Regulation of growth saturation and development of necrosis in EMT6/Ro multicellular spheroids by the glucose and oxygen supply

Abstract

To investigate the effects of glucose and oxygen on spheroid growth, EMT6/Ro mouse mammary carcinoma cell spheroids were cultured in suspension in either 0.28 mM (20%) or 0.07 mM (5%) oxygen and 16.5, 5.5, 1.7, and 0.8 mM glucose. The spheroids initially grew at the same exponential rate in all culture conditions, with spheroid volume and cell number doubling times of 20-24 h. The growth rates slowed as the spheroids grew, and the maximum volume and cell number attained at growth saturation were proportional to the oxygen and glucose concentrations in the medium. There was a 500-fold difference in saturation sizes comparing spheroids cultured in the highest oxygen and glucose concentrations to those grown in the lowest. The thickness of the viable cell rims was also positively correlated with the oxygen and glucose concentrations in the medium. Comparison of the growth saturation and viable cell rim data showed an excellent correlation between the onset of central necrosis and the cessation of spheroid growth. A model is presented to explain the observed spheroid growth characteristics by proposing a competition between externally supplied growth and viability-promoting factors and internally generated inhibitory factors produced by the process of necrosis. This model has critical implications for the use of spheroids as models of cellular growth in tumors.