Die vorliegende Arbeit befasst sich dem Glykosaminoglykan (GAG) Hyaluronan (HA) und dessen Einfluss auf die Entwicklung des malignen Melanoms (MM). Das in der extrazellulären Matrix (ECM) reichlich vorkommende HA wird schon seit dem späten 20. Jahrhundert genauerer im Zusammenhang mit der Tumorentwicklung untersucht. Gegensätzliche Eigenschaften von HA, die zum einen Tumore fördern und zum anderen ihnen entgegenwirken, wurden seither veröffentlicht. Allgemeiner Konsens ist, dass das HA-Molekulargewicht über die verschiedenen Effekte von HA entscheidet. Momentan ist jedoch nicht ausreichend belegt, wie HA auf das Tumorwachstum einwirkt und welche HA-Größen dies speziell begünstigen. Diese Untersuchung basiert auf einem in vivo Knockout von der Hyaluronan Synthase 2 (Has2) in der Maus, der die Produktion von hoch-molekulargewichtigen HA (HMW-HA) weitestgehend einschränkte. Das Wachstum vom experimentellem MM wurde in Abwesenheit der meisten HMW-HA untersucht. Diese Arbeit versuchte den Beweis zu erbringen, dass das lokale Wachstum und die Metastasenbildung der MM-Zellen abhängig von der vorhandenen HMW-HA in der Nähe des Tumors ist.
Der Has2-knockout in der Mauslinie C57BL6 wurde verifiziert und nach einem veränderten Phänotyp der Mäuse untersucht. Obwohl nahezu dreiviertel der absoluten HA Menge durch den Knockout fehlte, zeigten die Mäuse keinen offensichtlichen Veränderungen. Erst eine Messung der Haut-Permeabilität deutete auf eine womöglich verstärkte Ausbildung der Stratum corneum hin. Eine direkte Auswirkung vom fehlendem HA auf das Tumorwachstum und der Metastasierungsrate konnte nicht ausreichend belegt werden. Das verwendete Mausmodell sowie die Wahl des experimentellen Tumors werden in dieser Arbeit kritisch diskutiert. Parallel durchgeführte in vitro Versuche mit teilweise artifiziellen 3D Matrizen, die unterschiedliche Mengen von HA enthielten, zeigten einen stärkeren Einfluss von niedermolekulare HA (LMW-HA) auf die Proliferation und Invasion von MM Zellen. Diese Beobachtungen stimmen mit dem derzeitigen Konsens überein, dass LMW-HA aktivierende Signaltransduktion auslöst und HMW-HA eher homöostatisch wirkt.:TABLE OF CONTENT
Zusammenfassung
Summary
Table of content
List of figures
List of tables
Abbreviations
1 Introduction
1.1 Structures of the skin
1.2 The malignant melanoma
1.3 The tumor microenvironment (TME)
1.4 Hyaluronan
1.4.1 HA Structure
1.4.2 HA anabolism
1.4.3 HA catabolism
1.4.4 HA binding proteins
1.4.5 HA cell surface receptors
1.5 Hyaluronan in malignant melanoma
1.6 Aim of the thesis
2 Methods
2.1 Murine malignant melanoma cell lines
2.2 Inducible Has2-knockout mice
2.2.1 The Cre/loxP System
2.2.2 Genetical modification for the Has2-knockout
2.2.3 4-Hydroxytamoxifen induction of knockout
2.2.4 Experimental tumor model in mouse
2.2.5 Intravenous tumor injection
2.3 Primary Has2-knockout fibroblasts for in vitro experiments
2.3.1 Isolation of primary fibroblast from mouse skin tissue
2.3.2 Induction of Has2-ko fibroblasts
2.4 Molecular analysis
2.4.1 PCR analysis of genome DNA
2.4.2 Quantification of gene expression
2.4.3 Microarray analysis
2.4.4 Quantification of Has2 protein levels
2.4.5 Quantification of HA
2.4.6 HA size analysis by agarose gel electrophoresis
2.5 Physical analysis of the skin
2.5.1 Skin elasticity measurement
2.5.2 Skin permeability measurement
2.5.3 Skin hydration and TEWL measurement
2.6 Histological analysis
2.6.1 Cryo-fixed samples
2.6.2 Immunofluorescence staining
2.6.3 FFPE samples
2.6.4 HA staining
2.6.5 Histochemical images
2.7 Physiological analysis of MM cell proliferation with BrdU-Assay
2.8 Statistical analysis
3 Materials
3.1 Mouse lines
3.2 Cell lines
3.3 Buffer and solution recipes
3.4 Chemicals
3.5 Molecular-biological reagents and enzymes
3.6 Primers
3.7 Antibodies
3.8 Kits
3.9 Devices and tools
3.10 Disposables
3.11 Software
4 Results
4.1 The Has2-knockout mouse model
4.1.1 Has2-knockout characterization
4.1.2 Incomplete Has2 deletion
4.1.3 Effects of the HA knockdown
4.1.4 Has2-knockout efficiency in other organs
4.1.5 Effects of Has2-knockout on gene expression
4.2 In vitro Has2-knockout and effect on MM cells
4.2.1 In vitro Has2- and HA-knockdown
4.2.2 Has2-ko fibroblast conditioned media decreased MM proliferation
4.2.3 MM conditioned media influenced fibroblast’s HA secretion
4.2.4 The transition towards in vivo tumor experiments
4.3 In vivo Tumor experiments
4.3.1 HA threshold, correlation, and exclusions
4.3.2 Effects of HA knockdown on primary tumor volume and weight
4.3.3 Tumor histology and HA localization
4.3.4 HA fragments in tumors, healthy-, and tumor-related-skin samples
4.3.5 Metastasis formation
5 Discussion
5.1 HA knockdown
5.2 HA knockdown phenotype
5.2.1 Skin stiffness
5.2.2 Skin water homeostasis
5.3 Paracrine interactions between MM and fibroblasts
5.4 HA thresholding
5.5 Tumor readouts
5.6 In vitro ECM models
5.7 Metastasis
5.8 Alternative tumor models with stronger stromal interaction
5.9 The presented results considering current HA-Tumor research
6 Conclusion
7 Literature
Danksagung
Lebenslauf
Akademischer Werdegang
Stipendium und Auszeichnung
Publikation und Posterpräsentation
Publikationen
Vorträge und Posterpräsentationen
Eigenständigkeitserklärung / The present work addresses the glycosaminoglycan (GAG) hyaluronan (HA) and its influence on the development of malignant melanoma (MM). HA, which is abundant in the extracellular matrix (ECM), has been studied closely in relation to tumor development since the late 20th century. Opposing properties of HA, on the one hand promoting tumors and the other hand counteracting them, have since been published. The general consensus is that HA molecular weight determines the various effects of HA. However, there is insufficient evidence on how HA affects tumor growth and which HA sizes specifically promote it. This study is based on an in vivo knockout of hyaluronan synthase 2 (Has2) in mice, which largely restricted the production of high molecular weight HA (HMW-HA). Growth from experimental MM was examined in the absence of most HMW-HA. This work sought to provide evidence that local growth and metastasis of MM cells is dependent on the presence of HMW-HA in the vicinity of the tumor.
Has2 knockout in the mouse line C57BL6 was verified and examined for an altered phenotype of the mice. Although nearly three-quarters of the absolute HA amount was absent due to the knockout, the mice showed no obvious change. Only a measurement of skin permeability indicated a possible increased barrier function of the stratum corneum. A direct effect of the lack of HA on tumor growth and metastasis rate could not be sufficiently demonstrated. The applied mouse model and the choice of experimental tumor are critically discussed in this work. Parallel in vitro experiments with partially artificial 3D matrices containing different amounts of HA showed a stronger influence of low molecular weight HA (LMW-HA) on proliferation and invasion of MM cells. These observations are consistent with the emerging consensus that LMW-HA triggers activating signal transduction and HMW-HA is more homeostatic.:TABLE OF CONTENT
Zusammenfassung
Summary
Table of content
List of figures
List of tables
Abbreviations
1 Introduction
1.1 Structures of the skin
1.2 The malignant melanoma
1.3 The tumor microenvironment (TME)
1.4 Hyaluronan
1.4.1 HA Structure
1.4.2 HA anabolism
1.4.3 HA catabolism
1.4.4 HA binding proteins
1.4.5 HA cell surface receptors
1.5 Hyaluronan in malignant melanoma
1.6 Aim of the thesis
2 Methods
2.1 Murine malignant melanoma cell lines
2.2 Inducible Has2-knockout mice
2.2.1 The Cre/loxP System
2.2.2 Genetical modification for the Has2-knockout
2.2.3 4-Hydroxytamoxifen induction of knockout
2.2.4 Experimental tumor model in mouse
2.2.5 Intravenous tumor injection
2.3 Primary Has2-knockout fibroblasts for in vitro experiments
2.3.1 Isolation of primary fibroblast from mouse skin tissue
2.3.2 Induction of Has2-ko fibroblasts
2.4 Molecular analysis
2.4.1 PCR analysis of genome DNA
2.4.2 Quantification of gene expression
2.4.3 Microarray analysis
2.4.4 Quantification of Has2 protein levels
2.4.5 Quantification of HA
2.4.6 HA size analysis by agarose gel electrophoresis
2.5 Physical analysis of the skin
2.5.1 Skin elasticity measurement
2.5.2 Skin permeability measurement
2.5.3 Skin hydration and TEWL measurement
2.6 Histological analysis
2.6.1 Cryo-fixed samples
2.6.2 Immunofluorescence staining
2.6.3 FFPE samples
2.6.4 HA staining
2.6.5 Histochemical images
2.7 Physiological analysis of MM cell proliferation with BrdU-Assay
2.8 Statistical analysis
3 Materials
3.1 Mouse lines
3.2 Cell lines
3.3 Buffer and solution recipes
3.4 Chemicals
3.5 Molecular-biological reagents and enzymes
3.6 Primers
3.7 Antibodies
3.8 Kits
3.9 Devices and tools
3.10 Disposables
3.11 Software
4 Results
4.1 The Has2-knockout mouse model
4.1.1 Has2-knockout characterization
4.1.2 Incomplete Has2 deletion
4.1.3 Effects of the HA knockdown
4.1.4 Has2-knockout efficiency in other organs
4.1.5 Effects of Has2-knockout on gene expression
4.2 In vitro Has2-knockout and effect on MM cells
4.2.1 In vitro Has2- and HA-knockdown
4.2.2 Has2-ko fibroblast conditioned media decreased MM proliferation
4.2.3 MM conditioned media influenced fibroblast’s HA secretion
4.2.4 The transition towards in vivo tumor experiments
4.3 In vivo Tumor experiments
4.3.1 HA threshold, correlation, and exclusions
4.3.2 Effects of HA knockdown on primary tumor volume and weight
4.3.3 Tumor histology and HA localization
4.3.4 HA fragments in tumors, healthy-, and tumor-related-skin samples
4.3.5 Metastasis formation
5 Discussion
5.1 HA knockdown
5.2 HA knockdown phenotype
5.2.1 Skin stiffness
5.2.2 Skin water homeostasis
5.3 Paracrine interactions between MM and fibroblasts
5.4 HA thresholding
5.5 Tumor readouts
5.6 In vitro ECM models
5.7 Metastasis
5.8 Alternative tumor models with stronger stromal interaction
5.9 The presented results considering current HA-Tumor research
6 Conclusion
7 Literature
Danksagung
Lebenslauf
Akademischer Werdegang
Stipendium und Auszeichnung
Publikation und Posterpräsentation
Publikationen
Vorträge und Posterpräsentationen
Eigenständigkeitserklärung
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:75732 |
| Date | 16 August 2021 |
| Creators | Nguyen, Khiet Tam Christoph |
| Contributors | Universität Leipzig |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | German |
| 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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