Multicolor flow cytometry allows for analysis of tens of cellular parameters in millions of cells at a single-cell resolution within minutes. The lack of technologies that would facilitate feasible and relatively cheap profiling of such a number of cells with an antibody-based approach led us to the development of a high-throughput cytometry-based platform for surface profiling. We coupled the fluorescent cell barcoding with preexisting, commercially available screening tools to analyze cell surface fingerprint at a large scale. This powerful approach will help to identify novel biomarkers and druggable targets and facilitate the discovery of new concepts in immunology, oncology, and developmental biology.

• Klíčová slova
• Cell surface phenotyping, Fluorescent cell barcoding, High-throughput screening, Multicolor flow cytometry,
• MeSH
• antigeny povrchové * MeSH
• biologické markery analýza   MeSH
• fluorescenční barviva MeSH
• průtoková cytometrie MeSH
• výzkum * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny povrchové * MeSH
• biologické markery MeSH
• fluorescenční barviva MeSH

Background:The intratumoural heterogeneity, often driven by epithelial-to-mesenchymal transition (EMT), significantly contributes to chemoresistance and disease progression in adenocarcinomas. Methods:We introduced a high-throughput screening platform to identify surface antigens that associate with epithelial–mesenchymal plasticity in well-defined pairs of epithelial cell lines and their mesenchymal counterparts. Using multicolour flow cytometry, we then analysed the expression of 10 most robustly changed antigens and identified a 10-molecule surface signature, in pan-cytokeratin-positive/EpCAM-positive and -negative fractions of dissociated breast tumours. Results:We found that surface CD9, CD29, CD49c, and integrin ß5 are lost in breast cancer cells that underwent EMT in vivo. The tetraspanin family member CD9 was concordantly downregulated both in vitro and in vivo and associated with epithelial phenotype and favourable prognosis. Conclusions:We propose that overall landscape of 10-molecule surface signature expression reflects the epithelial–mesenchymal plasticity in breast cancer.

• MeSH
• antigeny CD9 biosyntéza   imunologie   MeSH
• antigeny nádorové biosyntéza   imunologie   MeSH
• antigeny povrchové biosyntéza   imunologie   MeSH
• epitelo-mezenchymální tranzice imunologie   MeSH
• genetická transkripce MeSH
• lidé MeSH
• metastázy nádorů MeSH
• nádorové biomarkery MeSH
• nádorové buněčné linie MeSH
• nádory prsu genetika   imunologie   patologie   MeSH
• plasticita buňky imunologie   MeSH
• přeprogramování buněk fyziologie   MeSH
• průtoková cytometrie MeSH
• rychlé screeningové testy MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny CD9 MeSH
• antigeny nádorové MeSH
• antigeny povrchové MeSH
• CD9 protein, human MeSH Prohlížeč
• nádorové biomarkery MeSH

In 67 cases of newly diagnosed blood malignancies, NonT-ALL, T-ALL, AMLL, AML, CML, CLL, HCL, PLL, MDS, B splenic lymphoma, AUL, as well as in 9 cell lines (U937, HEL, Jurkat, HL60, UHKT2, KG1, Raji, K562, REH), we have analysed the expression and distribution of 2 relatively incompletely studied antigenic markers from the CD nomenclature: CDw12 and CD17, individually and in combination with well characterized ones. We present our data for the usefulness of these molecules in immunodiagnosis of leukemias and lymphomas.

• MeSH
• antigeny povrchové analýza   MeSH
• CD antigeny analýza   MeSH
• imunofenotypizace MeSH
• kostní dřeň patologie   MeSH
• krevní nemoci metabolismus   MeSH
• leukemie imunologie   patologie   MeSH
• lidé MeSH
• lymfom imunologie   patologie   MeSH
• nádorové biomarkery MeSH
• nádorové buňky kultivované MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny povrchové MeSH
• CD antigeny MeSH
• nádorové biomarkery MeSH

Glycosylated sphingolipids (GSLs) are a diverse group of cellular lipids typically reported as being rare in normal mammary tissue. In breast cancer (BCa), GSLs have emerged as noteworthy markers associated with breast cancer stem cells, mediators of phenotypic plasticity, and contributors to cancer cell chemoresistance. GSLs are potential surface markers that can uniquely characterize the heterogeneity of the tumor microenvironment, including cancer cell subpopulations and epithelial-mesenchymal plasticity (EMP). In this study, mass spectrometry analyses of the total sphingolipidome in breast epithelial cells and their mesenchymal counterparts revealed increased levels of Gb3 in epithelial cells and significantly elevated GD2 levels in the mesenchymal phenotype. To elucidate if GSL-related epitopes on BCa cell surfaces reflect EMP and cancer status, we developed and rigorously validated a 12-color spectral flow cytometry panel. This panel enables the simultaneous detection of native GSL epitopes (Gb3, SSEA1, SSEA3, SSEA4, and GD2), epithelial-mesenchymal transition markers (EpCAM, TROP2, and CD9), and lineage markers (CD45, CD31, and CD90) at the single-cell level. Next, the established panel was used for the analysis of BCa primary tumors and revealed surface heterogeneity in SSEA1, SSEA3, SSEA4, GD2, and Gb3, indicative of native epitope presence also on non-tumor cells. These findings further highlighted the phenotype-dependent alterations in GSL surface profiles, with differences between epithelial and stromal cells in the tumor. This study provides novel insights into BCa heterogeneity, shedding light on the potential of native GSL-related epitopes as markers for EMP and cancer status in fresh clinical samples. The developed single-cell approach offers promising avenues for further exploration.

• Klíčová slova
• breast cancer, epithelial cells, glycosphingolipids, phenotypic plasticity, stromal-like cells, surface profiling,
• MeSH
• analýza jednotlivých buněk * metody   MeSH
• epitelo-mezenchymální tranzice * MeSH
• fenotyp MeSH
• glykosfingolipidy * metabolismus   analýza   MeSH
• lidé MeSH
• nádory prsu * metabolismus   patologie   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• glykosfingolipidy * MeSH

Saccharomyces cerevisiae strain MW11 is a temperature-sensitive mutant which exports twenty times more proteins at 37 degrees C than parental or wild-type strains do. To understand the mechanism underlying the protein overexport in the mutant the possibility of an altered cell-wall structure leading to facilitated release of cell-surface proteins was studied. Data on calcofluor white and zymolyase sensitivities, resistance to killer 1 toxin and determination of exported acid phosphatase and invertase did not provide evidence for alterations in the cell-wall structure that could explain the protein overexport phenotype. The results were obtained in experiments when transcription of mutated gene was discontinued which permits the full expression of the protein overexport phenotype.

• MeSH
• benzensulfonáty farmakologie   MeSH
• buněčná stěna chemie   metabolismus   MeSH
• fungální proteiny metabolismus   MeSH
• genetická transkripce MeSH
• hydrolasy farmakologie   MeSH
• killer faktory kvasinek MeSH
• membránové proteiny metabolismus   MeSH
• mutace * MeSH
• periplazma enzymologie   MeSH
• proteiny farmakologie   MeSH
• Saccharomyces cerevisiae účinky léků   genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• benzensulfonáty MeSH
• C.I. Fluorescent Brightening Agent 28 MeSH Prohlížeč
• fungální proteiny MeSH
• hydrolasy MeSH
• killer faktory kvasinek MeSH
• membránové proteiny MeSH
• proteiny MeSH
• zymolyase MeSH Prohlížeč

The adaptive immune response critically hinges on the functionality of T cell receptors, governed by complex molecular mechanisms, including ubiquitination. In this study, we delved into the role of in T cell immunity, focusing on T cell-B cell conjugate formation and T cell activation. Using a CRISPR-Cas9 screening approach targeting deubiquitinases genes in Jurkat T cells, we identified BAP1 as a key positive regulator of T cell-B cell conjugate formation. Subsequent investigations into BAP1 knockout cells revealed impaired T cell activation, evidenced by decreased MAPK and NF-kB signaling pathways and reduced CD69 expression upon T cell receptor stimulation. Flow cytometry and qPCR analyses demonstrated that BAP1 deficiency leads to decreased surface expression of T cell receptor complex components and reduced mRNA levels of the co-stimulatory molecule CD28. Notably, the observed phenotypes associated with BAP1 knockout are specific to T cells and fully dependent on BAP1 catalytic activity. In-depth RNA-seq and mass spectrometry analyses further revealed that BAP1 deficiency induces broad mRNA and protein expression changes. Overall, our findings elucidate the vital role of BAP1 in T cell biology, especially in T cell-B cell conjugate formation and T cell activation, offering new insights and directions for future research in immune regulation.

• Klíčová slova
• BAP1, CRISPR-Cas9 screening, T cell activation, T cell receptor (TCR), T cell-B cell conjugates,
• MeSH
• aktivace lymfocytů * imunologie   MeSH
• B-lymfocyty * imunologie   metabolismus   MeSH
• Jurkat buňky MeSH
• lidé MeSH
• nádorové supresorové proteiny * metabolismus   genetika   MeSH
• receptory antigenů T-buněk * metabolismus   MeSH
• signální transdukce MeSH
• T-lymfocyty * imunologie   metabolismus   MeSH
• thiolesterasa ubikvitinu * genetika   metabolismus   nedostatek   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• BAP1 protein, human MeSH Prohlížeč
• nádorové supresorové proteiny * MeSH
• receptory antigenů T-buněk * MeSH
• thiolesterasa ubikvitinu * MeSH

• MeSH
• antigeny CD34 metabolismus   MeSH
• fenotyp MeSH
• hematopoetické kmenové buňky imunologie   MeSH
• imunofenotypizace MeSH
• leukemie klasifikace   imunologie   MeSH
• lidé MeSH
• lymfom klasifikace   imunologie   MeSH
• nádorové kmenové buňky imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny CD34 MeSH

• MeSH
• antigeny nádorové analýza   MeSH
• buněčná adheze MeSH
• imunofenotypizace MeSH
• leukemie metabolismus   patologie   MeSH
• lidé MeSH
• lymfom metabolismus   patologie   MeSH
• molekuly buněčné adheze metabolismus   MeSH
• myelodysplastické syndromy metabolismus   patologie   MeSH
• nádorové buňky kultivované MeSH
• nádorové kmenové buňky metabolismus   MeSH
• nádorové proteiny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• antigeny nádorové MeSH
• molekuly buněčné adheze MeSH
• nádorové proteiny MeSH

• MeSH
• buněčná stěna metabolismus   MeSH
• buněčné dělení MeSH
• buněčné klony MeSH
• buněčné linie MeSH
• fenotyp MeSH
• film jako téma MeSH
• kontaktní inhibice * MeSH
• krysa rodu Rattus MeSH
• kultivační média MeSH
• kultivované buňky růst a vývoj   MeSH
• mikromanipulace MeSH
• mitóza MeSH
• permeabilita buněčné membrány MeSH
• pinocytóza MeSH
• povrchové vlastnosti MeSH
• ptačí sarkom patologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kultivační média MeSH

BACKGROUND: Multiple myeloma (MM) is characterized by accumulation of pathological plasma cells (PCs) in bone marrow (BM) as a result of deregulation of B-cell development. To clarify its pathophysiology it is necessary to investigate in detail the developmental stages of B-cells. MATERIALS AND METHODS: Enumeration of total CD19-positive (CD19(+)) cells and their subpopulations together with PCs was done in peripheral blood (PB) and BM of newly diagnosed monoclonal gammopathy patients and control subjects. Representation of subsets was compared among groups and relationships between subset percentage and cytogenetic/biochemical findings were analyzed. RESULTS: A lower number of total CD19(+) cells was found in MM, particularly in advanced stages of disease. Reduction of naive (P < .01) and transitional B-cells (P < .05) and increase of switched memory and switched CD27(-) B-cells and germinal center founder cells were detected in PB of MM compared with controls (P < .01). Similar results were found in BM. β2 microglobulin level in MM positively correlated with the number of PCs and negatively with percentage of naive B-cells (P < .05). CONCLUSION: Our results provided a detailed phenotypic profile and enumeration of B and PC subpopulations in monoclonal gammopathy patients. A reduced number of B-cells and particularly a differentiation shift to more numerous antigen-stimulated forms was observed in MM. This might indicate a potential source of myeloma-initiating cells in one of these subpopulations.

• Klíčová slova
• B-cell, Flow Cytometry, Monoclonal Gammopathy, Multiple Myeloma, Plasma Cell,
• MeSH
• antigeny povrchové metabolismus   MeSH
• chromozomální aberace MeSH
• diferenciální diagnóza MeSH
• dospělí MeSH
• fenotyp MeSH
• imunofenotypizace MeSH
• lidé středního věku MeSH
• lidé MeSH
• mnohočetný myelom diagnóza   genetika   metabolismus   MeSH
• monoklonální gamapatie nejasného významu diagnóza   genetika   metabolismus   MeSH
• paraproteinemie diagnóza   genetika   metabolismus   MeSH
• podskupiny B-lymfocytů metabolismus   patologie   MeSH
• průtoková cytometrie MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny povrchové MeSH