Fluorescence lifetime-based pH mapping of tumors in vivo using genetically encoded sensor SypHerRed

Changes in intracellular pH (pHi) reflect metabolic states of cancer cells during tumor growth and dissemination. Therefore, monitoring of pHi is essential for understanding the metabolic mechanisms that support cancer progression. Genetically encoded fluorescent pH sensors have become irreplaceable tools for real-time tracking pH in particular subcellular compartments of living cells. However, ratiometric readout of most of the pH probes is poorly suitable to measure pH in thick samples ex vivo or tissues in vivo including solid tumors. Fluorescence lifetime imaging (FLIM) is a promising alternative to the conventional fluorescent microscopy. Here, we present a quantitative approach to map pHi in cancer cells and tumors in vivo, relying on fluorescence lifetime of a genetically encoded pH sensor SypHerRed. We demonstrate the utility of SypHerRed in visualizing pHi in cancer cell culture and in mouse tumor xenografts using fluorescence lifetime imaging microscopy and macroscopy. For the first time to our knowledge, the absolute pHi value is obtained for tumors in vivo by an optical technique. In addition, we demonstrate the possibility of simultaneous detection of pHi and endogenous fluorescence of metabolic cofactor NADH, which provides a complementary insight into metabolic aspects of cancer. Fluorescence lifetime-based readout and red-shifted spectra make pH sensor SypHerRed a promising instrument for multiparameter in vivo imaging applications.