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Pancreatic ductal adenocarcinoma (PDAC) remains a treatment-refractory disease. Characterizing PDAC by mRNA profiling remains particularly challenging. Previously identified bulk expression subtypes were influenced by contaminating stroma and have not yet informed clinical management, whereas single cell RNA-seq (scRNA-seq) of fresh tumors under-represented key cell types. Here, we developed a robust single-nucleus RNA-seq (snRNA-seq) technique for frozen archival PDAC specimens and used it to study both untreated tumors and those that received neoadjuvant chemotherapy and radiotherapy (CRT). Gene expression programs learned across untreated malignant cell and fibroblast profiles uncovered a clinically relevant molecular taxonomy with improved prognostic stratification compared to prior classifications. Moreover, in the increasingly-adopted neoadjuvant treatment context, there was a depletion of classical-like phenotypes in malignant cells in favor of basal-like phenotypes associated with TNF-NFkB and interferon signaling as well as the presence of novel acinar and neuroendocrine classical-like states, which may be more resilient to cytotoxic treatment. Spatially-resolved transcriptomics revealed an association between malignant cells expressing these basal-like programs and higher immune infiltration with increased lymphocytic content, whereas those exhibiting classical-like programs were linked to sparser macrophage-predominant microniches, perhaps pointing to susceptibility to distinct therapeutic strategies. Our refined molecular taxonomy and spatial resolution can help advance precision oncology in PDAC through informative stratification in clinical trials and insights into differential therapeutic targeting leveraging the immune system. ### Competing Interest Statement A.R. is a co-founder and equity holder of Celsius Therapeutics, an equity holder in Immunitas, and was an SAB member of ThermoFisher Scientific, Syros Pharmaceuticals, Neogene Therapeutics and Asimov. From August 1, 2020, A.R. is an employee of Genentech. T.J. is a member of the Board of Directors of Amgen and Thermo Fisher Scientific. He is also a co-Founder of Dragonfly Therapeutics and T2 Biosystems. T.J. serves on the Scientific Advisory Board of Dragonfly Therapeutics, SQZ Biotech, and Skyhawk Therapeutics. None of these affiliations represent a conflict of interest with respect to the design or execution of this study or interpretation of data presented in this manuscript. T.J. laboratory currently also receives funding from the Johnson & Johnson Lung Cancer Initiative, but this funding did not support the research described in this manuscript. D.T.T. has received consulting fees from ROME Therapeutics, Foundation Medicine, Inc., EMD Millipore Sigma, and Pfizer that are not related to this work. D.T.T. is a founder and has equity in ROME Therapeutics, PanTher Therapeutics and TellBio, Inc., which is not related to this work. D.T.T. receives research support from ACD-Biotechne, PureTech Health LLC, Ribon Therapeutics, which was not used in this work. M.M.K. has served as a compensated consultant for H3 Biomedicine and AstraZeneca and received a research grant (to institution) from Novartis that is not related to this work. The interests of D.T.T. and M.M.K. were reviewed and are managed by Massachusetts General Hospital and Mass General Brigham in accordance with their conflict of interest policies. J.R., D.R.Z., K.F., R.F., and J.B. are employees of NanoString Technologies. A.R., W.L.H., K.A.J., J.A.G., and T.J. submitted a provisional patent application based on this work. All other authors declare no competing interests.

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