Media based off the metabolome of tumor interstitial fluid reveals metabolic checkpoints and novel oncometabolites in cancer immunity

Cancer evades immunity by establishing an immunosuppressive tumor microenvironment (TME). The metabolic composition of the TME represents a ‘checkpoint’ on cancer immunity as the local milieu, the tumor interstitial fluid (TIF), contains metabolites which may render T cells metabolically deficient and alter immune function. We employed media based on a metabolomic profile of TIF (Tumor Interstitial Fluid Medium; TIFM) to define how T cells behave under TME metabolic stress alone. We found that passage of T cells post-stimulation in TIFM, both limited T cell expansion and imprinted lasting effector dysfunction that persisted even after extended expansion in metabolically replete conditions. And we identified phosphoethanolamine (pEtn), a Kennedy pathway intermediate that accumulates in the TME in many cancers, as a driver of T cell dysfunction. Here, bulk RNA-seq was performed on OT-I CD8 T cells activated with peptide and cultured either in RPMI based media or RPMI based media supplemented with pEtn. We identified several key immunologic genes altered by pEtn treatment. However, there was no notable differentiation pattern associated with pEtn treatment as determined through enrichment analysis of T cell differentiation gene sets, suggesting these cells did not necessarily seem to differentiate into canonical exhaustion or anergic states. We further found in other studies that pEtn, as a precursor for the membrane phospholipid phosphatidylethanolamine (PE), drove elevated PE:PC ratios and modulated TCR signaling, in part by depleting T cells of diacylglycerol required for TCR signal transduction, thus contributing to the observed T cell dysfunction.