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.

Breast Cancer Centre Masaryk Memorial Cancer Institute Brno Czech Republic; Prostate Cancer Centre Masaryk Memorial Cancer Institute Brno Czech Republic

Department of Clinical and Molecular Pathology Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacký University Olomouc Czech Republic

Department of Clinical and Molecular Pathology Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacký University Olomouc Czech Republic; Department of Clinical and Molecular Pathology University Hospital Olomouc Czech Republic

Department of Cytokinetics Institute of Biophysics of the Czech Academy of Sciences Brno Czech Republic

Department of Cytokinetics Institute of Biophysics of the Czech Academy of Sciences Brno Czech Republic; Department of Experimental Biology Faculty of Science Masaryk University Brno Czech Republic

Department of Cytokinetics Institute of Biophysics of the Czech Academy of Sciences Brno Czech Republic; International Clinical Research Center St Anne's University Hospital Brno Brno Czech Republic

Department of Cytokinetics Institute of Biophysics of the Czech Academy of Sciences Brno Czech Republic; International Clinical Research Center St Anne's University Hospital Brno Brno Czech Republic; Department of Experimental Biology Faculty of Science Masaryk University Brno Czech Republic

Department of Pharmacology and Toxicology Veterinary Research Institute Brno Czech Republic

Institute of Biostatistics and Analyses Faculty of Medicine Masaryk University Brno Czech Republic

Prostate Cancer Centre Masaryk Memorial Cancer Institute Brno Czech Republic

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