Identification of Distinct N-Glycosylation Patterns on Extracellular Vesicles from Small-Cell and Non-Small-Cell Lung Cancer Cells

Asparagine-linked glycosylation (N-glycosylation) of proteins in the cancer secretome has gained increasing attention as a potential biomarker for cancer detection and diagnosis. Small extracellular vesicles (sEVs) constitute a large part of the cancer secretome, yet little is known about whether their N-glycosylation status reflects cancer characteristics. Here we investigated the N-glycosylation of sEVs released from small-cell lung carcinoma (SCLC) and non-small-cell lung carcinoma (NSCLC) cells. The N-glycans of SCLC-sEVs were characterized by the presence of structural units found in the brain N-glycome, while NSCLC-sEVs were dominated by typical lung-type N-glycans with NSCLC-associated core fucosylation. We pulled down these glycoproteins from the detergent-solubilized sEVs with SSA-conjugated beads for H520-sEVs and WGA-conjugated beads for H446-sEVs, and the protein bands were subjected to shotgun proteomics. The analysis revealed that several integrin subunits were enriched in the sEVs: V, 6, 1, and 5 subunits in H520-sEVs and V and 1 subunits in H446-sEVs.