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Background Lactate produced by glycolytic tumor cells can drive pro-oncogenic processes, including neo-angiogenesis and immune evasion. We investigated whether correcting tumor vascular abnormalities restores anti-tumor immunity and response to immune checkpoint blockade depending on the tumor glycolytic state.Methods We used LDHA knock-down (LDHA-KD; glycolysis-defective) and scramble shRNA control (Sc; glycolytic) metabolic variants of murine 4T1 breast cancer and B16F10 melanoma models. 1 2 Tumors were orthotopically implanted in syngeneic mice and treated with anti-CTLA-4 therapy +/- anti-VEGFR2.3 4 Tumor growth, metastasis, vasculature, and immune infiltration were assessed by flow cytometry, immunofluorescence staining, and immunoassays. To study the impact of tumor glycolysis and vasculature normalization on T-cell recirculation, we used photoconvertible KAEDE mice5 and measured T-cell egress from the tumor microenvironment to draining lymph nodes (dLNs). Vasculature, metabolic, and immune signatures were scored and integrated to estimate outcomes in human TCGA data.Results Metabolic rewiring in LDHA-KD tumors corresponded to improved vascular architecture and increased T-cell infiltration. Endothelial cells (ECs) and angiogenic pericytes (PCs) were decreased, and quiescent PCs and MECA-79+ high endothelial venules (HEVs) increased in LDHA-KD versus Sc tumors. LDHA-KD tumor vasculature had greater PC coverage and reduced leakiness. Moreover, soluble VEGF-A and Ang-2 were decreased, while VEGF-C and VEGF-D increased in LDHA-KD tumors, suggesting lymphangiogenic remodeling. Accordingly, CD8+ T-cell egress to dLNs was higher in LDHA-KD tumors, indicating functional lymphatic drainage. Vascular normalization in LDHA-KD tumors was associated with enhanced responses to anti-CTLA-4. We hypothesized that vasculature targeting could restore similar responses in glycolytic tumors. Indeed, low-dose anti-VEGFR2 3 combined with anti-CTLA-4 improved tumor vasculature and immune infiltration in glycolytic tumors, reducing circulating tumor cells and achieving complete remissions in the neoadjuvant setting. Mechanistically, the combination increased intratumoral MECA-79+ HEVs, which recruited CD62L+CD8+ T cells developing into cytotoxic GzmB+VEGFR2+CD62L+CD44+CD8+ effector cells in tumors.Notably, the same combination therapy diminished the vascular and immune benefits of LDHA-KD tumors. By leveraging the human TCGA data set, we found that glycolysis correlates with abnormal vasculature and immune evasion and that these features together predict poor outcomes across aggressive tumor types.Conclusions Our data indicate that the tumor glycolytic state controls vascular architecture and immune infiltration. We identified novel cooperative effects of anti-VEGFR2 with anti-CTLA-4 therapy occurring in glycolysis-high but not glycolysis-defective tumors, including vascular normalization, HEV formation, and increased infiltration and activation of memory CD8+ T cells. This suggests that tailoring anti-angiogenic and immunotherapy combinations to the tumor glycolytic state may restore immune surveillance and overcome resistance.References Zappasodi R, et al. CTLA-4 blockade drives loss of Treg stability in glycolysis-low tumours. Nature 2021;591:652–658.Serganova I, et al. LDH-A regulates the tumor microenvironment via HIF-signaling modulates the immune response. PLoS One 2018;13.Huang Y, et al. Vascular normalizing doses of antiangiogenic treatment reprogram the immunosuppressive tumor microenvironment and enhance immunotherapy. PNAS 2012;109(43):17561–6.Asrir A, et al. Tumor-associated high endothelial venules mediate lymphocyte entry into tumor and predict response to PD-1 plus CTLA-4 combination immunotherapy. Cancer Cell 2022;40:318–334.Steele MM, et al. Quantifying leukocyte egress via lymphatic vessels from murine skin and tumors. J Vis Exp. 2019 Jan 7:(143).