Proliferative and clonogenic heterogeneity of cells from EMT6/Ro multicellular spheroids induced by the glucose and oxygen supply.

The culture of EMT6/Ro multicellular tumor spheroids in a 20-fold range of glucose concentrations and a 4-fold range of oxygen tensions had profound effects on the cell subpopulations which developed in the spheroids. As the spheroids increased in diameter the rate of accumulation of cells with a G1-phase DNA content was much greater for those spheroids cultured in the lower oxygen and glucose concentrations. Growth fractions of 600- to 800-micron-diameter spheroids, calculated from either DNA content or [3H]thymidine labeling data, showed a direct correlation with the glucose concentrations in the culture medium. The cells from spheroids cultured in low glucose concentrations also showed a more rapid loss in clonogenicity as a function of increasing spheroid diameter. Lowering the oxygen tension enhanced these effects of reduced glucose concentrations. Selective dissociation of 600- to 800-micron-diameter spheroids demonstrated that both the proliferation index and the clonogenic capacity of the spheroid cells decreased with increasing depth into the spheroid cell rim. The rates of decrease of both of these parameters as a function of depth into the spheroid were greater for spheroids cultured in the lower oxygen and glucose concentrations. These results indicate that both the glucose and oxygen supply are critical in the development of nonproliferating and nonclonogenic cell subpopulations in spheroids.