Feasibility of modeling human intraductal papillary mucinous neoplasms of the pancreas using an organoid approach

Abstract

Presenter: Nicholas Peters MD | Yale University School of Medicine Background: Intraductal papillary mucinous neoplasm (IPMN) is a mucin-producing, premalignant cystic lesion arising from the pancreatic ducts that can result in invasive pancreatic adenocarcinoma. The molecular origin of IPMN and etiology of malignant degeneration remains poorly understood, in part due to the lack of an in vitro model for study. Organoid-based systems have emerged as a genetically-tractable approach for tumor study. We sought to assess the feasibility of creating an organoid-based model system for the study of human IPMN. Methods: Patients undergoing surgical resection for presumed IPMN of any histological subtype or category of dysplasia were eligible for study inclusion. Within 30 minutes of resection, fresh aliquots of IPMN and grossly normal pancreatic duct were obtained. Specimens were enzymatically digested to oligo-cell clusters, embedded in solubilized basement membrane (Matrigel), and grown in permissive media promoting enrichment of the ductal epithelial fraction. Creation of spherical organoids capable of sustained growth, defined as 3 sequential culture passages, was defined as a successfully propagated culture. Microscopy, immunohistochemistry (IHC), and genotypic analysis of known highly-penetrant IPMN mutations were then performed on mature organoid cultures when possible. Results: 29 unique cyst wall or adjacent, grossly normal pancreatic ductal tissue sections from 11 patients (range, 1-6 sections/patient) were freshly harvested from surgical pancreatectomy specimens. The source tissues were then subsequently confirmed as IPMN via histologic examination by an experienced pancreatic surgical pathologist. The harvested tissue subjected to enzymatic digestion and culture ranged from 18 – 164mg. Spheroid formation and successful, sustained growth for at least three passages was attained in 11 organoid cultures (38%) as demonstrated by phase-contrast microscopy (Figure 1A). Continuous cultures were sustained in successful isolates from 22 - 385 days (median, 92 days). Genomic DNA was successfully isolated from 10 organoid cultures. Targeted sequencing was completed in 4 samples and revealed KRAS G12x/Q61x or GNAS R201x mutations in 3/3 IPMN samples, and 0/1 adjacent normal samples. To date, IHC analysis of 1 organoid demonstrated recapitulation of pathognomonic IPMN-defining protein expression consistent with the clinical tissue of origin (CDX2, MUC2, MUC5AC shown in Figure 1 B, C, and D, respectively). Conclusion: Creation of sustained human-derived IPMN organoids is feasible and the current data shows recapitulation of key driver mutations and IPMN-specific protein expression in vitro. Future improvements in organoid generation efficiency, durability and characterization will enhance the applicability of this approach.