DiscussionConn Carey1, Alex Sim2, James Hynes1
1Luxcel Biosciences, Cork, Ireland,& 2AMSBIO , Abingdon, , U.K.
Knowledge of oxygen availability and metabolism is key to a detailed understanding of tumour biology. As well as transcriptional responses and genetic instability, tumour hypoxia is known to cause an increase in glycolytic flux despite the availability of oxygen, a phenomenon known as the Warburg effect. However, although control of ambient oxygen concentrations is easily achievable, the key information required is the level of tissue oxygenation and the subsequent relationship between available oxygen and these key metabolic alterations. Such knowledge has been quiet limited due in large part to the lack of in vitro tools capable of measuring these parameters robustly. This is particularly true of 3D models where relative deoxygenation can be even more pronounced. Here we describe a novel fluorometric multiplexed plate-reader assay that facilitates the parallel monitoring of intracellular oxygen (MitoXpress Intra®) and glycolytic flux (pH Xtra™) in the same well. Tumour deoxygenation is monitored using a plate reader equipped with an atmospheric control unit, and an O2 & pH calibration function allowing conversion of fluorescence signal to an O2 scale & a pH scale for each assay. The data presented reveals that significant differences can exist between the oxygen concentration that pertains at the metabolically competent tissue/cells and to the applied ambient oxygen concentration, a difference rarely accounted for in standard in vitro analysis. The impact of metabolic stimulation and inhibition is also examined, as is the effect of oxygen concentration on the glycolytic activity of test cells, allowing any impact on the balance between oxidative and glycolytic ATP generating pathways to be identified. This allows a detailed analysis of the correlation between actual intracellular oxygen concentrations and concomitant alterations to glucose metabolism which is key to a detailed understanding of tumour metabolism.