Single cell migration in artificial collagen gels as an in vitro model system in the context of cancer are studied. Cell and matrix mechanical properties are determined using atomic force microscopy and an advanced analysis method. Matrix pore-size is studied using a novel approach and analysis method. A novel, minimally invasive approach to determine the amount of displacement of the cell microenvironment due to force generation of single cells during migration in artificial 3D collagen gels is introduced. An automated analysis and user friendly software to analyze high-throughput cell invasion is introduced. These methods are used to study cell migration and mechanical properties of the breast cancer cell lines MDA-MB-231 and MCF-7 and the influence of cell nuclear elasticity is investigated. Using mouse embryonic fibroblasts, the role of focal adhesion kinase (FAK) during cell migration is studied using FAK deficient knock-out cell lines FAK-/- and control FAK+/+ as well as kinase-dead mutants FAKR454/R454 and control FAKWT/WT.:Abstract i
Acknowledgements iii
1 Introduction 1
2 Background 5
2.1 Cancer — An ever-changing Disease 5
2.1.1 Carcinogenesis and Neoplasm 6
2.1.2 Hallmarks of Cancer 7
2.1.3 Metastasis— The malignant Progression of Cancer 7
2.1.4 Metastatic Cascade 9
2.2 The Cell— Where it begins 10
2.2.1 Actomyosin Complex 12
2.2.1.1 Actin Monomer 12
2.2.1.2 Polymerization 12
2.2.1.3 Structures 14
2.2.1.4 Actin Cortex 15
2.2.1.5 Filopodia 16
2.2.1.6 Lamellipodium 16
2.2.1.7 Invadopodium 17
2.2.1.8 Stress Fibers 17
2.2.1.9 Actin in Cancer and Metastasis 17
2.2.1.10 Myosin and Actin 18
2.2.2 Focal Adhesions 19
2.2.3 Microtubules 20
2.2.4 Intermediate Filaments 21
2.2.5 Cellular Stiffness 22
2.2.6 Nuclear Deformability 23
2.3 The Extracellular Matrix— Where it happens 24
2.3.1 Components and Structure 25
2.3.2 Collagen as a Model System 26
2.3.2.1 Collagen I Fibril Formation 27
2.3.2.2 The Rat/Bovine-Collagen-Mix Model System 28
2.4 Single Cell Migration— Why it spreads 29
3 Materials and Methods 31
3.1 Cell Culture 31
3.1.1 Cancer Cells 31
3.1.2 Mouse fibroblasts 32
3.1.3 Pharmacological treatment 34
3.2 Collagen matrices 34
3.3 Cell Elasticity 36
3.3.1 Atomic Force Microscopy 36
3.3.2 Preparation 37
3.3.3 Data Aquisition 38
3.3.4 Data Analysis 38
3.4 Matrix Stiffness 40
3.4.1 Preparation 40
3.4.2 Data Aquisition 41
3.4.3 Data Analysis 41
3.5 Invasion Assay 42
3.5.1 Preparation 42
3.5.2 Data aquisition 44
3.5.3 Data Analysis 44
3.6 Matrix Topology 48
3.6.1 Preparation 49
3.6.2 Data Acquisition 50
3.6.3 Data Analysis 51
3.6.3.1 Binarization 51
3.6.3.2 Pore-Size 53
3.6.3.3 Fiber Thickness 54
3.7 Fiber Displacement 55
3.7.1 Preparation 56
3.7.2 Data Aquisition 56
3.7.3 Data analysis 57
3.7.3.1 Fiber Displacement 59
3.7.3.2 Cell Segmentation 60
3.7.3.3 Shell Analysis 61
3.8 A toolset to understand Single Cell Migration and what influences it 62
4 Results 65
4.1 Cell Elasticity 65
4.1.1 Example Force-Distance Curves 66
4.1.2 Single Cell Elasticity 67
4.2 Matrix Stiffness 69
4.3 Invasion 71
4.4 Matrix Topology 75
4.5 Influence of Cell Nucleus on Cell Migration 79
4.5.1 Cellular Elasticity 79
4.5.2 Invasion 81
4.6 Fiber Displacement 89
4.7 Effect of FAK on Cell Invasion and Fiber Displacement 93
4.7.1 FAK Knock-Out 93
4.7.2 Kinase-dead FAK Mutant 96
5 Discussion 103
References 107 / Die Einzelzellmigration in künstlichen Kollagennetzwerken als ein in vitro Modellsystem im Kontext von Krebs wurde studiert. Mechanische Eigenschaften von Zellen und der verwendeten Kollagennetzwerke wurden mithilfe der Atomic Force Microscopy (AFM) und weiterentwickelten Analysemethoden bestimmt. Die Porengröße der verwendeten Kollagennetzwerke wurde mit einer neuentwickelten Auswertemethode analysiert. Eine neuartige, minimal-invasive Methode zur Bestimmung der Verformung der Mikroumgebung von Zellen während der Migration verursacht durch Kräftegenerierung der Zelle wird beschrieben. Die Analyse des Invasions-Assays wurde automatisiert und eine nutzerfreundliche Software entwickelt, mit der große Datenmengen ausgewertet werden können. Diese Methoden wurden verwendet, um mechanische Eigenschaften und Migration der humanen Brustkrebszellinien MDA-MB-231 und MCF-7 zu studieren. Die Rolle der focal adhesion kinase (FAK) wurde mithilfe von embryonalen Maus-Fibroblasten studiert. Sowohl eine FAK knock-out Zellinie FAK-/- und Kontrolle FAK+/+, als auch eine kinase-dead Mutante FAKR454/R454 und Kontrolle FAKWT/WT wurden hinsichtlich ihrer Invasion und Verformung der Mikroumgebung analysiert.:Abstract i
Acknowledgements iii
1 Introduction 1
2 Background 5
2.1 Cancer — An ever-changing Disease 5
2.1.1 Carcinogenesis and Neoplasm 6
2.1.2 Hallmarks of Cancer 7
2.1.3 Metastasis— The malignant Progression of Cancer 7
2.1.4 Metastatic Cascade 9
2.2 The Cell— Where it begins 10
2.2.1 Actomyosin Complex 12
2.2.1.1 Actin Monomer 12
2.2.1.2 Polymerization 12
2.2.1.3 Structures 14
2.2.1.4 Actin Cortex 15
2.2.1.5 Filopodia 16
2.2.1.6 Lamellipodium 16
2.2.1.7 Invadopodium 17
2.2.1.8 Stress Fibers 17
2.2.1.9 Actin in Cancer and Metastasis 17
2.2.1.10 Myosin and Actin 18
2.2.2 Focal Adhesions 19
2.2.3 Microtubules 20
2.2.4 Intermediate Filaments 21
2.2.5 Cellular Stiffness 22
2.2.6 Nuclear Deformability 23
2.3 The Extracellular Matrix— Where it happens 24
2.3.1 Components and Structure 25
2.3.2 Collagen as a Model System 26
2.3.2.1 Collagen I Fibril Formation 27
2.3.2.2 The Rat/Bovine-Collagen-Mix Model System 28
2.4 Single Cell Migration— Why it spreads 29
3 Materials and Methods 31
3.1 Cell Culture 31
3.1.1 Cancer Cells 31
3.1.2 Mouse fibroblasts 32
3.1.3 Pharmacological treatment 34
3.2 Collagen matrices 34
3.3 Cell Elasticity 36
3.3.1 Atomic Force Microscopy 36
3.3.2 Preparation 37
3.3.3 Data Aquisition 38
3.3.4 Data Analysis 38
3.4 Matrix Stiffness 40
3.4.1 Preparation 40
3.4.2 Data Aquisition 41
3.4.3 Data Analysis 41
3.5 Invasion Assay 42
3.5.1 Preparation 42
3.5.2 Data aquisition 44
3.5.3 Data Analysis 44
3.6 Matrix Topology 48
3.6.1 Preparation 49
3.6.2 Data Acquisition 50
3.6.3 Data Analysis 51
3.6.3.1 Binarization 51
3.6.3.2 Pore-Size 53
3.6.3.3 Fiber Thickness 54
3.7 Fiber Displacement 55
3.7.1 Preparation 56
3.7.2 Data Aquisition 56
3.7.3 Data analysis 57
3.7.3.1 Fiber Displacement 59
3.7.3.2 Cell Segmentation 60
3.7.3.3 Shell Analysis 61
3.8 A toolset to understand Single Cell Migration and what influences it 62
4 Results 65
4.1 Cell Elasticity 65
4.1.1 Example Force-Distance Curves 66
4.1.2 Single Cell Elasticity 67
4.2 Matrix Stiffness 69
4.3 Invasion 71
4.4 Matrix Topology 75
4.5 Influence of Cell Nucleus on Cell Migration 79
4.5.1 Cellular Elasticity 79
4.5.2 Invasion 81
4.6 Fiber Displacement 89
4.7 Effect of FAK on Cell Invasion and Fiber Displacement 93
4.7.1 FAK Knock-Out 93
4.7.2 Kinase-dead FAK Mutant 96
5 Discussion 103
References 107
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:36293 |
| Date | 27 November 2019 |
| Creators | Fischer, Tony |
| Contributors | Universität Leipzig |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/acceptedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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