Hyperpolarized [2-¹³C]Pyruvate Identifies a Mitochondria-Active Hepatocellular Carcinoma Phenotype With Vulnerability...

Hepatocellular carcinoma (HCC) exhibits metabolic heterogeneity that is not fully characterized by glycolysis-focused spectroscopic profiling. This study investigated whether in vitro hyperpolarized (HP) [2-¹³C]pyruvate NMR spectroscopy can identify a mitochondria-active HCC phenotype and assess its association with sensitivity to mitochondrial metabolic inhibition. HP [2-¹³C]pyruvate NMR spectroscopy was used to evaluate mitochondrial metabolism in McA-RH7777 HCC cells, with N1S1 cells serving as a glycolysis-dominant reference. Cell viability following treatment with the glutaminase inhibitor BPTES and the mitochondrial metabolic inhibitor CPI-613 was assessed by MTT assay, and metabolic changes following CPI-613 treatment were further evaluated using HP [2-¹³C]pyruvate. HP [2-¹³C]pyruvate demonstrated enhanced pyruvate-to-glutamate conversion in McA-RH7777 cells, whereas N1S1 showed minimal glutamate labeling. CPI-613 treatment resulted in a dose-dependent reduction in cell viability, while BPTES produced limited effects. Although pyruvate-to-glutamate conversion did not significantly decrease following CPI-613 treatment, pyruvate-to-lactate conversion increased, indicating metabolic adaptation. These findings demonstrate that HP [2-¹³C]pyruvate enables functional identification of a mitochondria-active HCC phenotype characterized by enhanced pyruvate-to-glutamate conversion. This approach may facilitate metabolic subtype classification, help identify tumors susceptible to mitochondrial metabolic inhibition, and enable non-invasive monitoring of treatment-induced metabolic adaptation.