We have established an in vitro clonogenic cell assay for a class of anaplastic rat prostatic tumor cells (R-3327-At). We used collagenase to isolate these cells from transplanted solid tumors growing in the flanks of mature male Copenhagen rats and cultured them in α-minimal essential medium supplemented with 10% fetal calf serum. A linear relationship was obtained between the number of cells plated and the number of colonies formed. R-3327-At clonogenic cells had a plating efficiency of about 4% when monodispersed cells were isolated from the primary tumor, and their cell doubling time was 24 hr. Cultured tumor cells have maintained their ability to induce tumors when retransplanted into normal male reciplent rats even after 30 weekly passages in vitro, although later passages were not as “tumorigenic.” The R-3327-At cells were readily distinguishable from fibroblasts on the basis of cellular morphology and growth kinetics and because only the R-3327-At cells were able to proliferate in semisolid (plasma) culture and induce tumors upon retransplantation in vivo.

The addition of conditioned media (CM) prepared from R-3327-At tumor cell cultures to low density R-3327-At cell cultures produced larger colonies compared to those cultures to which we added either no CM or CM from Copenhagen rat skin fibroblasts. These data indicated that the growth-promoting activity present in the CM of R-3327-At cultures can increase the extent of proliferation, although it is not essential for its initiation. The addition of sera from rats bearing either the R-3327-At or the hormone-dependent (R-3327-H) tumor was “cytotoxic” to R-3327-At clonogenic cells. Not only were fewer colonies formed, but also individual colonies contained fewer cells when compared to control cultures to which normal rat serum was added. These findings are potentially important since they may reflect a host immune response to the tumor.

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This investigation was supported by Grant 5PO1CA13053-05 and awarded by the National Cancer Institute, Department of Health, Education, and Welfare.

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