Small extracellular vesicles (sEVs) are a significant component of broader cell secretomes, emerging as carriers of crucial regulators of cell-cell communication in monitoring disease onset, progression, and treatment response through their cargo. Additionally, current sEVs biogenesis models suggest that sEVs transport cargo from the cell membrane to the target cells and determine the cell’s state; we aimed to characterize the comprehensive N-glycomic profiles of sEVs derived from two distinct breast cancer cell lines, namely, triple-negative (MDA-MB-231) and hormone-dependent (MCF-7) breast cancer, in two different cell-culture models (2D and 3D). These sEVs N-glycan profiles were then compared with their isolated cell membrane fractions, along with their sEV-depleted cell secretome.
Both cancer cell lines, in 2D and 3D “on-top Agar” models, were cultured and passaged in serum-free defined cell culture media supplemented with growth factors i.e., insulin, hydrocortisone, transferrin, b-estradiol and sodium selenite. sEVs from both cell lines were isolated from the defined media using differential ultracentrifugation and characterized for their size distribution, surface morphology, and concentration. The sEV-depleted cell culture media was defined as the secretome and the isolated cell membrane fractions were used to characterize the membrane proteins. N-glycans were released enzymatically by PNGase F and reduced from equal protein amounts of isolated sEVs, membrane fractions and secretomes. The glycans were desalted, resolved using porous graphitized carbon chromatography, and analyzed by mass spectrometry. Our results indicate that the glycans in sEVs closely resemble the glycans from the cell membrane proteins, correlating with the sEV biogenesis models. Additionally, intriguing and distinctive isomer variances were present between 2D and 3D cell culture models of the same cell lines. Fascinatingly, our study also outlines distinct glycan feature differences between sEVs and their secretome in both cell lines, despite originating from the same cellular machinery, grown in their respective culture flasks. Henceforth, we were able to unravel six unequivocal distinct N-glycome signature profiles across the intracellular domain, spanning sEVs and their corresponding secretome. This revelation transcends individual cell lines, boldly highlighting discernible disparities between the two cell lines in both 2D and 3D cell culture models.