Extracellular Vesicles from Oral Squamous Cell Carcinoma Carry OGT/OGA with Possible Implications in Tumor O-GlcNAcylation

ABSTRACT: Oral squamous cell carcinoma (OSCC) is a malignant epithelial neoplasm characterized by high aggressiveness and limited options for early diagnosis. In recent years, extracellular vesicles (EVs) have gained attention as mediators of intercellular communication in cancer, contributing to tumor progression and remodeling of the microenvironment. O-GlcNAcylation, regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), participates in multiple tumor processes; however, its association with EVs in OSCC has not yet been explored. In this study, EVs were isolated from SCC-152, SCC-25, and HaCaT cell lines using differential centrifugation, and their identity was confirmed by detection of CD63 and TSG101 markers and by transmission electron microscopy (TEM). Immunocytochemistry revealed the nuclear and cytoplasmic localization of OGT and OGA in all analyzed cell lines. Importantly, both enzymes were detected in EVs cargo by Western blot analysis, with significant differences between tumor and non-tumor lines as determined by densitometric and fluorescence intensity analyses. Quantitative analysis indicated a higher relative signal for OGA compared with OGT across all cell lines (with an approximate ~1.5–2.2-fold difference depending on the cell line, p < 0.05), and cell line-derived samples showed a higher relative signal than non-tumoral HaCaT (corresponding to an approximate ~1.2–1.3-fold increase under the experimental conditions evaluated). All experiments were performed using three independent biological replicates (n = 3), and statistical significance was assessed using one-way or two-way ANOVA followed by Tukey’s post hoc test. These findings suggest that OSCC-derived EVs carry enzymatic components of the O-GlcNAcylation machinery as vesicular protein cargo, potentially influencing tumor microenvironment regulation and cancer progression. Overall, these results should be considered hypothesis-generating, opening new perspectives for their use as vesicular biomarkers.