糖基化鞘脂(GSL)是一组不同的细胞脂质,通常报道在正常乳腺组织中罕见。在乳腺癌(BCa)中,GSL已经成为与乳腺癌干细胞相关的值得注意的标志物,表型可塑性的介体,以及癌细胞化学抗性的贡献者。GSL是潜在的表面标记,可以唯一地表征肿瘤微环境的异质性,包括癌细胞亚群和上皮间质可塑性(EMP)。在这项研究中,对乳腺上皮细胞和它们的间充质对应物中的总鞘凝集层的质谱分析显示,上皮细胞中Gb3水平升高,间充质表型中GD2水平显著升高.为了阐明BCa细胞表面的GSL相关表位是否反映EMP和癌症状态,我们开发并严格验证了12色光谱流式细胞仪面板.该面板能够同时检测天然GSL表位(Gb3,SSEA1,SSEA3,SSEA4,GD2),上皮间质转化(EMT)标志物(EpCAM,TROP2,CD9),和谱系标记(CD45,CD31,CD90)在单细胞水平。作为下一步,已建立的小组用于BCa原发性肿瘤的分析,并揭示了SSEA1,SSEA3,SSEA4,GD2和Gb3的表面异质性,这表明非肿瘤细胞上也存在天然表位.这些发现进一步强调了GSL表面特征的表型依赖性改变,肿瘤中上皮细胞和基质细胞之间的差异。这项研究为BCa异质性提供了新的见解,揭示了天然GSL相关表位作为新鲜临床样品中EMP和癌症状态标志物的潜力。开发的单细胞方法为进一步探索提供了有希望的途径。
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.