Project Summary Pancreatic ductal adenocarcinoma (PDA) is a deadly malignancy, and only 8% of patients diagnosed with this cancer survive 5 years after diagnosis. The poor outcomes of patients with PDA are attributed to the advanced stage of disease at the time of diagnosis and the lack of effective therapies, highlighting the need for new approaches. PDA is characterized by an abundance of extracellular matrix (ECM) around the cancer cells, referred to as a desmoplastic reaction. The desmoplasia provides a niche for different types of stromal cells, including immune cells and cancer-associated fibroblasts (CAFs), which are derived primarily from pancreatic stellate cells (PSCs). PDA CAFs have recently been found to display heterogeneity with distinct phenotypes and spatial distributions: myofibroblastic CAFs (myCAFs) express high levels of α-smooth muscle actin (αSMA) and are in direct physical contact with cancer cells, while inflammatory CAFs (iCAFs) are farther away from tumor cells and secrete inflammatory cytokines. Although this finding has provided opportunities for new therapeutic approaches, their interaction with other cell types within the immunosuppressive, tumor-promoting microenvironment of PDA requires further study. Tumor-associated macrophages (TAMs) are prominent in PDA tumors and are known to have an immunosuppressive function, making them an attractive therapeutic target. However, the mechanisms through which TAMs signal to and impact the biology of iCAFs and myCAFs remains to be explored. This study aims to understand the crosstalk between macrophages and the two CAF subtypes and how these cell populations fluctuate during PDA progression. My preliminary studies show that PSCs promote the proliferation and alternative activation of macrophages in vitro. In addition, in a genetically engineered mouse model (GEMM) of pancreatic cancer, PanIN lesions (precursors of PDA) display an abundance of both αSMA+ fibroblasts and alternatively activated macrophages in close proximity to each other. These results suggest that the fibroblasts and macrophages signal to each other, contributing to the CAF and macrophage phenotypes in PDA. To identify factors responsible for such crosstalk, I will use an innovative, three- dimensional triple co-culture system that combines pancreatic ductal organoids, PSCs, and macrophages. To better understand how the CAF subtypes impact tumor progression and macrophage phenotype in vivo, I will engineer iCAF- and myCAF-conditional knockout mice and perturb each subtype in an orthotopically transplanted organoid mouse model. Ultimately, the identification of iCAF/myCAF and macrophage dynamics and interactions will illuminate the role these cells play in the disease, opening avenues for new and much- needed therapeutic targets.