Head and neck squamous cell carcinomas are still challenging despite progress in the oncological treatment. Study of the molecular biology allows to deeply characterize tumor properties and to predict the prognosis for affected patients. Nowadays there are many drugs clinically tested in the group of targeted therapy medicine Experimental work comprised both in vitro and in situ assays, being performed thanks to the collaboration between a number of departments of the 1st Faculty of Medicine of the Charles University in Prague, Academy of Sciences of the Czech Republic, Institute of Hematology and Blood Transfusion and Faculty of Veterinary Medicine of the Ludwig-Maxmillian University Munich. Galectin-1 is important inductor of the myofibroblasts/cancer associated fibroblasts. These fibroblasts are regarded as negative prognostic markers thanks to their capability of invasive cancer cells induction. On the other hand, Galectin-9 is not present in the carcinoma and in the case of dysplasia, its expression indicate aberrant features together with aberrant expression of keratin 14 and 19. Except from galectins using as prognostic markers, we focused on the galectins as a therapeutics instruments as well. Presented work with mutant variants of galectin-2 proved their effect on both pharmacodynamics and...

No description available.

The majority of cancer research projects mainly focus on the epithelial cancer cell, while the role of the tumour stroma has been largely neglected. Conventional 2D techniques, such as well plates and other kinds of tissue culture plastic, and animal models are mainly used to broaden our understanding of how tumours arise, develop, and induce metastasis. However, there is accumulating evidence suggesting a tremendous impact of the non‐cancerous tumour stroma on carcinogenesis, while other publications illustrate the great importance of advanced 3D in vitro models for cancer research. The overall goal of this work was to investigate how cancer associated fibroblasts (CAFs; the most abundant component in the tumour stroma) and normal prostate fibroblasts (NPFs), isolated from patients diagnosed with aggressive forms of prostate cancer, contribute to angiogenesis, an important hallmark of cancer progression. For this purpose, a 3D in vitro angiogenesis co‐culture model was established. At first, two (semi‐) synthetic hydrogel platforms, gelatine methacrylate (GelMA) and star‐shaped (star)PEG‐heparin hydrogels were characterised and their physicochemical properties were compared with each other. Interestingly, GelMA gels shrank while starPEG‐heparin gels swelled in cell culture medium over the course of 24 hours. The cell concentration, in addition to the stiffness, was critical for the formation of endothelial networks, and the knowledge of swelling behaviour enabled the adjustment of initial cell density to ensure the density between both gel types was comparable. Moreover, preliminary tests with mesenchymal stem cells demonstrated that the hydrogel can be actively remodelled, as evaluated by stiffness parameters at day one and seven of incubation. Growth factors (GFs) affect cellular fate and behaviour, and storage, presentation and administration of such chemokines can be critical for certain cellular applications. Due to the high anionic charge density of heparin, starPEG‐heparin hydrogels are known to reversibly immobilise several GFs and thereby might mimic the GF reservoir of the extra cellular matrix. Thus, transport processes of GFs with low and high heparin affinity inside these hydrogels were analysed by fluorescence correlation spectroscopy and a bulk diffusion approach. Results indicated that diffusion constants were synergistically decreased with increasing size and heparin affinity of the diffusant. Next, the capability of endothelial cells (ECs) to self‐assemble and organise into 3D capillary networks was tested in GelMA, starPEG‐heparin and Matrigel hydrogels. Only starPEG‐heparin hydrogels allowed the formation of interconnected capillaries in macroscopic hydrogel samples. However, as it is widely used to test for pro‐ and anti‐angiogenic agents, the 2D Matrigel angiogenesis assay was included for subsequent co‐culture experiments of ECs and fibroblasts in order to investigate how the stromal cells influence the formation of endothelial networks. For a detailed characterisation of 3D structures, a conventionally applied 2D method (Maximum Intensity Projection for 3D reconstructed images, MIP) was compared to an optimised 3D analysing tool. As a result, it was discovered that MIP analysis did not allow for an accurate determination of 3D endothelial network parameters, and can result in misleading interpretations of the data set. Indirect co‐cultures of hydrogel‐embedded ECs with a 2D layer of fibroblasts showed that fibroblast‐derived soluble factors, including stromal cell‐derived factor 1 and interleukin 8, affected endothelial network properties. However, only co‐encapsulation of ECs and fibroblasts in starPEG‐heparin hydrogel discs revealed remarkable changes in endothelial network parameters between CAF and NPF samples. In detail, the total length and branching of the capillaries was increased. For two donor pairs, the diameter of capillaries was decreased in CAF samples compared to NPF samples, underlining the high physiological relevance of this model. In contrast, significant differences in 2D Matrigel assays were not detected between, CAF, NPF and control (ECs only) samples. In summary, a 3D angiogenesis co‐culture system was successfully developed and used to characterise stromal‐endothelial interactions in detail. The combination of advanced biomaterials (starPEG‐heparin) and 3D analysing techniques goes beyond conventional 2D in vitro cancer research, and opens new avenues for the development of more complex models to further improve the acquisition of more biologically relevant data.

Die Expression homöostatischer Chemokinrezeptoren auf hämatopoetischen Neoplasien wird zunehmend mit tumorpathogenen Funktionen in Zusammenhang gebracht. In der Arbeit wurden Funktionen der Rezeptoren CXCR7 und CCR7 in der Pathogenese lymphatischer Erkrankungen anhand von Mausmodellen charakterisiert. Für CXCR7 konnte in der normalen Differenzierung von T-Zellen eine schwache Expression in murinen Thymozyten, dagegen eine verstärkte, vor allem intrazellulär lokalisierte Expression in peripheren aktivierten T-Zellen identifiziert werden. Eine aberrante Überexpression lag in humanen Zelllinien, aber auch in primären Fällen von T-ALL und klassischen Hodgkin-Lymphomen vor. Die Analyse eines retroviralen Überexpressionsmodells ergab für CXCR7 die Funktion als anti-apoptotischer Kostimulator während der thymischen beta-Selektion. Im Signaltransduktionskomplex mit CXCR4 und dem präTCR vermittelte CXCR7 einen effizienteren DN3-zu-DN4 Übergang. Unreife Thymozyten waren durch eine verstärkte Apoptoseresistenz und Expression von anti-apoptotischen Bcl2-Molekülen charakterisiert. Dies könnte CXCR7 überexprimierende Thymozyten putativ empfänglicher für die Entwicklung von T-ALLs machen. Für CCR7 konnten in der Arbeit bedeutende Funktionen in der organspezifischen Dissemination von B-Zelllymphomen identifiziert werden. Unter Verwendung des Eµ-Myc-Mausmodells wurde gezeigt, dass Eµ-Myc Lymphomzellen CCR7-abhängig in die T-Zellzone von Milz und Lymphknoten einwandern und dort durch reziproke Interaktionen mit gp38+ FRCs und DCs entscheidende Überlebenfaktoren, darunter Ihh, Igf-1 und VCAM-1, erhalten. Die Lymphomzellen vermittelten darüber hinaus eine aktive Veränderung der Stromazellzusammensetzung, welche durch ein expandiertes FRC-Netzwerk, durch die Induktion putativ immunsupprimierender DCs und durch ein inflammatorisches Milieu charakterisiert war. Eine Inhibition der Lymphom-Stroma-Interaktionen könnte daher eine neue Strategie der Lymphomtherapie darstellen. / In recent years the expression of homeostatic chemokine receptors on hematological tumors was increasingly associated with tumor pathogenic functions. Within this thesis, functions of the chemokine receptors CXCR7 und CCR7 in the pathogenesis of lymphoid diseases were characterized using different mouse models. For CXCR7, low expression levels were detected in murine thymocytes during normal T cell development. Enhanced expression was found mainly intracellularly in peripheral activated T cells. An aberrant overexpression was identified in human cell lines and primary cases of T-ALL and classical Hodgkin lymphoma. The analysis of a retroviral overexpression model suggested a function of CXCR7 as an anti-apoptotic costimulator during thymic beta-selection. In a functional complex with CXCR4 and the preTCR CXCR7 mediated a more efficient DN3-to-DN4 transition. CXCR7 expressing thymocytes were characterized by enhanced apoptosis resistance and expression of anti-apoptotic Bcl2-family genes. Thus, CXCR7 could putatively make immature thymocytes more susceptible to develop T-ALL. In addition, new insights into the function of CCR7 in the context B cell lymphoma dissemination were gained within this thesis. Applying the Eµ-Myc mouse model, CCR7 was shown to mediate the specific homing of Eµ-Myc lymphoma cells into the T cell zone of spleen and lymph nodes. Here, lymphoma cells received pivotal survival signals following reciprocal interactions with gp38+ FRCs and DCs, amongst them Ihh, Igf-1 and VCAM-1. Moreover, the lymphoma cells induced a survival promoting active remodelling of the T cell zone stroma, which was characterized by the expansion of the FRC network, by the induction of putatively immune suppressive DCs and by the induction of a pro-inflammatory milieu. Therefore, an inhibition of lymphoma-stroma interactions could provide a new strategy in lymphoma therapy.

Melanomas are one of the most aggressive types of tumours, with increasing incidence, high mortality and high potential to metastasize to a variety of diverse locations. The aim of this thesis was to study the tumour as a complex structure consisting not only of tumour cells but also of tumour stroma. Stromal cells play a major role in cancer biology. This is well documented for example in squamous cell epithelium tumours of the head and neck. Similar mechanisms can be expected to occur in melanomas. In the first experiment, we simulated the conditions in vivo during the metastatic process and studied the influence of non-adhesive environment both with and without the influence of stromal fibroblasts. The presented data demonstrates a change of tumour cells' phenotype leading to increased plasticity of the melanoma cells in these conditions. It also indicates the crucial role of stromal fibroblasts in interactions with melanoma cells. Cancer cell lines show variability in their behaviour, which is in accordance with well-known melanoma heterogeneity in clinical practice. The previous experiments in our laboratory indicate that cancer associated fibroblasts are able to influence the phenotype of a tumour cell line and this effect is based on a tumour type-unspecific mechanism. In the second part of...

In dieser Arbeit wurden Genexpressionsprofile diffuser und intestinaler Magenadenokarzinome mittels Microarray-Technik erstellt. Der intestinale Typ konnte als stark proliferierender Tumor mit signifikanter Überexpression von zellzyklusrelevanten Genen definiert werden, während der diffuse Typ als stark stromaabhängig mit signifikanter Überexpression von Genen der extrazellulären Matrix hervortrat. Thrombospondin 4 (THBS4) wurde dabei als das am stärksten differentiell exprimierte Gen identifiziert, wobei seine mRNA in diffusen Tumoren eminent überexprimiert wird. Immunhistochemische Studien bestätigten diese starke Überexpression auf Proteinebene und zeigten, dass THBS4 eine übermäßig angereicherte extrazelluläre Komponente des Tumorstromas ist. Kolokalisierungsstudien zeigten zudem, dass THBS4-positive Zellen auch positiv für Vimentin und Smooth muscle actin (alpha) sind. Diese Ergebnisse belegen, dass THBS4 von Tumor-assoziierten Fibroblasten (TAF) exprimiert wird. Dies konnte durch zusätzliche in vitro Experimente bestätigt werden, die aufzeigten, dass TAF von diffusen Tumoren eine stärkere THBS4-mRNA Expression aufweisen als normale Fibroblasten des Magens. Abschließend konnten in vitro Kokultur-Studien aufdecken, dass die THBS4-Expression in Fibroblasten durch Tumorzellen diffuser Magentumore transkriptionell stimuliert wird. Metastasenbefall regionaler Lymphknoten (N+) ist bei den meisten Magenadenokarzinomdiagnosen bereits vorhanden. Dieser ist der stärkste derzeit verfügbare Parameter zur Abschätzung der Prognose, reicht aber für eine eindeutige Vorhersage nicht aus. Um ergänzende molekulare Prognoseindikatoren zu identifizieren, wurden aus den Microarray-Daten Gene, deren Expression mit dem klinischen Verlauf von N+ Patienten korreliert, extrahiert. Einige dieser Gene, z.B. RAN binding protein 17 und ras-related associated with diabetes, konnten mittels quantitativer real-time PCR als Marker für verkürztes progressionsfreies Überleben validiert werden. / In this work, gene expression profiles of diffuse and intestinal-type gastric adenocarcinomas were established using the microarray technique. The intestinal type was identified to be a highly proliferative entity with significant overexpression of cell cycle-relevant genes, whereas the diffuse type was proven to be strongly stroma-dependent with significant overexpression of extracellular matrix genes. Thrombospondin 4 (THBS4) was identified as the gene most differentially expressed between the two types with vast mRNA overexpression in diffuse-type tumors. Immunohistochemical studies proved overexpression on protein level and elucidated that THBS4 is a heavily accumulated extracellular constituent of the tumor stroma. Colocalization studies uncovered that THBS4-positive cells are also positive for vimentin and alpha-smooth muscle actin. These data signify that THBS4 is expressed by subpopulations of cancer-associated fibroblasts (CAFs). This was further evidenced by in vitro experiments demonstrating that THBS4 mRNA expression is increased in CAFs of diffuse-type tumors compared to normal gastric fibroblasts. Finally, in vitro coculture studies revealed that transcriptional THBS4 expression in fibroblasts is stimulated by diffuse-type gastric tumor cells. Metastatic involvement of regional lymph nodes (N+) usually accompanies diagnosis of gastric adenocarcinoma and is currently considered the most important parameter for assessment of prognosis. However, estimation of prognosis based on this parameter alone is not sufficiently reliable. In order to identify additional molecular prognosis markers, genes whose expression correlates with clinical outcome of N+ patients were extracted from the microarray data. Via quantitative real-time PCR, several genes, e.g. RAN binding protein 17 and ras-related associated with diabetes, were successfully validated to allow an expression-based stratification of patients with respect to disease-free survival.

No description available.

Beside tumor cells themselves, tumors consist of many non-malignantly transformed cellular elements and an extracellular matrix. This so-called tumor microenvironment, or stroma, significantly influences the biological properties of the tumor through intercellular interactions. In this thesis I have focused on the study of tumor-associated fibroblasts in squamous cell carcinomas of the head and neck, malignant melanoma and glioblastoma. The data show the presence of cells with mesenchymal characteristics, present even in the glioblastoma stroma, which could potentially have a positive effect on proliferative activity and invasiveness of glioblastoma cells. In malignant melanoma, the presence of keratinocytes should also be considered, as they are the major cells of the epidermis influencing tumor melanocytes. The conditioned medium from UVB irradiated keratinocytes and non-irradiated fibroblasts stimulates the invasion of malignant melanoma cells. Targeting the tumor stroma may be a new direction in oncological therapy, so we have focused on the influence of synthetic polyamine on the formation of myofibroblasts, which are an active part of the population of tumor-associated fibroblasts. The tested polyamine prevents the formation of myofibroblasts but has no effect on those already formed nor on...

The majority of cancer research projects mainly focus on the epithelial cancer cell, while the role of the tumour stroma has been largely neglected. Conventional 2D techniques, such as well plates and other kinds of tissue culture plastic, and animal models are mainly used to broaden our understanding of how tumours arise, develop, and induce metastasis. However, there is accumulating evidence suggesting a tremendous impact of the non‐cancerous tumour stroma on carcinogenesis, while other publications illustrate the great importance of advanced 3D in vitro models for cancer research. The overall goal of this work was to investigate how cancer associated fibroblasts (CAFs; the most abundant component in the tumour stroma) and normal prostate fibroblasts (NPFs), isolated from patients diagnosed with aggressive forms of prostate cancer, contribute to angiogenesis, an important hallmark of cancer progression. For this purpose, a 3D in vitro angiogenesis co‐culture model was established. At first, two (semi‐) synthetic hydrogel platforms, gelatine methacrylate (GelMA) and star‐shaped (star)PEG‐heparin hydrogels were characterised and their physicochemical properties were compared with each other. Interestingly, GelMA gels shrank while starPEG‐heparin gels swelled in cell culture medium over the course of 24 hours. The cell concentration, in addition to the stiffness, was critical for the formation of endothelial networks, and the knowledge of swelling behaviour enabled the adjustment of initial cell density to ensure the density between both gel types was comparable. Moreover, preliminary tests with mesenchymal stem cells demonstrated that the hydrogel can be actively remodelled, as evaluated by stiffness parameters at day one and seven of incubation. Growth factors (GFs) affect cellular fate and behaviour, and storage, presentation and administration of such chemokines can be critical for certain cellular applications. Due to the high anionic charge density of heparin, starPEG‐heparin hydrogels are known to reversibly immobilise several GFs and thereby might mimic the GF reservoir of the extra cellular matrix. Thus, transport processes of GFs with low and high heparin affinity inside these hydrogels were analysed by fluorescence correlation spectroscopy and a bulk diffusion approach. Results indicated that diffusion constants were synergistically decreased with increasing size and heparin affinity of the diffusant. Next, the capability of endothelial cells (ECs) to self‐assemble and organise into 3D capillary networks was tested in GelMA, starPEG‐heparin and Matrigel hydrogels. Only starPEG‐heparin hydrogels allowed the formation of interconnected capillaries in macroscopic hydrogel samples. However, as it is widely used to test for pro‐ and anti‐angiogenic agents, the 2D Matrigel angiogenesis assay was included for subsequent co‐culture experiments of ECs and fibroblasts in order to investigate how the stromal cells influence the formation of endothelial networks. For a detailed characterisation of 3D structures, a conventionally applied 2D method (Maximum Intensity Projection for 3D reconstructed images, MIP) was compared to an optimised 3D analysing tool. As a result, it was discovered that MIP analysis did not allow for an accurate determination of 3D endothelial network parameters, and can result in misleading interpretations of the data set. Indirect co‐cultures of hydrogel‐embedded ECs with a 2D layer of fibroblasts showed that fibroblast‐derived soluble factors, including stromal cell‐derived factor 1 and interleukin 8, affected endothelial network properties. However, only co‐encapsulation of ECs and fibroblasts in starPEG‐heparin hydrogel discs revealed remarkable changes in endothelial network parameters between CAF and NPF samples. In detail, the total length and branching of the capillaries was increased. For two donor pairs, the diameter of capillaries was decreased in CAF samples compared to NPF samples, underlining the high physiological relevance of this model. In contrast, significant differences in 2D Matrigel assays were not detected between, CAF, NPF and control (ECs only) samples. In summary, a 3D angiogenesis co‐culture system was successfully developed and used to characterise stromal‐endothelial interactions in detail. The combination of advanced biomaterials (starPEG‐heparin) and 3D analysing techniques goes beyond conventional 2D in vitro cancer research, and opens new avenues for the development of more complex models to further improve the acquisition of more biologically relevant data.

L’Adénocarcinome Canalaire Pancréatique (ACP), la forme majeure de cancer du pancréas, est un des cancers les plus mortels du fait de son agressivité d’invasion locale et de dissémination vasculaire et de l’absence de méthode de détection précoce de la maladie. Nous avons développé un nouveau modèle d’invasion in vivo, sur la membrane chorio-allantoïdienne de l’embryon de poulet, permettant d’analyser les mécanismes d’interactions entre les cellules tumorales pancréatiques et leur microenvironnement. Nous avons montré que dans ce modèle les gènes codant pour les protéines sécrétées nétrine-1 et CXCL4L1/PF4v1 sont surexprimés dans les cellules tumorales au cours du processus d’invasion et que cette surexpression est également retrouvée dans les échantillons de patients humains. Nos études fonctionnelles ont indiqué que la nétrine-1 et CXCL4L1 pourrait jouer le rôle de régulateurs de la progression tumorale selon le modèle suivant : a) la chimiokine CXCL4L1 exercerait de façon paracrine une activité angiostatique sur les cellules endothéliales de l’ACP alors que b) la nétrine-1 aurait une activité pro-tumorale en agissant à la fois sur les cellules tumorales et sur les cellules endothéliales. Ces résultats ont permis d’une part de valider notre modèle en confirmant que les gènes surexprimés sélectionnés peuvent être impliqués dans la progression tumorale chez les patients. D’autre part, notre étude a permis de démontrer que les protéines CXCL4L1 et nétrine-1 constituent de nouvelles cibles thérapeutiques et/ou diagnostiques potentielles pour le cancer du pancréas. / Pancreatic Ductal Adenocarcinoma (PDAC), the major form of pancreatic cancer, is one of the deadliest cancers because of its propensity for local invasion and vascular dissemination and the lack of early diagnostic strategy. We have developed a new in vivo invasion model, on the chick embryo chorioallantoic membrane, allowing the analyze of mechanisms governing interactions between pancreatic tumor cells and their host microenvironment. We showed in its model that the genes encoding netrin-1 and CXCL4L1/PF4v1 secreted proteins are up-regulated in tumor cells in the course of the invasion process and we confirmed these up-regulation was also observed in human patients. Our functional studies indicated that netrin-1 and CXCL4L1 may play regulator roles in tumor progression according to the following model: a) CXCL4L1 chimiokine may have an angiostatic activity on endothelial cells by a paracrine mechanism of action whereas b) netrin-1 may have a pro-tumoral activity by acting on both endothelial and tumor cells. These results allowed in one hand to validate our model by showing that selected up-regulated genes may be involved in PDAC progression in human patients. On the other hand, our work provided evidence that CXCL4L1 and netrin-1 constitute new potential therapeutic and/or diagnostic targets for pancreatic cancer.