PROJECT SUMMARY/ABSTRACT With a dismal 5-year survival rate of ~8%, pancreatic cancer is projected to become the second leading cause of all cancer-related deaths by 2030. While many therapeutics against pancreatic cancer have been identified, treatment of this disease remains challenging owing to the exceptionally dense and hypovascularized stromal tissue. Therefore, deeper understanding of tumor-stroma interactions in a viscoelastic matrix will facilitate the identification of novel molecular targets against this deadly disease. Polymeric hydrogels capable of recapitulating aspects of the extracellular matrix (ECM) are ideal for studying cancer cell fate as these gels can be engineered with precise biophysical and/or biochemical properties that emulate the tumor ECM. The long- term objective of this project is to use bio-inspired, responsive, and viscoelastic (BRAVE) matrices for elucidating molecular mechanisms governing progression of pancreatic cancer cells (PCCs), as well as for identifying novel molecular targets to improve treatment outcome. In this R01 project, we will develop BRAVE hydrogels composed of functionalized hyaluronic acid and gelatin, as well as collagen and fibronectin for interrogating matrix factors guiding pancreatic cell behaviors. In Aim 1, we will develop the said BRAVE hydrogels to define the synergisms between major matrix components (e.g., HA, FN, and matrix viscoelasticity) on PCC progression. In Aim 2, we will prepare BRAVE hydrogels compatible with high-content assay (HCA) of cell fate processes. We will also design dual-layer BRAVE gels with identical biochemical compositions but spatially graded mechanics for co-culturing PCCs and patient-derived cancer associated fibroblasts (PD-CAFs), the major tumor stromal cells. This platform will provide direct experimental evidence regarding the necessity of a stiffened matrix on cell-cell crosstalk and EMT in PCCs. In Aim 3, we will develop BRAVE gel with gradient stiffness to interrogate PCC migration (i.e., durotaxis) under the influence of various matrix factors (e.g., cytokines, CAFs, and inhibitors). The results will provide insights regarding PCC migration/invasion and potential molecular targets against PCC metastasis. The BRAVE hydrogels and durotaxis device developed in this proposal will allow us to answer many pancreatic cancer relevant questions that are otherwise difficult to address. Furthermore, the outcome of this project will have impact on basic and applied research in other cancers, as well as on directed stem cell differentiation for tissue regeneration.