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
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:84753 |
| Date | 18 April 2023 |
| Creators | Rao, Venkatesh Sadananda |
| Contributors | Kahlert, Christoph, Füssel, Susanne, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English, German |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | 10.1038/s41388-022-02218-9 |
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