2531 - Harnessing Lactate-Efflux Inhibition to Sensitize Radiotherapy: A Metabolic-Immune Dual-Targeting Strategy for NSCLC

Purpose/Objective(s): Radiotherapy remains a cornerstone in lung cancer treatment, yet radioresistance limits its clinical efficacy. Current strategies predominantly focus on restricting DNA damage repair process, overlooking radiotherapy's role as a metabolic reprogrammer. Although tumor metabolic heterogeneity is known to sustain malignancy and shape microenvironments, the metabolic consequences of radiotherapy remain poorly defined. Our metabolic profiling revealed lactate accumulation in post-irradiation TME. Thus, this study aims to interrogate the roles of lactate-enriched TME in radioresistance and identify targetable metabolic vulnerabilities, thereby exploring metabolism-targeted therapies to achieve radiosensitization.

Materials/Methods: Metabolic perturbations were investigated through metabolomic profiling of tumor interstitial fluid (TIF) collected at 48h post-irradiation. Glycolytic flux was quantitatively assessed via ¹³C₆-glucose-based metabolic flux analysis. Lactate dynamics were assessed through ECAR measurements using Seahorse XF24 system. Protein interaction of MCT4 were deciphered through CO-IP-MS. Mct4-knockout cells were established via CRISPR-Cas9 technology. ScRNA sequencing was conducted on dissociated tumors from subcutaneous Lewis models.

Results: Metabolomic profiling of irradiated tumors revealed a significantly elevation of lactate in TIF. Subsequent functional analyses demonstrated that radiation induces metabolic reprogramming in NSCLC cells, characterized by amplified glycolytic flux, elevated ECAR, and enhanced lactate secretion. Proteomic screening identified MCT4 as the most prominently upregulated glycolytic regulators post-irradiation. Further investigation into mechanism revealed that radiation triggers RAB3B expression, which competes with Hsc70 for binding to MCT4, inhibiting the Hsc70-mediated chaperone-mediated autophagy of MCT4. In vitro, MCT4 blockade attenuated radiation-induced glycolytic potentiation, suppressed lactate efflux, and intensified cytoplasmic acidification, ultimately synergizing with radiation to promote apoptosis. In vivo, Gene depletion of Mct4 reversed lactate-enriched acidic TME. ScRNA-sec analysis demonstrated that Mct4 deficiency suppresses polarization of immunosuppressive N2-neutrophils. Flow cytometry validated that genetic or pharmacological (VB124) inhibition of tumor-derived lactate export significantly reduced N2 neutrophil infiltration, thereby potentiating radiotherapy efficacy.

Conclusion: Our study demonstrates that radiation reprograms tumor metabolism by enhancing glycolytic capacity and driving lactate accumulation in the TME via RAB3B-mediated recycling of MCT4. This lactate surge polarizes neutrophils toward N2 phenotype. Our findings propose that targeting MCT4 exerts dual therapeutic effects: reversing radiation-induced metabolic adaptation and disrupting immunosuppressive neutrophil remodeling.