Abstract B019: GABA signaling in CAFs as a novel regulator of pancreatic cancer immunosuppression

Abstract

Cancer-associated fibroblasts (CAFs) are one of the major cell types that populate the stroma of PDAC tumors and are extremely immunosuppressive because they produce large amounts of inhibitory cytokines, growth factors and metabolites. Thus, understanding how CAFs impart severe immunosuppression in the tumor microenvironment is critical to make PDAC amenable to immunotherapies. Herein, our group uncovered that the neurotransmitter gamma-aminobutyric acid (GABA), is present in PDAC patient tumors, and acts as an immunosuppressive factor, promoting tumor growth. Through a novel tumor interstitial fluid (TIF) isolation protocol, we detected GABA at ∼25µM, among around 150 polar metabolites, and GABA was significantly more concentrated in TIF when compared with patient matched plasma, highlighting that GABA likely exerts its effects on the local tumor microenvironment. Furthermore, with heavy isotope tracing of glucose and pyruvate, we found that CAFs generated GABA de novo. Lastly, using a large single cell RNAseq ATLAS, we profiled ∼65,000 fibroblasts across 135 PDAC tumors, and found that CAFs expressed all non-canonical GABA synthesis enzymes, as well as GABA receptors. Thus, we hypothesized that GABA was promoting tumor growth in PDAC through its recently established immunosuppressive action on anti-tumor immune cells. Using a novel 3D culturing method, we demonstrate that CAFs express all components of non-canonical GABA synthesis (ARG/ODC1/OAT/DAO/ALDH1A1), as well as GABAA and GABAB receptors, as determined by western blotting. Additionally, CAFs produced significantly more GABA than normal pancreatic fibroblasts isolated from donor tissue. Normal and cancer associated fibroblasts treated with GABA had increased production of TGFβ, IL8, and IL6 cytokines, in a dose dependent manner. Mechanistically, genetic and pharmacological blockade of ALDH1A1 reduced GABA production in CAFs during 3D growth. In parallel, we began to analyze the effect of GABA on downstream signaling pathways. We found that treating CAFs with GABA modulated the expression of p-p70, p-JNK, p-MTOR and p-AKT proteins in CAFs, key signaling hubs in PDAC. Therapeutically, treatment with pharmacological inhibitors against ALDH1A1 and GABAB Receptor 1 in orthotopic PDAC models significantly decreased tumor burden and increased infiltration of anti-tumor CD8 T-cells and natural killer cells. Overall, we show different ways in which modulation of GABA synthesis through CAFs can influence the immunosuppressive profile observed in PDAC. Moving forward, we will further dissect the importance of GABA signaling in the PDAC tumor microenvironment in spontaneous models of PDAC, combined with conditional KO of GABA production enzymes and receptors in mice. It is our hope that the insights gained from this study can lead to a better understanding of the influence neurotransmitter signaling has on immunosuppression in PDAC, as well as generate new targeted therapies to improve the efficacy of immunotherapies against this devasting disease.