Wenying Li, Jiannan Chen, Jiayi Li, Shuai Wang, Zhengliang Chen, Lianfeng Zhao, Yaoyao Zhao, Lili Gu, Jiaqi Liu, Yan Zhang, Xinhao Yang, Tianyu Chen, Zhigang Guo
BACKGROUND: Chimeric antigen receptor (CAR)-T cell therapy has demonstrated remarkable success in hematologic malignancies; however, its efficacy in solid tumors remains limited. A major barrier is the immunosuppressive tumor microenvironment (TME), which is characterized by hypoxia and nutrient deprivation, leading to impaired CAR-T cell proliferation, persistence, and cytotoxic function. To address these barriers, we designed a dual-regulatory CAR-T strategy that integrates hypoxia-responsive control with metabolic enhancement to improve therapeutic efficacy in solid tumors. METHODS: to drive sustained CAR expression under hypoxic conditions. Concurrently, we overexpressed the glutamine transporter SLC38A2 to enhance glutamine uptake and metabolic fitness in nutrient-deprived environments. RESULTS: Compared with conventional CAR-T cells, our engineered CAR-T cells exhibited superior antitumor activity under hypoxia and nutrient stress, with enhanced proliferation, elevated memory phenotype, and reduced exhaustion markers. Mechanistically, quantitative PCR demonstrated upregulation of glutamine metabolic and glycolytic pathways, while Seahorse assays confirmed enhanced oxidative phosphorylation and glycolysis. SLC38A2 knockout reversed these enhancements, highlighting its role in sustaining CAR-T metabolic fitness. CONCLUSION: Our findings establish SLC38A2 as a critical metabolic regulator that enhances CAR-T antitumor efficacy, providing a promising strategy to improve the durability and efficacy of CAR-T cell therapies in TME.