Real time detection of extracellular ATP in tumor microenvironment.

Background: The tumor microenvironment plays a critical role in tumor initiation, progression and invasion. Nucleotides, such as ATP, have a surprisingly wide range of modulatory effects on tumor cells, however techniques for measuring ATP in the extracellular environment are still rudimentary. Here we show an in vivo method for measuring extracellular ATP release in tumor microenvironment. Methods: We stably expressed pmeLUC, a chimeric luciferase targeted to the extracellular side of the plasma membrane, in HEK293 cell lines. A stable cell clone (HEK293-pmeLUC) was then injected anesthetized nude/nude mice bearing or not a tumor. Bioluminescence was then monitored by IVIS imaging system (Xenogen). Results: Endovenous, intraperitoneal and subcutaneous inoculation of HEK293-pmeLUC cells in healthy nude mice caused a modest, barely detectable, increase in luminescence. On the contrary, bioluminescence observed after injection of the probe into mice bearing the OVCAR-3 human ovarian carcinoma or the MZ2-MEL human melanoma increased strongly. We then validated HEK293-pmeLUC cells as ATP reporters using the ATP-hydrolyzing enzyme, potato apyrase. Apyrase injection cause a significative drop in luminescence. We then performed an in vitro calibration which revealed that ATP in the tumor interstitium is in the hundrend micromolar range, while it is basically undetectable in healthy tissues. Our approach offers a new tool for the investigation of the biochemical composition of tumor milieu and for development of novel therapies based on the modulation of extracellular purine-based signalling.