Single-Nucleus Rna-Seq Of Frozen Archival Primary Pancreatic Ductal Adenocarcinoma Uncovers Multi-Compartment Intratumoral Heterogeneity Associated With Neoadjuvant Treatment.

4633 Background: Pancreatic ductal adenocarcinoma (PDAC) remains a treatment-refractory disease and existing molecular subtypes do not inform clinical decisions. Previously identified bulk transcriptomic subtypes of PDAC were often unintentionally driven by “contaminating” stroma. RNA extraction from pancreatic tissue is difficult and prior single-cell RNA-seq efforts have been limited by suboptimal dissociation/RNA quality and poor performance in the setting of neoadjuvant treatment. We developed a robust single-nucleus RNA-seq (sNuc-seq) technique compatible with frozen archival PDAC specimens. Methods: Single nuclei suspensions were extracted from frozen primary PDAC specimens (n = 27) derived from patients with (borderline)-resectable PDAC who underwent surgical resection with or without neoadjuvant chemoradiotherapy (CRT). Approximately 170,000 nuclei were processed with the 10x Genomics Single Cell 3’ v3 pipeline and gene expression libraries were sequenced (Illumina HiSeq X). Results: Distinct nuclei clusters with gene expression profiles/inferred copy number variation analysis consistent with neoplastic, acinar, ductal, fibroblast, endothelial, endocrine, lymphocyte, and myeloid populations were identified with proportions similar to corresponding multiplexed ion beam imaging. Non-negative matrix factorization revealed intra-tumoral heterogeneity shared across patients. Neoplastic cells featured eight distinct transcriptional topics characterized by developmental (epithelial, mesenchymal, endoderm progenitor, neural progenitor) and environmental (anabolic, catabolic, cycling, hypoxic) programs. CAFs exhibited four different transcriptional topics (activated/desmoplastic, myofibroblast, neurogenic, osteochondral). Differential gene expression and gene set enrichment analyses demonstrated that CRT was associated with an enrichment in myogenic programs in CAFs, activation pathways in immune cells, and type I/II interferons in malignant cells. CRT was also associated with a depletion in developmental programs within malignant cells. Conclusions: We uncovered significant intratumoral heterogeneity and treatment-associated differences in the malignant, fibroblast, and immune compartments of PDAC using sNuc-seq. Deconvolution of clinically-annotated bulk RNA-seq cohorts and characterization of intercellular interactions with receptor-ligand analysis and spatial transcriptomics are ongoing.