Ovarian Cancer is the 5th most common cause of cancer death in women and the second most common gynaecological cancer in the UK. Worldwide, around 152,000 women were estimated to have died from ovarian cancer in 2012. Survival rates for women with epithelial ovarian cancer have not significantly changed since platinum-based treatment was introduced over 30 years ago. This is particularly disconcerting considering the fact that there is a less than 5% five year survival rate for patients diagnosed with late stage high grade serous ovarian cancer. This thesis examines the role of BMP signalling in ovarian cancer using in vitro cancer cell models. It builds upon the initial published work by the Inman lab identifying autocrine BMP9 as a promoter of ovarian cancer cell proliferation. The findings of Chapters 3-5 provide strong evidence indicating BMP9 as a context specific modulator of ovarian cancer cell proliferation. This significantly builds upon on the sole pro-proliferative BMP9 growth response previously described. Responding cell lines were subjected to a microarray with and without BMP9 treatment In order to determine early BMP target genes which were subsequently transiently knocked down in order to determine their role in the aetiology of said growth phenotype. ID1 gene expression was found to significantly contribute to the BMP9 proproliferative phenotype. Moreover several other BMP genes identified significantly alter basal cell proliferation. It was subsequently determined that BMP9 implemented a cell growth phenotype by negating apoptosis. .Excitingly, preliminary evidence suggests a marked reduction in detectable levels of a recently described Bax isoform, Bax β that coincide with BMP9 addition and the resultant anti-apoptotic phenotype observed. This is very interesting as no prior evidence correlating the BMP family and Bax β currently exists. These findings provide an enhanced understanding of BMP9s contribution to ovarian cancer pathogenesis that may result in the development of effective and targeted therapeutic interventions upon further stratification of the contextuality of the BMP induced growth response.

Thesis (Ph.D. (Biochemistry)) --University of Limpopo, 2009 / Refer to document / National Research Foundation (NRF) and University of Limpopo

Thioredoxin is a small ubiquitous oxido-reductase found in all species. The highly conserved active site, which facilitates thioredoxins redox activity, contains two redox active cysteine residues. Thioredoxin has numerous protein substrates to which it donates H+ ions and it can also function as a free radical scavenger. Through these activities thioredoxin is able to influence the redox state of not only its protein targets, but also the entire cellular environment. Thioredoxin has been implicated in many biological functions, and one mechanism by which it influences these functions is through interactions with a number of transcription factors including NF-kappa-B and p53. Thioredoxin also has numerous extracellular biological roles. It has been shown that thioredoxin is actively secreted from a number of normal and transformed cell lines including fibroblasts and activated B and T cells. This study investigates the role of thioredoxin in embryonic implantation and cancer cell metastasis, two physiological functions which rely on the same basic processes. Thioredoxin expression has previously been shown to be increased in many cancers. However it has not yet been established whether this increase is a causative or a side effect of the cancerous phenotype. Similarly thioredoxin expression has previously been shown to be increased during different phases of the oestrus cycle and pregnancy. This thesis describes the role of thioredoxin in embryonic implantation using a marmoset model. A thioredoxin cDNA was isolated and subsequently sequenced. Preliminary antibody experiments indicated that the anti human thioredoxin monoclonal antibodies available in our laboratory would recognise marmoset thioredoxin. Subsequently immunocytochemistry using anti human thioredoxin antibodies was carried out on sectioned marmoset uterus and embryonic tissue. The results indicated that thioredoxin is expressed by cells at the embryonic-maternal interface of early implantation sites. Further studies demonstrated that thioredoxin is also expressed and secreted by cultured blastocysts in vitro. This thesis also describes the role of thioredoxin in cancer cell metastasis. Results of this study indicate that thioredoxin is actively involved in facilitating the invasive phenotype of breast cancer cells. The two cell lines utilised were MCF-7, a well differentiated, relatively non-invasive breast cancer cell line; and MDA-MB-231, a poorly differentiated, highly invasive breast cancer cell line. The cell lines were transfected with thioredoxin sense, antisense and 1SS (encodes thioredoxin with both active cysteine residues mutated to serine residues and is thus redox inactive) constructs. The results demonstrate that when endogenous thioredoxin levels are increased, i.e. transfected with a sense thioredoxin construct, the invasive breast cancer cell line MDA-MB-231 becomes more invasive, conversely when endogenous levels are decreased, i.e. transfected with antisense or 1SS constructs, the invasive capacity of these cells decreases. However, when the endogenous level of thioredoxin was manipulated in the relatively non-invasive cell line MCF-7 very little effect was observed. Results also indicate that thioredoxin has the ability to act as a chemoattractant for actively invading breast cancer cells. Both of these functions appear to be dependent on thioredoxin's redox activity. Additional studies described in this thesis have shown that thioredoxin is involved in the regulation of Sp1 in vitro. Sp1 is a transcription factor known to regulate the transcription of a number of genes whose products are intimately involved in the invasive phenotype. The results in this study suggest that Sp1 DNA binding is regulated by thioredoxin such that when reduced by the enzyme its binding to DNA is facilitated. Results also indicate that Sp1 may regulate the transcription of thioredoxin by binding to Sp1 sites within the thioredoxin promoter.

Intestinal epithelial differentiation entails the formation of highly specialized cells with specific absorptive, secretory, digestive and immune functions. Cell-cell and cell-microenvironment interactions appear to be crucial in determining the outcome of the differentiation process. Using the Caco-2 cell line that can undergo spontaneous differentiation when grown past confluency, we observed a loss of VCAM1 (vascular cell adhesion molecule-1) expression while ICAM1 (intercellular cell adhesion molecule-1) expression was seen to be stable in the course of differentiation. Protein kinase C theta (PKC&theta / ) acted downstream of PKC to inactivate Inhibitor of kappa B (IB) and activate NF-&kappa / B in the undifferentiated cells and this axis was inhibited in the differentiated cells. The increase in ICAM1 expression in the differentiated cells was due to a transcriptional upregulation by C/EBP. The protein expressions of both ICAM-1 and VCAM-1, however, were found to decrease in the course of differentiation, with both proteins getting post-translationally degraded in the lysosome. Functionally, a decrease in adhesion to HUVEC cells was observed in the differentiated Caco-2 cells. Thus, the regulation of ICAM-1 and VCAM-1, although both NF-B target genes, appear to be different in the course of epithelial differentiation. microRNAs are known to regulate many cellular pathways. miR-146a, which is known to target NF-&kappa / B, was shown to be highly upregulated in differentiated Caco-2 cells. As a predicted target of miR-146a, mRNA and protein expression of MMP16 was inversely correlated with miR-146a during differentiation of Caco-2 cells. miR-146a could bind to the 3&rsquo / UTR of MMP16 and ectopic expression of miR-146a resulted in a decreased mRNA and protein expression of MMP16 in the undifferentiated Caco-2 and HT-29 cells. Functionally, decreased gelatinase activity determined by gelatin zymography and reduced invasion and migration through Transwells was observed. In the final part of the thesis, the inhibition of NF-&kappa / B via PPAR&gamma / in 15-Lipoxygenase-1 (15LOX1) expressing cells was investigated. The expression of 15LOX1, a member of the inflammatory arachidonate cascade, could lower phosphorylation of I&kappa / B&alpha / and NF-&kappa / B DNA binding activity which was reversed with a 15LOX1 inhibitor. This inhibition was mediated by phospho-PPAR&gamma / , which in turn was phosphorylated by ERK1/2.

The ultimate objective of this research was to develop a low cost, reliable system that would lead to early detection of lung cancer. Tests involved the quantitation of gaseous metabolic emissions from immortalized lung cancer cell lines in order to correlate the chemical markers to be of cancerous origin. The specific aims of the project were the study of gas emissions in selected cancer cell lines and identification of volatile organic compounds (VOCs) in them. Disadvantages of earlier studies were that the measurements were not real time or state specific so that molecular identification was often inconclusive. Furthermore the methods of study used in the past were not quantitative, which limited their practicality for medical applications. We felt the need to prove or disprove these earlier results using a new technique. The method we proposed is different and unique when compared to previous methods because cell lines have not been studied extensively for cancer markers. We have studied cancer cell lines which are adherent, immortalized cultures originating from primary tumors obtained from patients with no prior treatment for lung cancer. We have used an alternative method for the spectrometric analysis and quantitation of the selected chemical markers. The pre-concentration method involved a Purge and Trap unit with a thermal desorber where the vapor concentration was enhanced. The concentrated head space gases were analyzed using a Gas Chromatograph ?? Mass Spectrometer setup. This setup eliminated the bulky apparatus used in earlier studies. It is simpler in design and more comprehensive so that external factors such as patient??s diet, habitat and lifestyle do not contribute to our study of recognition of cancer markers. Based on the results obtained in the above experiments, a more comprehensive, inexpensive study of lung cancer related markers could be made. The first section, after giving an introduction to lung cancer, goes on to explain the background work done by other researchers on cancer. The third section gives a detailed explanation of the experimental setup. This is followed by all the tests conducted with corresponding results. The final section deals with the conclusions drawn from all experiments.

Le récepteur-canal P2X7 est fortement exprimé et est fonctionnel dans la lignée de cellules cancéreuses mammaires humaines hautement invasives MDA-MB-435s. L’activation de P2X7 par l’ATP extracellulaire est responsable de l'émission des prolongements cellulaires et l'augmentation de la migration cellulaire. En outre, l’activation de P2X7 augmente l’invasion cellulaire à travers la matrice extracellulaire et fait intervenir la libération de forme mature de cathepsines à cystéine dans le milieu extracellulaire. L’inhibition pharmacologique de P2X7 diminue l’invasivité des cellules cancéreuses dans un modèle de micrométastases chez le poisson zèbre. Nous avons également montré que l’émodine (1,3,8-trihydroxy-6-méthylanthraquinone) une anthraquinone isolée de Rheum officinale Baill (Rhubarbe chinoise) inhibe l’invasivité des cellules cancéreuses humaines via l’antagonisme de P2X7 et n’as pas d’effet sur les autres récepteurs P2X. Nos résultats démontrent un nouveau mécanisme entre la fonctionnalité de P2X7 dans les cellules cancéreuses et l’invasivité cellulaire, un paramètre clé dans la croissance tumorale et le développement des métastases. Ceci suggère également un rôle thérapeutique potentiel pour les antagonistes des P2X7. / P2X7 receptor channel is highly expressed and fully functional in the highly invasive human breast cancer cell line MDA-MB-435s. Its activation by extracellular ATP is responsible for the extension of neurite-like cellular prolongations, and the increase in cell migration. Furthermore, P2X7 activation enhanced invasion through the extracellular matrix and was related to the increase of mature forms of cysteine cathepsins in the extracellular medium. Pharmacological targeting of P2X7 decreases cancer cell invasiveness in a zebrafish model of micrometastases. We also showed that emodin (1,3,8-trihydroxy-6-methylanthraquinone) an anthraquinone derivative originally isolated from Rheum officinale Baill (Chinese Rhubarb) inhibits human cancer cell invasiveness by specifically antagonizing the P2X7 and not the other members of the P2X family. Our results demonstrate a novel mechanistic link between P2X7 functionality in cancer cells and invasiveness, a key parameter in tumour growth and in the development of metastases. These results also suggest a potential therapeutic role for the newly developed P2X7 antagonists.

abstract: Relapse after tumor dormancy is one of the leading causes of cancer recurrence that ultimately leads to patient mortality. Upon relapse, cancer manifests as metastases that are linked to almost 90% cancer related deaths. Capture of the dormant and relapsed tumor phenotypes in high-throughput will allow for rapid targeted drug discovery, development and validation. Ablation of dormant cancer will not only completely remove the cancer disease, but also will prevent any future recurrence. A novel hydrogel, Amikagel, was developed by crosslinking of aminoglycoside amikacin with a polyethylene glycol crosslinker. Aminoglycosides contain abundant amount of easily conjugable groups such as amino and hydroxyl moieties that were crosslinked to generate the hydrogel. Cancer cells formed 3D spheroidal structures that underwent near complete dormancy on Amikagel high-throughput drug discovery platform. Due to their dormant status, conventional anticancer drugs such as mitoxantrone and docetaxel that target the actively dividing tumor phenotype were found to be ineffective. Hypothesis driven rational drug discovery approaches were used to identify novel pathways that could sensitize dormant cancer cells to death. Strategies were used to further accelerate the dormant cancer cell death to save time required for the therapeutic outcome. Amikagel’s properties were chemo-mechanically tunable and directly impacted the outcome of tumor dormancy or relapse. Exposure of dormant spheroids to weakly stiff and adhesive formulation of Amikagel resulted in significant relapse, mimicking the response to changes in extracellular matrix around dormant tumors. Relapsed cells showed significant differences in their metastatic potential compared to the cells that remained dormant after the induction of relapse. Further, the dissertation discusses the use of Amikagels as novel pDNA binding resins in microbead and monolithic formats for potential use in chromatographic purifications. High abundance of amino groups allowed their utilization as novel anion-exchange pDNA binding resins. This dissertation discusses Amikagel formulations for pDNA binding, metastatic cancer cell separation and novel drug discovery against tumor dormancy and relapse. / Dissertation/Thesis / Doctoral Dissertation Bioengineering 2016

Orientador: Christiane Pienna Soares / Banca: Christiane Pienna Soares / Banca: Haroldo Wilson Moreira / Banca: Ana Marisa Fusco Almedia / Banca: Raquel Alves dos Santos / Banca: Maria Regina Torqueti Toloi / Resumo: O câncer de mama é a maior causa de morbidade e mortalidade entre as mulheres no mundo. Pesquisas revelam vários fatores prognósticos e preditivos para a identificação de pacientes com alto risco de agressividade, metastases e doença recorrente na condição de combater estas estatísticas. Por esta razão, é evidente a necessidade do desenvolvimento de estratégias de tratamento mais eficazes. Estudos prévios com Pterogyne nitens Tul. (Fabaceae-Caesalpinioideae), uma planta nativa do Brasil resultou o isolamento de dois alcalóides guanidínicos. Exibiram atividade seletiva direcionada a DNA deficiente de reparo, sugerindo potencial atividade anticâncer. Objetivo: O objetivo do presente estudo foi avaliar a citotoxicidade e apoptose induzidas pelos alcalóides pteroginina (PGN) and pteroginidina (PGD) em linhagem de adenocarcinoma (MCF-7) e carcinoma ductal invasivo (ZR-7531). Materiais e Métodos: As duas linhagens celulares foram tratadas pelos alcalóides em várias concentrações (0.25 - 10 mM) em dois tempos, t0 (24h) e t24 (24h seguido por 24h pós-tratamento). O ensaio de citotoxicidade foi determinado pelo teste de MTT; a morte celular (apoptose e necrose) foi analisada usando os métodos Hoechst 33342/iodeto propídio, Kit de Anexina V-FITC e atividade de Caspases 3/7. Resultados: Os tratamentos com os alcalóides demonstraram citotoxicidade concentração-resposta nas linhagens de câncer de mama. Para avaliação da apoptose foi observado um intense efeito concentraçãoresposta em apoptose tardia/necrose e discreto sinal para apoptose precoce em todas concentrações (p<0,01). No ensaio Hoechst/iodeto, observou diferença significante entre os estágios de apoptose precoce e tardia de ambas linhagens. A pteroginina no período t0 e t24, e pteroginidina no período t0 demonstraram possuir intenso efeito concentração... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Breast cancer is a major cause of morbidity and mortality among women worldwide. Research has elucidated several specific prognostic and predictive factors to identify patients at high risk of the aggressive disease, metastasis and recurrence of the disease in order to combat these statistics. For this reason, there is an obvious need to develop more efficacious treatment strategies. Previous studies on Pterogyne nitens Tul. (Fabaceae-Caesalpinioideae), a native plant of Brazil, resulted in the isolation of two guanidine alkaloids, exhibited selective activity towards a DNA repair-deficient, suggesting potential anti-cancer activity. Objective: The aim of the present study was to evaluate the citotoxicity and apoptosis induced by alkaloids pterogynine (PGN) and pterogynidine (PGD) in human adenocarcinoma cell line (MCF-7) and human invasive ductal carcinoma cell line(ZR-7531). Material and Methods: The two cell lines were treated by both alkaloids at several concentrations (0.25 - 10 mM) and two time points, 24h (t0) and 24h followed by 24h pos-treatment (t24). The cytotoxicity assay was determined by MTT assay; the cell death (apoptosis and necrosis) were analyzed using the dye Hoechst 33342/propidium iodide, Annexin V-FITC and Caspase 3/7 activity. Results: The treatments with the alkaloids demonstrated citotoxicity effect concentrationresponse in breast cell lines. Apoptosis evaluation, pterogynine and pterogynidine has an intense effect concentration-response of late apoptosis/necrosis and a discrete signal of early apoptosis in all of the concentrations (p<0.01). Hoechst/iodide assay, it was observe significant difference among the stages of early and late apoptosis in the both cells lines. Pterogynine for the period of t0 and t24, and pterogynidine for the period of t0 demonstrated to possess an intense concentrationresponse... (Complete abstract click electronic access below) / Doutor

Em mulheres, o câncer de mama é o tipo mais incidente depois do tumor de pele não melanoma e é a principal causa de morte por câncer. Apesar dos avanços já alcançados na caracterização da doença, a busca por novos marcadores moleculares para diagnóstico, tratamento e entendimento molecular da doença é de extrema importância. Estudos em nosso laboratório apontaram a proteína EMC1 (do inglês, Endoplasmic Reticulum Complex 1) como relacionada a propriedades malignas em linhagens celulares de câncer de mama e melanoma, assim como aumento no crescimento tumoral em ensaios in vivo. Despertou-se, então, o interesse em nosso laboratório, no estudo das outras proteínas do complexo EMC. Este estudo atual, busca analisar dados em larga escala do banco TCGA em um painel de 32 tipos tumorais, e aponta associação da expressão de diversas proteínas EMCs a pior sobrevida dos pacientes. O gene EMC2, que se localiza na região cromossômica altamente amplificada em diversos tumores (8q23.1), se destaca pela intensidade de pacientes com superexpressão em câncer de mama (40%). Em linhagens desse tipo tumoral, o knockdown de EMC2 aponta redução na taxa proliferativa, assim como associação à progressão do ciclo celular na fase M, quando feito o protocolo de sincronização utilizando a droga nocodazol. Em conjunto, nossos dados sugerem que o complexo EMC pode favorecer o desenvolvimento de tumores e influenciar em sua malignidade. Além disso, a proteína EMC2 parece apresentar funções relacionadas a proliferação e possivelmente, ciclo celular. / In woman, the breast cancer is the most incidence type after skin tumor non melanoma and it is a main cause of cancer death. Despite the advances already achieved in the disease caracterization, the search for novel molecular biomarkers to diagnostic, treatment and disease knowledge is extremely importante. In vitro approaches pointed Endoplasmic Reticulum Complex 1 (EMC1) involvement in malignant properties in breast cancer and melanoma cell lines, and increase in tumor growth in a in vivo assay. It highlighted the study of the other members of EMC complex. In this study, we used a large-scale database from TCGA in a range of 32 tumoral types, and showed association in EMC expression and poor surviving curve. The EMC2 gene, localized in a high amplified chromosome region in cancer (8q23.1), have a interesting upregulation level in breast cancer (40%). In knockdown EMC2 cell lines, the proliferation is less intense and progression in M phase, in a nocodazole sincronization assay, is impaired. Together, the data suggest that the EMC complex favors the tumour development and the EMC2 protein seens to play a role in proliferation and maybe in cell cycle.

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