In Alzheimer’s disease (AD), different types of neurons and different brain areas show differential patterns of vulnerability towards neurofibrillary degeneration, which provides the basis for a highly predictive profile of disease progression throughout the brain that now is widely accepted for neuropathological staging. In previous studies we could demonstrate that in AD cortical and subcortical neurons are constantly less frequently affected by neurofibrillary degeneration if they are enwrapped by a specialized form of the hyaluronan-based extracellular matrix (ECM), the so called ‘perineuronal net’ (PN). PNs are basically composed of large aggregating chondroitin sulphate proteoglycans connected to a hyaluronan backbone, stabilized by link proteins and cross-linked via tenascin-R (TN-R). Under experimental conditions in mice, PN-ensheathed neurons are better protected against iron-induced neurodegeneration than neurons without PN. Still, it remains unclear whether these neuroprotective effects are directly mediated by the PNs or are associated with some other mechanism in these neurons unrelated to PNs. To identify molecular components that essentially mediate the neuroprotective aspect on PN-ensheathed neurons, we comparatively analysed neuronal degeneration induced by a single injection of FeCl3 on four different mice knockout strains, each being deficient for a different component of PNs. Aggrecan, link protein and TN-R were identified to be essential for the neuroprotective properties of PN, whereas the contribution of brevican was negligible. Our findings indicate that the protection of PN-ensheathed neurons is directly mediated by the net structure and that both the high negative charge and the correct interaction of net components are essential for their neuroprotective function.

The bone marrow (BM) niche provides hematopoietic stem (HSC) and progenitor cells with many exogenous cues that tightly regulate homeostasis. These cues orchestrate cellular decisions, which are difficult to dissect and analyze in vivo. This thesis introduces a novel in vitro platform that permits systematic studies of BM-relevant factors that regulate homeostasis. Specifically, the role of 3D patterned adhesion ligands and soluble cytokines were studied in a combinatorial fashion. Analysis of human HSC differentiation and proliferation at both population and single cell level showed synergistic and antagonistic effects of adhesion- and cytokine-related signals. Those effects were dependent on the cytokine concentration and the distribution and number of adhesion ligands. The aim of this thesis was to model the in vivo bone marrow with its porous 3D structure and different sized niche compartments using a microcavity culture carrier. The developed culture system presented extracellular matrix (ECM) adhesion ligands to the HSCs in various defined dimensions ranging from single- to multi-cell capacity. The 3D open well geometry of the microcavity carriers also allowed HSCs to freely explore different scenarios including homing, migration, adhesion, or suspension. Furthermore, the developed setup offered straightforward accessibility to analytical methods like cytometry and quantitative microscopy. Single cell analysis of adherent HSCs showed decreased DNA synthesis and higher levels of stem cell marker expression within single cell microcavities under low cytokine conditions . This effect was reflected in a decline of proliferation and differentiation with decreasing microcavity size. When the cytokine concentration was increased2 beyond physiological levels the inhibitory effect on proliferation and differentiation due to single-cell-microcavity adherence was diminished. This result highlighted the fine balance between adhesion related and soluble cues regulating HSC fate. Within small microcavities more adhesion related receptors were engaged due to the 3D character of the culture carrier compared to multi-cell wells or conventional 2D cell culture plates. This study demonstrated that adhesion-related signal activation leads to reduced proliferation and differentiation. This geometry-based effect could be reversed by increased cytokine supplementation in the culture media. For plane substrates, HSCs attachment to fibronectin or heparin initiated early cell cycle entry compared to non-adherent cells during the initial 24h. Cytokine supplemented media favored integrin activation that induced fast adhesion, ultimately leading to early cell cycle activation. However, after prolonged cell culture the system balanced itself with a lower cycling rate of adherent versus non-adherent HSCs. Furthermore, HSCs within the 3-dimensionality of the microcavities cycled less than 2D adherent cells. These findings additionally supported the above stated idea of limited HSC proliferation as a consequence of more adhesion-related signals overwriting cytokine driven expansion. To complement the various in vitro studies, an in vivo repopulation study was performed. Cultured HSCs derived from single cell microcavities outperformed freshly isolated HSCs in a competitive repopulation assay, indicating that carefully engineered substrates are capable of preserving stem cell potential. Overall the reported findings provide a promising in vitro culture strategy that allows the stem cell field to gain a better understanding of the impact of distinct exogenous signals on human HSCs, which discloses new concepts for the wide scientific community working towards tissue engineering and regenerative medicine.:Kurzbeschreibung 4 Abstract 6 1 Introduction 8 1.1 Motivation 8 1.2 Objective 8 2 Basics 10 2.1 Stem Cells and their Role in Life 10 Stem Cells and their Niches 12 2.1.1 Hematopoietic Stem Cells 12 2.1.2 Hematopoietic Stem Cell Niche 14 2.1.3 The ECM Relevancy 16 2.1.4 HSC Relevant Cytokines 19 2.2 Cell Culture Scaffolds 21 2.2.1 General 2D, 3D 21 2.2.2 Substrate Engineering 22 2.2.3 Co-Culture versus the Artificial 3D Niche 23 3 Materials and Methods 25 3.1 Chemicals, Reagents and Equipment 25 3.2 Wafer Design and Surface Functionalization 29 3.3 Cell Culture and Analysis 31 3.3.1 HSC Culture in ECM-functionalized Microcavities 32 3.4 Surface Passivation 33 3.5 Mouse Bone Marrow Preparation 35 4 Results and Discussion 37 4.1 Scaffold Design and Preparation 37 4.1.1 Surface Characterization 37 4.1.2 Surface Passivation 39 Approaches for Surface Passivation 39 Efficiency of Surface Passivation 39 4.1.3 Redesigned Microcavities 43 4.2 Summarized Discussion of the Surface Passivation 44 4.3 HSC Culture inside Microcavities 45 4.3.1 HSC-ECM Interaction Reduces Proliferation 45 4.3.2 Population-wide Proliferation and Differentiation of Spatially Constrained HSCs . … 46 HSCs within Redesigned Microcavities 48 4.3.3 Colony-forming Ability of Microcavity Cultures 50 4.4 Single Cell Analysis of Differentiation 52 4.5 Cell Cycling Dependency on Cytokine Level 53 4.5.1 Plane Surfaces 54 4.5.2 Microcavities Reduce Cycling Frequency 57 4.6 Mice Repopulation of Microcavity Cultured HSCs 58 4.7 Summarized Discussion of the HSC–ECM Relation 60 4.8 Future Prospects 62 5 Summary 63 References 64 Figure Legend 73 Tables 73 Theses 74 6 Appendices I 6.1 FACS Principle I 6.1.1 HSC Staining for CD Marker and Cell Cycle Kinetics I 6.1.2 Apoptosis Test II 6.2 Differentiation and Proliferation on Redesigned Microcavities III 6.3 Colony-forming Capability of Microcavity Cultured Cells IV 6.4 Effect of Trypsin on HSC Properties in Long Term Culture IV 6.5 Surface Functionalization with SCF V 6.5.1 Analysis of the HSCs Grown on Immobilized SCF VI 6.5.2 SCF Immobilization and its Kinetics VII 6.5.3 c-kit Expression Kinetics and HSC Differentiation VIII Short Discussion on the Growth Factor Immobilization IX Publications X Posters X Proceedings XI Talks XI Patents XI Papers XI Awards XI 7 Danksagung: XII Selbstständigkeitserklärung: XIII / Die Homöostase der Hämatopoietischen Stamm- und Vorläuferzellen (HSC) in der Knochenmark Nische wird von einer Vielzahl exogener Faktoren gezielt reguliert. Diese Faktoren orchestrieren intrazelluläre Vorgänge, deren in vivo Analyse kompliziert ist. Die vorliegende These widmet sich einem neuen biotechnologischen Ansatz, der systematische Studien von Knochenmark-relevanten Faktoren ermöglicht. Im Speziellen wurde die Rolle 3D-präsentierter Zell Adhäsionsliganden in Kombination mit verschiedenen Konzentrationen löslicher Zytokine untersucht. Die Auswertung der Proliferation und Differenzierung von humanen HSC auf Einzelzell- und Populationsebene offenbarte die synergistischen und antagonistischen Effekte von Adhäsions- und Zytokinsignalen in ihrer Abhängigkeit von der Verteilung und der Anzahl von Adhäsionsliganden sowie der Zytokinkonzentration. Um die poröse Struktur des Knochenmarks in vivo-ähnlich darzustellen, wurde eine Zellkultur Plattform mit Mikrokavitäten verschiedenster Dimensionen von Multi- bis Einzelzellgröße entwickelt und mit Molekülen der extrazellulären Matrix beschichtet. Die Vorteile dieser Plattform liegen in der offenen 3D-Geometrie dieses mikrokavitäten Kultursystems, die den Zellen ermöglichte verschiedene Wachstumsbedingungen bezüglich Homing, Migration, Adhäsion oder Suspension frei zu erkunden. Das leicht zugängliche Setup eignete sich zudem hervorragend für die zytometrische Analyse der Zellen oder die quantitative Mikroskopie. Die Einzelzellanalyse adhärenter HSC ergab eine Reduktion von DNA Synthese und eine höhere Expression von Stammzelloberflächenfaktoren innerhalb der Einzelzell-Mikrokavitäten bei niedrigen Zytokinkonzentrationen . Dieser Effekt spiegelte sich auch auf Populationsebene in verminderter Proliferation und Differenzierung mit abnehmender Größe der Mikrokavitäten wider. Wurde die Zytokinkonzentration jedoch weit über physiologische Bedingungen erhöht, verminderte sich der Effekt (reduzierte DNA Synthese und höhere Stammzellfaktorexpression) beschrieben für die Einzelzellmikrokavitäten. Dieses Ergebnis verdeutlicht die empfindliche intrazelluläre Balance, vermittelt durch Adhäsionsignale und löslichen Faktoren, die das Verhalten von HSCs regulieren. Aufgrund des 3D-Charakters des Zellkulturträgers wurden innerhalb kleiner Mikrokavitäten mehr Adhäsionsrezeptoren ringsum die Zelle aktiviert. Dieser Vorteil gegenüber den Multizellkavitäten oder der herkömmlichen 2D–Zellkultur ermöglichte eine hohe Anzahl adhäsionsvermittelter Signale mit entsprechend höherer Proliferations-inhibitorischer Wirkung. Je höher die Konzentration der Zytokine war, desto stärker erfolgte die Stimulation der Proliferation und Differenzierung. Auf 2D Substraten, initiierte Adhäsion zu Fibronektin und Heparin innerhalb der ersten 24h einen frühen Zell-Zyklus-Start im Gegensatz zu nicht adhärenten Zellen. Die Zytokine im Zellmedium förderten die Integrin Aktivierung, was zu einer schnellen Zelladhäsion führte. Die Adhäsionsrezeptoren wiederum kooperieren mit Zytokinrezeptoren im Zellinneren und begünstigten damit einen zeitigeren Zell-Zyklus- Start. Allerdings stellte sich danach ein Gleichgewicht im Kultursystem ein, wobei weniger adhärente Zellen als nicht-adhärente Zellen den Zellzyklus durchliefen. Des Weiteren war die Zellzyklusrate innerhalb von 3D Mikrokavitäten niedriger verglichen mit herkömmlichen 2D Substraten. Diese Ergebnisse bestätigen ferner obenstehende These, dass Zytokin-induzierte Zellexpansion durch erhöhte Zelladhäsions-vermittelte Signale überschrieben wird. Um die in vitro Studien zu komplettieren wurde ein in vivo Repopulationsversuch durchgeführt. HSC kultiviert auf Einzel-Zell-Mikrokavitäten übertrafen frisch isolierte Konkurrenz-Zellen in einem kompetitiven Repopulationsversuch. Dieses erste Ergebnis zeigt, dass sich der Zellgröße entsprechende Biomaterialien für die erfolgreiche Stammzell-Kultur eignen. Die Ergebnisse dieser Arbeit bieten eine vielversprechende in vitro Zellkulturstrategie, die ein besseres Verständnis der Einflüsse von exogenen Signalen auf HSC erlaubt und damit eine Grundlage für neue Erkenntnisse in Richtung erfolgreicheres Tissue Engineering und klinische Anwendungen im Bereich der regenerativen Medizin bildet.:Kurzbeschreibung 4 Abstract 6 1 Introduction 8 1.1 Motivation 8 1.2 Objective 8 2 Basics 10 2.1 Stem Cells and their Role in Life 10 Stem Cells and their Niches 12 2.1.1 Hematopoietic Stem Cells 12 2.1.2 Hematopoietic Stem Cell Niche 14 2.1.3 The ECM Relevancy 16 2.1.4 HSC Relevant Cytokines 19 2.2 Cell Culture Scaffolds 21 2.2.1 General 2D, 3D 21 2.2.2 Substrate Engineering 22 2.2.3 Co-Culture versus the Artificial 3D Niche 23 3 Materials and Methods 25 3.1 Chemicals, Reagents and Equipment 25 3.2 Wafer Design and Surface Functionalization 29 3.3 Cell Culture and Analysis 31 3.3.1 HSC Culture in ECM-functionalized Microcavities 32 3.4 Surface Passivation 33 3.5 Mouse Bone Marrow Preparation 35 4 Results and Discussion 37 4.1 Scaffold Design and Preparation 37 4.1.1 Surface Characterization 37 4.1.2 Surface Passivation 39 Approaches for Surface Passivation 39 Efficiency of Surface Passivation 39 4.1.3 Redesigned Microcavities 43 4.2 Summarized Discussion of the Surface Passivation 44 4.3 HSC Culture inside Microcavities 45 4.3.1 HSC-ECM Interaction Reduces Proliferation 45 4.3.2 Population-wide Proliferation and Differentiation of Spatially Constrained HSCs . … 46 HSCs within Redesigned Microcavities 48 4.3.3 Colony-forming Ability of Microcavity Cultures 50 4.4 Single Cell Analysis of Differentiation 52 4.5 Cell Cycling Dependency on Cytokine Level 53 4.5.1 Plane Surfaces 54 4.5.2 Microcavities Reduce Cycling Frequency 57 4.6 Mice Repopulation of Microcavity Cultured HSCs 58 4.7 Summarized Discussion of the HSC–ECM Relation 60 4.8 Future Prospects 62 5 Summary 63 References 64 Figure Legend 73 Tables 73 Theses 74 6 Appendices I 6.1 FACS Principle I 6.1.1 HSC Staining for CD Marker and Cell Cycle Kinetics I 6.1.2 Apoptosis Test II 6.2 Differentiation and Proliferation on Redesigned Microcavities III 6.3 Colony-forming Capability of Microcavity Cultured Cells IV 6.4 Effect of Trypsin on HSC Properties in Long Term Culture IV 6.5 Surface Functionalization with SCF V 6.5.1 Analysis of the HSCs Grown on Immobilized SCF VI 6.5.2 SCF Immobilization and its Kinetics VII 6.5.3 c-kit Expression Kinetics and HSC Differentiation VIII Short Discussion on the Growth Factor Immobilization IX Publications X Posters X Proceedings XI Talks XI Patents XI Papers XI Awards XI 7 Danksagung: XII Selbstständigkeitserklärung: XIII

Dynamic alterations of composition and mechanics of the extracellular matrix (ECM) are suggested to modulate cellular behavior including plasticity of macrophages (MPhs) during wound healing. In this study, engineered 3D fibrillar matrices based on naturally occurring biopolymers (collagen I, glycosaminoglycans (GAGs)) were used to mimic matrix stiffening as well as modification by sulfated and non-sulfated GAGs at different stages of wound healing. Human MPhs were found to sensitively respond to these microenvironmental cues in terms of polarization towards pro-inflammatory or wound healing phenotypes over 6 days in vitro. MPhs exhibited a wound healing phenotype in stiffer matrices as determined by protein and gene expression of relevant cytokines (IL10, IL12, TNF). Presence of sulfated and non-sulfated GAGs inhibited this polarization effect. Furthermore, control experiments on 2D matrices stressed the relevance of using stiffness-controlled 3D matrices, as MPhs showed a reciprocal polarization behavior depending on GAG presence. Hence, the results indicate a strong influence of dimensionality, stiffness, and GAG presence of the biomaterial scaffold on MPh polarization and emphasize the need for matrices closely mimicking the 3D in vivo context with a variable stiffness and GAG composition in in vitro studies.

Despite progress in our understanding of the molecular drivers that propagate the overall process of metastasis, the adaptation of specific organs upon these molecular interactions for metastatic entry remains poorly understood. This is particularly true for liver metastases, the liver being a common site for metastatic disease, and metastatic hepatic tumors are more prominent than primary hepatocellular or biliary tumors. Liver metastases most commonly arise from colorectal cancer than any other cancer and constitute one of the most detrimental outcomes of cancer, characterized by poor prognosis, high mortality, and no effective therapies available other than surgical interventions. Since interactions between tumour cells and the tumour microenvironment play an important part in the engraftment, survival, and progression of the metastases, the discovery of new drivers of liver metastasis with the potential to become therapeutic and preventive targets is required to advance the care of liver metastasis patients as well as cancer patients at risk of metastatic spread to the liver. The alteration of the physical structure of the tissue is extremely important in the progression of malignant diseases, such as cancer metastasis, as it directly affects the extravasation and colonization of tumour cells. The major hurdles in liver metastasis research, stem not only from our insufficient understanding of the molecular mechanisms directing and mediating metastasis particularly to the liver but also from the limited number of pre-clinical models available that mimic human disease and enable the study of the complex interactions between tumor cells and the liver microenvironment. The liver metastatic process underlies the acquisition of key adaptations by tumor-derived factors and is determined by both tumour-intrinsic properties and the crosstalk between tumour cells and stromal cells in the liver. A normal functioning and structurally intact extracellular matrix (ECM) constitute a hostile “soil” for seeding tumor cells to colonize. Eventually, it is the ability of tumor cells to remodel the liver microenvironment and create a supportive niche for metastatic tumor cell survival and outgrowth that determines successful metastatic colonization. Among tumour-secreted factors, which are recognized as major contributors to the formation of pre-metastatic and metastatic niches, tumor-derived extracellular vesicles (EVs) have recently arisen as crucial players in cell-to-cell communication and in the remodeling of distant microenvironments that favor organ-specific metastasis. Therefore, we sought to determine the role of tumor-derived EVs in the modulation of the liver microenvironment and their specific contribution to supporting metastatic colonization of the liver. The preliminary step to this process was to establish a model system to identify EV-associated targets and their effect on the ECM remodelling. Immunohistochemical analyses of primary colon tumour (CRC) and secondary liver metastases (CRC liver MET) tissue samples from patients with CRC revealed higher stromal TIMP1 levels in CRC liver MET than in CRC. The elevated stromal TIMP1 signature in the invasive front was associated with poor progression-free survival in patients with CRC liver MET. Our characterisation of the CRC tumour-derived EVs showed TIMP1 enrichment in the EVs (TIMP1EV) compared to its parental cell. Using cultures of primary liver fibroblasts, we could demonstrate that TIMP1 enrichment in the CRC-EVs was associated with regulation of TIMP1 levels in the EV-conditioned liver fibroblasts. Using our optimized ex vivo 3D ECM remodelling assay, we observed that pre-conditioning the liver fibroblasts with EVs from CRC cells promotes ECM remodelling. In accordance with our cell line model, we showed that serum-derived TIMP1EV from CRC patients promotes ECM remodelling. Moreover, high serum TIMP1EV expression in CRC liver MET patients was significantly associated with poor overall survival. In addition, our data also indicated that the determination of EV-associated TIMP1 is superior for non-invasive diagnosis than the analysis of soluble TIMP1 from total serum. Finally, we showed that HSP90AA is constitutively bound to TIMP1EV and that targeting HSP90AA leads to TIMP1 downregulation and inhibits ECM-mediated remodelling. This study defining the contribution of extravesicular TIMP1 to liver metastasis brings a novel insight into the molecular mechanisms through which tumor-secreted factors packaged via EVs promote remodelling of the liver microenvironment. The clinical significance of overexpression of extravesicular TIMP1 in patients with colorectal liver metastases highlights its potential as a prognostic biomarker and therapeutic target. With further clinical studies, Heparin and HSP90 inhibitors targeting the EV mediated TIMP1 regulation could be a putative treatment strategy to treat colorectal liver metastases.:Table of Contents Abbreviations v 1. Introduction 1 1.1 Colorectal cancer 1 1.1.1. Incidence and mortality 1 1.1.1. Tumor staging 2 1.1.1. Pattern of distant metastases in colorectal cancer 5 1.2 Colorectal liver metastases 6 1.2.1 Current evaluation and treatment strategies for colorectal liver metastases 7 1.2.2 The liver metastasis cascade - a multi-step process 10 1.3 Tumor microenvironment 12 1.3.1 Tumour-stroma interactions 15 1.3.2 ECM remodelling and its role in CRC tumor progression 17 1.4 Extracellular vesicles 21 1.4.1 EV types 21 1.4.2 Biogenesis and secretion of EVs 22 1.4.3 Molecular composition of EVs 24 1.4.4 Biological functions of EVs 26 1.4.5 EVs in Tumor microenvironment 28 1.4.6 EVs in Tumor-fibroblast communication 29 1.4.7 Role of EVs in colorectal cancer 31 1.5 Tissue inhibitor of metalloproteinases (TIMP1) 35 1.5.1 TIMP1 in cancer 37 2. Background and Research Aims 39 3. Material and Methods 40 3.1 Material 40 3.1.1 Devices 40 3.1.2 Additional material and equipment 42 3.1.3 Fine chemicals 43 3.1.4 Biochemicals 45 3.1.5 Primary antibodies 46 3.1.6 Secondary antibodies 47 3.1.7 Nucleic acids 47 3.1.8 Consumables 50 3.1.9 Softwares 51 3.2 Methods 52 3.2.1 Patients 52 3.2.2 Immunohistochemistry 52 3.2.3 Hematoxylin eosin staining 54 3.2.4 Cell lines 54 3.2.5 Primary liver fibroblast cell lines 54 3.2.6 Passaging and freezing of cells 55 3.2.7 Revival of frozen cells 55 3.2.8 Cell counting 56 3.2.9 EV Isolation from CRC cell lines 56 3.2.10 Isolation of serum-derived EVs from liquid biopsies 56 3.2.11 Characterisation of EVs 57 3.2.12 Treatment of Fibroblasts with EVs 58 3.2.13 Stimulation of PFs with recombinant TIMP1 59 3.2.14 RNA isolation 59 3.2.15 cDNA synthesis 59 3.2.16 Quantitative Real-Time PCR (qRT-PCR) 60 3.2.17 Protein quantification 61 3.2.18 Immunoblotting and co-immunoprecipitation 61 3.2.19 ELISA 62 3.2.20 TIMP1 Knock-Out (KO) and Over-Expression (OE) 62 3.2.21 17 AAG and HSP90AA antibody treatment 63 3.2.22 3D ECM-remodelling assay 63 3.2.23 PKH staining 65 3.2.24 In vivo experiments 65 3.2.25 DAPI staining 66 3.2.26 Tissue explant model 66 3.2.27 Statistical analysis and reproducibility 67 4. Results 68 4.1 Identification of TIMP1 as target molecule 68 4.1.1 Identification of TIMP1 as a target through data mining 68 4.1.2 Localization pattern of TIMP1 in CRC and CRC liver MET 70 4.1.3 Invasion front-specific overexpression of TIMP1 in the stroma of patients with CRC liver MET is associated with poor progression-free survival (PFS) 72 4.2 Model system to study CRC-EV mediated ECM remodelling 73 4.2.1 Investigating the role of CRC- derived EVs in the evolution of colorectal liver metastases 73 4.2.2 Characterizsation of isolated EVs from the CRC cell lines 74 4.2.3 TIMP1 enrichment in EVs derived from CRC cell lines 75 4.2.4 CRC-derived TIMP1EV regulates TIMP1 levels in recipient fibroblasts 76 4.2.5 TIMP1EV mediated TIMP1 upregulation in the recipient fibroblast is an EV-mediated effect 79 4.2.6 Recombinant TIMP-1 induces TIMP1 levels in recipient pFs in a time- and concentration-dependent manner 81 4.2.7 Alteration of TIMP1 levels in HCT 116 cells translates into EVs but does not affect EV packaging. 83 4.2.8 TIMP1EV levels in CRC EVs determine TIMP1 levels in recipient fibroblasts 85 4.2.9 EV-mediated TIMP1 upregulation in pFs induces ECM remodelling 86 4.2.10 TIMP1 levels in the PFs influence the extent of ECM remodelling 88 4.3 Clinical significance of TIMP1EV 89 4.3.1 TIMP1 enriched in serum-derived EVs of CRC patients compared to healthy controls 89 4.3.2 Serum derived TIMP1EV from CRC patients regulate TIMP1 levels in primary liver fibroblasts 91 4.3.3 Serum derived TIMP1EV from CRC patients promote ECM remodelling 93 4.3.4 TIMP1EV exhibits superior stratification power compared to soluble TIMP1 in liquid biopsies 93 4.3.5 TIMP1EV is a non-invasive independent prognostic marker in colorectal liver metastases 94 4.4 Targeting TIMP1EV mediated ECM remodelling 97 4.4.1 TIMP1EV binds to HSP90AA 97 4.4.2 HSP90 inhibition interferes with TIMP1 protein stabilisation 99 4.4.3 17AAG attenuates TIMP1EV-mediated ECM remodelling 101 4.5 EVs derived from murine CRC cell lines regulate TIMP1 levels in recipient fibroblasts 104 4.6 Increased homing of CRC EVs to the liver compared to other organs 106 4.7 TIMPEV regulates TIMP1 levels in liver tissues 108 5. Discussion 112 5.1 TIMP1 Localization and its significance in liver metastases 112 5.2 Model system to study the role of CRC-EVs in liver metastasis 113 5.3 In-vitro model to study the pro-metastatic effects of TIMP1EV 114 5.4 Serum-derived extravesicular TIMP1 and its pro-metastatic functions underlying remodeling of the extracellular matrix 116 5.5 Clinical significance of TIMP1EV in colorectal liver metastases 117 5.6 Scope of HSP90 inhibitors in the prevention and treatment of CRC liver metastases...……………………………………………………………………………………..118 6. Future perspectives and concluding remarks 120 7. Graphical summary of the findings 122 Zusammenfassung 123 Summary 125 List of figures 127 List of Tables 129 References 130 Acknowledgements 163 Appendix 165

Im Gehirn gemessene Extrazelluläre Feldpotentiale (EFPs) sind ein wichtiges Maß für neuronale Aktivität. In vielen Fällen ist der genaue physiologische Ursprung dieser Potentiale unbekannt oder umstritten. Der auditorische Hirnstamm der Schleiereule bietet eine ausgezeichnete Möglichkeit, die EFPs und ihren Ursprung zu untersuchen. Der Hirnstamm der Eule ist ideal, weil das Feldpotential in ihm sehr stark ist, weil die zugrundeliegende Anatomie wohl-untersucht ist, und weil das Potential sehr einfach durch auditorische Stimulation gesteuert werden kann. In dieser Arbeit präsentiere ich zwei Beispiele, in welchen ich mir die einzigartigen Eigenschaften der Schleiereule zunutze mache, um das EFP zu erforschen. Das erste Beispiel behandelt Axone, und ich zeige, dass neuronale Aktivität in Axonbündeln, welche eine charakteristische Endzone besitzen, ein starkes Dipolmoment erzeugen kann. Im zweiten Beispiel behandele ich Synapsen. Aus den EFPs der Synapsen konnte ich die Merkmale der synaptischen Kurzzeitplastizität extrahieren. Die Methoden und Erkenntnisse die ich entwickelt habe sind auf andere Organismen übertragbar und erweitern das Verständnis vom Einfluss unterschiedlicher anatomischer Strukturen auf das EFP. / Extracellular field potentials (EFPs) recorded in the brain are an important indicator of neural activity for neuroscientists. In many cases, their physiological basis is unknown or debated. The barn owl auditory brainstem provides an excellent opportunity to study these EFPs and their origins. The barn owl auditory brainstem is ideal because the field potentials are very large and very easily controlled by the auditory stimulus, and the underlying anatomy is well known. Here I present two examples of exploiting the unique properties of the EFP in the barn owl auditory brainstem. The first is concerned with axons, where I show that activity in axon bundles with characteristic termination zones generates strong dipole moments. The second example is concerned with synaptic currents, from which I was able to extract a signature of short-term plasticity. The methods and insights I developed are applicable to other organisms as well, and contribute to the general understanding of the roles different anatomical structures can play in the generation of EFPs.

Les pathologies musculo-squelettiques affectant les os et les articulations demeurent un défi pour la médecine régénératrice. Les difficultés retrouvées sont liées aux besoins d’une vascularisation tissulaire optimale pour les substituts osseux et à l’obtention d’un cartilage de qualité pour les substituts articulaires. Les objectifs de ces travaux de thèse étaient de parvenir à différencier des cellules souches mésenchymateuses (CSM) ombilicales humaines – outil de choix en médecine régénérative - vers un phénotype vasculaire et un phénotype chondrogénique articulaire pour répondre aux besoins de l’ingénierie tissulaire musculo-squelettique. La différenciation de CSM ombilicales en cellules musculaires lisses vasculaires a été montrée après 12 jours de stimulation par des facteurs solubles comme le transforming growth factor (TGF)-beta1 et l’acide ascorbique. Face aux limites des supports de culture en deux dimensions in vitro, un modèle de culture tridimensionnel a été mis en place à l’aide de CSM cultivées en “pellets” en présence ou non d’une solution matrice extracellulaire (MEC) ombilicale pour les expériences suivantes. Les facteurs solubles disposant d’une efficacité limitée et étant souvent onéreux, une transduction des “pellets” de CSM-GW a été réalisée à l’aide de virus adéno-associés recombinants (rAAV) permettant une synthèse de facteurs par les cellules à plus long terme. Ici, les pellets ont été transduits à l’aide de rAAV contenant le gène du facteur de transcription Sox9 afin d’induire une différenciation chondrogénique articulaire. Ces travaux démontrent l’intérêt d’associer la MEC et les CSM ombilicales humaines dans un modèle de culture en trois dimensions, ainsi que les outils de thérapie génique comme les rAAV pour constituer des implants utilisables pour régénérer les tissus musculo-squelettiques / Musculoskeletal conditions affecting bones and joints remain a challenge for regenerative medicine. The difficulties found are related to the needs of an optimal tissue vascularization for bone substitutes and to obtain a cartilage of quality for articular substitutes. The objectives of this PhD work were to differentiate human umbilical mesenchymal stem cells (MSC) - a tool of choice in regenerative medicine - towards a vascular phenotype and a joint chondrogenic phenotype to meet the needs of musculoskeletal tissue engineering. The differentiation of umbilical MSC into vascular smooth muscle cells was shown after 12 days of stimulation by soluble factors such as transforming growth factor (TGF)-beta1 and ascorbic acid. Facing the limits of two-dimensional culture supports in vitro, a three-dimensional culture model was set up using MSC cultured in pellets with or whitout an umbilical extracellular matrix (ECM) solution for the following experiences. Soluble factors have a limited efficacy and are often expensive, a transduction of MSC pellets was performed using recombinant adeno-associated viruses (rAAV) allowing a long term synthesis of factors by the cells. Here, the pellets were transduced using rAAV containing the Sox9 transcription factor gene to induce articular chondrogenic differentiation. This work demonstrates the interest of associating ECM and human umbilical MSC in a three-dimensional culture model, as well as gene therapy tools such as rAAVs to provide grafts that can be used to regenerate musculoskeletal tissues / Muskel- und Skeletterkrankungen an Knochen und Gelenken bleiben eine Herausforderung für die regenerative Medizin. Die Schwierigkeiten stehen im Zusammenhang mit den Ansprüchen einer optimalen Gewebevaskularisierung der Knochenersatzstoffe und damit, einen qualitativ hochwertigen Knorpel für den Gelenkersatz zu erhalten. Das Ziel dieser Doktorarbeit war es, humane mesenchymale Stammzellen (MSZ) aus der Nabelschnur, die das Werkzeug der Wahl in der regenerativen Medizin sind, in einen vaskulären und in einen chondrogenen Phänotyp zu differenzieren, um den Anforderungen des muskuloskeletalen Tissue Engineerings zu entsprechen. Die Differenzierung von Nabelschnur-MSZ in vaskuläre glatte Muskelzellen konnte durch Stimulation mit löslichen Faktoren wie dem Transforming Growth Factor (TGF)-beta1 und Ascorbinsäure nach 12 Tagen gezeigt werden. Angesichts der Grenzen einer zweidimensionalen Zellkultur in vitro, wurde ein dreidimensionales Kulturmodell, in dem MSZ in Pellets mit oder ohne extrazelluläre Matrix der Nabelschnur (EZM) kultiviert wurden, für die weiteren Versuche erstellt. Lösliche Faktoren haben eine limitierte Wirksamkeit und sind häufig teuer. Deshalb wurden die MSZ-Pellets mit rekombinanten Adeno-assoziierte Viren (rAAV) transduziert, was eine Synthese der Faktoren durch die Zellen über einen längeren Zeitraum ermöglicht. In unserem Fall wurden die Pellets mit rAAV, die das Transkriptionsgen Sox9 enthalten, tranzduziert, um eine artikuläre chondrogene Differenzierung zu induzieren. Diese Arbeit zeigt, dass es von Interesse ist EZMs und humane Nabelschnur-MSZ in einem dreidimensionalen Kulturmodell zu vereinen. Auch wird gezeigt, dass Werkzeuge der Gentherapie wie rAAVs, die den Transplantaten zugeführt werden, helfen können muskuloskeletales Gewebe zu regenerieren

Gewebezellen sammeln ständig Informationen über die mechanischen Eigenschaften ihrer Umgebung, indem sie aktiv an dieser ziehen. Diese Kräfte werden an Zell-Matrix-Kontakten übertragen, die als Mechanosensoren fungieren. Jüngste Experimente mit Zellen auf elastischen Substraten zeigen, dass Zellen sehr empfindlich auf Veränderungen der effektiven Steifigkeit ihrer Umgebung reagieren, die zu einer Reorganisation des Zytoskeletts führen können. In dieser Arbeit wird ein theoretisches Model entwickelt, um die Selbstorganisation von Zellen in weichen Materialien vorherzusagen. Obwohl das Zellverhalten durch komplexe regulatorische Vorgänge in der Zelle gesteuert wird, scheint die typische Antwort von Zellen auf mechanische Reize eine einfache Präferenz für große effektive Steifigkeit der Umgebung zu sein, möglicherweise weil in einer steiferen Umgebung Kräfte an den Kontakten effektiver aufgebaut werden können. Der Begriff Steifigkeit umfasst dabei sowohl Effekte, die durch größere Härte als auch durch elastische Verzerrungsfelder in der Umgebung verursacht werden. Diese Beobachtung kann man als ein Extremalprinzip in der Elastizitätstheorie formulieren. Indem man das zelluläre Kraftmuster spezifiziert, mit dem Zellen mit ihrer Umgebung wechselwirken, und die Umgebung selbst als linear elastisches Material modelliert, kann damit die optimale Orientierung und Position von Zellen vorhergesagt werden. Es werden mehrere praktisch relevante Beispiele für Zellorganisation theoretisch betrachtet: Zellen in externen Spannungsfeldern und Zellen in der Nähe von Grenzflächen für verschiedene Geometrien und Randbedingungen des elastischen Mediums. Dafür werden die entsprechenden elastischen Randwertprobleme in Vollraum, Halbraum und Kugel exakt gelöst. Die Vorhersagen des Models stimmen hervorragend mit experimentellen Befunden für Fibroblastzellen überein, sowohl auf elastischen Substraten als auch in physiologischen Hydrogelen. Mechanisch aktive Zellen wie Fibroblasten können auch elastisch miteinander wechselwirken. Es werden daher optimale Strukturen als Funktion von Materialeigenschaften und Zelldichte bzw. der Geometrie der Zellpositionen berechnet. Schließlich wird mit Hilfe von Monte Carlo Simulationen der Einfluss stochastischer Störungen auf die Strukturbildung untersucht. Das vorliegende Model trägt nicht nur zu einem besseren Verständnis von vielen physiologischen Situationen bei, sondern könnte in Zukunft auch für biomedizinische Anwendungen benutzt werden, um zum Beispiel Protokolle für künstliche Gewebe im Bezug auf Substratgeometrie, Randbedingungen, Materialeigenschaften oder Zelldichte zu optimieren. / Adherent cells constantly collect information about the mechanical properties of their extracellular environment by actively pulling on it through cell-matrix contacts, which act as mechanosensors. In recent years, the sophisticated use of elastic substrates has shown that cells respond very sensitively to changes in effective stiffness in their environment, which results in a reorganization of the cytoskeleton in response to mechanical input. We develop a theoretical model to predict cellular self-organization in soft materials on a coarse grained level. Although cell organization in principle results from complex regulatory events inside the cell, the typical response to mechanical input seems to be a simple preference for large effective stiffness, possibly because force is more efficiently generated in a stiffer environment. The term effective stiffness comprises effects of both rigidity and prestrain in the environment. This observation can be turned into an optimization principle in elasticity theory. By specifying the cellular probing force pattern and by modeling the environment as a linear elastic medium, one can predict preferred cell orientation and position. Various examples for cell organization, which are of large practical interest, are considered theoretically: cells in external strain fields and cells close to boundaries or interfaces for different sample geometries and boundary conditions. For this purpose the elastic equations are solved exactly for an infinite space, an elastic half space and the elastic sphere. The predictions of the model are in excellent agreement with experiments for fibroblast cells, both on elastic substrates and in hydrogels. Mechanically active cells like fibroblasts could also interact elastically with each other. We calculate the optimal structures on elastic substrates as a function of material properties, cell density and the geometry of cell positioning, respectively, that allows each cell to maximize the effective stiffness in its environment due to the traction of all the other cells. Finally, we apply Monte Carlo simulations to study the effect of noise on cellular structure formation. The model not only contributes to a better understanding of many physiological situations. In the future it could also be used for biomedical applications to optimize protocols for artificial tissues with respect to sample geometry, boundary condition, material properties or cell density.

Der Erfolg neuer Strategien in der Regenerativen Medizin und im Tissue Engineering hängt maßgeblich von einem gut entwickeltem vaskulären Netzwerk ab, welches die auf den Implantaten wachsenden Zellen und Gewebe versorgen. Oberflächeneigenschaften der Implantate sowie die Präsentation verschiedener Liganden für extrazelluläre Matrixproteine spielen bei der Besiedlung der Implantate, als auch bei der Bildung versorgender Blutgefäße durch die Endothelzellen eine wesentliche Rolle. In dieser Arbeit konnte durch Variation der Anbindungsstärke (kovalent oder physisorptiv) des extrazellulären Matrixproteins Fibronektins an die MSA-Copolymere der Einfluss des Aufbaus der extrazellulären Matrix auf das Differenzierungsverhalten der Endothelzellen gezeigt werden. Auch die initiale Konzentration von Adhäsionsproteinen an der Substratoberfläche zeigte sich bedeutend für das Verhalten der Zellen. Optimal für eine gute Adhäsion, native Entwicklung und Kapillarbildung der Endothelzellen war die stabile (kovalente) Anbindung weniger Adhäsionsproteine (hier Fibronektin) an die Substratoberfläche, so dass die Zellen problemlos adhärieren konnten. Erfolgte die weiter Proteinadsorption an die Oberflächen in einem nativen Zustand (hier auf den hydrophilen Oberflächen) so waren die Endothelzellen in der Lage, die extrazelluläre Matrix zu reorganisieren und ein dem in vivo Zustand ähnlicher Aufbau der extrazellulären Matrix konnte realisiert werden. Dies ermöglichte den Zellen wiederum ein natürliches Verhalten. Die Ausbildung einer moderaten Anzahl von Adhäsionsstellen der Zellen, sowie der in vivo ähnliche Aufbau der Adhäsionspunkte ermöglichte den Zellen einen eher lockeren Kontakt zum Substrat. Daher waren sie sehr flexibel in ihrer Morphologieanpassung. Unter diesen Bedingungen war es möglich, dass die Endothelzellen bei Stimulierung der Angiogenese kapillarähnliche Strukturen ausbildeten. Die Verwendung dreidimensionaler Zellkulturträger zeigte eine Unterstützung der Kapillarbildung der Endothelzellen in Abhängigkeit unter den beschrieben Bedingungen. / The success of tissue engineering strategies using artificial scaffolds crucially depends on a controlled formation of well-developed vascular networks in growing tissues. The presentation of extracellular matrix ligands on scaffolds is often envisioned as an appropriate strategy to support capillary formation. We show that the control of primary coupling mode — covalent versus physisorbed — as well as of secondary interactions of cell-secreted extracellular matrix proteins have a strong impact on endothelial cell development. A set of maleic anhydride copolymer thin films was used as planar model substrates. They exhibit a switchable mode of primary matrix coupling combined with a gradation of secondary matrix–substrate interactions due to a variation of surface hydrophobicity and polarity. We found that the cells adhere in a more native state at a low amount of covalent primary coupled fibronectin ligands in conjunction with weak interactions of secondarily adsorbed adhesion ligands on hydrophilic surfaces. These substrates allow for a formation of capillary-like networks of endothelial cells. High ligand densities and strong secondary hydrophobic interactions inhibit a pronounced capillary formation. The composition and structure of the formed extracellular matrix correlates well with the specific integrin expression pattern. From these results it is concluded that the formation of blood capillaries in artificial scaffolds can be triggered by controlling primary and secondary coupling of cell adhesion ligands to implant materials. 2

Interactions of cells with the extracellular matrix (ECM) have important roles in various physiological and pathological processes, including tissue morphogenesis during embryonic development, wound healing and tumor invasion. Although most of the proteins involved in cell-ECM interactions have been identified, the underlying mechanisms and involved signaling pathways are incompletely understood. Here, atomic force microscope-based imaging and single-cell force measurements were used to characterize the interactions of different cell types with ECM proteins. The interplay between cells and ECM is complex. However, two interaction types, protein-protein and protein-carbohydrate, predominate. Integrins, adhesion receptors for ECM, mediate the former, galectins, a family of animal lectins, the latter. In the second chapter of this thesis, the contributions of both receptor families to the interactions of epithelial MDCK cells with ECM proteins are presented. It was found that galectins-3 and 9 are highly expressed in MDCK cells and required for optimal long-term adhesion (90 minutes) to ECM proteins collagen-I and laminin-111. Interestingly, early adhesion (< 2 minutes) to laminin-111, was integrin-independent and instead mediated by carbohydrate interactions and galectins. In contrast, early adhesion to collagen-I was exclusively mediated by integrins. Moreover, cells frequently entered an enhanced adhesion state, marked by a significant increase in the force required for cell detachment. Although adhesion was mediated by integrins, adhesion enhancement was especially observed in cells depleted for galectin-3. It was proposed that galectin-3 influences integrin-mediated adhesion complex formation by altering receptor clustering. To control their attachment to ECM proteins, cells regulate integrin receptors. One regulatory process is integrin crosstalk, where the binding of one type of integrin influences the activity of another type. In the third chapter, the implementation of a single-cell force spectroscopy assay to identify such crosstalks and gain insight into their mechanisms is described. In this assay the interactions of integrin receptors being specifically attached to one ligand are characterized in dependence of another ligand-bond receptor pair. With this assay a crosstalk between collagen-binding integrin α1β1 and fibronectin-binding integrin α5β1 was identified in HeLa cells. This crosstalk was directional from integrin α1β1 to integrin α5β1 and appeared to regulate integrin α5β1 by inducing its endocytosis. In the fourth and final chapter, mechanisms of matrix-induced cell alignment were studied by imaging cells on two-dimensional matrices assembled of highly aligned collagen fibrils. Integrin α2β1 was identified as the predominant receptor mediating cell polarization. Time-lapse AFM demonstrated that during alignment cells deform the matrix by reorienting individual collagen fibrils. Cells deformed the collagen matrix asymmetrically, revealing an anisotropy in matrix rigidity. When matrix rigidity was rendered uniform by chemical cross-linking or when the matrix was formed from collagen fibrils of reduced tensile strength, cell polarization did not occur. This suggested that both the high tensile strength and pliability of collagen fibrils contribute to the anisotropic rigidity of the matrix and lead to directional cellular traction and cell polarization. During alignment, cellular protrusions contacted the collagen matrix from below and above. This complex entanglement of cellular protrusions and collagen fibrils may further promote cell alignment by maximizing cellular traction. The work presented here adds to the understanding of cell-ECM interactions. Atomic force microscopy imaging allowed characterizing the behavior of cells on nanopatterned collagen matrices whereas single-cell force spectroscopy revealed insights into the regulation of cell adhesion by galectins. Furthermore, methodological advances in the single-cell force spectroscopy assay allowed the intracellular regulation of receptor molecules to be studied. The work demonstrates that atomic force microscopy is a versatile tool to study cell-ECM interactions.

Das Zytokin Interleukin-10 (IL-10) ist ein Schlüsselspieler in der Regulation des Immunsystems mit pro- und anti-inflammatorischen Funktionen. Es spielt eine wichtige Rolle bei der Terminierung und Unterdrückung einer Entzündungsantwort, die ansonsten zu einer bleibenden Schädigung des Gewebes führen kann. Eine Dysregulation von IL-10 ist mit verschiedenen Krankheitsbildern wie chronischen Entzündungen, Autoimmunerkrankungen und Krebs assoziiert. IL-10 wird von einem breiten Spektrum von Zelltypen, darunter hauptsächlich hämatopoetische Zellen, aber auch epitheliale und mesenchymale Zellen, gebildet und in den extrazellulären Raum freigesetzt, wo es mit Komponenten der extrazellulären Matrix in Kontakt kommt. Es ist bekannt, dass IL-10 an Glykosaminoglykane (GAGs) binden kann und dass diese Interaktion seine biologische Aktivität beeinflusst. GAGs sind eine Klasse linearer Polysaccharide der extrazellulären Matrix. Sie bestehen aus wiederholenden Disaccharideinheiten und haben einen hoch negativ geladenen Charakter, welcher durch einen hohen Grad an Sulfatierung in der Zuckerkette zustandekommt. Sie binden eine Vielzahl an Signalproteinen und regulieren deren biologische Funktionen, etwa indem sie Einfluss auf die Rezeptorbindung oder die räumliche Verteilung des Proteins im Gewebe nehmen. Die molekularen Mechanismen, wodurch GAGs die biologische Aktivität von IL-10 beeinflussen, sind bisher unbekannt. Insbesondere ist nichts über die strukturellen Grundlagen der Interaktion bekannt, die Voraussetzung für ihr funktionelles Verständnis sind. In dieser Arbeit wurden daher die Bindungseigenschaften von IL-10 und GAGs sowie der strukturelle Aufbau ihres molekularen Komplexes unter Verwendung von NMR-Spektroskopie in Lösung charakterisiert. Es wurde eine definierte GAG-Bindungsstelle in IL-10 identifiziert und die Bindungsepitope und Bindungsaffinitäten von GAGs bestimmt. Die Ergebnisse dieser Arbeit weisen auf eine wichtige Rolle, die GAGs in der Biologie von IL-10 spielen können, hin – etwa für seine Speicherung im Gewebe oder für die IL-10-Rezeptorbindung.