Abstract 2439: The importance of cellular oxygenation measurements in the analysis of hypoxia-induced signaling and related metabolic adaptation

Abstract

Abstract There is a growing appreciation that typical cell culture conditions reflect a hyperoxic condition for the majority of cell types. In an effort to more accurately reflect the in vivo condition, a growing number of researchers are conducting in vitro measurements at what are deemed to be more physiologically relevant oxygen concentrations (1-8% O2). The importance of oxygen concentration in in vitro model design is underscored by observations that O2 levels influence a wide range of cellular phenomena, including metabolic poise, differentiation and senescence while the development of localised hypoxic environments are implicated in a variety of diseases including stroke, and cancer. Impaired O2 supply can reduce O2 concentrations to below normal physiological levels, initiating a graded series of complex interconnected adaptive responses mediated by regulators including the hypoxia-inducible factor (HIF). These processes are particularly relevant within solid tumours, where oncogene-driven proliferation causes nutrient and oxygen deprivation, aberrant angiogenesis, and the activation of O2-sensitive survival pathways. Despite the importance of cellular oxygenation, research using in vitro models makes the assumption that oxygenation can be defined by simply altering the oxygen condition applied to the in vitro model. To test this key assumption, cellular oxygenation measurements are performed using a nanoparticulate intracellular oxygen probe (MitoXpress®-Intra), while tumour hypoxia/oygenation is modelled on a fluorescence plate reader equipped with an atmospheric control unit (CLARIOstar®). Data is presented illustrating that the depth of hypoxia experienced by the cell model is impacted significantly by respiratory activity, and that this additional oxygen deprivation is a dynamic process, effected by respiratory substrate availability and related metabolic poise. Data is also presented demonstrating that, in in vitro models, additional respiration-induced hypoxia directly impacts glycolytic flux and other relevant metabolic pathways. Together these data invalidate the assumption that cellular oxygenation can be inferred from ambient oxygen measurements. If ignored, the significant and dynamic deviations between ambient O2 and oxygenation at the cellular level leads to erroneous collusions regarding the relationship between oxygen concentration, HIF stabilisation and related metabolic adaptations. This in turn can impair the physiological relevance of experimental observations. Citation Format: Michelle Potter, James Hynes, Anke Nijhuis, Conn Carey, Christos Zois, Adrian Harris, Hector Keun, Karl Morten. The importance of cellular oxygenation measurements in the analysis of hypoxia-induced signaling and related metabolic adaptation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2439.