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Tumor microenvironment of soft tissue sarcomas and it's predictive significance in modern oncological treatment
Ozaniak, Andrej ; Ozaniak Střížová, Zuzana (advisor) ; Büchler, Tomáš (referee) ; Posová, Helena (referee)
Soft tissue sarcomas (STSs) are malignant tumors of mesenchymal origin, characterized by an extreme heterogeneity in histological composition, biological behavior, and clinical manifestation. Most STSs are chemo- and radiotherapy resistant. A key prognostic factor predicting the risk of distant metastases and affecting the overall survival is the tumor grade. However, grade has not been associated with the risk of local recurrence. Radical surgical procedure is in many cases the only possible treatment modality or at least plays a main role in the multimodal treatment. For patients with distant metastases, the treatment options are very limited. The chemosensitivity of STSs is generally very low, with the exception of several less common subtypes, and accounts for only 5-10% of the cases. In many cases, radiotherapy is a standard part of the treatment protocol. It is usually given in either neoadjuvant or adjuvant settings. However, radiotherapy administration in generalized patients does not improve the prognosis. Cancer immunotherapy is a therapeutic modality that utilizes the physiological cytotoxic antitumoral abilities of the immune cells. Therefore, it does not target the rapidly proliferating tumor cells but rather stimulates the immune cells. A wide variety of different strategies have been...
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Regulatory mechanisms of CD47 surface expression
Jakubec, Martin ; Drbal, Karel (advisor) ; Dibus, Michal (referee)
CD47 glycoprotein can be found on the surface of all healthy cells in our body. The interaction of CD47 with inhibitory receptor SIRPα on the macrophage leads to the inhibition of phagocytosis. This makes CD47 irreplaceable for the safe recognition of own cells and removal of aged or apoptotic cells. Apart from this, CD47 plays a major role in several essential signalling pathways, such as cell adhesion and motility or calcium homeostasis. The level of CD47 expression and its presence on the cell membrane depends not only on the type of tissue, but also on the age of a cell. An increased expression of CD47 protein has also been observed in the cells undergoing tumorigenic transformation, allowing them to escape from tumour immunosurveillance. Spontaneous regulation of the CD47 gene expression is achieved via regulatory transcription factors, such as NF-κB or HIF-1. Another mechanism of CD47 regulation includes the 3'UTR of CD47 mRNA, which serves as a binding site for either regulatory proteins, such as HuR, or miRNAs. CD47 expression can thus be regulated on both transcriptional, as well as translational level. However, appropriate topological CD47 localization within the cell and on the cell surface has also an important effect of its physiological function. Our in depth understanding of key regulatory...
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Exprese CD47 a jeho topologie na povrchu primárních buněk karcinomu močového měchýře při interakci s makrofágy
Rajtmajerová, Marie ; Drbal, Karel (advisor) ; Brdička, Tomáš (referee)
CD47 is a so-called "don't eat me" signal, which protects cells from phagocytosis. Its high expresion on tumor cells brings new perspective to the tumor therapy. Monoclonal antibodies, which are these days undergoing clinical trials, prevent CD47 binding to the SIRPA inhibitory receptor on macrophages, and so they enhance their phagocytic functional capacity. In this way they enable phagocytic removal of tumor cells. Overall expression, structural conformation and stoichiometry of CD47 on a particular cell predestine whether it will be phagocytised. The aim of the thesis is to develop and test methods to characterise expression parameters of CD47 via flow cytometry (FCM), quantitative PCR (qPCR) and microscopy. To achieve this goal I performed competition tests of commercially available antibodies in order to characterise their binding epitopes on cell lines. After performing tSNE analysis of primary BCa patient samples I correlated CD47 expression with other cell surface markers. I focused on CD47 expression in various differentiation stages of the tumor. To better understand the relationship between CD47 expression and differentiation status of cells I performed qPCR analysis of particular transcription factors. Using cell lines I examined method for phagocytosis quantification, which will be...
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Exprese CD47 a jeho topologie na povrchu primárních buněk karcinomu močového měchýře při interakci s makrofágy
Rajtmajerová, Marie ; Drbal, Karel (advisor) ; Brdička, Tomáš (referee)
CD47 is a so-called "don't eat me" signal, which protects cells from phagocytosis. Its high expresion on tumor cells brings new perspective to the tumor therapy. Monoclonal antibodies, which are these days undergoing clinical trials, prevent CD47 binding to the SIRPA inhibitory receptor on macrophages, and so they enhance their phagocytic functional capacity. In this way they enable phagocytic removal of tumor cells. Overall expression, structural conformation and stoichiometry of CD47 on a particular cell predestine whether it will be phagocytised. The aim of the thesis is to develop and test methods to characterise expression parameters of CD47 via flow cytometry (FCM), quantitative PCR (qPCR) and microscopy. To achieve this goal I performed competition tests of commercially available antibodies in order to characterise their binding epitopes on cell lines. After performing tSNE analysis of primary BCa patient samples I correlated CD47 expression with other cell surface markers. I focused on CD47 expression in various differentiation stages of the tumor. To better understand the relationship between CD47 expression and differentiation status of cells I performed qPCR analysis of particular transcription factors. Using cell lines I examined method for phagocytosis quantification, which will be...
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Regulatory mechanisms of CD47 surface expression
Jakubec, Martin ; Drbal, Karel (advisor) ; Dibus, Michal (referee)
CD47 glycoprotein can be found on the surface of all healthy cells in our body. The interaction of CD47 with inhibitory receptor SIRPα on the macrophage leads to the inhibition of phagocytosis. This makes CD47 irreplaceable for the safe recognition of own cells and removal of aged or apoptotic cells. Apart from this, CD47 plays a major role in several essential signalling pathways, such as cell adhesion and motility or calcium homeostasis. The level of CD47 expression and its presence on the cell membrane depends not only on the type of tissue, but also on the age of a cell. An increased expression of CD47 protein has also been observed in the cells undergoing tumorigenic transformation, allowing them to escape from tumour immunosurveillance. Spontaneous regulation of the CD47 gene expression is achieved via regulatory transcription factors, such as NF-κB or HIF-1. Another mechanism of CD47 regulation includes the 3'UTR of CD47 mRNA, which serves as a binding site for either regulatory proteins, such as HuR, or miRNAs. CD47 expression can thus be regulated on both transcriptional, as well as translational level. However, appropriate topological CD47 localization within the cell and on the cell surface has also an important effect of its physiological function. Our in depth understanding of key regulatory...
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Cell surface CD47 expression in cancer stem cell-targeted tumor therapy
Kuzmík, Ján ; Drbal, Karel (advisor) ; Černý, Jan (referee)
CD47 is a transmembrane glycoprotein with a high expression in both, healthy and cancer (stem) cells. Level of the CD47 expression is negatively correlated with survival of cancer patients. Binding of CD47 to SIRPα, localized on a phagocyte, triggers intracellular signaling cascade. The final effect of this cascade is dephosphorylation of nonmuscle myosin-IIA, which disrupts its function and accumulation to phagocytic synapse. The blockage of CD47-SIRPα signaling pathway in a presence of the pro-phagocytic signal induces phagocytosis of cancer cells. Afterwards, phagocytes can serve as the antigen presenting cells and prime T cell response. Role of CD47-SIRPα signaling pathway in immunity has established this pathway as a target of cancer therapy testing. Preclinical research has identified a positive therapeutic effect of blocking this signaling pathway. Nowadays, the first phase of clinical trials is being conducted. The most prevalent approach of blocking CD47-SIRPα signaling pathway in therapy is the use of anti-CD47 blocking monoclonal antibodies, which cause mild anemia. However, alternative approaches of blocking this pathway are also being developed. In this bachelor thesis, I have summarized the research related to the blockage of CD47-SIRPα signaling pathway as a cancer therapy.
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