cells. We found that the presence of 10(5) B16F1 stimulator cells during the growth of responder B16F1 cells from approximately 10(4) to approximately 10(6) cells resulted in cells with an increased metastatic phenotype (greater than 8-fold increase in median number of lung tumors relative to untreated B16F1 parental cells). The presence of stimulator cells also increased the metastatic phenotype of nascent clones, which were grown to a population size of less than 10(6) cells, suggesting that the rate of generation of metastatic variants of the responder B16F1 clones was affected by the stimulator cells. Other cell lines, including highly metastatic B16F10 and BL6 melanoma cells, and KHT35-L1 fibrosarcoma cells, were effective stimulator cells when as few as 10(4) cells were added to transwells. In addition, normal immortalized NIH 3T3 cells were effective stimulator cells only at 10(5) cells/transwell. The cell density at which untreated parental B16F1 cells were harvested (3 x 10(3)-3 x 10(5) cells/cm2) did not affect the median number of lung tumors significantly. These results suggest that factors released from both tumor and immortalized normal cells can modulate epigenetic changes in the metastatic phenotype of B16F1 melanoma cells.