Flux analysis shows that hypoxia-inducible-factor-1-alpha minimally affects intracellular metabolism in tumor spheroids.

Heterogeneous metabolic microenvironments in tumors affect local cell growth, survival, and overall therapeutic efficacy. Hypoxia-inducible-factor-1alpha (HIF-1alpha) is a transcription factor that responds to low-oxygen environments by upregulating genes for cell survival and metabolism. To date, the metabolic effects of HIF-1alpha in three-dimensional tissue have not been investigated. Preliminary experiments have shown that the effects of HIF-1alpha are dependent on glucose availability. Based on this observation, we hypothesized that HIF-1alpha would not affect cell survival and metabolism in the center of spheroids, where the concentrations of oxygen and glucose are low, similar to hypoxic regions found in tumors. To test this hypothesis we used fluorescence microscopy and the tumor cylindroid model to quantify cellular viability in three-dimensional tissue. Isotope labeling and metabolic flux analysis were also used to quantity the intracellular metabolism of wild-type and HIF-1alpha-null spheroids. As hypothesized, cell survival and intracellular metabolism were not different between wild-type and HIF-1alpha-null tissues. In addition, small spheroids, which contain less quiescent cells and are less nutritionally limited, were found to have increased carbon flux through the biosynthetic pentose phosphate and pyruvate carboxylase pathways. These results show how nutrient gradients affect cell growth and metabolism in spheroids and suggest that metabolic microenvironment should be taken into account when developing HIF-1alpha-based therapies.