ABSTRACT Pancreatic ductal adenocarcinoma (PDA) is characterized by an extensive desmoplastic stroma that represents a major challenge for its effective treatment and improved survival of patients. We seek to define the composition and roles of cancer-associated fibroblasts (CAFs), the most abundant cell population in the PDA stroma, as a potential avenue for the development of new therapeutic strategies. Although CAFs have been historically considered tumor-promoting components, their ablation in pre-clinical and clinical studies have led to mixed outcomes, indicating the poorly understood complexity of CAFs. To investigate CAF biology, we previously established a pancreatic tumor organoid/fibroblast co-culture model. In addition, we performed single cell RNA- sequencing (scRNA-seq) of murine and human PDA specimens to characterize CAF composition at single-cell resolution. These analyses have revealed that fibroblasts are heterogeneous and comprised of at least three distinct subtypes with unique transcriptional and functional features. More so, this heterogeneity emphasizes the need to design therapies that selectively target the tumor-promoting CAF populations. Although our work has started to reveal the complex heterogeneity of fibroblasts in primary or metastatic PDA tissues, iterative developments in scRNA-seq and analysis methods have revealed four additional CAF subtypes in primary and metastatic PDA. To comprehensively define the CAF repertoire in primary and metastatic PDA, we will systematically differentiate CAF subtypes and their regulatory elements using scRNA-seq, scATAC-seq, machine-learning computational methods, and in situ tissue analytic methods that detect RNA and protein species, such as imaging mass cytometry (Aim 1). We hypothesize that a deeper understanding of the dynamic CAF states that occurs in primary and metastatic PDA will guide the selection of specific therapeutic regimens. To complement this analysis, we will identify new CAF subtypes and study their dynamics in a novel murine model with a reversible mutant Kras allele (Aim 2). Furthermore, we will study CAF-activating pathways by investigating several genes implicated in stromal activation that were revealed using our recently developed intraductal transplantation model of PDA (Aim 2). These new mediators appear to have roles in PDA progression, immunosuppression, and stromal activation, and may represent new PDA therapeutic targets. Finally, we have demonstrated different immunomodulatory functions of distinct CAF subtypes. We will test combinatorial strategies to target distinct CAF subtypes in the PDA microenvironment, and study the effect of these strategies on tumor progression in a murine PDA model (Aim 3). In addition, we will assess the role of macrophage-CAF crosstalk in promoting and maintaining CAF identify. Overall, this project will clarify the diversity of PDA CAFs and the role of cancer cells in regulating CAF subtypes. Our results will provide ...