Progressive state selection of cells in low serum promotes high density growth and neoplastic transformation in NIH 3T3 cells.

A subline of NIH 3T3 cells maintained by frequent passage (every 2 to 3 days) in 10% calf serum (CS) at low population density reached a low saturation density in 2% CS and produced no transformed foci on prolonged incubation at confluency in 2% CS. Within 3 frequent low density passages in 2% CS, the saturation density and focus-forming capacity in that serum concentration began an increase which was continued in subsequent passages. The saturation density and focus-forming capacities of the cells in both 2% and 1% CS were further enhanced by passage in 1% CS. The cells could then be passaged in 0.5% CS and then in 0.25% CS, which would support no multiplication of cells previously passaged only in 10% CS. The cells passaged in 0.25% CS gradually increased their saturation density and focus-forming capacity in that extremely low serum concentration during 24 low density passages, although their initial growth rate did not increase. They also attained a colony-forming efficiency in 0.25% CS of about 30%, as compared to less than 1% for cells passaged in 10% CS. Cells passaged, cloned, and passaged again in 2% CS yielded clonal populations which differed from one another in saturation density and focus-forming capacity in 2% CS. We conclude that NIH 3T3 cells diversify phenotypically at a high rate in their capacity to multiply and produce foci in limiting concentrations of serum, and we propose that progressive selection of these heterogeneous states accounts for the acquired capacity to function effectively in low concentrations of serum growth factors. Since lymph and presumably extracellular fluid in vivo contain low concentrations of growth factors which govern the multiplication of normal cells, the adaptation we observe in vitro may be related to tumor production in the animal.