P09.01 Adapting immune cells to the hypoglycemic tumor microenvironment by solute carrier 2a1 (Slc2a1/GLUT1) overexpression

Background In recent years, T cell-based immunotherapies have shown promising results in hematologic malignancies. However, these strategies seem to be limited in solid cancers, posing more complex challenges including a hostile TME with nutrient deprivation and tissue hypoxia 1. Additionally, metabolic reprogramming has been identified as a crucial factor for proper cytotoxic T-cell functions upon their activation. Such energy demands are answered by the upregulation of glycolysis, oxidative phosphorylation, and upregulation of nutrient transporters represented by SLCs 2,3. Within the TME, tumor and immune cells compete for nutrients and shape a distinct metabolic milieu, resulting in an ineffective effector function 4. Herein, we aim to metabolically engineer T cells to improve their fitness in the glucose-deprived TME and optimize ACT. Materials and Methods We retrovirally overexpressed the glucose transporter Slc2a1/GLUT1 in murine CD8+ T cells (CD8+Slc2a1). To assess T-cell fitness we conducted experiments in physiologic (5mM) and hypoglycemic (0.5mM) media conditions. CellTraceTM-based proliferation experiments and killing assays in the OT1-OVA model are used to examine differences to MOCK in functionality and were analyzed via flow cytometry and microscopy, respectively. Furthermore, Seahorse analyses, bulk RNA-Seq, and metabolomic analyses were performed to examine the mechanical background. Murine in vivo studies are performed to approach the translatability of this system into living organisms. Results CD8+Slc2a1 cells possessed a higher proliferative capacity in all conditions tested but most prominently in hypoglycemic (0.5mM) media. This better functional activity of CD8+Slc2a1 was also translated to higher killing rates in coculture assays with tumor cells, especially in low-glucose environments. Metabolic flux analyses and multi-omics suggested greater metabolic activity of CD8+Slc2a1 and revealed higher ROS production and upregulation of correlating anti-oxidative pathways, especially the pentose-phosphate pathway. Preliminary in vivo studies support the in vitro killing in a syngeneic tumor model. Furthermore, signs of altered memory formation were visible, expressed in a higher proportion of effector memory cells. Conclusions Our data point to the role of GLUT1 overexpression in T cells for improved cytotoxic activity, proliferation, and long-term persistence. Therefore, combinatorial approaches with GLUT1 overexpression could serve as a potential approach to increase efficacy in ACT against solid cancer. We also identified GLUT1-dependent reprogramming in CD8+Slc2a1 cells which is further investigated in ongoing studies. Additionally, we are evaluating the potential risk of this approach to neoplastic formation. References Treating hematological malignancies with cell therapy: where are we now? Landoni E, Savoldo B.; Expert Opin Biol Ther. 2018. Anticancer targets in the glycolytic metabolism of tumors: a comprehensive review; Paolo E. Porporato et al. Frontiers in Pharmacology 2011. Glucose Metabolism on Tumor Plasticity, Diagnosis, and Treatment; Lin Xiaoping et al. Frontiers in Oncology 2020 Fighting in a wasteland: deleterious metabolites and antitumor immunity. Watson MJ, Delgoffe GM. J Clin Invest. 2022. M.E. Kirmaier: None. B.L. Cadilha: None. A. Hadzic: None. W. Schmitz: None. M.R. Benmebarek: None. D. Briukhovetska: None. S. Michaelides: None. V. Buschinger: None. B. Tast: None. C.H. Bönigk: None. S. Oganesian: None. L. Vona: None. A. Tischmacher: None. V. Heissmeyer: None. M. Vaeth: None. V.R. Buchholz: None. M. Eilers: None. M. von Bergwelt-Baildon: None. M. Subklewe: None. S. Kobold: None. S. Theurich: None.