Helix pomatia agglutinin bound to surface glycans of small extracellular vesicles in-vitro and in-vivo increases in early and late stage breast cancer.

BACKGROUND: Breast cancer is the most frequently diagnosed cancer and a leading cause of cancer-related mortality in women globally. Small extracellular vesicles (sEVs) play a crucial role in cell communication and cancer progression. This study aimed to investigate the glycosylation patterns of sEVs derived from breast epithelial cells and plasma samples from breast cancer patients, focusing on the presence of truncated O-linked glycans, such as the Tn antigen, using Helix pomatia agglutinin (HPA). METHODS: Breast cancer cell lines were investigated for HPA lectin surface binding by confocal microscopy and flow cytometry. The sEVs of these were tested for surface HPA and tetraspanin binding using imaging-flow cytometry, single particle interferometry, and direct stochastical optical reconstruction microscopy. Plasma from healthy and stage II-IV breast cancer patients were tested by imaging-flow cytometry for HPA binding and analyzed for the source of HPA + EVs using 37 colocalised markers by multiplex flow cytometry . RESULTS: Quantitative analysis revealed elevated HPA binding in sEVs from metastatic MCF-7 cells compared to that in non-metastatic BT-474 and immortalized healthy normal hTERT-HME1 cells, suggesting a correlation between HPA binding and metastatic potential. Analysis of sEVs revealed differential glycan presentation with CD81-positive sEVs from MCF-7 cells compared to CD63. In patient-derived plasma sEVs, HPA binding was significantly higher in patients with breast cancer than in healthy individuals, highlighting its potential as a biomarker for cancer detection. CONCLUSIONS: These findings highlight the complex glycosylation of sEVs and their potential early diagnostic utility in breast cancer for HPA positive sEVs.