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Current methods to monitor cellular ATP do not provide spatial or temporal localization of ATP in single cells in real time or they display imperfect specificity to ATP. Here, we have developed a single cell, Enhanced Acceptor Fluorescence (EAF)-based ATP biosensor to visualize ATP in real time. This biosensor utilizes a modified mimic of the ε-subunits of the Bacillus subtilis F(0)F(1) synthase and is coupled to the EAF fluorophores pairs, GFP and YFP. The sensor was then used to monitor ATP in a heterogeneous glioblastoma multiform cancer cell population. We anticipate that this innovative technology and our better understanding of the ATP machinery will have substantial influence on future translational studies.

Gliomablastoma multiforme (GBM) is the most aggressive of brain cancers in humans. Response to current therapies remains extremely poor, with dismal survival statistics. Recently, the endoplasmic reticulum UDPase, ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5), was identified as a key component in the Akt/phosphatidylinositol 3-kinase/phosphatase and tensin homolog regulatory loop, capable of synergizing aerobic glycolysis and cancer cell proliferation in vitro. Utilizing a novel enhanced acceptor fluorescence-based single-cell adenosine 5'-triphosphate (ATP) biosensor, we analyzed ENTPD5-mediated modulation of cytosolic ATP. Here, ENTPD5-dependent modulation of cellular ATP in GBM results in altered metabolic kinetics in vitro, increasing the catabolic efficiencies of aerobic glycolysis and fatty acid oxidation. Additionally, an upregulation of ENTPD5 in both GBM mouse xenografts and in GBM patient tumors was identified, resulting in dramatically reduced survival. Therefore, these results not only provide new tools to monitor ATP flux and cellular metabolism kinetics but also identified a novel therapeutic target for GBM.

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