| Popis: |
Background and Aims Despite years of research, mechanisms of pancreatic injury and healing remain poorly understood. Acute pancreatitis is a painful and debilitating condition; chronic pancreatitis may be asymptomatic, but greatly enhances the risk for development of pancreatic ductal adenocarcinoma (PDAC). Acinar to ductal metaplasia (ADM), or the transdifferentiation of digestive enzyme producing acinar cells to ductal cells, is an early event in both conditions. While ADM is thought to function in healing and regeneration under conditions of injury, it also represents a possible first step in tumorigenesis, demonstrating the duplicitous nature of this inherent plasticity. The aims of this study were to identify ADM-derived populations in pancreatic injury and to characterize their role in disease progression. Methods We performed lineage tracing and single cell RNA sequencing (scRNAseq) on injury-induced pancreatic ADM using acinar cell-specific Ptf1aCreERTM/+;RosaYFP/+(CY) mice. We used the bioinformatics techniques Monocle 2, Pseudotime, and p-Creode to assess lineage progression. Results were confirmed using lineage tracing with Ptf1aCreERTM/+;RosaBrainbow2/+ (CBbow) mice. Populations identified by scRNA-seq were confirmed using multiplex immunofluorescence, immunohistochemistry (IHC), and electron microscopy. The role of ADM in pancreatic injury was tested using Aclyfl/fl;Ptf1aCre/+mice and we examined the role of ADM-derived tuft cells in pancreatic injury using Pou2f3fl/fl;Ptf1aCre/+(tuft cell knockout) mice. Weconducted IHC to evaluate the formation of ADMpopulations discovered in mice, in human disease. Results Using lineage tracing and 10x scRNA-seq we identified and characterized over 15,000 single ADM cells from chronically injured CY pancreata. Using known markers, we discovered that ADM results in the formation of significant epithelial heterogeneity, including the formation of mucin-producing cells, tuft cells, and various subtypes of enteroendocrine cells. Monocle 2, Pseudotime, and p-Creode all agree that acinar cells pass through a mucinous (gastric spasmolytic polypeptide expressing metaplasia-like) intermediate before forming either tuft or enteroendocrine cells. Lineage tracing with CBbow demonstrates that ADM cells have the ability to clonally expand. Pancreatic injury in either ADM-impaired Aclyfl/fl;Ptf1aCre/+mice, or tuft cell knockout Pou2f3fl/fl;Ptf1aCre/+mice results in significantly more injury as compared to wild type littermates. Conclusions The carefully orchestrated plasticity in ADM generates myriad epithelial cell types with the ability to mitigate injury, providing protection from the formation of pancreatitis and PDAC. By uncovering the inherent disease suppressive mechanisms of ADM-derived cell types, we can evaluate the therapeutic benefit of co-opting or targeting these pathways in patients. Download : Download high-res image (265KB) Download : Download full-size image |
