TECHNISCHE UNIVERSITÄT MÜNCHEN Fakultät für Medizin Institut für Allgemeine Pathologie und Pathologische Anatomie (Direktor: Prof. Dr. W. Weichert) Characterization of Pancreatic Stellate Cells in Pancreatic Ductal Adenocarcinoma and Cases of Chronic Pancreatitis of Various Etiologies Lena Julia Häberle Vollständiger Abdruck der von der Fakultät für Medizin der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Medizin genehmigten Dissertation. Vorsitzender: Prof. Dr. Ernst J. Rummeny Prüfer der Dissertation: 1. Prof. Dr. Irene Esposito 2. Prof. Dr. Bernhard Holzmann Die Dissertation wurde am 12.12.2016 bei der Technischen Universität München eingereicht und durch die Fakultät für Medizin am 08.11.2017 angenommen. Index 1 Abbreviations .................................................................................................................................. 1 2 Introduction ..................................................................................................................................... 5 2.1 Stromal reaction ...................................................................................................................... 5 2.1.1 Production of connective tissue in physiological wound healing ................................... 5 2.1.2 Fibrosis in chronic inflammatory diseases ...................................................................... 5 2.1.3 Desmoplasia in malignant diseases ................................................................................. 6 2.2 Chronic pancreatitis ................................................................................................................ 6 2.2.1 Subtypes .......................................................................................................................... 6 2.2.1.1 Alcohol-induced chronic pancreatitis ...................................................................... 6 2.2.1.2 Autoimmune pancreatitis ........................................................................................ 7 2.2.1.3 Hereditary pancreatitis ............................................................................................ 8 2.2.1.4 Others ...................................................................................................................... 8 2.2.2 Epidemiology and risk factors ....................................................................................... 10 2.2.3 Diagnostics and therapy ................................................................................................ 10 2.2.4 Role of stromal reaction in chronic pancreatitis ........................................................... 11 2.3 Pancreatic ductal adenocarcinoma ....................................................................................... 12 2.3.1 Epidemiology and risk factors ....................................................................................... 12 2.3.2 Diagnostics and therapy ................................................................................................ 12 2.3.3 Role of stromal reaction in PDAC .................................................................................. 13 2.4 Pancreatic stellate cells ......................................................................................................... 14 2.4.1 Phenotype and function ................................................................................................ 14 2.4.2 Similarities with hepatic stellate cells ........................................................................... 15 2.4.3 Role of pancreatic stellate cells in chronic pancreatitis ................................................ 17 2.4.4 Role of pancreatic stellate cells in pancreatic ductal adenocarcinoma ........................ 17 3 Aim of the study ............................................................................................................................ 18 4 Material and Methods ................................................................................................................... 19 4.1 Material ................................................................................................................................. 19 4.1.1 Reagents ........................................................................................................................ 19 4.1.2 Consumables ................................................................................................................. 22 4.1.3 Equipment ..................................................................................................................... 23 4.1.4 Kits ................................................................................................................................. 25 4.1.5 Tissue samples ............................................................................................................... 26 2 4.1.6 Cell lines ......................................................................................................................... 27 4.1.7 Antibodies ...................................................................................................................... 29 4.1.8 Buffers ........................................................................................................................... 30 4.1.9 Software ........................................................................................................................ 32 4.1.10 Primer pairs ................................................................................................................... 32 4.2 Methods ................................................................................................................................ 33 4.2.1 Tissue collection ............................................................................................................ 33 4.2.2 Ethic vote ....................................................................................................................... 33 4.2.3 Hematoxylin & Eosin stain ............................................................................................. 33 4.2.4 Movat’s pentachrome stain .......................................................................................... 34 4.2.5 Immunohistochemistry ................................................................................................. 35 4.2.6 Immunofluorescence ..................................................................................................... 36 4.2.7 Cell culture ..................................................................................................................... 37 4.2.7.1 Maintenance of cell lines ....................................................................................... 37 4.2.7.2 Counting of cells .................................................................................................... 38 4.2.7.3 Splitting of cells...................................................................................................... 38 4.2.7.4 Freezing of cells ..................................................................................................... 38 4.2.7.5 Thawing of cells ..................................................................................................... 38 4.2.7.6 Mycoplasma test ................................................................................................... 38 4.2.7.7 Senescence test ..................................................................................................... 40 4.2.7.8 MatrigelTM coating ................................................................................................. 40 4.2.7.9 Oil Red O staining .................................................................................................. 40 4.2.8 Protein extraction .......................................................................................................... 40 4.2.9 Protein quantification .................................................................................................... 41 4.2.10 Western blot .................................................................................................................. 41 4.2.11 Statistical analysis .......................................................................................................... 41 5 Results ........................................................................................................................................... 42 5.1 Histology ................................................................................................................................ 42 5.1.1 Hematoxylin & Eosin stain ............................................................................................. 42 5.1.2 Movat’s pentachrome stain .......................................................................................... 43 5.1.3 Immunohistochemistry ................................................................................................. 46 5.1.3.1 α-Crystallin B ......................................................................................................... 46 5.1.3.2 α-SMA .................................................................................................................... 49 5.1.3.3 CD34 ...................................................................................................................... 52 5.1.3.4 CD56 (NCAM) ........................................................................................................