We have established that spontaneous metastasis of the mouse B16 melanoma proceeds in two distinct stages: initially to the lungs as a generalizing site, and secondarily from established lung metastases to systemic organs and tissues. Like advanced metastasis of human malignant melanoma, systemic B16 melanoma metastasis is widespread but exhibits reproducible patterning: the kidneys, adrenals, brain and ovaries are frequently involved, whereas the spleen, liver and bones never develop visible metastases. Preliminary studies indicate that metastases are disseminated randomly to systemic sites, but metastasis growth may be determined mainly by positive or negative proliferative stimuli due to soluble factors or to resident non-parenchymal cells in organs. Utilizing quiescent or proliferating monolayer cultures of a metastatic clone, and derived variants with enhanced capacity to establish systemic metastases, organ-conditioned medium from """"""""favorable"""""""" and """"""""hostile"""""""" organs will be assessed for growth-promoting or -inhibiting effects. Similarly, effects of possible proliferative factors produced in organ sites favorable for metastasis growth (e.g., progesterone and beta-estradiol from the ovarian corpus luteum; various corticosteroids from the adrenal cortex) will be investigated. Growth-modulating influences of isolated organ cells (parenchymal/stromal cells and lymphoid/reticuloendothelial cells) will also be examined. Positive or negative effects will be assessed for possible relevance to metastasis in vito tests using a subcutaneous """"""""artificial organ"""""""" implant as an experimentally modifiable environment for development of spontaneous systemic metastasis. Soluble preparations and factors will be delivered to implants from incorporated sustained-release polymer pellets or from osmotic pumps, and cells will be introduced through an external port and connecting tubing. Effects on metastasis will be correlated with changes in host cell infiltration of implants, monitored by histology and histochemistry. The number of metastatic cells reaching an organ is likely to influence proliferative or inhibitory effects. To examine this issue, variable numbers of cultured cells, immobilized on pieces of plastic or filter substrate, will be introduced into kidneys, liver, and spleen in situ to establish relative thresholds of tumorigenicity in each organ. Also, organs removed from mice subsequent to the onset of systemic metastatic dissemination will be cultured ex vivo as fragments or slices to determine if metastatic foci will emerge from their original arrested locations. If so, approximate numbers of metastatic emboli entrapped in each organ will be estimated.
