Brain metastases are a very common and serious complication of oncological diseases. Despite the vast progress in multimodality treatment, brain metastases significantly decrease the quality of life and prognosis of patients. Therefore, identifying new targets in the microenvironment of brain metastases is desirable. Fibroblast activation protein (FAP) is a transmembrane serine protease typically expressed in tumour-associated stromal cells. Due to its characteristic presence in the tumour microenvironment, FAP represents an attractive theranostic target in oncology. However, there is little information on FAP expression in brain metastases. In this study, we quantified FAP expression in samples of brain metastases of various primary origin and characterised FAP-expressing cells. We have shown that FAP expression is significantly higher in brain metastases in comparison to non-tumorous brain tissues, both at the protein and enzymatic activity levels. FAP immunopositivity was localised in regions rich in collagen and containing blood vessels. We have further shown that FAP is predominantly confined to stromal cells expressing markers typical of cancer-associated fibroblasts (CAFs). We have also observed FAP immunopositivity on tumour cells in a portion of brain metastases, mainly originating from melanoma, lung, breast, and renal cancer, and sarcoma. There were no significant differences in the quantity of FAP protein, enzymatic activity, and FAP+ stromal cells among brain metastasis samples of various origins, suggesting that there is no association of FAP expression and/or presence of FAP+ stromal cells with the histological type of brain metastases. In summary, we are the first to establish the expression of FAP and characterise FAP-expressing cells in the microenvironment of brain metastases. The frequent upregulation of FAP and its presence on both stromal and tumour cells support the use of FAP as a promising theranostic target in brain metastases.

• MeSH
• fibroblasty patologie   MeSH
• individualizovaná medicína MeSH
• karcinom z renálních buněk * patologie   MeSH
• kvalita života MeSH
• lidé MeSH
• membránové proteiny metabolismus   MeSH
• nádorové mikroprostředí MeSH
• nádory ledvin * patologie   MeSH
• nádory mozku * patologie   MeSH
• serinové endopeptidasy metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The proline-specific serine protease fibroblast activation protein (FAP) can participate in the progression of malignant tumors and represents a potential diagnostic and therapeutic target. Recently, we demonstrated an increased expression of FAP in glioblastomas, particularly those of the mesenchymal subtype. Factors controlling FAP expression in glioblastomas are unknown, but evidence suggests that transforming growth factor beta (TGFbeta) can trigger mesenchymal changes in these tumors. Here, we investigated whether TGFbeta promotes FAP expression in transformed and stromal cells constituting the glioblastoma microenvironment. We found that both FAP and TGFbeta-1 are upregulated in glioblastomas and display a significant positive correlation. We detected TGFbeta-1 immunopositivity broadly in glioblastoma tissues, including tumor parenchyma regions in the immediate vicinity of FAP-immunopositive perivascular stromal cells. Wedemonstrate for the first time that TGFbeta-1 induces expression of FAP in non-stem glioma cells, pericytes, and glioblastoma-derived endothelial and FAP+ mesenchymal cells, but not in glioma stem-like cells. In glioma cells, this effect is mediated by the TGFbeta type I receptor and canonical Smad signaling and involves activation of FAP gene transcription. We further present evidence of FAP regulation by TGFbeta-1 secreted by glioma cells. Our results provide insight into the previously unrecognized regulation of FAP expression by autocrine and paracrine TGFbeta-1 signaling in a broad spectrum of cell types present in the glioblastoma microenvironment.

• MeSH
• endopeptidasy genetika   metabolismus   MeSH
• fluorescenční protilátková technika MeSH
• fosforylace MeSH
• glioblastom etiologie   metabolismus   patologie   MeSH
• imunohistochemie MeSH
• kultivované buňky MeSH
• lidé MeSH
• membránové proteiny genetika   metabolismus   MeSH
• nádorové buněčné linie MeSH
• nádorové mikroprostředí účinky léků   genetika   MeSH
• regulace genové exprese u nádorů * účinky léků   MeSH
• transformující růstový faktor beta1 metabolismus   farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

• Publikační typ
• abstrakt z konference MeSH

BACKGROUND AND AIMS: Proteolytic enzymes contribute to the progression of various cancers. We previously reported increased expression of the proline specific peptidases dipeptidyl peptidase-IV (DPP-IV) and its closest paralogue fibroblast activation protein (FAP) in human glioblastomas. Here we analyze the molecular heterogeneity of DPP-IV and FAP in glioblastomas. METHODS: ELISA, isoelectric focusing, 1D and 2D electrophoresis followed by WB or enzyme overlay assay were utilized to analyze DPP-IV and FAP isoforms. Cell fractionation using a Percoll gradient and deglycosylation with PNGase F were performed to analyze the possible basis of DPP-IV and FAP microheterogeneity. RESULTS: Molecular forms of DPP-IV with an estimated molecular weight of 140-160 kDa and a pI predominantly 5.8 were detected in human glioblastoma; in some tumors additional isoforms with a more acidic (3.5-5.5) as well as alkaline (8.1) pI were revealed. Using 2D electrophoresis, two to three molecular forms of FAP with an alkaline (7.0-8.5) pI and an estimated MW of 120-140 kDa were identified in glioblastoma tissues. In glioma cell lines in vitro, several isoforms of both enzymes were expressed, however the alkalic forms present in glioblastoma tissues were not detected. Removal of N-linked oligosaccharides decreased the estimated molecular weight of both enzymes; the overall pattern of molecular forms nevertheless remained unchanged. CONCLUSION: Several isoforms of DPP-IV and FAP are present in glioblastoma tissue. The absence of alkaline isoforms of both enzymes in glioma cell lines however suggests that isoforms from other, most likely stromal, cell types contribute to the overall pattern seen in glioblastoma tissues.

• MeSH
• dipeptidylpeptidasa 4 fyziologie   MeSH
• elektroforéza MeSH
• ELISA MeSH
• frakcionace buněk MeSH
• genetická heterogenita MeSH
• glioblastom enzymologie   MeSH
• imunohistochemie MeSH
• isoelektrická fokusace MeSH
• lidé MeSH
• membránové proteiny fyziologie   MeSH
• mozek enzymologie   patologie   MeSH
• nádorové buňky kultivované MeSH
• serinové endopeptidasy fyziologie   MeSH
• stabilita enzymů MeSH
• želatinasy fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Glioblastomas are deadly neoplasms resistant to current treatment modalities. Fibroblast activation protein (FAP) is a protease which is not expressed in most of the normal adult tissues but is characteristically present in the stroma of extracranial malignancies. FAP is considered a potential therapeutic target and is associated with a worse patient outcome in some cancers. The FAP localization in the glioma microenvironment and its relation to patient survival are unknown. By analyzing 56 gliomas and 15 non-tumorous brain samples, we demonstrate increased FAP expression in a subgroup of high-grade gliomas, in particular on the protein level. FAP expression was most elevated in the mesenchymal subtype of glioblastoma. It was neither associated with glioblastoma patient survival in our patient cohort nor in publicly available datasets. FAP was expressed in both transformed and stromal cells; the latter were frequently localized around dysplastic blood vessels and commonly expressed mesenchymal markers. In a mouse xenotransplantation model, FAP was expressed in glioma cells in a subgroup of tumors that typically did not express the astrocytic marker GFAP. Endogenous FAP was frequently upregulated and part of the FAP(+) host cells coexpressed the CXCR4 chemokine receptor. In summary, FAP is expressed by several constituents of the glioblastoma microenvironment, including stromal non-malignant mesenchymal cells recruited to and/or activated in response to glioma growth. The limited expression of FAP in healthy tissues together with its presence in both transformed and stromal cells suggests that FAP may be a candidate target for specific delivery of therapeutic agents in glioblastoma.

• MeSH
• apoptóza MeSH
• buňky stromatu metabolismus   patologie   MeSH
• dospělí MeSH
• fibroblasty metabolismus   patologie   MeSH
• glioblastom genetika   metabolismus   patologie   MeSH
• imunoenzymatické techniky MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé středního věku MeSH
• lidé MeSH
• membránové proteiny genetika   metabolismus   MeSH
• messenger RNA genetika   MeSH
• mezoderm metabolismus   patologie   MeSH
• míra přežití MeSH
• myši inbrední NOD MeSH
• myši MeSH
• nádorové biomarkery metabolismus   MeSH
• nádorové buňky kultivované MeSH
• následné studie MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• prognóza MeSH
• proliferace buněk MeSH
• senioři MeSH
• serinové endopeptidasy genetika   metabolismus   MeSH
• staging nádorů MeSH
• studie případů a kontrol MeSH
• transformované buněčné linie metabolismus   patologie   MeSH
• western blotting MeSH
• xenogenní modely - testy antitumorózní aktivity MeSH
• želatinasy genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• senioři MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

Malignant gliomas represent tumors with dismal prognosis. Previous work of the applicant demonstrated a grade dependent increase of dipeptidyl peptidase (DPP)-IV enzymatic activity in human astrocytic tumors. This enzymatic activity is a common attribute of several multifunctional molecules belonging to the “DPP-IV activity and/or structure homologues” (DASH). These include among others fibroblast activation protein (FAP), whose pathogenetic role is presumed in several human malignancies. In order to determine the expression, localization and molecular forms present in the human astrocytic tumors, bioptic material obtained from patients undergoing therapeutic tumor resection will be used, and the data will be correlated with the clinical and histopathological parameters. The study on the pathogenetic role of FAP and its enzymatic activity will utilize an orthotopic xenotransplantation model and glioma cell lines transfected with wild type and mutated, enzymatically inactive FAP.

Maligní gliomy představují nádorová onemocnění se špatnou prognózou. Předchozí práce navrhovatele prokázala závislost dipeptidylpeptidase (DPP)-IV podobné aktivity na stupni malignity astrocytárních tumorů. Tato aktivita je společným atributem skupiny „DPP -IV aktivitou a/nebo strukturou homologních“ (DASH) molekul. Mezi tyto molekuly patří mj. fibroblastový aktivační protein (FAP), jehož patogenetický význam je předpokládán u řady humánních malignit. K určení exprese, tkáňové lokalizace a molekulových forem FAP přítomných v lidských astrocytárních tumorech bude použit bioptický materiál odebraný od pacientů podstupujících terapeutickou resekci nádoru, data budou korelována s klinickými a histopatologickými parametry. Ke studiu patogenetických mechanismů účasti FAP a jeho enzymové aktivity na gliomagenezi bude využito ortotopického xenotransplantačního modelu a gliomových linií transfekovaných enzymově aktivní a mutovanou, enzymově neaktivní formou FAP.

• MeSH
• analýza přežití MeSH
• astrocytom patofyziologie   MeSH
• cílená molekulární terapie MeSH
• dipeptidylpeptidasa 4 MeSH
• ELISA MeSH
• enzymová indukce MeSH
• fibroblasty asociované s nádorem MeSH
• glioblastom patofyziologie   MeSH
• messenger RNA MeSH
• proliferace buněk MeSH
• serinové proteasy MeSH
• western blotting MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• molekulární biologie, molekulární medicína
• onkologie
• neurologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

Ribonucleic acid (RNA) represents an important target of a wide array of laboratory anal yses. Thus, RNA purification is a critical first preceding step of a number of preparative and analytical methods, important particularly in diagnostics of dozens of viral, bacterial, and parasitic diseases, dia gnosis of inherited disorders, and tumours, as well as in basic research. To provide relevant and reliable results, techniques of molecular biology used for such purposes require pure and intact molecules of purified RNA. Moreover, RNA has to be purified effectively and reproducibly from various heterogeneous materials such as fresh or frozen tissues, cell lines, PCR products or long-term chemically preserved samples. Principally, methods of RNA purification can be divided into three groups. The first group of methods is based on organic phenol:chloroform extraction. The second group encompasses methods of RNA purification by means of its ability to bind specific surfaces in the presence of chaotropic salt, and the third group includes methods exploiting RNA isolation on isopycnic gradients. Although RNA can be isolated from either prokaryotic or eukaryotic organisms, this review is to give out a basic outline of methods available for eukaryotic, with emphasis on mammalian, tissues.

• MeSH
• adsorpce MeSH
• lidé MeSH
• RNA chemie   izolace a purifikace   MeSH
• rozpouštědla chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• přehledy MeSH