Tumour-specific immune responses and tumour stroma analysis in a murine model for pancreatic adenocarcinoma [Elektronische Ressource] / von Benjamin Vermeer

Benjamin Vermeer

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• Gesundheit

Informations

• Publié par: gottfried_wilhelm_leibniz_universitat_hannover
• Publié le: 01 janvier 2007
• Nombre de lectures: 7
• Langue: English
• Poids de l'ouvrage: 7 Mo

Extrait

Tumour-specific immune responses and tumour
stroma analysis in a murine model for pancreatic
adenocarcinoma



Von der Naturwissenschaftlichen Fakultät der
Gottfried Wilhelm Leibniz Universität Hannover
zur Erlangung des Grades

Doktor der Naturwissenschaften
Dr. rer. nat.
genehmigte Dissertation



von
M.Sc. Benjamin Vermeer
geboren am 21. August 1972
in Amstelveen, Die Niederlande



2007



























st1 Referee: Prof. Dr. T. F. Greten
nd
2 Referee: Prof. Dr. W. Müller
Date of defence: July 19, 2007
















I dedicate this thesis to my Oma and my parents
Table of Contents 4
Kurzzusammenfassung ............................................................................................8
Abstract......................................................................................................................9
1 Introduction.......................................................................................................10
1.1 Tumour immunology .................................................................................10
1.1.1 Adaptive and innate effector mechanisms in cancer immunity.....10
1.1.2 Tumour antigens ............................................................................11
1.1.3 Tumour immunotherapy ................................................................13
1.1.3.1 Cancer vaccines ..............................................................14
1.1.3.2 Adoptive transfer ............................................................14
1.1.4 Tumour immune escape mechanisms and possible therapeutic
approaches ..................................................................................14
1.1.4.1 Alterations of the antigen processing machinery............14
1.1.4.2 Soluble immunosuppressive factors ...............................15
1.1.4.3 Tumour counterattack .....................................................16
1.1.4.4 Tumour-derived exosomes and microvesicles................19
1.1.5 Tumour microenvironment............................................................19
1.2 Pancreas and pancreatic cancer..................................................................21
1.2.1 Genes associated with the development of pancreatic cancer .......23
1.3 Cancer and inflammation...........................................................................26
1.3.1 Pancreatic cancer and chronic pancreatitis ....................................28
1.4 Mouse models for cancer29
1.4.1 Mouse models for human pancreatic cancer..................................29
2 Materials and Methods.....................................................................................32
2.1 Materials ....................................................................................................32
2.1.1 Mice ...............................................................................................32
2.1.2 Chemicals and enzymes.................................................................32
2.1.3 Cell culture media, buffers, and solutions .....................................34
2.1.4 Antibodies for FACS, immunohistochemistry and
immunofluorescence analysis .....................................................36
2.1.5 Cytokines, consumed materials and kits........................................37
2.1.6 Devices...........................................................................................38
2.2 Methods .....................................................................................................40
2.2.1 Methods for mice experiments ......................................................40
2.2.1.1 Tumour transplantation...................................................40
2.2.1.2 Adoptive Transfer ...........................................................40
2.2.1.3 Induction of chronic pancreatitis ....................................41 Table of Contents 5
2.2.1.4 Cyclophosphamide treatment .........................................41
2.2.2 Methods of the cell biology ...........................................................41
2.2.2.1 General cell culture.........................................................41
2.2.2.2 mPAC tumour cell lines generation and other
tumour cell lines41
2.2.2.3 Cell count........................................................................42
2.2.2.4 Cell freezing....................................................................42
2.2.2.5 Cell thawing42
2.2.2.6 Preparation of single cell suspensions ............................42
2.2.2.7 Flow cytometry and depletions.......................................42
2.2.2.8 IFN-γ capture assay ........................................................42
2.2.2.9 Apoptosis induction by mitomycin C treatment .............43
2.2.2.10 Intracellular cytokine staining ........................................43
2.2.2.11 Serology..........................................................................43
2.2.2.12 Cytometric bead array (CBA).........................................44
2.2.3 Histological methods .....................................................................44
2.2.3.1 Haematoxylin & Eosin (H&E) staining..........................44
2.2.3.2 Immunohistochemistry staining of TILs.........................44
2.2.3.3 Immunofluorescence stain of TILs .................................45
2.2.4 Molecular biological methods .......................................................45
2.2.4.1 PCR screening.................................................................45
2.2.4.2 Preparation of RNA and reverse transcription (RT)
PCR..............................................................................45
2.2.4.3 Real time PCR ................................................................46
2.2.4.4 Agarose gel electrophoresis............................................46
2.2.4.5 Quantification of RNA concentration by
spectrophotometer analysis..........................................46
3 Results................................................................................................................47
3.1 Subcutaneous and spontaneous pancreatic tumours ..................................47
3.1.1 Expression of cytokines in spontaneous and subcutaneous
pancreatic tumours......................................................................47
3.1.2 Histological analysis of the microenvironment of the
spontaneous and subcutaneous pancreatic tumour .....................49
3.1.3 Activation- and migration-status of tumour-specific CTLs in
spontaneous or subcutaneous tumour .........................................55
3.1.4 Analysis of tumour-specific humoral immune responses in
mice with subcutaneous and spontaneous pancreatic tumours...62 Table of Contents 6
3.2 Pancreatic tumour cell lines with the same origin behave differently in
vivo..........................................................................................................65
3.2.1 Expression of cytokines in regressive and progressive
subcutaneous pancreatic tumours ...............................................66
3.2.2 Functional analysis of splenocytes from regressor and
progressor tumour bearing mice .................................................75
3.3 Chronic pancreatitis and tumour-specific immune responses ...................79
3.3.1 Influence of chronic pancreatitis on the tumour-specific
immune responses and tumour growth of mice with
premalignant lesions or spontaneous tumours............................79
3.3.2 Influence of chronic pancreatitis on the tumour-specific
immune responses in regressor tumour bearing mice.................80
3.3.3 Specificity of the pancreatic tumour-specific immune reduction
in mice suffering from chronic pancreatitis................................81
3.3.4 Tumour growth kinetics of subcutaneous regressor and
spontaneous tumour bearing mice suffering from chronic
pancreatitis..................................................................................82
3.4 Influence of tumour stroma on the growth of subcutaneous pancreatic
tumours ...................................................................................................84
3.4.1 Regressor and progressor tumour growth in different tissues .......84
3.4.2 Growth kinetics of regressor or spontaneous tumours in
C57BL/6 wt mice........................................................................85
3.4.3 Growth kinetics of transplanted tumour pieces in C57BL/6 wt
mice.............................................................................................86
3.4.4 Influence of cyclophosphamide on the in vitro and in vivo
tumour growth of pancreatic tumours.........................................87
3.4.5 Influence of cyclophosphamide on immune cells in the
pancreatic tumour model ............................................................89
4 Discussion ............................................................................