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Pancreatic ductal adenocarcinoma (PDAC) is projected to be the second leading cause of cancer mortality in the United States by 2030. Given that resistance to cytotoxic therapy is pervasive, there is a critical need to elucidate salient gene expression programs and spatial relationships among malignant and stromal cells in the tumor microenvironment (TME), particularly in residual disease. We developed and applied a single-nucleus RNA-seq (snRNA-seq) technique to 43 banked frozen primary PDAC specimens that either received neoadjuvant therapy (n=25) or were treatment-naïve (n=18). We discovered expression programs across malignant cell and fibroblast profiles that formed the basis for a refined molecular taxonomy, including a novel neural-like progenitor (NRP) malignant program enriched with neoadjuvant treatment in tumors and organoids, and associated with the worst prognosis in bulk profiles from independent cohorts. To elucidate how neoadjuvant treatment and cancer cell- and fibroblast-intrinsic programs modulate the composition of multicellular neighborhoods, we performed spatial profiling with the GeoMx[1] platform (NanoString) on 21 formalin-fixed paraffin-embedded sections using the human whole transcriptome atlas (WTA). Each tumor showed intra-tumoral heterogeneity in tissue architecture and regions of interest (ROIs) with diverse patterns of neoplastic cells, cancer-associated fibroblasts (CAFs), and immune cells were selected for profiling. We deconvolved the WTA data with our snRNA-seq cell type signatures and mapped expression programs onto the tumor architecture to reveal three distinct multicellular neighborhoods, which we annotated as classical, squamoid-basaloid, and treatment-enriched. The observed enrichment in post-treatment residual disease of multiple spatially-defined receptor-ligand interactions and a neighborhood featuring the NRP program, neurotropic CAF program, and CD8+ T cells may open new therapeutic opportunities. Next, we mapped malignant/CAF programs and immune cell subsets at single-cell spatial resolution by performing spatial molecular imaging (SMI[2]; NanoString CosMx) using a panel of 960 RNA targets on a subset of seven tumors (2 untreated, 5 treated) and captured over 200,000 cells with an average of more than 450 transcripts detected per cell. Correlating ROIs from whole-transcriptome DSP to matched fields of view in kiloplex SMI enabled further dissection of PDAC architecture and treatment-associated remodeling of cell type distributions and receptor-ligand interactions. Ongoing functional studies have begun to elucidate the key regulatory elements underlying the distinct treatment-associated NRP malignant program and its interactions with the TME. Overall, the complementary combination of snRNA-seq, whole-transcriptome DSP, and kiloplex SMI provides a high-resolution molecular framework that can be harnessed to augment precision oncology efforts in pancreatic cancer. [1] GeoMx DSP is for Research Use Only and not for use in diagnostic procedures. [2] CosMx SMI is for Research Use Only and not for use in diagnostic procedures. Citation Format: William L. Hwang, Karthik A. Jagadeesh, Jimmy A. Guo, Hannah I. Hoffman, Carina Shiau, Jennifer Su, Payman Yadollahpour, Jason W. Reeves, Youngmi Kim, Sean Kim, Mark Gregory, Prajan Divakar, Eric Miller, Michael Rhodes, Sarah Warren, Erroll Rueckert, Kit Fuhrman, Daniel R. Zollinger, Robin Fropf, Joseph M. Beechem, Arnav Mehta, Toni Delorey, Cristin McCabe, Jaimie L. Barth, Piotr Zelga, Cristina R. Ferrone, Motaz Qadan, Keith D. Lillemoe, Rakesh K. Jain, Jennifer Y. Wo, Theodore S. Hong, Ramnik Xavier, Orit Rozenblatt-Rosen, Andrew J. Aguirre, Carlos Fernandez-Del Castillo, Andrew S. Liss, Mari Mino-Kenudson, David T. Ting, Tyler Jacks, Aviv Regev. Multicellular spatial community featuring a novel neuronal-like malignant phenotype is enriched in pancreatic cancer after neoadjuvant chemotherapy and radiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr SY12-04.