Abstract A081: Single-cell transcriptomic analysis of organoids derived from normal pancreata reveals epithelial cell heterogeneity and expression of PanIN-associated genes

Abstract Pancreatic intraepithelial neoplasia (PanIN) lesions are the microscopic pre-malignant precursor to pancreatic ductal adenocarcinoma (PDAC). Animal models have demonstrated that PanINs arise from acinar cells which have undergone acinar-to-ductal metaplasia (ADM), but the exact molecular mechanisms which govern this transformation remain poorly understood. Novel in vitro models of PanIN genesis are necessary to better characterize pancreatic carcinogenesis and identify clinically actionable biomarkers of early disease. Organoids cultured from normal human pancreata have generated promise in this regard, but the cellular identity and malignant potential of their component cells have not been comprehensively characterized. We performed single-cell RNA sequencing (10X Genomics) on 6 normal pancreatic organoid lines maintained in culture for 3 weeks as well as their parent pancreata obtained from donors with no history of PDAC or other exocrine pancreatic pathology. Integration of whole pancreatic and organoid data using the R package Seurat generated a UMAP with clearly defined “ductal” and “acinar” clusters based on marker gene expression. The majority of organoid cells (47.3-81.5%) colocalized to the “ductal” cluster, and expressed SOX9, KRT19, and PDX1. However, a subset of cells (1.0-33.3%) present in each organoid sample aligned with the “acinar” cluster and expressed PRSS1, PRSS3, REG3A, MUC1, and SPINK1. In accordance with prior reports, organoid cells did not meaningfully express the acinar/endocrine markers PTF1A, CPA1, CHGA, or INS. Further examination of the “acinar” organoid cells revealed heightened expression of CLDN18, TFF1, TFF2, CTSE, CA2, and LYZ, genes associated with PanIN in prior spatial RNA sequencing studies. Other genes significantly enriched in this “acinar” organoid cluster include MUC5AC, a known PanIN marker, OLFM4, a tumor suppressor implicated in intestinal carcinogenesis, and the chemokine CXCL17, which is expressed in cells undergoing ADM. Overall, this data suggests that organoids derived from normal pancreata represent a heterogeneous population of epithelial cells with both acinar and ductal features. Furthermore, these organoids may represent an in vitro model of ADM and/or early PanIN formation, though additional functional mechanistic studies are required for confirmation. Citation Format: Alexander W Bray, Eileen S Carpenter, Timothy L Frankel, Marina Pasca di Magliano. Single-cell transcriptomic analysis of organoids derived from normal pancreata reveals epithelial cell heterogeneity and expression of PanIN-associated genes [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A081.