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很少见。在乳腺癌 (BCa) 中，GSL 已成为与乳腺癌干细胞相关的值得注意的标记物、表型可塑性的介质以及癌细胞化疗耐药性的贡献者。 GSL 是潜在的表面标志物，可以独特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，对乳腺上皮细胞及其间充质细胞中总鞘脂组的质谱分析显示，上皮细胞中的 Gb3 水平增加，而间充质表型中的 GD2 水平显着升高。为了阐明 BCa 细胞表面上的 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 色光谱流式细胞术面板。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) 标记物（EpCAM、TROP2、CD9）和谱系标记物（CD45、CD31、CD90）单细胞水平。下一步，建立的面板用于分析 BCa 原发性肿瘤，并揭示了 SSEA1、SSEA3、SSEA4、GD2 和 Gb3 的表面异质性，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 表面轮廓的表型依赖性改变，以及肿瘤中上皮细胞和基质细胞之间的差异。这项研究提供了对 BCa 异质性的新见解，揭示了天然 GSL 相关表位作为新鲜临床样本中 EMP 和癌症状态标记的潜力。开发的单细胞方法为进一步探索提供了有希望的途径。版权所有 © 2024 作者。由爱思唯尔公司出版。保留所有权利。

Glycosylated sphingolipids (GSLs) are a diverse group of cellular lipids, typically reported as 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 whether 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, GD2), epithelial-mesenchymal transition (EMT) markers (EpCAM, TROP2, CD9), and lineage markers (CD45, CD31, CD90) at the single-cell level. As a next step, 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.Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.