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

The distribution of fluorescence signals measured with flow cytometry can be influenced by several factors, including qualitative and quantitative properties of the used fluorochromes, optical properties of the detection system, as well as the variability within the analyzed cell population itself. Most of the single cell samples prepared from in vitrocultures or clinical specimens contain a variable cell cycle component. Cell cycle, together with changes in the cell size, are two of the factors that alter the functional properties of analyzed cells and thus affect the interpretation of obtained results. Here, we describe the association between cell cycle status and cell size, and the variability in the distribution of fluorescence intensity as determined with flow cytometry, at population scale. We show that variability in the distribution of background and specific fluorescence signals is related to the cell cycle state of the selected population, with the 10% low fluorescence signal fraction enriched mainly in cells in their G0/G1 cell cycle phase, and the 10% high fraction containing cells mostly in the G2/M phase. Therefore we advise using caution and additional experimental validation when comparing populations defined by fractions at both ends of fluorescence signal distribution to avoid biases caused by the effect of cell cycle and cell size.

• MeSH
• buněčné dělení MeSH
• buněčný cyklus fyziologie   MeSH
• G2 fáze * MeSH
• průtoková cytometrie metody   MeSH
• velikost buňky MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

The epithelial-mesenchymal plasticity, in tight association with stemness, contributes to the mammary gland homeostasis, evolution of early neoplastic lesions and cancer dissemination. Focused on cell surfaceome, we used mouse models of pre-neoplastic mammary epithelial and cancer stem cells to reveal the connection between cell surface markers and distinct cell phenotypes. We mechanistically dissected the TGF-β family-driven regulation of Sca-1, one of the most commonly used adult stem cell markers. We further provided evidence that TGF-β disrupts the lineage commitment and promotes the accumulation of tumor-initiating cells in pre-neoplastic cells.

• MeSH
• ataxin-1 metabolismus   MeSH
• epitelo-mezenchymální tranzice genetika   MeSH
• epitelové buňky patologie   MeSH
• experimentální nádory mléčných žláz genetika   patologie   MeSH
• lidé MeSH
• mléčné žlázy zvířat patologie   MeSH
• myši MeSH
• nádorové buněčné linie transplantace   MeSH
• nádorové kmenové buňky patologie   MeSH
• nádory prsu genetika   patologie   MeSH
• plasticita buňky genetika   MeSH
• receptor erbB-2 genetika   MeSH
• regulace genové exprese u nádorů MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• signální transdukce genetika   MeSH
• transformující růstový faktor beta genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ataxin-1 MeSH
• Atxn1 protein, mouse MeSH Prohlížeč
• Erbb2 protein, mouse MeSH Prohlížeč
• receptor erbB-2 MeSH
• rekombinantní proteiny MeSH
• transformující růstový faktor beta MeSH

Skp2 is a crucial component of SCFSkp2 E3 ubiquitin ligase and is often overexpressed in various types of cancer, including prostate cancer (PCa). The epithelial-to-mesenchymal transition (EMT) is involved in PCa progression. The acquisition of a mesenchymal phenotype that results in a cancer stem cell (CSC) phenotype in PCa was described. Therefore, we aimed to investigate the expression and localization of Skp2 in clinical samples from patients with PCa, the association of Skp2 with EMT status, and the role of Skp2 in prostate CSC. We found that nuclear expression of Skp2 was increased in patients with PCa compared to those with benign hyperplasia, and correlated with high Gleason score in PCa patients. Increased Skp2 expression was observed in PCa cell lines with mesenchymal and CSC-like phenotype compared to their epithelial counterparts. Conversely, the CSC-like phenotype was diminished in cells in which SKP2 expression was silenced. Furthermore, we observed that Skp2 downregulation led to the decrease in subpopulation of CD44+CD24- cancer stem-like cells. Finally, we showed that high expression levels of both CD24 and CD44 were associated with favorable recurrence-free survival for PCa patients. This study uncovered the Skp2-mediated CSC-like phenotype with oncogenic functions in PCa.

• MeSH
• antigen CD24 genetika   MeSH
• antigeny CD44 genetika   MeSH
• buňky PC-3 MeSH
• epitelo-mezenchymální tranzice * MeSH
• lidé MeSH
• myši nahé MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádorové kmenové buňky metabolismus   fyziologie   MeSH
• nádory prostaty genetika   metabolismus   patofyziologie   MeSH
• proteiny asociované s kinázou S-fáze genetika   MeSH
• regulace genové exprese u nádorů * MeSH
• stupeň nádoru MeSH
• xenogenní modely - testy protinádorové aktivity MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigen CD24 MeSH
• antigeny CD44 MeSH
• CD24 protein, human MeSH Prohlížeč
• CD44 protein, human MeSH Prohlížeč
• proteiny asociované s kinázou S-fáze MeSH
• SKP2 protein, human MeSH Prohlížeč

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