A bioluminescent ratiometric sensor for the measurement of extracellular ATP concentration in inflammation and cancer.

ATP is not only the key intracellular energy currency but also a ubiquitous extracellular messenger. Large increases in its extracellular concentration occur at sites of infection and inflammation, in the tumor microenvironment and virtually in any disease states. However, the accurate measurement of the extracellular ATP (eATP) is still a crucial issue. We describe here a novel ratiometric bioluminescent indicator suitable to accurately measure the eATP concentration in vitro and in vivo. This sensor, named pmeLUC/nilla, is an evolution of the pmeLUC probe previously developed by us, and allows faithful monitoring of eATP changes, independently of the level of probe expression, thus overcoming a key limitation of existing probes. The pmeLUC/nilla consists of two luciferases expressed as a polycistronic mRNA. PmeLUC, the real ATP sensor, is synthetized into the endoplasmic reticulum, delivered to the cell surface and anchored on the plasma membrane. The Renilla luciferase, which is ATP insensitive, is translated thanks to an internal ribosomal binding site, and remains cytosolic. The luminescence coming from pmeLUC is normalized by the signal from the Renilla luciferase. The performance of the sensors was analyzed in vitro, in different cell types and upon the application of various stimuli. Moreover, its ability to monitor eATP in the tumor microenvironment was assessed in mice. Our findings demonstrate the capability of pmeLUC/nilla for real-time and precise monitoring of eATP dynamics both in vitro and in vivo. This sensor could pave the way for advanced luminescent diagnostics, enhancing our understanding and detection of inflammation and cancer.