Monitoring Glycolysis by Endogenous 31P CEST Magnetic Resonance Imaging

: In this study, we present a novel approach to investigate glycolysis by means of the 3¹P CEST technique applied to phosphate-containing substrates at their endogenous concentration. The method relies on the assessment of the saturation transfer (ST) observed on the 3¹P signals of inorganic phosphate (Pi) or phosphocreatine (PCr) following the selective irradiation of phosphate groups of endogenous molecules exchanging with ATP, Pi, and indirectly with PCr in enzyme-catalyzed reactions. The concentrations of these substrates often fall below the threshold for direct detection. The 3¹P CEST technique amplifies their responses, making them detectable via the ST effect to the 3¹P resonance of the selected reference signal. The method was first validated in vitro on mouse breast adenocarcinoma cell pellets (TS/A), where the intracellular Pi signal was monitored to assess the ST effect associated with the saturation of phosphoester-containing molecules. The use of a glycolysis inhibitor and different experimental temperatures (37 °C or 4 °C) provided insights supporting the rationale behind the method. A comparison of 3¹P Z-spectra was carried out on murine breast cancer cell lines with different degrees of aggressiveness, showing the ability to assess metabolic differences. Finally, in vivo experiments on mice models of mammary adenocarcinoma demonstrated that 3¹P CEST can differentiate tumor and healthy tissue based on their metabolic characteristics.