3-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 cul tures 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 cell ular growth, proliferation, cell viability and oxygenation was investi gated using spontaneously immortalized (Rat1) and c-myc-transfected (M 1) Fisher 344 rat embryo fibroblasts and a tumorigenic T24Ha-ras-trans fected clone of each (Rat1-T1 and MRI). Spheroid volume growth curves and [H-3]thymidine autoradiographs clearly demonstrated that spheroids better reflect the degree of tumorigenicity in vivo as opposed to mon olayer cultures. Studies on Rat1 and M1 aggregates showed that the pot ential for tumor formation of Rat1 cells might be manifested in vitro as an increased capability of the cells to survive in 3D culture. pO(2 ) measurements confirmed that neither cell quiescence nor cell death i n the pseudo-normal cell aggregate types is due to an oxygen deficienc y. In contrast, depletion of oxygen coincided with necrotic cell death in Rat1-T1 spheroids and proliferation arrest in MR1 cultures. Cell-l ine-specific attributes in 3D culture that were not specifically relat ed to ros transfection of the cells included histological structure, d evelopment of necrosis and thickness of viable cell rim. However, grow th behavior, proliferation characteristics and their association with the oxygen supply might be correlated with the extent of transformatio n. (C) 1996 Wiley-Liss, Inc.