PROJECT SUMMARY – Core 1: Tumor and Microenvironment Heterogeneity Core The central hypothesis driving efforts of the TMH Core is that immune and other stromal cells in the tumor microenvironment (TME) contribute to both inherent and acquired therapeutic resistance in PDA. Such cells vary phenotypically, functionally, metabolically, and spatially within the TME, creating distinct niches. For example, functionally specialized immune cells (e.g., macrophages, T cells, neutrophils) are common within the TME and can promote or suppress tumor growth. Likewise, cancer associated fibroblasts (CAFs) contribute to extracellular matrix formation, tumor invasion/metastasis, and T cell exclusion, and can be subdivided into myofibroblastic CAFs (myCAFs) and inflammatory CAFs (iCAFs) that differentially affect tumor growth and therapeutic resistance. Thus, characterizing cellular and functional heterogeneity and TME localization, and understanding how they contribute to therapeutic resistance is key to developing effective therapies. Utilizing expertise in single cell transcriptomics, epigenomics, functional assays, and digital spatial profiling (DSP), the Core will interrogate how cellular diversity and localization contribute to therapeutic resistance in PDA. The TMH Core will support the individual Projects in their efforts to interrogate the TME and tumor-TME crosstalk with state-of-art approaches and by providing expertise in genomics, bioinformatics, and immunology. The TMH Core will provide services that are unavailable in existing Salk Institute Cores, and therefore fulfill an unmet need. In Aim 1, the TMH Core will map the spatial heterogeneity of tumor and stromal cells in human PDA via DSP (NanoString) to correlate phenotypes with specific tumor niches. In Aim 2, the Core will functionally characterize immune cells and inflammatory cytokines in the TME via flow cytometry and use Isoplexis IsoSpark for single cell-secretome analysis and multiplex cytokine assays. In Aim 3, the Core will provide an integrated platform of single-cell RNA and ATAC sequencing to define the functional heterogeneity of tumor and stromal cells in PDA and to understand how this heterogeneity contributes to and/or is affected by therapeutic responses. These aims will support the goals of the projects to elucidate mechanisms of therapeutic resistance in PDA through epigenetic changes (Project 1), extracellular signaling (Project 2), and AMPK driven autophagy (Project 3). The TMH Core will collaborate with the Mouse Models Core and the Human Specimen and Organoid Core to design novel experiments and incorporate mouse and human organoid datasets to decipher the role of stromal and tumor cells in PDA resistance to therapy. Moreover, the TMH Core will work closely with the Biostatistics Unit within the Administrative and Biostatistics Core to ensure rigorous state-of-the-art preprocessing and analysis pipelines. In combination with the efforts of the Projects and other Cores, the ...