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Pancreatic ductal adenocarcinoma is a lethal disease with limited treatment options and poor survival. We studied 83 spatial samples from 31 patients (11 treatment-naïve and 20 treated) using single-cell/nucleus RNA sequencing, bulk-proteogenomics, spatial transcriptomics and cellular imaging. Subpopulations of tumor cells exhibited signatures of proliferation, KRAS signaling, cell stress and epithelial-to-mesenchymal transition. Mapping mutations and copy number events distinguished tumor populations from normal and transitional cells, including acinar-to-ductal metaplasia and pancreatic intraepithelial neoplasia. Pathology-assisted deconvolution of spatial transcriptomic data identified tumor and transitional subpopulations with distinct histological features. We showed coordinated expression of TIGIT in exhausted and regulatory T cells and Nectin in tumor cells. Chemo-resistant samples contain a threefold enrichment of inflammatory cancer-associated fibroblasts that upregulate metallothioneins. Our study reveals a deeper understanding of the intricate substructure of pancreatic ductal adenocarcinoma tumors that could help improve therapy for patients with this disease. A multi-omic analysis of pancreatic cancer identifies spatially resolved, heterogeneous cell populations including transitional cell types. Analysis of primary samples identifies treatment-related changes in cross-talk between tumor and stromal cells.

KRAS, SMAD4, and GNAQ, were associated with differential phosphosignaling and metabolic responses compared to wild type. Single cell subtyping discovered 12 of 21 tumors with mixed basal and classical features. Trefoil factor family members were upregulated in classical populations, while the basal populations showed enhanced expression of mesenchymal genes, including VIM and IGTB1. Acinar-ductal metaplasia (ADM) populations, present in 95% of patients, with 46% reduction of driver mutation fractions compared to tumor populations, exhibited suppressive and oncogenic features linked to morphologic states. We identified coordinated expression of TIGIT in exhausted and regulatory T cells and Nectin receptor expression in tumor cells. Higher expression of angiogenic and stress response genes in dendritic cells compared to tumor cells suggests they have a pro-tumorigenic role in remodeling the microenvironment. Treated samples contain a three-fold enrichment of inflammatory CAFs when compared to untreated samples, while other CAF subtypes remain similar. A subset of tumor and/or ADM-specific biomarkers showed differential expression between treatment groups, and several known drug targets displayed potential cross-cell type reactivities. This resolution that spatially defined single cell omics provides reveals the diversity of tumor and microenvironment populations in PDAC. Such understanding may lead to more optimal treatment regimens for patients with this devastating disease. HIGHLIGHTS 1. Acinar-ductal metaplasia (ADM) cells represent a genetic and morphologic transition state between acinar and tumor cells. 2. Inflammatory cancer associated fibroblasts (iCAFs) are a major component of the PDAC TME and are significantly higher in treated samples 3. Receptor-ligand analysis reveals tumor cell-TME interactions through NECTIN4-TIGIT 4. Tumor and ADM cell proteogenomics differ between treated and untreated samples, with unique and shared potential drug targets Competing Interest Statement The authors have declared no competing interest. Footnotes * ↵¶ Lead contact