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

Oxygen (O) tension plays a key role in tissue function and pathophysiology. O-controlled cell culture, in which the O concentration in an incubator's gas phase is controlled, is an indispensable tool to study the role of O . For this technique, it is presumed that the incubator setpoint is equal to the O tension that cells experience (., pericellular O). We discovered that physioxic (5% O) and hypoxic (1% O) setpoints regularly induce anoxic (0.0% O) pericellular tensions in both adherent and suspension cell cultures. Electron transport chain inhibition ablates this effect, indicating that cellular O 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 tensions, we developed a reaction-diffusion model that predicts pericellular O tension . This model revealed that the effect of cellular O consumption is greatest in smaller volume culture vessels ( 96-well plate). By controlling pericellular O tension in cell culture, we discovered that MCF7 cells have stronger glycolytic and glutamine metabolism responses in anoxia . hypoxia. MCF7 also expressed higher levels of , , , . and lower levels of , , . in response to hypoxia . 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 . anoxia. Collectively, these results reveal that breast cancer cells respond non-monotonically to low O, suggesting that anoxic cell culture is not suitable to model hypoxia. We demonstrate that controlling atmospheric O tension in cell culture incubators is insufficient to control O in cell culture and introduce the concept of .