PROJECT SUMMARY/ABSTRACT Pancreatic ductal adenocarcinoma (PDA), is a lethal malignancy, due in part to a dense, desmoplastic (fibrotic) tumor microenvironment which aids tumor growth and inhibits drug delivery. Poor survival rates, harsh treatments, and tumor intrinsic aspects of the disease such as pain and altered cytokine pools, promote high levels of anxiety in PDA patients. Nearly 25% of pancreatic cancer patients are prescribed benzodiazepines (BZDs) to treat anxiety, insomnia, or to relieve nausea. Epidemiological studies indicate that BZDs may increase the risk of cancer development but the role of BZDs in modifying the tumor microenvironment is unknown. Recent studies indicate that n-unsubstituted BZDs promote the signaling of the proton-sensing G protein-coupled receptor, GPR68, under acidic conditions, such as those present in the PDA tumor microenvironment. GPR68 is overexpressed by PDA cancer-associated fibroblasts (CAFs), and its expression drives desmoplasia, fibrosis, and inflammation, microenvironmental features associated with impaired drug delivery and chemoresistance. To establish clinical relevance, covariate adjusted analyses was conducted of pancreatic cancer patients who received chemotherapy at Roswell Park from 2004 to 2020. Patients receiving lorazepam (LOR), a strong GPR68 activator, had significantly decreased progression-free survival (PFS) relative to non-users (HR 3.83 (1.53,9.57)), while patients receiving alprazolam (ALP), a GPR68 non-activator, had significantly improved PFS (HR 0.38 (0.16-0.92)). Preliminary studies using subcutaneous allografts derived from KPC mice, a genetically engineered mouse model of PDA, indicated that LOR promoted collagen deposition, desmoplasia, and ischemic necrosis in vivo, supporting that this BZD may promote an unfavorable tumor microenvironment that could negatively impact PDA patient survival. Additionally, LOR-treated CAFs had increased expression of pro- inflammatory and pro-fibrotic genes, suggesting that CAFs may be driving the observed phenotype. These findings led to the central hypothesis that GPR68-activating BZDs modulate CAF signaling, which will increase desmoplasia, subsequently constricting the tumor vasculature. The long-term goal is to determine the impact of BZDs on the PDA tumor microenvironment and chemotherapeutic efficacy. This hypothesis will be tested with two aims. Aim 1 will determine the impact of LOR/ALP on the PDA vasculature and desmoplasia in tumor-bearing KPC mice, using imaging and histological methods. Aim 2 will evaluate the role of BZDs in modifying CAF signaling and activity. Changes in the expression/secretion of pro-fibrotic proteins, inflammatory cytokines, and GPR68 downstream signaling molecules by BZD-treated immortalized CAFs will be quantified. Alterations in PDA organoid proliferation and collagen contraction by BZD-treated CAFs will also be quantified. This research is clinically significant because it will indicate if BZDs commonly...