Intravital Multiphoton Autofluorescence Imaging is Sensitive to Changes in T Cell and Melanoma Tumor Cell Metabolism During Immunotherapy

Background Intravital multiphoton autofluorescence microscopy provides in vivo, 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 of tumor and immune cells is closely associated with cancer progression and cell phenotype.1–3 We aim to study metabolic changes during administration of an effective, triple-combination immunotherapy within murine melanoma tumors.4 This therapy includes external beam radiation, intratumoral hu14.18-IL2 immunocytokine (anti-GD2 mAb fused to IL2), and intraperitoneal anti-CTLA-4 leading to in situ vaccination and cure of GD2+ murine tumors.5 Previous work has shown that a T cell response is critical to the efficacy of this therapy4–5, so we created an mCherry-labeled T cell mouse model to study this response. Here, intravital multiphoton metabolic imaging was used to image concurrent tumor and CD8+ T cell metabolic trends during administration of immunotherapy. Methods We implanted syngeneic B78 (GD2+) melanoma cells into the flanks of mCherry-labeled CD8+ T cell reporter mice (C57Bl/6 background) to induce tumors. Under anesthesia, skin flap surgery was performed and tumors were imaged at several time points during therapy. Multiphoton imaging was performed to collect NAD(P)H, FAD, mCherry, and collagen signal through a 40X objective (figure 1A). Fluorescence lifetime data was collected using time correlated single photon counting electronics. Tissues were harvested and analyzed via flow cytometry and multiplex immunofluorescence to corroborate intravital imaging findings and characterize the immune infiltrate. Results Here we demonstrate that our intravital imaging is sensitive to metabolic changes in both B78 melanoma and CD8 T cells from immunotherapy treated tumors versus control tumors. These metabolic differences include changes in protein binding and redox balance (figure 1B) within treated tumors. We also show remodeling of collagen (figure 1C), a major component of the extracellular matrix, during immunotherapy that may be explained by an observed decrease in macrophages. Flow cytometry and multiplex immunofluorescence illustrate changes in the immune infiltrate composition, activation, and cytotoxicity during therapy (figure 1D). Conclusions These results show that intravital metabolic imaging enables single cell quantification of metabolic changes in tumor and immune cells during therapy. Combined with other traditional assays, we can elucidate key immune cell populations and the crucial timepoints during therapy where changes are occurring. With continued efforts, this imaging platform may be leveraged to develop new combinations of immunotherapies. Acknowledgements This work is supported by the Morgridge Institute for Research (Interdisciplinary Fellowship awarded to A.R.H.) and the NIH (R01 CA205101 and R35 CA197078). The authors thank the University of Wisconsin Carbone Cancer Center (UWCCC) Support Grant P30 CA014520, the UWCCC Translational Research Initiatives in Pathology laboratory – supported by the UW Department of Pathology and Laboratory Medicine and the Office of The Director NIH (S10OD023526), the UWCCC Flow Cytometry Laboratory, and the Genome Editing and Animal Models Laboratory for core services. References Renner K, Singer K, Koehl GE, Geissler EK, Peter K, Siska PJ, Kreutz M. Metabolic Hallmarks of Tumor and Immune Cells in the Tumor Microenvironment. Front. Immunol. 2017, 8 (MAR), 1–11. Mockler MB, Conroy MJ, Lysaght J. Targeting T Cell Immunometabolism for Cancer Immunotherapy; Understanding the Impact of the Tumor Microenvironment. Front. Oncol. 2014, 4 (May), 1–11. Ghesquière B, Wong BW, Kuchnio A, Carmeliet P. Metabolism of Stromal and Immune Cells in Health and Disease. Nature 2014, 511 (7508), 167–176. Morris ZS, Guy EI, Francis DM, Gressett MM, Werner LR, Carmichael LL, Yang RK, Armstrong EA, Huang S, Navid F, Gillies SD, Korman AJ, Hank JA, Rakhmilevich AL, Harari PM, Sondel PM. In Situ Tumor Vaccination by Combining Local Radiation and Tumor-Specific Antibody or Immunocytokine Treatments. Cancer Res. 2016, 76 (13), 3929–3941. Morris ZS, Guy EI, Werner LR, Carlson PM, Heinze CM, Kler JS, Busche SM, Jaquish AA, Sriramaneni RN, Carmichael LL, Loibner H, Gillies SD, Korman AJ, Erbe AK, Hank JA, Rakhmilevich AL, Harari PM, Sondel PM. Tumor-Specific Inhibition of In Situ Vaccination by Distant Untreated Tumor Sites. Cancer Immunol. Res. 2018, 6 (7), 825–834. Ethics Approval All animal work was approved by the University of Wisconsin Institutional Animal Care and Use Committees.