Abstract 5989: Metabolic heterogeneity of the tumor microenvironment
Authors: Reinfeld Bradley I. , Madden Matthew Z. , Ali Ahmed , Wolf Melissa M. , Young Kirsten L. , Hongo Racheal , Beckermann Kathryn E. , Heiden Matthew G. Vander , Muir Alex , Rathmell Jeffrey C. , Rathmell W. Kimryn 
Published: September 2020

Abstract
Background: Single cell technologies have provided insight into the cellular and phenotypic heterogeneity of the tumor microenvironment (TME). However, major technical hurdles exist to elucidate the metabolic fate of a given metabolite in the cellularly diverse TME.
Methods: By collecting tumor interstitial fluid from human kidney tumors and matched normal tissue, our group has gained valuable insight into the metabolic content of the Renal Cell Carcinoma microenvironment and the normal kidney parenchyma. Given the high concentration of glucose in both of these tissue environments (&gt;4 mM), we hypothesized that infiltrating immune cells and transformed cells can use this widely available sugar as a fuel source. To test this hypothesis, we labeled glycolytic cells in the tumor microenvironment using the widely clinically adopted F18 fluoro-2-deoxyglucose (FDG) Positron Emission Tomography (PET) tracer. By conducting successive magnetic bead separation, highly enriched immune cell and non-immune cell fractions were analyzed for radioactive, transcriptomic and metabolic analysis.
Results: This work has led to the intriguing discovery that infiltrating CD45+ immune cells are more actively glucose consuming than CD45- tumor cells on a per cell basis. Additionally, even though these cells make up a minority of the population of the entire TME (&lt;15%), they contribute to &gt; 65% of the total glucose uptake in the tumor microenvironment. Subsequent metabolic studies have shown that they are also more actively glycolytic. This phenomenon holds true across multiple orthotopic tumor models in multiple mouse genetic backgrounds. Further characterization of this glycolytic CD45+ fraction illustrates that the CD11b+, F4/80+ tumor associated macrophages (TAM) fraction is the most avid. Additionally, the tumor infiltrating T cells have much higher FDG avidity than the naïve splenocytes and surprisingly the transformed cancer cells. In support of this data, ex vivo seahorse analysis using these fractions demonstrates that the most metabolically active fraction is TAM-rich F4/80+ fraction. Infiltrating CD45+ cells appear to be metabolically fixed, as demonstrated by their lack of glycolytic reserve when compared to transformed tumor cells in the glycostress test. In order to examine the immunogenicity of F18 based tracers, we have traced the fate of F18-Glutamine (Q). Interestingly, the F18-Q accumulates to a much greater extent in the CD45- cells (on a per cell basis), illustrating the anaplerotic need of cancer cells.
Conclusion: Ultimately, this work reveals the nutrient environment of the tumor interstitial space at a resolution not previously revealed, and uncovers the diversity of metabolic utilization across cell types in the heterogenous tumor microenvironment.
Citation Format: Bradley I. Reinfeld, Matthew Z. Madden, Ahmed Ali, Melissa M. Wolf, Kirsten L. Young, Racheal Hongo, Kathryn E. Beckermann, Matthew G. Vander Heiden, Alex Muir, Jeffrey C. Rathmell, W. Kimryn Rathmell. Metabolic heterogeneity of the tumor microenvironment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5989.