Abstract Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal malignancies due largely to their lack of response to current cytotoxic, targeted, and immune therapies. PDAC tumor tissues harbor an expansive, desmoplastic stroma that both suppresses angiogenesis and limits perfusion and diffusion. Consequently, delivery of therapeutic agents through systemic administration is impeded, lowering drug efficacy and increasing general toxicity. Indeed, multiple components of the PDAC stroma support its survival and growth, for example by conditioning a locally immunosuppressed microenvironment that facilitates tumor survival. Conversely, we previously showed that at least some elements of the tumor stromal restrain PDAC growth and progression, for example Hedgehog pathway-responsive myofibroblasts. Early attempts to modulate the PDAC stroma in order to facilitate drug delivery failed upon clinical translation. Post-clinical trials ultimately demonstrated that stromal remodulation upon inhibition of individual pathways can lead to unpredictable consequences in multiple additional cell types. Based on these data, we hypothesize that individual paracrine pathways typically link together to form “paracrine cascades” that propagate through multiple pathways and cell types. We advance that reconstructing these paracrine cascades offers both the opportunity to better understand the consequences of therapeutic intervention and also to infer candidate targets that act on a broad range of cell types within the PDAC TME to enact stromal remodulation. In order to test this, we will make use of a series of innovative systems biology tools built by members of our transdisciplinary team. These include a suite of algorithms leveraging the computational field of regulatory network analysis, as well as technically innovative techniques for studying outcomes in single cell datasets. Moreover, we will acquire unique dataset from samples collected by members of our multidisciplinary clinical service at the Pancreas Center of New York Presbyterian Hospital. These include acquiring human PDAC tumor interstitial fluid and generating matched sets of tumor, normal pancreas, spleen, and blood samples from PDAC patients. We will also routinely utilize fresh PDAC tissue samples to make tumor “explants” a novel ex vivo model system for the short-term, medium throughput study of PDAC. This new model system enables the dissection of complex multi-cellular phenotypes in ways that are not possible through study of intact tumors or co-cultures of purified cell types Using these approaches, we will reconstruct the network of paracrine cascades in PDAC and validate selected candidates experimentally. We will also test a specific candidate pathway uncovered through study of the Hh pathway that connects myofibroblasts to myeloid derived suppressor cells and cytotoxic lymphocytes. We expect that the proposed studies will provide an expansive understanding of paracrine crosstalk...