Controlling pericellular oxygen tension in cell culture reveals distinct breast cancer responses to low oxygen tensions

Oxygen (O(2)) tension plays a key role in tissue function and pathophysiology. O(2)-controlled cell culture, in which the O(2) concentration in an incubator’s gas phase is controlled, is an indispensable tool to study the role of O(2) in vivo. For this technique, it is presumed that the incubator setpoint is equal to the O(2) tension that cells experience (i.e., pericellular O(2)). We discovered that physioxic (5% O(2)) and hypoxic (1% O(2)) setpoints regularly induce anoxic (0.0% O(2)) pericellular tensions in both adherent and suspension cell cultures. Electron transport chain inhibition ablates this effect, indicating that cellular O(2) consumption is the driving factor. RNA-seq revealed that primary human hepatocytes cultured in physioxia experience ischemia-reperfusion injury due to anoxic exposure followed by rapid reoxygenation. To better understand the relationship between incubator gas phase and pericellular O(2) tensions, we developed a reaction-diffusion model that predicts pericellular O(2) tension a priori. This model revealed that the effect of cellular O(2) consumption is greatest in smaller volume culture vessels (e.g., 96-well plate). By controlling pericellular O(2) tension in cell culture, we discovered that MCF7 cells have stronger glycolytic and glutamine metabolism responses in anoxia vs. hypoxia. MCF7 also expressed higher levels of HIF2A, CD73, NDUFA4L2, etc. and lower levels of HIF1A, CA9, VEGFA, etc. in response to hypoxia vs. anoxia. Proteomics revealed that 4T1 cells had an upregulated epithelial-to-mesenchymal transition (EMT) response and downregulated reactive oxygen species (ROS) management, glycolysis, and fatty acid metabolism pathways in hypoxia vs. anoxia. Collectively, these results reveal that breast cancer cells respond non-monotonically to low O(2), suggesting that anoxic cell culture is not suitable to model hypoxia. We demonstrate that controlling atmospheric O(2) tension in cell culture incubators is insufficient to control O(2) in cell culture and introduce the concept of pericellular O(2)-controlled cell culture.