Separation Methods Affect Glycan Patterns in Extracellular Vesicle Preparations From Canine Osteoblasts and Osteosarcoma Cells

Extracellular vesicles (EVs) are nanosized, membrane-enclosed particles released by cells, facilitating intercellular communication via the transfer of bioactive molecules from producing to recipient cells. EVs are involved in several physiological and pathological processes, including cancer progression. The glycosylation of EVs influences the attachment and uptake by recipient cells, among other processes. In this study, we investigated the glycan profile of EVs from canine osteosarcoma (OS) cells and non-cancerous canine osteoblasts in vitro, using lectin blots, following EV separation with ultracentrifugation (UC), size exclusion chromatography (SEC), and a commercial precipitation kit (TEI). Beyond phenotypic differences, purity and yield, separation methods led to heterogeneous protein glycosylation patterns of EV preparations, with the least reproducibility in TEI preparations. In cellular comparison, SEC revealed contrasting results, most consistently showing higher levels of specific sugars in EVs from canine osteoblasts, including the detection of a unique glycoprotein absent in UC and TEI preparations. Osteosarcoma cells and their EVs exhibited a relative reduction of glycoproteins compared to osteoblasts and their EVs. Additionally, specific sugars were more abundant in EV preparations relative to their corresponding cell lysates. In conclusion, this study highlights the critical influence of separation methods on EV characteristics, leading to differing glycan patterns.