Hyperpolarized [2– ¹³ C]Pyruvate Identifies a Mitochondria‐Active Hepatocellular Carcinoma Phenotype With Vulnerability to Mitochondrial Inhibition

ABSTRACT

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‐ ¹³ C]pyruvate NMR spectroscopy can identify a mitochondria‐active HCC phenotype and assess its association with sensitivity to mitochondrial metabolic inhibition. HP [2‐ ¹³ C]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‐ ¹³ C]pyruvate. HP [2‐ ¹³ C]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‐ ¹³ C]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.