Abstract 4543: Low dose radiation enhanced antitumor immune response by reprogramming T cell and CAR-T cell metabolism in a tumor immune microenvironment

Abstract Background: The infiltration of T cells and chimeric antigen receptor T (CAR-T) cells into solid tumors is essential for robust antitumor response and therapeutic outcomes. While cell therapies succeeded in hematologic malignancies, their efficacy in solid tumors is limited due to poor tumor penetration and the immunosuppressive tumor immune microenvironment (TIME). Our previous studies showed that combining low-dose radiotherapy (LDRT) with T-cell therapy synergistically enhanced tumor control and survival. Nanostring metabolic gene analysis indicated that LDRT modulates T-cell metabolism within TIME. Therefore, we hypothesized that LDRT enhances T-cell metabolism, favoring anticancer potency. Methods & Results: To investigate the role of LDRT in T-cell metabolism, we established a TIME using co-culture of cancer cells and PBMCs in xenograft NSG mice, followed by LDRT. Using nCounter Metabolic Pathways, we quantified the expression of genes associated with core metabolic and immune metabolic processes in intratumoral T cells. Nanostring analysis revealed key pathways and genes that regulate T-cell effector functions, further underscoring LDRT's impact on T-cell metabolism. Seahorse assays highlighted that LDRT positively influenced the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), favoring improved metabolic and effector functions in T/ CAR-T cells. Mitotracker analysis revealed that LDRT-treated T/ CAR-T cells exhibited enhanced mitochondrial membrane potential (Mitotracker Deep Red) and mitochondrial mass (Mitotracker Green), suggesting robust mitochondrial health and functionality. Transmission electron microscopy (TEM) showed improved mitochondrial morphology of T cells following LDRT. Additionally, Incucyte live-cell analysis demonstrated that CAR-T cells treated with LDRT exhibited significantly enhanced cancer cell killing (P ≤ 0.001) compared to controls. CAR-T cells conditioned with LDRT showed increased tumor cell killing in vivo, further validating the potentiating effect of LDRT on cell-based therapies. These findings collectively highlight the pivotal role of LDRT in modulating T-cell metabolism and enhancing antitumor immune responses. Conclusion: The study highlights the translational potential of combining LDRT with cell therapies in solid tumors. By reprogramming T-cell metabolism within TIME, LDRT significantly enhanced tumor control and improved survival outcomes, establishing a promising foundation for metabolism-targeted therapies. These findings provide compelling evidence to support further exploration and clinical investigation of this synergistic approach, aiming to optimize the efficacy of cell-based immunotherapies and improve outcomes for patients with challenging solid tumor malignancies. Citation Format: Zahid Rafiq, Puebla Nahum Osorio, Weiqin Lu, James Welsh. Low dose radiation enhanced antitumor immune response by reprogramming T cell and CAR-T cell metabolism in a tumor immune microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4543.