Failure of traditional therapy in pancreatic ductal adenocarcinoma (PDAC) is due to our limited understanding of how the tumor microenvironment (TME) can facilitate the rapid progression or recurrence of PDAC. In PDAC, the stromal cells identified as cancer associated fibroblasts (CAFs) account for up to 90% of tumor volume. Recent studies have revealed the significance of branched chain amino acids (BCAAs) in cancer. However, the role of stromal cells in support of BCAA metabolism in tumors is still poorly understood. Understanding cancer-stromal ecosystem requires insight into the intersection of cancer- associated transformations in the stroma with reprogramming of their BCAA metabolism. SMAD4 deletion is a frequent event in PDAC. However, the metabolic role of SMAD4-deleted PDAC cells in modulating activation of stromal cell transformation is unknown. Our proposed aims here have aimed to bridge this knowledge gap. We propose a differential BCAA metabolism in cancer and stromal compartments of PDAC tumors and uncover a heavy reliance of PDAC cells on CAF-secreted branched chain keto acids (BCKAs) in stromal- rich tumors. We hypothesize that reactive stromal BCAA metabolism is altered from quiescent stroma and is the driver for regulating PDAC cell growth by secreting BCKAs. Second, SMAD4-deleted PDAC cells induces metabolic alterations in stromal cells. First, we will validate stromal BCKA synthesis metabolic pathway as a lethal target in patient-derived circulating tumor cell (CTC)-organoids and patient-derived tissue slices, using 13C-based isotope tracing and genetic tools and unravel that CAF-secreted extracellular matrix (ECM) is a source for BCKA. To understand the mechanism behind ECM degradation, we will regulate proteasomal proteolysis of ECM in CTC-organoids and patient-derived tissue slices. Second, to elucidate SMAD4-deletion in PDAC cells induced metabolic rewiring of CAFs we have proposed loss-of-function studies and use CTC- organoids and tissue slices to demonstrate increased stromal-reliance of SMAD4-deleted PDAC cells on stromal BCAT1 and elucidate temporal metabolic reprogramming in SMAD4-deleted CTC-organoids over time course of organoid formation. Third, we will test the efficacy of targeting BCKA anabolism in CAFs with conditional knockout mice and perform in vivo studies targeting stromal BCKA anabolic pathway using patient- derived low-passage cell lines and the syngeneic allograft model. Importantly, we will inhibit stromal BCAT1 in the proposed Aims using Gabapentin, Erg 240 (BCAT1 inhibitor from Ergon Pharma), ERG 245 (BCAT1/2 inhibitor from Ergon Pharma), & CRISPR targeting of BCAT1 in CAFs. Notably, our proposed aims will reveal a novel BCAA metabolism-centric regulatory role of reactive stroma in cancers and will uncover the underlying mode of action of this regulation. These findings can lead to novel therapeutics targeting communication between cancer cells and their microenvironment.