Glycosylation is a prevalent PTMs involving the enzymatic addition of glycan moieties to proteins, profoundly influencing their structure and function [11]. This complex process primarily occurs in the Golgi apparatus and endoplasmic reticulum, where glycosylation takes place using nucleotide-sugar donors that are synthesized in cytosol from monosaccharides derived through dietary intake or intracellular metabolic pathways [12]. Glycans are categorized into several major types. N-glycans are attached to asparagine (Asn) residues within a consensus sequence of Asn-X-Ser/Thr, where X represents any amino acid except proline [13, 14]. In contrast, O-glycans are linked to the hydroxyl groups of serine or threonine residues 15]. Another important class, glycosaminoglycans (GAGs), consists of linear polysaccharides formed by repeating disaccharide units and is commonly associated with proteoglycans [15]. Specific modifications within glycosylation, such as sialylation and fucosylation, further diversify glycoprotein function. Sialylation involves the addition of sialic acid residues to the terminal positions of glycan chains, playing a crucial role in immune modulation and cellular recognition. Fucosylation, mediated by fucosyltransferases (FUTs), introduces fucose residues and is implicated in processes such as cell signaling and adhesion [16, 17]. Given the extensive distribution of the glycosylation machinery, the process is tightly regulated at multiple levels and can be influenced by both intracellular and extracellular signals to maintain homeostasis [18]. Key factors governing glycosylation include the availability of nucleotide-sugar donors, the spatial localization of glycosyltransferases and glycosidases within cellular compartments, and the expression levels and enzymatic activity of these proteins [19, 20].

2.1 Glycosylation of EV proteins

EVs are membrane-bound vesicles released by cells that play a critical role in intercellular communication. Their biogenesis involves the selective sorting glycosylated molecules, such as glycoproteins and glycolipids, into intracellular compartments like endosomes or the Golgi apparatus (GA) before release [18, 21]. These organelles are key to the processing and trafficking of proteins destined for secretion or membrane localization. In particular, the GA is the central site for protein glycosylation, where N- and O-linked glycans are added and modified, shaping the final glycan structures presented on EV cargo. Meanwhile, the endosomal system, especially multivesicular bodies (MVBs), plays a pivotal role in sorting cargo into intraluminal vesicles (ILVs), which are later released as exosomes upon MVB fusion with the plasma membrane [18, 21, 22]. A subtype of EVs, exosomes, is formed through the inward budding of endosomal membranes, with cargo—including proteins, lipids, and nucleic acids being sorted via the ESCRT complex, lipid rafts, or other cellular mechanisms [1]. During this process, the glycosylation patterns from the parent cells are transferred to the vesicles, shaping the functional characteristics of EVs [18, 23]. Aberrant glycosylation, frequently observed in cancer cells, alters the glycan profiles of the EVs they release, resulting in cancer-derived EVs. These modifications affect the composition and surface properties of EVs, impacting intercellular communication, immune evasion, and tumor progression [9,10,11]. Altered glycan structures on EV surfaces are associated with changes in receptor tyrosine kinase (RTK) activation, immune response modulation, and adhesion-related protein regulation [24]. Understanding these glycosylation patterns offers insights into cancer biology, but the mechanisms underlying glycosylated protein packaging into EVs remain incompletely understood.

In this review, we suggest that cellular glycosylation may influence both the total and surface glycosylation of EVs during their biogenesis and release into biofluids, shaping interactions that impact cancer development. Supporting this idea, recent studies have revealed that EVs acquire a “protein corona” upon release into the extracellular environment, comprising proteins, protein complexes, nucleic acids, and lipoproteins from biofluids [25, 26] This corona, formed through electrostatic and hydrophobic interactions, varies according to the cellular origin of EVs and the biofluid composition [25, 27]. In cancer, it may reflect tumor-associated alterations that enable EVs to mediate cancer specific communication and promote metastasis. For instance, Tóth’s group found that certain corona proteins are distributed in a “patchy” manner along the EV surface, influencing how EVs interact with surrounding cells and molecules [26]. Their study showed that EVs exposed to EV depleted blood plasma became denser and enriched with newly associated proteins, suggesting that large protein aggregates in biofluids can significantly modify the EV corona [26]. Notably, this suggests that the corona of EVs from cancer patients may contain unique proteins not present in EVs from healthy individuals, potentially reflecting altered cellular environments that support cancer progression. The EV corona also incorporates biomolecules like lipids and glycans, forming a broader “EV surface interactome” that could facilitate adaptive responses of cancer cells to external stresses. These unique proteins and associated biomolecules, such as specific glycans and lipids, may influence tumor-related processes by modifying how EVs interact with cells and the extracellular matrix [25, 28, 29]. Furthermore, the presence of various enzymes within the EV corona enables cleavage of protein or glycan substrates, potentially aiding in matrix degradation and recipient cell modification, processes central to cancer invasion and metastasis [25]. Elevated levels of DNA within the corona, particularly observed after treatments such as antibiotics, suggest that the EV corona might reflect adaptive changes in the tumor microenvironment, further supporting cancer cell survival and dissemination [30]. By highlighting these aspects, this review underscores the role of EVs and their corona as active players in the tumor microenvironment, influencing cellular processes key to cancer development and progression.

2.2 Altered glycosylation in cancer-derived EV proteins

Although the mechanisms underlying the packaging of glycosylated proteins into EVs and their subsequent surface presentation remain incompletely understood, increasing evidence indicates that glycosylation occurs intracellularly during the biogenesis of EVs, particularly within the endosomal and Golgi compartments [8]. In addition, EVs can acquire cancer-associated glycan features through interactions with tumor-derived molecules in the extracellular environment. These interactions may occur at the cell surface or within the tumor microenvironment, where enzymes or glycan-binding proteins can modify EV surface glycans post-release. Such extracellular remodeling complements the intracellular glycosylation that occurs during EV biogenesis. Together, these complementary mechanisms contribute to the unique glycan profiles of cancer-derived EVs, which mirror the altered glycosylation landscape of their disease-specific origins [31]. In this section, we focus on the distinct glycosylation patterns in cancer-derived EVs by analyzing findings from studies using in vitro cell lines and biofluids, such as blood (plasma and serum) and urine, as summarised in Table 1. These models provide insights into cancer-specific alterations in EV glycosylation, offering a non-invasive means to detect disease-related changes [5, 7].

2.2.1 Glycosylation of EVs protein in vitro cell lines studies

While comprehensive studies on the full spectrum of glycosylation types in EVs remain limited, available in vitro cell line models provide valuable insights into specific glycan alterations associated with cancer. In this section, we highlight two representative examples observed in cancer cell-derived EVs: (i) intracellular O-linked-N-acetylglucosaminylation (O-GlcNAcylation), and (ii) N-glycosylation patterns inferred from studies of LGALS3BP-enriched EVs. These examples illustrate the diversity of glycosylation changes in EV proteins and their potential implications in cancer progression.

O-GlcNAcylation is the attachment of a single sugar either serine and/or threonine residues on intracellular proteins. It was investigated by Chaiyawat’s team that O-GlcNAc modification on EVs proteins showed an increase in metastatic cells using colorectal cancer (CRC) cell lines (HT29, SW480 and SW620) [32]. Particularly, O-GlcNAc alterations were confirmed to occur on transitional endoplasmic reticulum ATPase (TER ATPase) and RuVB-like 1 proteins at higher levels in EVs of metastatic CRC cell lines [32]. These results are consistent with a recent study by Netsirisawan’s team that studied extracellular O-GlcNAcylation in released substances from breast cancer (BrCa) [32, 33]. Appealingly, increased levels of O-GlcNAc were discovered in TER ATPase and heat-shock protein 70 (HSP70) of BrCa EVs [33]. High levels of HSP70 modified by O-GlcNAc detected in cancer cells serve to protect cells from cells and confer resistance to apoptosis [34]. The elevated presence of this type of glycosylation pattern of O-GlcNAc in tumoral EVs compared to normal control (NC) reveals the potential use as a for metastatic CRC and BrCa [32, 33].

Previously, one key sialoglycoprotein that was found in cancer exomere particles was the LGALS3BP, an immune response and cell communication regulator [35]. The study by Zhang et al. (2018) presented distinct glycosylation profiles observed for exomeres and exosome subpopulations from various cancers including melanoma, breast, and pancreatic [35]. Intriguingly, LGALS3BP strongly enriched in EVs from ovarian carcinoma [36, 37] and uveal melanoma [38] demonstrated similar glycosylation profiles. EVs from all these samples displayed complex-type N-glycans, fucose, mannose, O-glycan, bisecting and branched N-glycans with α(2,6)- or α(2,3)-linked sialic acid [35,36,37,38]. Further supporting this, immunoblotting has shown increased expression of LGALS3BP in small EVs isolated from colon cancer cell lines, with Western blotting revealing distinct differences in LGALS3BP levels between small EVs and the originating cells [39].

2.2.2 Glycosylation of EVs protein in blood samples

Blood is an abundant source of disease biomarkers and is the primary biological fluid where circulating EVs have been identified in plasma and serum. Studies on the glycosylation patterns of circulating EVs in blood are uncommon, despite the tremendous potential of EVs glycosylation in disease. Exosomes from human serum were isolated using a reverse capture method, and their N-glycome was analyzed [40]. When compared to N-glycans of exosomes from healthy samples, N-glycan analysis of hepatocellular carcinoma (HCC) patient samples showed that most of these individuals had complex-type N-glycans modified with fucose, mannose, and sialic acid. N-glycome of exosomes was found to have substantial abundance variation between HCC patients and healthy controls was found in which 13 out of 24 of these glycans are specifically altered in exosomes [40]. Sun’s group developed an innovative HCC-EV-based surface protein assay aimed at the early detection of HCC [41]. This assay targets several key proteins: epithelial cell adhesion molecule (EpCAM), CD147, glypican 3 protein (GPC3), and asialoglycoprotein receptor 1 (ASGPR1). The assay demonstrated significantly higher sensitivity and specificity compared to serum alpha-fetoprotein (AFP), maintaining exceptional performance across various subgroup analyses [41]. AFP-L3, a core-fucosylated variant of AFP, is the first recognized oncofetal biomarker in HCC and continues to be a valuable tool for early cancer detection. However, the findings from Sun’s group suggest that EV surface protein markers are highly promising for the early identification of HCC, outperforming AFP-L3 in clinical settings [41,42,43]. Similarly, employing a lectin microarray, differential glycomic profiling of EVs produced from serum in 117 pancreatic cancer patients (PC) and 98 NC revealed an elevation of O-glycosylated EVs [44]. Specifically, PC EVs had considerably higher signal intensities for the lectins Amaranthus caudatus agglutinin (ACA) and Agaricus bisporus agglutinin (ABA) than NC. This substantial increase in EVs with ACA and ABA positive PC sera provides a quantification method and differential glycomic profiling report that can be used in the development of the PC diagnostic test [44]. Following the discovery of LGALS3BP in cancer exomere particles as mentioned in the in vitro studies above [35], it was discovered that plasma-derived EVs from ovarian cancer accumulate LGALS3BP [37, 45]. LGALS3BP accumulated in EVs derived from cancer cells displays an enhanced level of sialylated α(2,3)-linked N-glycans, along with mannose and bisecting N-acetylglucosamine (GlcNAc) structures. This altered glycosylation profile suggests that LGALS3BP in cancer-derived EVs undergoes tumor-specific glycan processing, potentially reflecting underlying changes in the glycosylation machinery of malignant cells [35, 37, 45].

In contrast, the ability to discriminate between benign and malignant EVs is held by the differences in overexpression of specific glycans or glycoconjugates. In fact, tumor-derived exosomes (TEX) from heterogeneous plasma samples of PC [46] or melanoma [47] were found to contain the tumor antigens chondroitin sulphate proteoglycan 4 (CSPG4) and glypican-1 (GPC1). In both studies, these glycoproteins were used to distinguish TEX from benign particles [46, 47]. Interestingly, a previous study showed exosomes from whole blood of PC patients exhibit elevated levels of free sialyl Lewis A antigen (CA19-9) than those from NC patients [48]. It was demonstrated that the exosome-based CA19-9 analysis from whole blood has higher sensitivity than that of its direct measurement from serum, since free CA19-9 in serum was measured and found to be positive in exosomes but to be falsely negative in serum [48]. It was known that EVs generated by nearby tumor cells as well as a range of other soluble chemicals may have an impact on stromal cells, contributing to a favourable tumor microenvironment [49, 50]. Highly glycosylated extracellular matrix metalloproteinase inducer (EMMPRIN) has been discovered as a determinant of pro-invasive MVs isolated from patients with metastatic breast cancer’s peripheral blood [51]. Results showed that EMMPRIN is highly glycosylated at N160 and N268 for tumor-promoting effects. The mechanism proposed was MV-bound EMMPRIN activates pro-invasive factors on the tumor surface and delivers tumor-promoting proteins [51].

2.2.3 Glycosylation of EVs protein in urine samples

Urine exosomes from expressed prostatic secretions (EPS) were used to create glycan profiles conducted by Nyalwidhe’s team in 2013 [52]. Three complementary methods, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) profiling, MS, and normal-phase HPLC separation were performed in this study. Profiling results of EPS urine exosomes showed increased N-linked glycans with an apparent increase in larger tetra-antennary glycans amount [52]. Apart from that, prostate cancer (PCa) patients were shown to demonstrate higher levels of urinary EVs expressing prostate-specific antigen (PSA) with increased N-glycosylation [53]. To assess the correlation with the urinary N-glycosylation profile, this study established a novel metric, the urinary EVs associated PSA extraction ratio. Results reveal that patients with PCa had a higher EVs association PSA extraction ratio than patients with benign prostatic hyperplasia (BPH) due to the alterations in N-glycome of PCa patients [53].

Additionally, extensive studies have been done on urinary EVs enriched with glycosylated proteins as potential indicators of urological malignancies [54]. In a recent study, integrin subunit alpha 3 (ITGA3) was identified as a key marker in urinary EVs and was detected using a fucose-specific lectin nanoparticle assay (ITGA3-UEA test) [55]. This assay combines ITGA3 detection with Ulex europaeus agglutinin (UEA), a lectin that selectively binds to fucosylated glycans, allowing for the simultaneous recognition of both protein and glycan signatures on EV surfaces [55]. The results demonstrated that aberrantly fucosylated urinary EVs and ITGA3 could effectively distinguish bladder cancer (BlCa) patients from those with benign prostatic hyperplasia (BPH) or PCa, using a simple, non-invasive bioaffinity assay applied directly to unprocessed urine [55]. HCC has been shown to exhibit distinct glycosylation patterns in urinary EVs, as revealed by comprehensive glycoproteomic profiling. In particular, HCC-derived EVs displayed higher levels of mannose-type N-glycosylation and alterations in the monosaccharide composition of intact N-glycopeptides compared to EVs from healthy controls [43]. A study by Li et al. (2023) further identified site-specific glycoforms in urinary EV glycoproteins that may serve as promising non-invasive biomarkers for HCC. These findings suggest that specific glycosylation changes at defined sites in EV-associated glycoproteins could enable the differentiation of HCC from non-malignant conditions, offering a valuable diagnostic tool [43].

2.2.4 Common patterns of altered glycosylation in cancer-derived EV proteins

Evidence from studies on in vitro cell lines and biofluids, including blood and urine, reveals a recurring pattern of aberrant glycosylation in cancer-derived EVs. For example, the sialoglycoprotein LGALS3BP has been consistently identified in EVs across various cancers, including melanoma, breast, prostate, uveal melanoma, and ovarian cancers [35,36,37,38]. This protein, widely expressed as a hyperglycosylated form in human cells, was initially recognized for its association with cancer metastasis [56]. Additionally, common glycan alterations in cancers such as melanoma, breast, prostate, and ovarian cancers exhibit α(2,3)- or α(2,6)-linked sialic acids, branched N-glycan, and bisected N-glycans [36, 37]. Altered N-glycan structures, including fucosylation, branched, bisected, complex-type, and high-mannose N-glycans, have also been observed in cancer such as melanoma, breast, prostate, ovarian, uveal melanoma, and hepatocellular cancers [35,36,37,38, 40, 45].

Table 1 Summary of glycosylation pattern of cancer-derived EVs found in vitro studies using cell lines and various biofluids (blood and urine)2.3 LGALS3BP in cancer-derived EVs

LGALS3BP has gained attention for its significant role in advancing tumor growth and metastasis. Increasing evidence points to this highly glycosylated protein as a crucial factor in the cellular mechanisms that contribute to malignant transformation [59,60,61]. In reviewing the literature, our findings document that LGALS3BP consistently appears as a prominent glycosylation protein pattern in EVs across various malignancies, including neuroblastoma [62], glioblastoma [63], colorectal [39], prostate, breast, ovarian cancers, and melanoma [35,36,