Cellular energetics measured by phosphorous nuclear magnetic resonance spectroscopy are not correlated with chronic nutrient deficiency in multicellular tumor spheroids

We have measured the 31P nuclear magnetic resonance spectra of EMT6/Ro multicellular tumor spheroids over a wide range of sizes under constant nutrient conditions which matched those used for culturing the spheroids. The amount of nucleotide triphosphate per cell decreased with spheroid growth, roughly in proportion to the decrease in cell volume. There was no correlation between the intracellular pH, the nucleotide triphosphate:Pi ratio, or the phosphocreatine:Pi ratio and either the spheroid cellularity, the mean cell volume, the S-phase fraction, the clonogenic capacity, or the amount of central necrosis. The phosphoryethanolamine:phosphorylcholine ratio also increased with increasing spheroid size. There was a negative correlation between the phosphoryethanolamine:phosphorylcholine ratio and the S-phase cell fraction or the mean cell volume; this ratio was positively correlated with the extent of central necrosis. The membrane degradation components glycerophosphorylcholine and glycerophosphorylethanolamine showed no significant changes with increasing spheroid size. These results imply that spheroid necrotic areas induced by chronic nutrient deficiencies are "invisible" to 31P nuclear magnetic resonance and that the development of cellular quiescence in spheroids is not caused by a decrease in the steady-state level of high-energy phosphates or a reduced intracellular pH. Together, these data support a model in which cells maintain normal steady-state levels of high energy phosphates until they are very close to necrotic cell death. This implies that the deterioration of 31P nuclear magnetic resonance spectra of tumors with increasing size is not caused by chronic nutrient deficiencies resulting from cells outgrowing the capillary supply, but rather is more related to transient nutrient deprivation phenomena.