Three-dimensional cell culture induces novel proliferative and metabolic alterations associated with oncogenic transformation.

To date, cell biological characteristics of oncogene-transfected cells have been investigated either in relatively homogeneous monolayer cultures or in heterogeneous tumors in vivo. To evaluate the emergence of cellular heterogeneity during tumor formation, we have established a multicellular spheroid system from an oncogene-dependent, genetically determined 2-stage carcinogenesis model for 3-dimensional growth under well-defined conditions. The effect of T24Ha-ras transfection on cellular growth, proliferation, cell viability and oxygenation was investigated using spontaneously immortalized (Rat1) and c-myc-transfected (M1) Fisher 344 rat embryo fibroblasts and a tumorigenic T24Ha-ras-transfected clone of each (Rat1-T1 and MR1). Spheroid volume growth curves and [3H]thymidine autoradiographs clearly demonstrated that spheroids better reflect the degree of tumorigenicity in vivo as opposed to monolayer cultures. Studies on Rat1 and M1 aggregates showed that the potential for tumor formation of Rat1 cells might be manifested in vitro as an increased capability of the cells to survive in 3D culture. pO2 measurements confirmed that neither cell quiescence nor cell death in the pseudo-normal cell aggregate types is due to an oxygen deficiency. In contrast, depletion of oxygen coincided with necrotic cell death in Rat1-T1 spheroids and proliferation arrest in MR1 cultures. Cell-line-specific attributes in 3D culture that were not specifically related to ras transfection of the cells included histological structure, development of necrosis and thickness of viable cell rim. However, growth behavior, proliferation characteristics and their association with the oxygen supply might be correlated with the extent of transformation.