1359 A tumor metabolism-angiogenesis-immune axis impacts the response to cancer immunotherapy

<h3>Background</h3> Metabolites in the tumor microenvironment (TME) can affect phenotype and function of stromal cells, including blood vessels and immune cells. This suggests that tumor metabolic rewiring can impact crucial pro-oncogenic processes – such as angiogenesis and immune evasion – in a way that is mechanistically linked. Here, we tested the hypothesis that correcting abnormal angiogenesis based on the tumor metabolic state improves anti-tumor immunity and response to immunotherapy. <h3>Methods</h3> We used metabolic variants of the murine triple negative breast cancer model 4T1 generated by LDHA knock-down (LDHA-KD; glycolysis-low) or by low-dose ethidium bromide treatment to eliminate mitochondrial DNA (Rho0; mitochondria-defective) in comparison with 4T1 cells bearing a scramble shRNA as negative control (NC).¹ These models were validated by Seahorse live cell metabolic assays, LDH assay, and LDHA western blot.^{1 2} We orthotopically implanted these tumor variants in syngeneic Balb/c mice and characterized the tumor vasculature and immune compartment during tumor progression using flow cytometry, multiplex immunofluorescence staining, RNA-sequencing, and Evans Blue extravasation assays. We compared the response of these models to immune checkpoint blockade therapy +/- vasculature-normalizing agents (VEGFR2 blocking or LTBR agonist antibodies)^{3 4} as neoadjuvant treatments before primary tumor resection. Since our tumor models express constitutive firefly luciferase (Fluc) and hypoxia-inducible surface-bound Gaussia luciferase (exGLuc),² we used FLuc and exGluc bioluminescence imaging (BLI) to monitor respectively tumor progression, metastasis formation, and tumor hypoxia. <h3>Results</h3> Metabolic rewiring in LDHA-KD (glycolysis-defective) and Rho0 (mitochondria-defective) tumors resulted in changes in (1) lympho-angiogenic potential, (2) <i>in vivo</i> tumor vasculature structures and functionality, and (3) immune infiltration. Specifically, LDHA-KD tumors displayed the most significant vasculature normalization effect and T-cell infiltration increases, associated with long-lasting complete remissions upon neoadjuvant CTLA-4 blockade immunotherapy. We found that these features are mechanistically linked, as treatment with vasculature-normalizing low-dose anti-VEGFR2³ restored anti-CTLA-4 responses in aggressive glycolysis-high 4T1-NC tumors. Rho0 tumors also showed reduced blood vessels and leakiness <i>in vivo</i>. However, this did not correspond to increased immune infiltration, or decreased tumor hypoxia (probably due to constant lactate production by Rho0 cells that solely rely on glycolysis to produce energy). Promoting the generation of high endothelial venules with LTBR agonism⁴ before surgical tumor resection enhanced survival in mice bearing Rho0 but not LDHA-KD or NC tumors. <h3>Conclusions</h3> The tumor metabolic state specifically shapes the tumor vasculature architecture, with consequences on immune infiltration and response to immunotherapy. Correcting vasculature defects based on the tumor metabolic state can restore immunosurveillance and overcome immunotherapy resistance. <h3>Acknowledgements</h3> We thank the Bristol Myers Squibb International Immuno-Oncology Network for supporting this work. <h3>References</h3> Zappasodi R,<i> et al</i>. CTLA-4 blockade drives loss of treg stability in glycolysis-low tumours. <i>Nature</i> 2021;<b>591</b>:652–658. Serganova I, <i>et al</i>. LDH-A regulates the tumor microenvironment via HIF-signaling and modulates the immune response. <i>PLoS One</i> 2018;<b>13</b>. Huang Y,<i> et al</i>. Vascular normalizing doses of antiangiogenic treatment reprogram the immunosuppressive tumor microenvironment and enhance immunotherapy. <i>Proc Natl Acad Sci U S A</i>. 2012;<b>109</b>(43):17561–6. Asrir A, <i>et al</i>. Tumor-associated high endothelial venules mediate lymphocyte entry into tumors and predict response to PD-1 plus CTLA-4 combination immunotherapy. <i>Cancer Cell</i> 2022;<b>40</b>:318–334.