Subtype-specific cell-surface glycocalyx–associated molecular expression programs revealed by public single-cell RNA sequencing in breast cancer.

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Background: The cell-surface glycocalyx (GCX) is a carbohydrate-rich layer covering epithelial cells and plays an important role in tumor–microenvironment interactions and immune modulation. In breast cancer, GCX remodeling has been implicated in tumor heterogeneity and disease progression. However, integrated analyses linking GCX-associated morphological features with underlying molecular expression programs at the single-cell level across breast cancer subtypes remain limited. We have previously investigated GCX alterations in breast cancer using lectin staining and scanning electron microscopy (SEM). Methods: Frozen sections of resected breast cancer tissues and adjacent normal mammary epithelium were analyzed using multiple fluorescently labeled lectins to profile cell-surface glycan patterns. GCX ultrastructure was further evaluated by SEM using lanthanum nitrate staining. To elucidate the molecular basis underlying GCX phenotypes, publicly available breast cancer single-cell RNA sequencing (scRNA-seq) datasets were analyzed. Tumor epithelial cell populations were extracted, and expression programs of GCX-associated molecules, including glycosylation-related enzymes, mucins, and proteoglycans, were evaluated in a subtype-specific manner. Transcriptomic findings were integrated with lectin-based glycan patterns observed in frozen tissue sections. Results: Lectin staining demonstrated altered cell-surface glycan distribution and marked intratumoral heterogeneity in breast cancer tissues compared with normal mammary epithelium. Reduced binding of O-glycan–associated lectins was observed in tumor regions, indicating GCX remodeling, with patterns differing among breast cancer subtypes. SEM analysis revealed thinning, discontinuity, and surface irregularities of the GCX in tumor areas, consistent with lectin-based observations. Analysis of public scRNA-seq data revealed subtype-associated differences in the expression of GCX-related genes within tumor epithelial cell populations, including genes involved in glycosylation, mucin production, and proteoglycan biology. These transcript-level differences paralleled the subtype-dependent glycan distributions identified by lectin staining. Conclusions: By integrating lectin-based GCX profiling and SEM with public single-cell transcriptomic analysis, we mapped cell-surface glycocalyx phenotypes to corresponding molecular expression programs in breast cancer. GCX remodeling identified by lectin staining was associated with subtype-specific transcriptional programs at the single-cell level. This integrative approach provides a framework for understanding GCX-related tumor heterogeneity and may facilitate future studies on subtype-specific tumor biology and tumor–immune interactions in breast cancer.