L-5--glutamine PET of Breast Cancer: Kinetic Analysis in Mouse Models to Evaluate Glutamine Metabolism

Abstract (250 words) Background: Glutamine addiction is a hallmark of aggressive tumors, yet glutaminase (GLS1) inhibitor CB-839 showed disappointing anti-tumor efficacy in clinical trials. L-5-[11C]-glutamine ([11C]glutamine) PET enables non-invasive assessment of glutamine metabolism in vivo, providing a tool to test mechanistic hypotheses, and identify tumors likely to respond to GLS1 inhibition: focusing on compartmentation of GLS1-derived glutamate, CB-839 impact on flux, and reciprocal glutamine synthesis. Methods: Glutaminolytic TNBC (HCC1806) and poorly glutaminolytic ER+ (MCF-7) xenograft mice with or without CB-839, underwent dynamic [11C]glutamine PET. HPLC quantified fractional radioactivity of [11C]glutamine, soluble metabolites ([11C]glutamate, [11C]CO2), and macromolecule-incorporated metabolites from blood and tumor. A four-tissue compartment model characterized GLS1 activity (k_GLS) and flux, glutamine synthetase activity (k_GS), and subcellular glutamate distribution by comparing single vs. dual glutamate pool models. Averaged tumor curves and HPLC-derived tumor metabolites were fit. Monte Carlo simulations assessed parameter estimation performance. Results: The single glutamate pool model showed high correlations between k_GLS and other parameters, yielding inflated k_GLS estimates. The dual glutamate pool model reduced correlations, improved k_GLS recovery, and yielded subcellular glutamate distributions consistent with in vitro measurements. In TNBC, k_GLS was 3-fold higher than ER+ tumors (non-overlapping 95% CI) with glutamate concentrated in the mitochondrial compartment. CB-839 reduced k_GLS in TNBC and depleted mitochondrial glutamate (non-overlapping 95% CI), though glutaminolytic flux showed no distinguishable change. ER+ tumors showed higher k_GS compared to TNBC. Conclusion: [11C]glutamine PET kinetic analysis reveals distinct glutamine metabolic phenotypes in breast cancer subtypes. Preserved glutaminolytic flux and cytosolic glutamate in TNBC provide mechanistic hypotheses for clinical failure of GLS1 inhibitors, informing ongoing studies.