55 Renal Cell Carcinoma Tumor Interstitial Fluid Possesses Immunosuppressive and GPCR Activating Properties

Vollständiger Abstract

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Abstract Background The tumor microenvironment (TME) is a niche composed of cellular and fluid compartments. Tumor interstitial fluid (TIF) is the liquid milieu surrounding malignant, immune, and stromal cells within the TME, and its metabolic composition alongside its impact on immune cell function, is largely understudied. Metabolites themselves can act in the extracellular environment as signaling ligands, regulating activation of many common protein families such as G protein-coupled receptors (GPCRs), which play an underexplored role in antitumor CD8 immunity. Renal cell carcinoma (RCC) serves as an ideal model system for understanding the impact of TIF on infiltrating immune cells, in part due to large tumor masses, documented responses to immunotherapy, and known metabolic alterations due to VHL loss and HIF-2α overactivation. Methods Using human RCC tumors alongside patient-matched non-malignant renal tissue, we carried out targeted and untargeted four-mode metabolomics on TIF and non-malignant kidney interstitial fluid (KIF), yielding readouts of 2,000+ metabolites. In addition to this, we carried out in vitro culture experiments of human peripheral blood mononuclear cell (PBMC)-derived T cells and monocyte-derived macrophages with patient TIF and KIF. Lastly, to interrogate signaling landscape changes induced by TIF compared to KIF, we utilized the PRESTO-TANGO (Kroeze et al., Nat Struct Mol Bio, 2015) GPCR screening system to determine if human RCC cell line conditioned media from 10 primary cell lines (representing our tumor interstitial fluid samples) were capable of activating 29 GPCRs selected for their expression on tumor-infiltrating T cells in RCC. Results Partial least squares discriminant analysis of data revealed separation of fluid groups, indicating that tumor and kidney interstitial fluid had distinct metabolic compositions. FDR-corrected paired t-test analysis indicated several significant metabolite alterations in TIF vs. KIF, in both valine metabolism and gamma-aminobutyric acid (GABA) signaling. KEGG-based over-representation analysis combined with pathway topology analysis indicated major alterations in metabolic pathways, including several amino acid biosynthesis pathways (tryptophan, phenylalanine). In vitro culture of TIF with CD28/3-stimulated T cells led to reduced IFNγ (p = 0.0072) and TNFα (p = 0.1029) secretion, alongside reduced proliferative capacity via CellTrace Violet staining (p = 0.0131). Culture of monocyte-derived macrophages with TIF increased expression of immunosuppressive markers such as CD206 and MerTK, and reduced antigen presentation and costimulatory machinery such as CD86 and HLA-DR expression. Results from PRESTO-TANGO screening revealed activation of approximately 12 GPCR family members, including chemokine receptor family members (CXCR2, CCR8), purinergic P2Y receptors (P2RY8, P2RY10), and several others. In addition to this, multiple GPCRs with shared ligand specificity were consistently activated in this screen, such as GPR174 and P2RY10, which are both activated by phosphatidylserine. Conclusions Our data highlights an underappreciated and underreported ability for TIF to suppress immune cell activation and function. This is potentially accomplished via changes in the metabolite population, driving changes through differential GPCR activation compared to that of KIF. Future studies will focus on mechanistic drivers of these changes, and how changes in the GPCR activation landscape of RCC can be therapeutically targeted. DOD CDMRP Funding yes