Enhancing CAR-T Cell Metabolic Fitness and Memory Phenotype for Improved Efficacy against Hepatocellular Carcinoma

The persistence of chimeric antigen receptor (CAR) T cells in the tumor microenvironment limits their antitumor effects against solid tumors. Many studies have reported that the <i>in vitro</i> phenotype and metabolism of CAR-T cells correlates with their <i>in vivo</i> antitumor activity. Herein, we constructed PD-1 scFv-secreting and CD133-specific CAR-T (referred to as CAR-T) cells based on our previous work. We found that suitable concentration metformin-treated CAR-T (mCAR-T) cells exhibited an increased memory phenotype and demonstrated stronger and faster antitumor abilities with a reduced exhaustion phenotype. Using RNA sequencing, transmission electron microscope, and metabolic analysis, we discovered enhanced mitochondrial biogenesis and metabolism in CAR-T cells treated with 10 μM metformin, is associated with increased peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1α) expression, promotion of signal transducer and activator of transcription (STAT)3 and inhibition of STAT5 phosphorylation. This resulted in enhanced antitumor effects of mCAR-T cells in both subcutaneous and orthotopic xenograft models. Importantly, in some relapsed hepatocellular carcinoma (HCC) patients, high CD133 expression was observed in their paired primary or metastatic tumor sections. Our study revealed that enhancing metabolic fitness and central memory by <i>in vitro</i> metformin treatment is an effective strategy to improve the efficacy of CAR-T cell therapy, potentially benefiting some relapsed HCC patients.