Abstract 2536: <i>In vivo</i> label-free imaging reveals distinct CD8 T cell metabolic changes during effective radio-immunotherapy in cold murine melanoma versus hot colon carcinoma

Abstract In vivo multiphoton autofluorescence microscopy provides label-free, single-cell imaging of metabolic changes. These metabolic changes are quantified via the metabolic coenzymes NAD(P)H and FAD which are autofluorescent molecules endogenous to all cells. Metabolic reprogramming is a hallmark of cancer. Immune cells also change their metabolism during cancer progression and this metabolic switch correlates to immune cell phenotype and function. Here, we study metabolic changes during administration of a potent radio-immunotherapy regimen in murine melanoma and colon carcinoma. This therapy includes external beam radiation, hu14.18-IL2 immunocytokine (anti-GD2 mAb fused to IL2) or free IL2, and anti-CTLA-4, leading to in situ vaccination and cure of established murine tumors. We created a transgenic mCherry-labeled T cell mouse to study the critical T cell response. We implanted syngeneic B78 (GD2+) melanoma or MC38 (GD2-) colon carcinoma cells into the flanks of mCherry-labeled CD8 T cell reporter mice to induce tumors. Under anesthesia, skin flap surgery was performed, and tumors were imaged at multiple timepoints during therapy. Multiphoton imaging was performed to collect NAD(P)H, FAD, and mCherry signal. Fluorescence lifetime images were collected using time correlated single photon counting electronics. Tissues were also harvested and analyzed via flow cytometry, single-cell RNA sequencing, and multiplex immunofluorescence to corroborate imaging findings and characterize the immune infiltrate. Our in vivo label-free imaging revealed that CD8 T cells from treated versus control tumors exhibit different metabolic phenotypes including changes in NAD(P)H and FAD protein binding and redox balance across these two solid tumors. In cold B78 melanoma tumors, in vivo metabolic imaging revealed that CD8 T cells are activated early but maintain an oxidative metabolic state consistent with exhaustion or a memory phenotype. In hot MC38 colon carcinoma, in vivo metabolic imaging revealed that CD8 T cells are activated early and then quickly transition to a glycolytic state that fuels their cytotoxic function. scRNAseq confirmed the metabolic changes we observed within our B78 model and provided additional insight into CD8 T and tumor cell changes during therapy. Flow cytometry illustrated significant changes in the immune infiltrates of both tumor types over time and treatment. These results show that label-free in vivo imaging enables single-cell quantification of metabolic changes during therapy - across multiple solid tumors. Combined with other traditional assays, we can elucidate key immune cell populations and the crucial timepoints during therapy where changes are occurring. This imaging platform may be leveraged to help develop new immunotherapy combinations and study other cancer types where cures have not been realized. Citation Format: Alexa R. Heaton, Anna Hoefges, Nathaniel J. Burkard, Arika S. Feils, Noah W. Tsarovsky, Garrett M. Lublin, Alina A. Hampton, Samantha K. Burkard, Aurora D'Amato, Mildred A. Felder, Anqi Gao, Huy Q. Dinh, Amy K. Erbe-Gurel, Alexander L. Rakhmilevich, Paul M. Sondel, Melissa C. Skala. In vivo label-free imaging reveals distinct CD8 T cell metabolic changes during effective radio-immunotherapy in cold murine melanoma versus hot colon carcinoma [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 2536.