Introduction: The formation of nutrient-restricted environment is a way tumors can escape immune surveillance. Detailed evidence on the availability and spatial distribution of various nutrients and their metabolites within the tumor microenvironment (TME) as well as their effect on antitumor immune responses are still lacking. Insights into the role of metabolism in tumor development and progression are being used to design new drug targets and cancer therapies. The objective of this study was to explore the TME immuno-metabolic landscape that was recently coined to depict that both types of cells (tumor and immune) largely depend on and compete for the same nutrients. To do so, we developed a workflow to detect and quantify cellular metabolism using a multimodal platform allowing T-lymphocytes (LTs) infiltration staining and label free metabolic hallmarks histological localization and quantification.

Methods: Melanoma D10 cell line was transplanted in immunodeficient (NSG) mice, followed by injection of MART1 TCR transduced CD8 T cells when the tumors reached 200 mm3. Tumors were then harvested, measured and snap frozen at day 0 (control without T cells), 2, 7 and 25 post-T cell injection. 1,5-DAN matrix was sprayed onto tumor sections using the TM Sprayer (HTX Technologies, LLC). Data acquisition was performed using 7T MALDI-FTICR (Bruker Daltonics, Germany) at 50 µm spatial resolution. Acquired data were treated with MultimagingTM software (ImaBiotech). LTs immunostaining was applied on adjacent sections and histological regions were selected prior molecular image overlay, for metabolites relative quantity extraction.

Results: First, the tumor volume was measured showing an increase until 1500 mm3 for the control group, compared to the CD8 injected group (less than 400 mm3). Then, molecular imaging analysis highlighted the molecular distribution of different amino acid, nucleotide, energetic, hexosamine and TCA cycle metabolites. In parallel, CD3-staining showed lymphocytic infiltration starting from day 7 with extensive areas at day 25. After histological and molecular images overlay, metabolites level was measured from different regions. Potential correlation between metabolites/nutrients abundance and tumor/immune cells presence was studied. A decrease of Malate, N-acetylglucosamine, GSH and GSSG was noticed in tumor cells when their level was stable or increasing in LT immune cells. Ornithine and Histidine were increasing in tumor when decreasing in LT immune cells. Finally, ADP, AMP, Inosine, UMP, UDP-N-acetylglucosamine, Phenylalanine and Tyrosine level was stable in tumor when changing in LT cells.

Conclusion: For the first time, we localized and quantified the cellular metabolism of the tumor in its complexity. The combination of all these data extended our understanding on the melanoma D10 TME metabolic hallmarks that could be followed to assess the interplay between tumor and immune cells.

Citation Format: Lauranne Poncelet, Pierre Levy, Rima Ait-Belkacem, Maarten Ligtenberg, Daniel Peeper, Jonathan Stauber. Tumor and CD8 T cells metabolism and consumption in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4557.