Extracellular vesicles from 2D and 3D culture models exhibit distinct glycan profiles

Introduction: Glycosylation, a crucial post-translational protein modification, plays a key role in modulation of protein function, contributing to various stages of tumourigenesis. Studies have shown that tumour-derived extracellular vesicles (tEVs) have an altered glycoprofile, including distinct glycosylation patterns which may have utility as tumour biomarkers. Current research predominantly relies on monolayer cultures (2D). This study investigates whether tEVs isolated from 3D culture models, which should be more like in vivo conditions, display distinct glycan profiles compared to those isolated from 2D ones. Methods: The human ovarian (ES-2) and breast (MDA-MB-231, MCF-7) cancer cell lines were cultured using 2D and 3D culture models. For the 3D cultures, spheroids were formed by the hanging drop method(MDA-MB-231) or ultra-low attachment plates (ES-2, MCF-7) and then moved to Petri dishes coated with an anti-adhesion solution. EVs were isolated from both culture models’ supernatants by differential centrifugation. Their characterization involved TEM, NTA, andWestern Blot as per MISEV 2023 guidelines. The EVs glycosylation profile was assessed by a lectin-based affinity approach using an adapted ELISA assay (lectins analysed: PNA, SBA, UEA I, GSL I, GSL II, PSA, WGA, WGA succynilated, DBA-LCA, PHA-L). Results: The presence of EVs in the supernatant from both cell culture models was verified for all cell lines, and the EV identity was confirmed by characterization. The ELISA assay showed significant differences in glycan lectin-soundedness, indicating distinct enrichment profiles in EVs derived from the different culturemodels: in 3D-EVs, compared to 2D-EVs, glycans recognized by PNA, UEA I, and succinylated WGA in MDA and UEA I, DBA, and GSL II in MCF-7 decreased by approximately 50%; conversely, an increase of roughly 30% was observed for succinylated WGA, WGA, and PHA-L in ES-2. Differences for PNA, UEA I, WGA succinylated, and LCA were also evident when comparing MDA (higher aggressiveness) to MCF-7 (lower aggressiveness) 3D- EVs. Summary/Conclusion: The data suggests that the different cellular organizations lead to different glycan profiles of isolated EVs, which may exert a variable impact on their evaluation as potential cancer biomarkers and, hypothetically, affect their biological functions. Funding: This study was supported by the Funded by FFORIC24.23 to IG, Department of Life, Health and Environmental Sciences