All authors have agreed and read towards the posted version from the manuscript. Funding This work was funded by FEDER funds through the Operational Programme for Competitiveness Factors COMPETE 2020 (POCI-01-0145-FEDER-016585; POCI-01-0145-FEDER-007274) and nationwide funds through the building blocks for Research and Technology (FCT), beneath the tasks: PTDC/BBB-EBI/0567/2014 to C.A.UID/BIM/04293/2013 and R; and the task NORTE-01-0145-FEDER-000029, backed by Norte Portugal Regional Program (NORTE 2020), beneath the PORTUGAL 2020 Relationship Contract, through the Western european Regional Development Finance (ERDF). Institutional Review Panel Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement Zero brand-new data had been created or analyzed within this scholarly research. able to differentiate tumor EVs from harmless EVs. Within this review, the use of EV glycosylation in the introduction of novel EV capture and detection methodologies is talked about. Furthermore, we high light the potential of EV glycosylation in the scientific placing for both tumor biomarker breakthrough and EV healing delivery strategies. gene, which codifies an integral molecular chaperone that alongside the C1GalT1 glycosyltransferase is in charge of the gene knockout will result in the shift of the (AIA or Jacalin), that specifically bind to T-antigen, and the lectin I Nomegestrol acetate (MAL-I), that has a high affinity for Gal or GalNAc residues with an (2,3)-linked sialic acid, allowed to isolate different sized urinary EVs from healthy samples based on their surface glycosylation profile, with increased yields and higher purity when compared to CD9/CD81/CD63 antibody-based isolation [144]. In addition, the STL lectin, recognizing the N-acetylglucosamine and lactosamine residues, also showed high affinity and specificity when Nomegestrol acetate isolating EVs from healthy urine samples [145]. One of the main challenges when using urine samples is the co-purification of the highly glycosylated TammCHorsfall protein (THP). This glycoprotein is able to form aggregates and capture EVs, hampering further EV biomarker downstream analysis [146]. However, the use of lectin microarrays allowed the distinction between EV glycosylation and THP glycosylation, which potentiated the isolation of urine EVs with minimal interference of this glycoprotein [70]. A similar approach was also applied to tumor EVs. By coupling a high mannose-type glycan-specific lectin to beads it was possible to capture small EVs from melanoma, glioblastoma, lung and colon cancer cells [147]. Interestingly, CD109, integrin 6 and ADAM10 present on melanoma small EVs were apparently responsible for the identified EV-lectin interaction, as they carry high mannose glycans [147]. These results demonstrated the potential of lectin-conjugated beads to detect and isolate different sub-populations Nomegestrol acetate of EVs within a sample, based on its glycosylation profile. In addition, a nanoparticle-based time-resolved fluorescence immunoassay (NP-TRFIA) showed to be able to capture EVs from urine samples and cell supernatants based on Rabbit polyclonal to HMGN3 the interaction with the tetraspanins and glycan antigens present at the EV surface [148]. This approach also provided a general EV surface glycan profiling, which revealed a differential expression pattern of tumor-associated proteins on more aggressive versus less aggressive prostate cancer cell line-derived EVs [148]. Apart from the affinity-based isolation methods, a commercially available precipitation kit, the ExoGAG, was also developed taking into consideration the glycosylation profile of EVs. This method precipitates EVs out of the solution due to the presence of negatively charged Nomegestrol acetate GAG at the EV surface [149]. Interestingly, ExoGAG has proved to effectively isolate EVs from liquid biopsy patient samples with higher yields and purity when compared with UC and allowed the identification of Annexin A2 as an EV marker associated with endometrial cancer staging and recurrence [149]. Despite the technological advances in the field, the study of EVs is still technically challenging. Particularly, innovative and less laborious detection and isolation methods are an urgent need to facilitate an in-depth study of the different EV cargos as a reliable source of biomarkers. The presence of specific patterns of glycans at the EV surface constitute valuable sources to potentiate the development of more sensitive and specific EV detection and isolation methodologies, with the potential to be translated into the clinical setting. 4.2. The Functional Roles of Extracellular Vesicle Glycosylation in Cancer Up to date, little is still.