THE ENGINEERING OF pHLUC, A RATIOMETRIC LUMINESCENT REPORTER FOR MONITORING TUMOR ACIDOSIS IN VIVO

Abstract

Even under normoxia, cancer cells exhibit increased glucose uptake and glycolysis, an occurrence known as the Warburg effect. This altered metabolism results in increased lactic acid production, leading to extracellular acidosis and contributing to metastasis and chemoresistance. Current pH imaging methods are invasive, costly, or require long acquisition times, and may not be suitable for high-throughput pre-clinical small animal studies. This thesis presents a ratiometric pH-sensitive bioluminescence reporter called pHLuc for in vivo monitoring of tumor acidosis. pHLuc consists of a pH-sensitive GFP (superecliptic pHluorin or SEP), a pH-stable OFP (Antares), and Nanoluc luciferase. The resulting reporter produces a pH-responsive green 510 nm emission (from SEP) and a pH-insensitive red-orange 580 nm emission (from Antares). The ratiometric readout (R580/510) is indicative of changes in extracellular pH (pHe). In vivo proof-of-concept experiments with NSG mice model bearing human lung cancer A549 xenografts that stably express the pHLuc reporter suggest that the level of acidosis varies across the tumor. Altogether, this study demonstrates the diagnostic value of pHLuc in reporting pH change in the tumor microenvironment, and indicates the potential of pHLuc in the development and testing of the efficacy of anti-cancer drugs in vivo.