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Mutual communication and relationship between tumor cells and the tumor microenvironment
Novák, Štěpán ; Szabo, Pavol (advisor) ; Krška, Zdeněk (referee) ; Grobárová, Valéria (referee)
The present dissertation addresses two main objectives. First, the interaction between four pancreatic ductal adenocarcinoma (PDAC) cell lines and conditioned media from normal/healthy fibroblasts was investigated, HF) and cancer- associated fibroblasts (CAFs) derived from PDAC (PDAC-derived CAFs, PANFs), cells derived from ascitic fluid of patients with end-stage PDAC and primary PDAC tumor. Subsequently, the genome-wide profile of PANFs was determined. The second aim was to differentiate squamous cell carcinoma (SCC) cells, surgical resection margin (MSR) cells and normal mucosa (NM) cells in patients operated for head and neck squamous cell carcinoma (HNSCC) by immunohistochemistry, using detection of tenascin (Ten), fibronectin (Fn) and galectin-1 (Gal-1), and to correlate these results with their clinical characteristics. Subsequently, whole-genome profiling of SCC, MSR and NM samples was performed based on Ten expression stratification. Results Cultivation of the four PDAC lines under the influence of conditioned media from HF and PANF was heterogeneous. After stimulation, the most aggressive behavior was obtained in the Panc-1 cell line while the PaTu-8902 line was rather inhibited by the media. The PANF transcriptome showed increased expression of some genes (e.g. IL-6, IL-8, MFGE8,...
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Role of Fibroblast activation protein expressing stromal mesenchymal cells in microenvironment of brain tumors
Výmola, Petr ; Balážiová, Eva (advisor) ; Szabo, Pavol (referee)
High-grade astrocytic tumours are aggressive primary brain malignancies, from which the most malignant and the most common is glioblastoma multiforme with median survival 15 months. Fibroblast activation protein (FAP) is a serine protease possessing both endo- and exopeptidase activity. Its restricted and increased expression in pathological conditions makes it a promising target for the development of new diagnostic or therapeutic approaches. Previous results of our laboratory and of other authors confirm expression of FAP in high-grade astrocytic tumours, where FAP is expressed by perivascular population of stromal mesenchymal cells. The aim of this diploma thesis was to analyse the role of FAP positive primary stromal mesenchymal cultures in neovascularisation and extracellular matrix (ECM) deposition in glioblastoma microenvironment. The results of this thesis describe proangiogenic effect of FAP positive stromal mesenchymal cells derived from human glioblastoma tissue. Using conditioned media, it was proven, that this effect is realized by factors secreted by these cells, even though results of in vivo assay on chorioallantoid membrane, where direct cell-to-cell contact of all involved cell types (tumor associated FAP positive mesenchymal cells, endothelial cells, glioma cells) is assumed,...
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Role fibroblastového aktivačního proteinu v růstu a invazivitě gliomových buněk
Fejfarová, Edita ; Bušek, Petr (advisor) ; Szabo, Pavol (referee)
High grade astrocytomas are very progressive brain tumors. Glioblastoma multiforme is the most frequent and the most malignant type with very infiltrative phenotype of the tumor cells. Fibroblast activation protein FAP is a predominantly membrane bound prolyl peptidase bearing exo- and endopeptidase hydrolytic activities. FAP is known to play a role in wound healing, cell migration and invasion and its expression is linked to the pathogenesis of several malignancies. mRNA expression of FAP is upregulated in 48% of glioblastomas according to The Cancer Genome Atlas microarray data. The involvement of FAP in the pathogenesis of astrocytic tumors is largely unknown. The aims of this work are to analyse the expression of FAP in primary cell cultures derived from high grade gliomas and to analyse the influence of FAP on the growth, migration and invasion of glioma cells. Our ELISA and western blot results showed heterogenous expression of FAP in the studied glioma primary cell cultures and cell lines. Both enzymatic activities characteristic of FAP were detected in the primary glioma cell culture P11 with high expression of FAP. In these cells, FAP was present not only in the typical plasma membrane localization, but also in the cytoplasm as demonstrated by immunofluorescence staining. The P11 cells...
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Glycobiology of the head and neck cancer
Szabo, Pavol
The cell surface is covered by different oligosaccharides, which are anchored in the plasmatic membrane by proteins and lipids. Oligosaccharides mediate mutual interactions either between cells or between cells and components of extracellular matrix. Galectins are animal lectins with affinity to oligosaccharides containing β-galactosides. Furthermore, galectins are multifactorial lectins that participate in the regulation and modulation of various biological processes including cell-cell and cell-matrix interactions, cell proliferation and differentiation as well as apoptosis and pre-mRNA splicing. After translation, proteins undergo different structural modifications with direct impact on their function. For example, galectin-3 as possible prognostic marker, well recognize cells of squamos cell carcinomas, is phoshorylated on N-terminus. However, we showed that posttranslational phosphorylation of galectin-3 does not have a direct impact on its binding activity. Another endogenous lectin, galectin-1 is a typical protein of cancer stroma and wound granulation tissue. In this context we also detected, that galectin-1 induces a TGF-β1 independent fibroblast-myofibroblast transition including production of a bioactive fibronectin and galectin-1 rich extracelluar matrix in vitro. This knowledge can...
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Glycobiology of the head and neck cancer
Szabo, Pavol ; Smetana, Karel (advisor) ; Říhová, Blanka (referee) ; Brábek, Jan (referee)
Povrch buněk je bohatě pokryt oligosacharidy, které jsou v plazmalemě ukotvené pomocí proteinů a lipidů. Oligosacharidy zprostředkují vzájemnou vazbu mezi buňkami nebo vazbu buněk k složkám extracelulární matrix. Galektiny jsou živočišné lektinů které mají afinitu k oligosacharidům obsahujícím β-galaktózu. Jsou to multifaktoriální proteiny, které se účastňují řady reakcí v organizmu, jako jsou mezibuněčné interakce, interakce buněk s mezibuněčnou hmotou, proliferace i apoptóza a sestřih pre-mRNA. Proteiny po translaci procházejí různými strukturálnimy úpravami, které mají vliv na jejich funkci. Galektin-3 je možný prognostický ukazatel u nádorů vycházejících z vrstevnatých dlaždicových epitelů je fosforylován na N-konci. Prokázali jsme, že tato posttranslační modifikace nemá vliv na jeho vazebnou reaktivitu. Jiný endogenní lektin, galektin-1 je charakteristickou molekulou nádorového stromatu a granulační tkáně hojícího se poranění. Zjistili jsme, že galektin-1 indukuje na TGF-β nezávislý in vitro přechod normálních fibroblastů na myofibroblasty včetně produkce sítě extracelulární matrix bohaté fibronektinem a galektinem-1. Tento poznatek je využitelný v terapii hojení ran a v tkáňovém inženýrství. Dnes je jasné, že nádorové stroma ovlivňuje i biologické vlastnosti nádoru (lokální agresivita,...
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Role of Fibroblast activation protein expressing stromal mesenchymal cells in microenvironment of brain tumors
Výmola, Petr ; Balážiová, Eva (advisor) ; Szabo, Pavol (referee)
High-grade astrocytic tumours are aggressive primary brain malignancies, from which the most malignant and the most common is glioblastoma multiforme with median survival 15 months. Fibroblast activation protein (FAP) is a serine protease possessing both endo- and exopeptidase activity. Its restricted and increased expression in pathological conditions makes it a promising target for the development of new diagnostic or therapeutic approaches. Previous results of our laboratory and of other authors confirm expression of FAP in high-grade astrocytic tumours, where FAP is expressed by perivascular population of stromal mesenchymal cells. The aim of this diploma thesis was to analyse the role of FAP positive primary stromal mesenchymal cultures in neovascularisation and extracellular matrix (ECM) deposition in glioblastoma microenvironment. The results of this thesis describe proangiogenic effect of FAP positive stromal mesenchymal cells derived from human glioblastoma tissue. Using conditioned media, it was proven, that this effect is realized by factors secreted by these cells, even though results of in vivo assay on chorioallantoid membrane, where direct cell-to-cell contact of all involved cell types (tumor associated FAP positive mesenchymal cells, endothelial cells, glioma cells) is assumed,...
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Glycobiology of the head and neck cancer
Szabo, Pavol
The cell surface is covered by different oligosaccharides, which are anchored in the plasmatic membrane by proteins and lipids. Oligosaccharides mediate mutual interactions either between cells or between cells and components of extracellular matrix. Galectins are animal lectins with affinity to oligosaccharides containing β-galactosides. Furthermore, galectins are multifactorial lectins that participate in the regulation and modulation of various biological processes including cell-cell and cell-matrix interactions, cell proliferation and differentiation as well as apoptosis and pre-mRNA splicing. After translation, proteins undergo different structural modifications with direct impact on their function. For example, galectin-3 as possible prognostic marker, well recognize cells of squamos cell carcinomas, is phoshorylated on N-terminus. However, we showed that posttranslational phosphorylation of galectin-3 does not have a direct impact on its binding activity. Another endogenous lectin, galectin-1 is a typical protein of cancer stroma and wound granulation tissue. In this context we also detected, that galectin-1 induces a TGF-β1 independent fibroblast-myofibroblast transition including production of a bioactive fibronectin and galectin-1 rich extracelluar matrix in vitro. This knowledge can...
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Glycobiology of the head and neck cancer
Szabo, Pavol ; Smetana, Karel (advisor) ; Říhová, Blanka (referee) ; Brábek, Jan (referee)
Povrch buněk je bohatě pokryt oligosacharidy, které jsou v plazmalemě ukotvené pomocí proteinů a lipidů. Oligosacharidy zprostředkují vzájemnou vazbu mezi buňkami nebo vazbu buněk k složkám extracelulární matrix. Galektiny jsou živočišné lektinů které mají afinitu k oligosacharidům obsahujícím β-galaktózu. Jsou to multifaktoriální proteiny, které se účastňují řady reakcí v organizmu, jako jsou mezibuněčné interakce, interakce buněk s mezibuněčnou hmotou, proliferace i apoptóza a sestřih pre-mRNA. Proteiny po translaci procházejí různými strukturálnimy úpravami, které mají vliv na jejich funkci. Galektin-3 je možný prognostický ukazatel u nádorů vycházejících z vrstevnatých dlaždicových epitelů je fosforylován na N-konci. Prokázali jsme, že tato posttranslační modifikace nemá vliv na jeho vazebnou reaktivitu. Jiný endogenní lektin, galektin-1 je charakteristickou molekulou nádorového stromatu a granulační tkáně hojícího se poranění. Zjistili jsme, že galektin-1 indukuje na TGF-β nezávislý in vitro přechod normálních fibroblastů na myofibroblasty včetně produkce sítě extracelulární matrix bohaté fibronektinem a galektinem-1. Tento poznatek je využitelný v terapii hojení ran a v tkáňovém inženýrství. Dnes je jasné, že nádorové stroma ovlivňuje i biologické vlastnosti nádoru (lokální agresivita,...
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Role fibroblastového aktivačního proteinu v růstu a invazivitě gliomových buněk
Fejfarová, Edita ; Bušek, Petr (advisor) ; Szabo, Pavol (referee)
High grade astrocytomas are very progressive brain tumors. Glioblastoma multiforme is the most frequent and the most malignant type with very infiltrative phenotype of the tumor cells. Fibroblast activation protein FAP is a predominantly membrane bound prolyl peptidase bearing exo- and endopeptidase hydrolytic activities. FAP is known to play a role in wound healing, cell migration and invasion and its expression is linked to the pathogenesis of several malignancies. mRNA expression of FAP is upregulated in 48% of glioblastomas according to The Cancer Genome Atlas microarray data. The involvement of FAP in the pathogenesis of astrocytic tumors is largely unknown. The aims of this work are to analyse the expression of FAP in primary cell cultures derived from high grade gliomas and to analyse the influence of FAP on the growth, migration and invasion of glioma cells. Our ELISA and western blot results showed heterogenous expression of FAP in the studied glioma primary cell cultures and cell lines. Both enzymatic activities characteristic of FAP were detected in the primary glioma cell culture P11 with high expression of FAP. In these cells, FAP was present not only in the typical plasma membrane localization, but also in the cytoplasm as demonstrated by immunofluorescence staining. The P11 cells...
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