Investigation of pancreatic tumorigenesis by derivation of organoids, their immortalization, and genetic modification in vitro

• Applicant: Dr. Michael Johannes Pflüger
• Subject Area: General and Visceral Surgery
• Term: from 2020 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 445331015

• Final Report Year: 2023

Final Report Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease with about 90% of patients dying within the first five years after diagnosis. An important reason for this dismal prognosis is late detection. Surgical resection plays a key role as it allows for tumor removal in patients with localized PDAC in the absence of distant metastasis and forms the basis for longterm survival. However, eligibility to undergo surgery requires early detection. It is critical to understand the molecular alterations in the early stages of pancreatic tumorigenesis and to comprehend the mechanisms behind invasion and metastasis to identify diagnostic and potentially therapeutic targets. Therefore, our goal was to prospectively model pancreatic tumorigenesis by stepwise insertion of pancreatic driver gene mutations in healthy human ductal epithelial cells and recapitulate the tumorigenic process, entertaining latest techniques of threedimensional cell culture and gene editing in vitro. In our experiments, we were able to process normal human pancreatic duct tissue and successfully isolate epithelial cells. Subsequently, the cells were propagated in a threedimensional matrix as organoids and lentivirally transduced with the goal to immortalize them and to allow for enrichment for subsequent targeted modification of pancreatic driver genes using CRISPR/Cas9. These experiments required strict selection criteria for patients, were dependent on the supply of freshly resected specimens from the operating room, and demanded daily maintenance of the cells. Given the impact of the unprecedented COVID-19 pandemic on lab access and surgical volume, we were forced to set up other innovative projects which allowed us to work independently from these limitations. As such, we decided to investigate the early genetic changes during pancreatic tumorigenesis in precursor lesions, particularly in intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystic neoplasms (MCNs). These precursors have the potential to progress to PDAC. Early interference with their neoplastic progression through surgical resection can prevent development of invasive disease and/or allow for resection of early-stage PDAC. Academically, these lesions enable valuable insights into the molecular changes of pancreatic tumorigenesis and allowed us to pursue our overarching project goal of exploring the effects of early genetic alterations during pancreatic tumorigenesis. Using laser-capture microdissection of fresh-frozen paraffin-embedded (FFPE) tissue and subsequent targeted next-generation sequencing (tNGS) and whole exome sequencing (WES), we were able to improve the genetic characterization of these lesions. Moreover, our findings have large translational relevance with important implications for the clinical management of these precursor lesions. More specifically, separate genetic analysis of IPMN and corresponding neoplastic tissue at the resection margin allowed us to determine their relationship and elucidate the role of the surgical margin for recurrence. In another project, we comprehensively analyzed somatic mutations in small MCNs, evaluating the mechanisms of clonal evolution and genetic alterations associated with increasing cellular dysplasia. With currently ongoing projects beyond the Walter Benjamin fellowship funding period, we continue to investigate pancreatic precursor lesions to optimize their clinical management, thereby understanding the critical genetic changes of malignant transformation in PDAC.

Publications

• Gastric cancer following pancreaticoduodenectomy: Experience from a high-volume center and review of existing literature. Surgery Open Science, 2(4), 32-40.
  Pflüger, Michael Johannes; Felsenstein, Matthäus; Schmocker, Ryan; Wood, Laura DeLong; Hruban, Ralph; Fujikura, Kohei; Rozich, Noah; van Oosten, Floortje; Weiss, Matthew; Burns, William; Yu, Jun; Cameron, John; Pratschke, Johann; Wolfgang, Christopher Lee; He, Jin & Burkhart, Richard Andrew
  
• Generation and characterization of a cell line from an intraductal tubulopapillary neoplasm of the pancreas. Laboratory Investigation, 100(7), 1003-1013.
  Felsenstein, Matthäus; Trujillo, Maria A.; Huang, Bo; Nanda, Neha; Jiang, Zhengdong; Jeong, Yea Ji; Pflüger, Michael; Goggins, Michael G.; Hruban, Ralph H.; Thompson, Elizabeth D.; Heaphy, Christopher M.; Roberts, Nicholas J. & Wood, Laura D.
  
• Pattern of Invasion in Human Pancreatic Cancer Organoids Is Associated with Loss of SMAD4 and Clinical Outcome. Cancer Research, 80(13), 2804-2817.
  Huang, Wenjie; Navarro-Serer, Bernat; Jeong, Yea Ji; Chianchiano, Peter; Xia, Limin; Luchini, Claudio; Veronese, Nicola; Dowiak, Cameron; Ng, Tammy; Trujillo, Maria A.; Huang, Bo; Pflüger, Michael J.; Macgregor-Das, Anne M.; Lionheart, Gemma; Jones, Danielle; Fujikura, Kohei; Nguyen-Ngoc, Kim-Vy; Neumann, Neil M.; Groot, Vincent P.; ... & Wood, Laura D.
  
• The Impact of Clinical and Pathological Features on Intraductal Papillary Mucinous Neoplasm Recurrence After Surgical Resection. Annals of Surgery, 275(6), 1165-1174.
  Pflüger, Michael J.; Griffin, James F.; Hackeng, Wenzel M.; Kawamoto, Satomi; Yu, Jun; Chianchiano, Peter; Shin, Eunice; Lionheart, Gemma; Tsai, Hua-Ling; Wang, Hao; Rezaee, Neda; Burkhart, Richard A.; Cameron, John L.; Thompson, Elizabeth D.; Wolfgang, Christopher L.; He, Jin; Brosens, Lodewijk A. A. & Wood, Laura D.
  
• Small Mucinous Cystic Neoplasms Often Lack Somatic Driver Gene Mutations. Poster Presentation, American Pancreatic Association (APA) 2021 Annual Meeting; Miami Beach, FL [abstract in Pancreas (Volume 50, Issue 7, page 1089)]
  Pflüger MJ, Braxton AM, Fujikura K., Wood LD
  
• Blood transfusion is associated with worse outcomes following pancreatic resection for pancreatic adenocarcinoma. Annals of Pancreatic Cancer, 5 (2022, 3), 1-1.
  Javed, Ammar A.; Ronnekleiv-Kelly, Sean M.; Hasanain, Alina; Pflüger, Michael J.; Habib, Joseph R.; Wright, Michael J.; He, Jin; Cameron, John L.; Wolfgang, Christopher L.; Frank, Steven M.; Weiss, Matthew J. & Burkhart, Richard A.
  
• Pancreatic Cancer. The Pancreas (2023, 8, 4), 969-981. American Geophysical Union (AGU).
  Pflüger, Michael J.; Noë, Michaël & Brosens, Lodewijk A.A.