Projekt vytvoří technologickou platformu pro výzkum komplexních tekutých tkání (periferní krev, kostní dřeň a CUSA aspirát) které jsou infiltrovány nádorovými buňkami. Přístup kombinuje dvě inovativní datově bohaté technologie: hmotnostní cytometrii („nádorová cytomika“), která zjišťuje změny v buněčném zastoupení infiltrovaných tkání a SEC-MAP array („proteomika“), která zjišťuje změny v proteomu nádorových buněk. Nejprve zmapujeme normální vývoj B-lymfocytů rezidentních v kostní dření, ke kterému bude možné vztahovat nádorem způsobené abnormity (potenciální biomarkery). Na modelu leukemických buněk a buněk mozkových nádorů dále určíme změny proteomu při maligním zvratu metodou SEC-MAP arrays. V další fázi budou biomarkery zabudované do panelů vyšetřovaných hmotnostní cytometrií („nádorová cytomika“). Vyvineme bioinformatické nástroje pro porozumění vzniklým vysoce kompexním datům.; Proposed project will develop a technological platform for investigation of complex suspension tissues (peripheral blood, bone marrow and CUSA aspirates) that contain a tumor infiltration. The approach would combine and complement two innovative high-content approaches: a single cell mass cytometry (“cancer cytomics”) to determine changes in cellular composition of tumor affected tissue and SEC-MAP array (“proteomics”) to determine changes in proteome of tumor cells. First, a reference of normal cellular development will be established for bone marrow resident B-cells to which abnormal (leukemic) cells could be compared in search for biomarkers. Next, SEC-MAP will be used to determine tumor specific proteome changes (in leukemia and in brain tumors as a model). Last, useful biomarkers will be built into single cells analysis by mass cytometry panels for “cancer cytomics” investigation. Bioinformatic tools will be developed to enable understanding the complex data.

• MeSH
• B-lymfocyty cytologie   MeSH
• cytodiagnostika metody   MeSH
• hmotnostní spektrometrie metody   MeSH
• leukemie MeSH
• nádorová transformace buněk MeSH
• nádorové biomarkery MeSH
• nádorové mikroprostředí MeSH
• nádory centrálního nervového systému MeSH
• nádory diagnóza   MeSH
• proteomika metody   MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• onkologie
• cytologie, klinická cytologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

PURPOSE: The aim of this study was to test the possibility of using specimens obtained by a cavitron ultrasonic surgical aspirator (CUSA) in flow and mass cytometry investigations of pediatric brain tumors. METHODS: CUSA specimens obtained from 19 pediatric patients with brain tumors were investigated. Flow and mass cytometry methods were applied to analyze the composition of material collected using the CUSA. Cell suspensions were prepared from CUSA aspirates. Then sample viability was assessed by conventional flow cytometry and subsequently stained with a panel of 31 metal-labeled antibodies. RESULTS: Viability assessment was performed using conventional flow cytometry. Viability of cells in the acquired samples was below 50% in 16 of 19 cases. A mass cytometry investigation and subsequent analysis enabled us to discriminate brain tumor cells from contaminating leukocytes, whose proportions varied across the specimens. The addition of the viability marker cisplatin directly into the mass cytometry panel gave the means to selecting viable cells only for subsequent analyses. The proportion of non-viable cells was higher among tumor cells compared leukocytes. CONCLUSIONS: When the analysis of the tumor cell immunophenotype is performed with markers for determining viability, the expression of the investigated markers can be evaluated. Suitable markers can be selected by high-throughput methods, such as mass cytometry, and those that are diagnostically relevant can be investigated using flow cytometry, which is more flexible in terms of time.

• MeSH
• analýza jednotlivých buněk MeSH
• cisplatina metabolismus   MeSH
• leukocyty metabolismus   patologie   MeSH
• lidé MeSH
• nádorové biomarkery metabolismus   MeSH
• nádory mozku diagnóza   metabolismus   patologie   chirurgie   MeSH
• neurochirurgické výkony přístrojové vybavení   MeSH
• průtoková cytometrie MeSH
• ultrazvuková terapie přístrojové vybavení   MeSH
• viabilita buněk MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

In mass cytometry, the isolation of pure lymphocytes is very important to obtain reproducible results and to shorten the time spent on data acquisition. To prepare highly purified cell suspensions of peripheral blood lymphocytes for further analysis on mass cytometer, we used the new CD81+ immune affinity chromatography cell isolation approach. Using 21 metal conjugated antibodies in a single tube we were able to identify all basic cell subsets and compare their relative abundance in final products obtained by density gradient (Ficoll-Paque) and immune affinity chromatography (CD81+ T-catch™) isolation approach. We show that T-catch isolation approach results in purer final product than Ficoll-Paque (P values 0.0156), with fewer platelets bound to target cells. As a result acquisition time of 105 nucleated cells was 3.5 shorter. We then applied unsupervised high dimensional analysis viSNE algorithm to compare the two isolation protocols, which allowed us to evaluate the contribution of unsupervised analysis over supervised manual gating. ViSNE algorithm effectively characterized almost all supervised cell subsets. Moreover, viSNE uncovered previously overseen cell subsets and showed inaccuracies in Maxpar™ Human peripheral blood phenotyping panel kit recommended gating strategy. These findings emphasize the use of unsupervised analysis tools in parallel with conventional gating strategy to mine the complete information from a set of samples. They also stress the importance of the impurity removal to sensitively detect rare cell populations in unsupervised analysis. © 2016 International Society for Advancement of Cytometry.

• MeSH
• antigeny CD81 chemie   metabolismus   MeSH
• Ficoll chemie   MeSH
• leukocyty mononukleární cytologie   MeSH
• lidé MeSH
• lymfocyty cytologie   imunologie   MeSH
• obrazová cytometrie metody   MeSH
• protilátky chemie   imunologie   MeSH
• separace buněk metody   MeSH
• viabilita buněk imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Development of hematopoietic populations through the process of differentiation is critical for proper hematopoiesis. The transcription factor CCAAT/enhancer binding protein alpha (C/EBPα) is a master regulator of myeloid differentiation, and the identification of C/EBPα target genes is key to understand this process. Here we identified the Ecotropic Viral Integration Site 2B (EVI2B) gene as a direct target of C/EBPα. We showed that the product of the gene, the transmembrane glycoprotein EVI2B (CD361), is abundantly expressed on the surface of primary hematopoietic cells, the highest levels of expression being reached in mature granulocytes. Using shRNA-mediated downregulation of EVI2B in human and murine cell lines and in primary hematopoietic stem and progenitor cells, we demonstrated impaired myeloid lineage development and altered progenitor functions in EVI2B-silenced cells. We showed that the compromised progenitor functionality in Evi2b-depleted cells can be in part explained by deregulation of cell proliferation and apoptosis. In addition, we generated an Evi2b knockout murine model and demonstrated altered properties of hematopoietic progenitors, as well as impaired G-CSF dependent myeloid colony formation in the knockout cells. Remarkably, we found that EVI2B is significantly downregulated in human acute myeloid leukemia samples characterized by defects in CEBPA. Altogether, our data demonstrate that EVI2B is a downstream target of C/EBPα, which regulates myeloid differentiation and functionality of hematopoietic progenitors.

• MeSH
• akutní myeloidní leukemie metabolismus   patologie   MeSH
• apoptóza MeSH
• buněčná diferenciace účinky léků   MeSH
• buňky kostní dřeně cytologie   MeSH
• down regulace účinky léků   MeSH
• estradiol farmakologie   MeSH
• faktor stimulující kolonie granulocytů farmakologie   MeSH
• granulocyty cytologie   metabolismus   MeSH
• hematopoetické kmenové buňky cytologie   metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• malá interferující RNA metabolismus   MeSH
• membránové glykoproteiny antagonisté a inhibitory   genetika   metabolismus   MeSH
• membránové proteiny antagonisté a inhibitory   genetika   metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• proliferace buněk účinky léků   MeSH
• promotorové oblasti (genetika) MeSH
• protein alfa vázající zesilovač transkripce CCAAT genetika   metabolismus   MeSH
• RNA interference MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Acute leukemia is a disease pathologically manifested at both genomic and proteomic levels. Molecular genetic technologies are currently widely used in clinical research. In contrast, sensitive and high-throughput proteomic techniques for performing protein analyses in patient samples are still lacking. Here, we used a technology based on size exclusion chromatography followed by immunoprecipitation of target proteins with an antibody bead array (Size Exclusion Chromatography-Microsphere-based Affinity Proteomics, SEC-MAP) to detect hundreds of proteins from a single sample. In addition, we developed semi-automatic bioinformatics tools to adapt this technology for high-content proteomic screening of pediatric acute leukemia patients.To confirm the utility of SEC-MAP in leukemia immunophenotyping, we tested 31 leukemia diagnostic markers in parallel by SEC-MAP and flow cytometry. We identified 28 antibodies suitable for both techniques. Eighteen of them provided excellent quantitative correlation between SEC-MAP and flow cytometry (p< 0.05). Next, SEC-MAP was applied to examine 57 diagnostic samples from patients with acute leukemia. In this assay, we used 632 different antibodies and detected 501 targets. Of those, 47 targets were differentially expressed between at least two of the three acute leukemia subgroups. The CD markers correlated with immunophenotypic categories as expected. From non-CD markers, we found DBN1, PAX5, or PTK2 overexpressed in B-cell precursor acute lymphoblastic leukemias, LAT, SH2D1A, or STAT5A overexpressed in T-cell acute lymphoblastic leukemias, and HCK, GLUD1, or SYK overexpressed in acute myeloid leukemias. In addition, OPAL1 overexpression corresponded to ETV6-RUNX1 chromosomal translocation.In summary, we demonstrated that SEC-MAP technology is a powerful tool for detecting hundreds of proteins in clinical samples obtained from pediatric acute leukemia patients. It provides information about protein size and reveals differences in protein expression between particular leukemia subgroups. Forty-seven of SEC-MAP identified targets were validated by other conventional method in this study.

• MeSH
• akutní lymfatická leukemie diagnóza   imunologie   metabolismus   MeSH
• diferenciální diagnóza MeSH
• dítě MeSH
• gelová chromatografie metody   MeSH
• imunofenotypizace metody   MeSH
• imunoprecipitace MeSH
• kojenec MeSH
• laboratorní automatizace MeSH
• lidé MeSH
• mladiství MeSH
• nádorové buněčné linie MeSH
• předškolní dítě MeSH
• proteomika metody   MeSH
• protilátky farmakologie   MeSH
• regulace genové exprese u leukemie MeSH
• Check Tag
• dítě MeSH
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• předškolní dítě MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH