Abstract C055: Activation of oncogenes within Tff2 expressing cells of the pancreatic ductal glands results in increased lineage stemness preceding tumorigenesis: Insights from an inducible mouse model and derived organoids

Abstract

Abstract Introduction: Adult stem cell dysregulation can initiate cancer. Pancreatic ductal glands (PDGs) act as a stem cell niche for the pancreatic main duct epithelium (MD). Previous studies have shown that Tff2+ cells within the PDGs give rise to progeny that migrate to the MD epithelium during inflammation. In human IPMN, the PDGs have been shown to comprise the basal proliferative compartment of these neoplasms. Here we investigate the effect of oncogenic activation and tumor suppressor deletion within Tff2+ PDG cells. Methods: We utilized Tff2CreERT2;Rosa26mT/mG;KrasLSL-G12D;P53fl/fl mice (TTKP) to activate oncogenes in Tff2+ cells and GFP tag this lineage in vivo and ex vivo. Cre activity was induced by tamoxifen injection in TTKP experimental or Tff2CreERT2;Rosa26mT/mG (TT) control mice. Pancreata were collected at pre-determined time points post-tamoxifen with GFP tagged, Tff2 lineage cells quantified and compared between groups at each timepoint. For ex vivo experiments, ductal cells were isolated from tamoxifen-naïve TTKP pancreata via magnetic-assisted cell sorting, grown in 3D culture and induced via 4OHT. GFP+ cells were sorted, cultured, and orthotopically implanted in athymic nude mice. Tumors were collected before reaching 20mm in diameter. Spatial RNA sequencing (spRNAseq) was performed on frozen tissue sections and analyzed with R package Seurat. Cross-species analysis utilized human PDAC scRNAseq reference datasets. Results: TTKP mice developed lineage-traced PDAC eight weeks after tamoxifen induction. In vivo lineage tracing showed a significant increase of GFP+ Tff2-lineage cells in PDGs and MD between TTKP and TT mice at all analyzed time points (4-, 6- and 8-weeks post tamoxifen, ≥ 3 mice each). GFP-positive PDGs exhibited progressive PanIN-like changes before PDAC formation. Tff2-PDG cells in TT mice function as transient amplifying cells, expanding within PDGs and to the MD during inflammation and contracting upon resolution. Oncogenic activation (sans inflammation) in Tff2-PDG cells led to sustained GFP+ cell presence in PDGs and MD, indicating increased stem-like behavior preceding neoplasia. Orthotopic organoid experiments resulted in GFP+ tumor formation. SpRNAseq analysis indicated enrichment of "Ductal cell type 2" in TTKP tumors, particularly overlying the main duct epithelium. Conclusion: Our study demonstrates that dysregulation of the Tff2-lineage in PDGs can initiate PDAC. Oncogenic activation in Tff2+ cells increases their stemness as indicated by lineage expansion in PDGs and MD preceding PanIN and PDAC formation. Orthotopic organoid implantation experiments further validate the tumor-initiating capacity of Tff2+ ductal cells. Spatial transcriptomics analysis confirms the presence of similar cell types identified in human scRNAseq studies within TTKP tumors. These findings strongly support the role of PDGs as a potential niche of origin for human PDAC and provide additional insights into an under-studied disease initiating process. Citation Format: Kyle L. McAndrews, Pinaki Mondal, Dulce Maroni, Dongdong Wang, Michael A. Hollingsworth, Sarah P. Thayer. Activation of oncogenes within Tff2 expressing cells of the pancreatic ductal glands results in increased lineage stemness preceding tumorigenesis: Insights from an inducible mouse model and derived organoids [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C055.