Pancreatic duct ligation reduces premalignant pancreatic lesions in a Kras model of pancreatic adenocarcinoma in mice

[EN] Pancreatic duct ligation (PDL) in the murine model has been described as an exocrine pancreatic atrophy-inducing procedure. However, its influence has scarcely been described on premalignant lesions. This study describes the histological changes of premalignant lesions and the gene expression in a well-defined model of pancreatic ductal adenocarcinoma by PDL. Selective ligation of the splenic lobe of the pancreas was performed in Ptf1a-Cre((+/ki)); K-ras LSLG12Vgeo((+/ki)) mice (PDL-Kras mice). Three experimental groups were evaluated: PDL group, controls and shams. The presence and number of premalignant lesions (PanIN 1-3 and Atypical Flat Lesions-AFL) in proximal (PP) and distal (DP) pancreas were studied for each group over time. Microarray analysis was performed to find differentially expressed genes (DEG) between PP and PD. Clinical human specimens after pancreaticoduodenectomy with ductal occlusion were also evaluated. PDL-Kras mice showed an intense pattern of atrophy in DP which was shrunk to a minimal portion of tissue. Mice in control and sham groups had a 7 and 10-time increase respectively of risk of high-grade PanIN 2 and 3 and AFL in their DP than PDL-Kras mice. Furthermore, PDL-Kras mice had significantly less PanIN 1 and 2 and AFL lesions in DP compared to PP. We identified 38 DEGs comparing PP and PD. Among them, several mapped to protein secretion and digestion while others such as Nupr1 have been previously associated with PanIN and PDAC. PDL in Ptf1a-Cre((+/ki)); K-ras LSLG12Vgeo((+/ki)) mice induces a decrease in the presence of premalignant lesions in the ligated DP. This could be a potential line of research of interest in some cancerous risk patients.This work was supported by the Spanish Ministerio de Economia, Industria y Competitividad under "Plan Estatal de Investigacion, Desarrollo e Innovacion Orientada a los Retos de la Sociedad", Grant No "RTI2018-094357-B-C22".Cáceres, M.; Quesada, R.; Iglesias, M.; Real, FX.; Villamonte, M.; Martinez De Villarreal, J.; Pérez, M.... (2020). Pancreatic duct ligation reduces premalignant pancreatic lesions in a Kras model of pancreatic adenocarcinoma in mice. Scientific Reports. 10(1):1-12. https://doi.org/10.1038/s41598-020-74947-4112101Mattiuzzi, C. & Lippi, G. Current cancer epidemiology. J. Epidemiol. Glob. Health 9, 217–222 (2019).Rahib, L. et al. Projecting cancer incidence and deaths to 2030: the unexpected burden of thyroid, liver, and pancreas cancers in the united states. Cancer Res. 74, 2913–2921 (2014).Han, H. & Von Hoff, D. D. SnapShot: Pancreatic cancer. Cancer Cell 23, 424-424.e1 (2013).Hingorani, S. R. et al. Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse. Cancer Cell 4, 437–450 (2003).Shen, R. et al. The biological features of PanIN initiated from oncogenic Kras mutation in genetically engineered mouse models. Cancer Lett. 339, 135–143 (2013).Aichler, M. et al. Origin of pancreatic ductal adenocarcinoma from atypical flat lesions: A comparative study in transgenic mice and human tissues. J. Pathol. 226, 723–734 (2012).Esposito, I., Konukiewitz, B., Schlitter, A. M. & Klöppel, G. New insights into the origin of pancreatic cancer. Role of atypical flat lesions in pancreatic carcinogenesis. Pathologe 33, 189–193 (2012).Esposito, I., Konukiewitz, B., Schlitter, A. M. & Klöppel, G. Pathology of pancreatic ductal adenocarcinoma: Facts, challenges and future developments. World J. Gastroenterol. 20, 13833–13841 (2014).Löhr, M., Klöppel, G., Maisonneuve, P., Lowenfels, A. B. & Lüttges, J. Frequency of K-ras mutations in pancreatic intraductal neoplasias associated with pancreatic ductal adenocarcinoma and chronic pancreatitis: A meta-analysis. Neoplasia 7, 17–23 (2005).Almoguera, C. et al. Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes. Cell 53, 549–554 (1988).Wilentz, R. E., Argani, P. & Hruban, R. H. Loss of heterozygosity or intragenic mutation, which comes first?. Am. J. Pathol. 158, 1561–1563 (2001).Schmitt, E., Paquet, C., Beauchemin, M. & Bertrand, R. DNA-damage response network at the crossroads of cell-cycle checkpoints, cellular senescence and apoptosis. J. Zhejiang Univ. Sci. B 8, 377–397 (2007).Schuler, M., Bossy-wetzel, E., Goldstein, J. C., Fitzgerald, P. & Green, D. R. P53 induces apoptosis by caspase activation through mitochondrial cytochrome C release. J. Biol. Chem. 275, 7337–7342 (2000).Brune, K. et al. Multifocal neoplastic precursor lesions associated with lobular atrophy of the pancreas in patients having a strong family history of pancreatic cancer. Am. J. Surg. Pathol. 30, 1067–1076 (2006).Maitra, A. et al. Multicomponent analysis of the pancreatic adenocarcinoma progression model using a pancreatic intraepithelial neoplasia tissue microarray. Mod. Pathol. 16, 902–912 (2003).Wada, M., Doi, R., Hosotani, R., Lee, J. U. & Imamura, M. Apoptosis of acinar cells in rat pancreatic duct ligation. J. Gastroenterol. 30, 813–814 (1995).Walker, N. I. Ultrastructure of the rat pancreas after experimental duct ligation. I. The role of apoptosis and intraepithelial macrophages in acinar cell deletion. Am. J. Pathol. 126, 439–451 (1987).Scoggins, C. R. et al. p53-Dependent acinar cell apoptosis triggers epithelial proliferation in duct-ligated murine pancreas. Am. J. Physiol. Gastrointest. Liver Physiol. 270, 827–836 (2000).Watanabe, S., Abe, K., Anbo, Y. & Katoh, H. Changes in the mouse exocrine pancreas after pancreatic duct ligation: A qualitative and quantitative histological study. Arch. Histol. Cytol. 58, 365–374 (1995).Quesada, R. et al. Radiofrequency pancreatic ablation and section of the main pancreatic duct does not lead to necrotizing pancreatitis. Pancreas 43, 1–7 (2014).Quesada, R. et al. Long-term evolution of acinar-to-ductal metaplasia and β-cell mass after radiofrequency-assisted transection of the pancreas in a controlled large animal model. Pancreatology 16, 1–6 (2015).Guerra, C. et al. Tumor induction by an endogenous K-ras oncogene is highly dependent on cellular context. Cancer Cell 4, 111–120 (2003).Guerra, C. et al. Chronic pancreatitis is essential for induction of pancreatic ductal adenocarcinoma by K-Ras oncogenes in adult mice. Cancer Cell https://doi.org/10.1016/j.ccr.2007.01.012 (2007).De Groef, S. et al. Surgical injury to the mouse pancreas through ligation of the pancreatic duct as a model for endocrine and exocrine reprogramming and proliferation. J. Vis. Exp. https://doi.org/10.3791/52765 (2015).Hruban, R. H. et al. Pathology of genetically engineered mouse models of pancreatic exocrine cancer: Consensus report and recommendations. Cancer Res. 66, 95–106 (2006).Hruban, R. H., Maitra, A. & Goggins, M. Update on pancreatic intraepithelial neoplasia. Int. J. Clin. Exp. Pathol. 1, 306–316 (2008).Irizarry, R. A. et al. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Select. Works Terry Speed 4, 601–616 (2012).Kauffmann, A., Gentleman, R. & Huber, W. arrayQualityMetrics—A bioconductor package for quality assessment of microarray data. Bioinformatics 25, 415–416 (2009).Carvalho, B. S. & Irizarry, R. A. A framework for oligonucleotide microarray preprocessing. Bioinformatics 26, 2363–2367 (2010).James W. MacDonald. Affycoretools: Functions useful for those doing repetitive analyses with Affymetrix GeneChips. R package version 1.56.0. (2010).Ritchie, M. E. et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015).Fromm, D. & Schwarz, K. Ligation of the pancreatic duct during difficult operative circumstances. J. Am. Coll. Surg. 197, 943–948 (2003).Sato, N. et al. Long-term morphological changes of remnant pancreas and biliary tree after pancreatoduodenectomy on CT. Int. Surg. 83, 136–140 (1998).Tomimaru, Y. et al. Comparison of postoperative morphological changes in remnant pancreas between pancreaticojejunostomy and pancreaticogastrostomy after pancreaticoduodenectomy. Pancreas 38, 203–207 (2009).Burdío, F. et al. Radiofrequency-induced heating versus mechanical stapler for pancreatic stump closure: In vivo comparative study. Int. J. Hyperth. 32, 2 (2016).Nusse, R. Molecular biology of cancer genes. Trends Genet. 7, 103 (2003).Bhatia, M. Apoptosis of pancreatic acinar cells in acute pancreatitis: Is it good or bad?. J. Cell Mol. Med. 8, 402–409 (2004).Chu, L. C., Goggins, M. G. & Fishman, E. K. Diagnosis and detection of pancreatic cancer. Cancer J. 23, 333–342 (2020).Takahashi, S. et al. Apoptosis and mitosis of parenchymal cells in the duct-ligated rat submandibular gland. Tissue Cell 32, 457–463 (2000).Shi, C. et al. Differential cell susceptibilities to Kras in the setting of obstructive chronic pancreatitis. Cell. Mol. Gastroenterol. Hepatol. https://doi.org/10.1016/j.jcmgh.2019.07.001 (2019).Cheng, T. et al. Ductal obstruction promotes formation of preneoplastic lesions from the pancreatic ductal compartment. Int. J. Cancer 144, 2529–2538 (2019).Makawita, S. et al. Validation of four candidate pancreatic cancer serological biomarkers that improve the performance of CA19.9. BMC Cancer https://doi.org/10.1186/1471-2407-13-404 (2013).Cheung, W. et al. Application of a global proteomic approach to archival precursor lesions: Deleted in malignant brain tumors 1 and tissue transglutaminase 2 are upregulated in pancreatic cancer precursors. Pancreatology 8, 608–616 (2008).Kontos, C. K., Mavridis, K., Talieri, M. & Scorilas, A. Kallikrein-related peptidases (KLKs) in gastrointestinal cancer: Mechanistic and clinical aspects. Thromb. Haemost. 110, 450–457 (2013).Hamidi, T. et al. Nuclear protein 1 promotes pancreatic cancer development and protects cells from stress by inhibiting apoptosis. J. Clin. Invest 122, 2092–3103 (2012).Cano, C. E. et al. Genetic inactivation of Nupr1 acts as a dominant suppressor event in a two-hit model of pancreatic carcinogenesis. Gut 63, 984–995 (2014).Grasso, D. et al. Genetic inactivation of the pancreatitis-inducible gene Nupr1 impairs PanIN formation by modulating KrasG12D-induced senescence. Cell Death Differ. https://doi.org/10.1038/cdd.2014.74 (2014).Basturk, O. et al. A revised classification system and recommendations from the Baltimore consensus meeting for neoplastic precursor lesions in the pancreas. Am. J. Surg. Pathol. 39, 1730–1741 (2015).Guerra, C. & Barbacid, M. Genetically engineered mouse models of pancreatic adenocarcinoma. Mol. Oncol. 7, 232–247 (2013).Sharma, S. & Green, K. B. The pancreatic duct and its arteriovenous relationship. Am. J. Surg. Pathol. 28, 613–620 (2004).Pérez-Mancera, P. A., Guerra, C., Barbacid, M. & Tuveson, D. A. What we have learned about pancreatic cancer from mouse models. Gastroenterology 142, 1079–1092 (2012).von Figura, G., Morris, J. P., Wright, C. V. E. & Hebrok, M. Nr5a2 maintains acinar cell differentiation and constrains oncogenic Kras-mediated pancreatic neoplastic initiation. Gut 63, 656–664 (2014).Andaluz, A. et al. Endoluminal radiofrequency ablation of the main pancreatic duct is a secure and effective method to produce pancreatic atrophy and to achieve stump closure. Sci. Rep. https://doi.org/10.1038/s41598-019-42411-7 (2019).Wood, L. D., Yurgelun, M. B. & Goggins, M. G. Genetics of familial and sporadic pancreatic cancer. Gastroenterology 156, 2041–2055 (2019)