PROJECT SUMMARY Tumor cells and infiltrating immune cells co-evolve during the course of tumor progression, with the immune system progressively losing its efficacy as tumors advance. Immune cells are highly dependent on cellular metabolism to manifest their effector functions, but in the tumor microenvironment (TME), ‘metabolic competition’ with rapidly proliferating cancer cells leads to nutrient deprivation alongside increased metabolic waste, both of which negatively impact immune cell function. Moreover, ‘metabolic symbiosis’ between cancer and immune cells can promote the acquisition of suppressive immune cell phenotypes, ultimately disfavoring anti-tumor immunity. These dynamic metabolic interactions are shaped by the niches occupied by cells within the TME. Thus, the metabolic cross talk between tumor and immune cells over the course of tumor progression can be a major determinant of immune cell function and, consequently, response to immune-targeted therapies. Unfortunately, the study of immune cell metabolism and crosstalk in the TME has been very challenging due to reliance on methods that analyze metabolism in bulk cell populations, which obscures individual cellular diversity, difficulties in predicting downstream outcomes of metabolic perturbations because of the complexity of the metabolic network, and the lack of tools to map metabolic alterations in situ. To overcome these challenges, we developed Compass, a flux balance analysis (FBA) algorithm that applies a network-based analytical approach to single-cell RNA-sequencing (scRNA-seq) data to predict metabolic states of individual cells in tissue1. We have applied Compass as well as standard computational methods to the analysis of longitudinal scRNA-seq data from a pre-clinical murine model of melanoma with the goal of determining the temporal- and tumor-size- based metabolic alterations in both tumor and infiltrating immune cells during tumor progression. Our preliminary data indicate that polyamine metabolism is a key hub of metabolic crosstalk between tumor and immune cells that are either static or dynamic over the course of melanoma tumor progression and that may occupy distinct tissue niches. We find that CD4+ regulatory T cells (Treg), exhausted CD8+ T cells, and suppressive myeloid cells upregulate spermine/spermidine acetyltransferase (Sat1), which catalyzes acetylation of polyamines, with tumor progression. Conversely, a subset of c-Met+ melanoma cells that has features of stemness is high for polyamine recycling genes. Based on these observations, we hypothesize that that systems-based analysis of the alterations and crosstalk involving polyamine metabolism in tumor and immune cells during tumor progression will uncover novel means for therapeutic intervention. We propose to: 1) Dissect the functional role of polyamine metabolism in immune cells and tumor cells during tumor progression; 2) Construct a high resolution spatial map of tumor:immune metabolic crosstalk via the polyamine pathway.

NARRATIVE STATEMENT Cancer is a significant cause of morbidity and mortality worldwide. The immune system has the capacity to fight cancer, but it becomes disabled as cancer progresses. The proposed studies will advance our knowledge of how tumor cells and immune cells influence each other’s metabolism over the course of tumor progression with the goal of devising new anti-cancer therapies.