BBA - Reviews on Cancer 1868 (2017) 157–166

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BBA - Reviews on Cancer

journal homepage: www.elsevier.com/locate/bbacan

Glycoconjugates from extracellular vesicles: Structures, functions and MARK

emerging potential as cancer biomarkers
Julia Costa
Laboratory of Glycobiology, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal

A R T I C L E I N F O A B S T R A C T

Keywords: Extracellular vesicles (EVs) are released by virtually all cells, carry cellular molecules to the extracellular
Cancer biomarkers environment, and may interact with other cells. They are found in body fluids, therefore, constituting useful
Exosomes target sources for the identification of disease biomarkers, for example, in cancer. EVs originate from the plasma
Extracellular vesicles membrane or from multivesicular endosomes. They have the same topology as the plasma membrane and are
Glycoconjugates
rich in glycoconjugates, displaying specific glycosignatures. Surface glycoconjugates play important roles in EVs
Glycoproteins
Glycosylation
biogenesis and in their interaction with other cells. Changes in glycosylation constitute a hallmark of different
types of cancer, therefore, the study of glycoconjugates and glycosignatures of EVs appear as promising
candidates to identify novel cancer biomarkers and to increase the specificity and sensitivity of the existing
clinical biomarkers, many of which are glycosylated.

1. Introduction 2. Glycosylation

Cells release vesicles to their surroundings known as extracellular 2.1. Glycoconjugates

vesicles (EVs). EVs may originate from plasma membrane budding
(microvesicles) or from fusion of multivesicular endosomes with the
plasma membrane (exosomes) [1]. Increasing evidence has shown that
EVs have a relevant functional role in transferring biomolecules from
cell to cell, thereby constituting vehicles of communication among
cells. This is relevant in the transmission of pathogenic, signal and
regulatory molecules in disease, such as cancer [1,2]. On the other
hand, they can also transfer beneficial factors involved in regeneration
[3].
EVs are found in biological fluids and are capable of crossing
biological barriers [4]. Since they carry cellular material to the
extracellular environment, they are useful targets for the identification
of disease biomarkers [5].
There is ample evidence in the literature showing that EVs are
strongly glycosylated being rich in specific glycoconjugates [6–9]. In
this article we address the importance of glycoconjugates and their
glycosylation in EVs properties and outline their emerging potential as
cancer biomarkers.
In eukaryotic cells several classes of glycoconjugates can be found,
which include glycoproteins and glycolipids. Glycosylation is a com-
mon post-translational modification of proteins, N- and O-glycosylation
being the most widely studied. In N-glycosylation, glycans are cova-
lently bound via a nitrogen atom to asparagine side chains of proteins
(Fig. 1). In animal cells, the sugar linked to the asparagine residue is N-
acetylglucosamine (GlcNAc). N-glycosylation is initiated in the endo-
plasmic reticulum with the transfer of an oligosaccharide precursor
onto the consensus acceptor sequence NXS/T in the nascent polypeptide
chain. This precursor is further processed by the action of glycosidases
and glycosyltransferases in the endoplasmic reticulum and the Golgi
apparatus concomitant to the trafficking of proteins to the plasma
membrane, lysosome or the extracellular environment. There are
several classes of N-glycans: i) high mannose-type that only contain
mannose (Man) and GlcNAc residues; ii) complex-type that also have
other monosaccharide residues, e.g., galactose (Gal), N-acetylneurami-
nic acid (NeuAc), fucose (Fuc); iii) hybrid-type in which one arm has
only Man and the other is processed to the complex type.
In O-glycosylation, glycans are bound via an oxygen atom to side
chains of serine, threonine, hydroxylysine or hydroxyproline residues.

Abbreviations: CA19-9, carbohydrate antigen 19-9; CEA, carcinoembryonic antigen; EV, extracellular vesicle; Fuc, fucose; Gal, galactose; GlcNAc, N-acetylglucosamine; GPI,
glycosylphosphatidylinositol; HER2, human epidermal growth factor receptor 2; HSPG, heparan sulfate proteoglycan; Man, mannose; MUC1, mucin 1; MUC16, mucin 16; NeuAc, N-
acetylneuraminic acid
E-mail address: jcosta@itqb.unl.pt.

http://dx.doi.org/10.1016/j.bbcan.2017.03.007
Received 3 February 2017; Received in revised form 20 March 2017; Accepted 21 March 2017
Available online 24 March 2017
0304-419X/ © 2017 Elsevier B.V. All rights reserved.
J. Costa BBA - Reviews on Cancer 1868 (2017) 157–166

Fig. 1. Schematic representation of composition and topology of glycoconjugates in EVs.N-glycans of the complex and high mannose type are found in EVs [8]. HSPGs are present in EVs
and a schematic representation of the glycan moiety [10] is shown. Mucins are found in EVs and a representation of O-linked glycans commonly attached to mucins are presented [18]. O-
GlcNAcylated glycoconjugates are detected in EVs [90]. Glycans are represented according to the nomenclature of the Consortium for Functional Glycomics.

A major class of O-linked glycoproteins consists of mucins, where the golipids or glycophospholipids, respectively. Glycosphingolipids con-
sugar linked to serine or threonine residues is N-acetylgalactosamine tain a high diversity of structures including the most common ganglio-
(Fig. 1). In mucin O-glycosylation sequential addition of monosacchar- sides (e.g., sialylated GM3, GM2, GM1, GD1a, GD2) (Fig. 1). Glyco-
ide residues by glycosyltransferases occurs in the Golgi apparatus. sphingolipids may associate in lipid microdomains in the membrane
Mucins are heavily glycosylated and they may be attached to the that serve as important signaling platforms [11]. Glycolipids based on
membrane or be secreted. Another important class of O-glycosylated diacylglycerol that mediate the anchoring of proteins to the membrane
proteins consists of proteoglycans, where the carbohydrate moiety are designated as GPI-anchors (Fig. 1). GPI-anchored proteins are
(glycosaminoglycan) bound to the polypeptide chain (usually serine- predominantly localized on the plasma membrane generally in lipid
glycine sequences) is comparably very large and may have up to microdomains.
hundred monosaccharide residues. Proteoglycans may be attached to
the plasma membrane or be deposited in the extracellular matrix. The
2.2. Analytical techniques
attached glycans are linear and composed of disaccharides of alternat-
ing residues of amino sugar and hexose derivatives. Similar to mucins,
Glycans have more complex structures than other macromolecules
proteoglycan glycosylation involves the sequential addition of mono-
since they can be branched, there are different types of possible
saccharides by glycosyltransferases in the Golgi; these glycans are
glycosidic bonds and the linkage at the anomeric carbon can either
commonly sulfated in the Golgi concomitantly to biosynthesis, and
be α or β. Furthermore, glycoproteins with interesting biological roles
proteoglycans sulfation may be further regulated at the cell surface or
are often much less abundant than other cellular proteins. In view of
after secretion by the action of sulfatases [10]. There are different
this, the development of diversified and highly specific techniques for
groups of glycosaminoglycans based on monosaccharide composition
the analysis of glycan structures is required. The systematic study of
and sulfation: heparan sulfate and heparin, chondroitin sulfate and
glycosylation is approached by glycomics where total glycans (gly-
dermatan sulfate, hyaluronan and keratan sulfate. Syndecans and
come) are analysed, or by glycoproteomics where glycoprotein identi-
glypicans are heparan sulfate proteoglycans (HSPGs) that are bound
fication, sites of glycosylation and their occupancy including the
to the cell surface, the first consist of transmembrane proteoglycans
corresponding glycan structures are of relevance. Comprehensive re-
whereas the second are anchored to the membrane via a glycosylpho-
views on glycomics and glycoproteomics also with implications in the
sphatidylinositol (GPI)-anchor. HSPG is composed of disaccharides of D-
cancer biomarker field can be found in the literature [12–14].
glucuronic acid/L-iduronic acid and N-acetylglucosamine that are
In brief, for general characterization of glycosylation lectins can be
bound to the polypeptide chain via an O-xylose residue (Fig. 1).
used, which are proteins that bind to specific glycan structures. Lectins
Nuclear, cytoplasmic and mitochondrial proteins can also be modified
can be applied in blots [15,16], lectin arrays [6,7] or in lectin affinity
by addition of O-linked N-acetylglucosamine to serine or threonine
purification [17].
residues (Fig. 1).
Anti-glycan antibodies that recognize specific glycoepitopes are also
Another class of glycoconjugates found in cellular membranes are
used in immunoblots, immunohistochemistry and flow cytometry (e.g.,
glycolipids. Glycolipids may be embedded in the membrane via
anti-sialyl-Tn, anti-Lewis structures, anti-polysialic acid) [18], with
ceramide or via phosphatidylglycerol being designated as glycosphin-
strong applications in cancer diagnosis and prognosis.

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J. Costa
Fig. 2. Diagrammatic representation of production of EVs and their interaction with recipient cells. Glycans from glycoconjugates are represented as hexagons.
For structure analysis N-glycans are in general released enzymati-
cally with peptide N-glycosidase F from glycoproteins or derived
glycopeptides [15], and also chemically by hydrazinolysis [19]. Con-
cerning O-glycans, they are generally cleaved from the protein back-
bone in alkaline solution by β-elimination. To increase the sensitivity of
detection, glycans may be derivatized, for example, with 2-aminoben-
zamide for fluorescence detection [8] or by permethylation [20] for
mass spectrometry.
The profiling and detailed structure analysis of glycans is usually
done by liquid chromatography and mass spectrometry techniques
including high/ultra performance liquid chromatography (H/UPLC),
HPAEC-PAD capillary electrophoresis and mass spectrometric (MS)
techniques [8,15,21]. For determination of the type of linkage tandem
mass spectrometry (MS/MS) [22], endo- and exoglycosidase digestion
[8,23] and/or methylation analysis [19] are performed. Although less
sensitive NMR spectroscopy is also useful for the determination of
glycan primary structure and elucidation of glycans conformation. This
later aspect may also be unveiled in some cases from X-ray crystal-
lography, although this is a challenging aspect due to the heterogeneity
and mobility of glycans at the surface of glycoproteins.
The assignment of structures to specific glycosylation sites relies on
mass spectrometry analysis (LC/MS/MS) [13].
For the study of glycolipids mass spectrometry is commonly
performed.
In general, complementary techniques are used to establish the
glycosylation profiles from glycoconjugates, cells or tissues including
body fluids, with the aim of elucidating specific glycosignatures
relevant in given biological or physiological contexts. When consider-
ing specific glycoproteins the establishment of glycosignatures includes
in addition to the repertoire of glycostructures (from the different
glycoforms) information about specific site occupancy. These issues are
highly relevant in context of potential cancer biomarker identification
(Section 4.3).
2.3. Changes of glycosylation in cancer
Changes in glycosylation occur in many diseases and they have been
widely studied in cancer where neoglycoepitopes are detected asso-
ciated with tumors and are used as disease biomarkers [18,24,25].
Concerning N-glycosylation, most remarkable is the increased
branching of N-glycans as the product of GlcNAc-transferase V, which
is observed in different types of cancer [26]. On the other hand,
GlcNAc-transferase III synthesizes bisecting-GlcNAc-containing N-gly-
cans, which usually prevents further branching of N-glycans. However,
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BBA - Reviews on Cancer 1868 (2017) 157–166
bisecting GlcNAc has been found associated with ovarian cancer cells
and tissues [8,27,28].
Proximal fucosylation of N-glycans and the corresponding enzyme
FUT8 are upregulated in several types of cancer including hepatocel-
lular carcinoma. As a consequence proximally fucosylated α-fetopro-
tein, AFP-L3 appeared as a biomarker for liver cancer diagnosis [29].
Changes in O-glycosylation of the mucin type have also been
reported in cancer. Truncated structures have been observed with
increased levels of the T and Tn epitopes and sialylated derivatives
(e.g., sialyl-Tn) (Fig. 1) in different types of cancer including, breast,
colorectal, gastric, pancreatic, lung and salivary gland cancer. The
potential of these altered structures for drug and vaccine development
has been discussed in the literature [18].
Major changes in peripheral sialylation and fucosylation of glycans
are associated with cancer and are currently evaluated as markers for
improved diagnosis, prognosis and in response to treatment. The
fucosylated and sialylated structures that compose the sialylated
Lewis determinants (Fig. 1) present in O-glycans and N-glycans
participate in cell adhesion events underlying the formation of metas-
tases via interaction with selectins [24]. Their synthesis involves the
action of α2,3-sialyltransferases and α1,3/4 fucosyltransferases
[19,30,31].
Glycosaminoglycans either O-linked to the polypeptide chain (pro-
teoglycans) or free play different roles in physiology through their
action on cell surface receptors and interaction with growth factors.
HSPGs in cancer cells are relevant for tumor growth, are associated
with bad prognosis and constitute targets for anti-cancer therapies [32].
The levels of gangliosides are often deregulated in cancer. For
example, the disialoganglioside GD2 was reported as marker for several
cancers [33].
3. Extracellular vesicles
3.1. Characteristics and biogenesis
EVs are produced by many types of mammalian cells, including
tumor cells, cells of the immune system, neurons and glial cells. They
are designated as exosomes when they have an endosomal origin and
are produced from the fusion of multivesicular endosomes with the
plasma membrane releasing the intraluminal vesicles to the extracel-
lular environment (Fig. 2). In contrast, EVs that are produced from
plasma membrane budding are currently termed microvesicles. Exo-
somes and microvesicles have the same topology as the plasma
membrane. Exosomes have diameters below 150 nm whereas micro-
J. Costa BBA - Reviews on Cancer 1868 (2017) 157–166

vesicles are more heterogeneous with sizes typically up to 1 μm [34].
However, the complexity is high and the classification of EVs is
currently a challenge; supporting this notion, recently, several sub-
populations of EVs from dendritic cells were defined based on
proteomic analysis [35]. In recent years, the literature on EVs has
greatly increased and in many papers the term “exosomes” has been
applied to a mixture of EVs from different cellular origins and with
different properties.
EVs have characteristic compositions in nucleic acids (mRNA,
miRNA), proteins, including glycoproteins, and lipids including glyco-
lipids. Data concerning the composition of EVs from various sources are
available in several databases: EVpedia (http://evpedia.info/) [36],
Vesiclepedia (http://www.microvesicles.org) [37] and Exocarta
(http://www.exocarta.org) [38].
Exosomes are formed after the invagination of the endosome
membrane in a process assisted by the proteins from the endosomal
sorting complex required for transport (ESCRT)-0, -I, -II, -III and the
associated proteins, which results in the formation of intraluminal
vesicles, and the appearance of multivesicular endosomes (also known
as multivesicular bodies based on electron microscopy observations).
Several proteins that are used as exosome markers common to many
mammalian cells are involved in exosome biogenesis, for example,
Tsg101 from ESCRT-I or Alix. Proteins that are involved in membrane
fusion and transport, such as RabGTPases, annexins or flotillin, are also
found in exosomes. When the endosome membrane invaginates several
cytoplasmic proteins, such as heat-shock proteins (hsc70 and hsc90) or
cytoskeleton proteins, are included into the exosomes [34]. Whether
there are specific mechanisms of enrichment of certain cytoplasmic
proteins is not clear at present.
EVs are enriched in tetraspanins, which are palmitoylated mem-
brane proteins that may be glycosylated and have four transmembrane
domains. They participate in protein-protein interactions, including
oligomerization and interaction with other proteins such as integrins,
immunoglobulin-superfamily receptors and metalloproteinases. They
also interact with specific lipids such as cholesterol and gangliosides.
Tetraspanins like CD63, CD9, CD81 and CD82 are currently used as EVs
markers [39]. Tetraspanins are also involved in protein sorting into
exosomes, for example, in CD81-deficient cells exosomes are depleted
of CD81 ligands [40].
EVs from different cells and body fluids contain specific glycocon-
jugates with characteristic glycan structures (Table 1, Fig. 1). Initial
studies showed the presence of distinct prion PrP glycoforms in
exosomes [41]. Furthermore, the enrichment in high mannose epitopes,
complex N-linked glycans, N-acetyllactosamine, sialic acid and fucosy-
lated epitopes concomitant to exclusion of blood group antigens A/B in
microvesicles were detected in microvesicles and HIV-1 from several T-
cell lines by lectin array technology [6]. In agreement, and using the
same strategy, specific glycosignatures were found in microvesicles of
T-cell lines, as well as skin cancer and colon cancer cell lines, with
enrichment in high mannose, polylactosamine, α2,6-sialic acid, and
complex N-glycans concomitant to depletion of terminal blood group A
and B antigens [7]. Lectin array technology allowed the detection of
glycan structures present in glycoconjugates from EVs surface. On the
other hand, in SKOV3 and OVMz ovarian carcinoma cells specific
glycoproteins and glycan profiles were also found for EVs using lectin
blotting, which allowed the detection of N- and O-glycans linked to
glycoproteins [8,16,42]. Furthermore, complex N-glycans of the di-,
tri-, and tetraantennary type with or without proximal fucose and
bisecting GlcNAc as well as high mannose glycans released with peptide
N-glycosidase F from glycoproteins of SKOV3 cells were detected by
NP-HPLC complemented with mass spectrometry [8]. In addition,
proteomic analysis allowed the identification of an abundant sialogly-
coprotein as galectin-3 binding protein (LGALS3BP also known as Mac-
2 binding protein) [8,16]; LGALS3BP was suggested as a potential
exosome marker.
N-glycoproteomic/glycomic analysis of urinary exosomes showed
the presence of complex, hybrid and high mannose-type N-glycans from
glycoproteins; glycans containing sialic acid-, bisecting GlcNAc and
peripheral as well as proximal Fuc were detected and evidence for
sulfated and/or phosphorylated glycans was found [43]. In other
studies, high mannose and sialylated complex glycans were detected
in urinary exovesicles [44,45] as well as GlcNAc-containing glycocon-
jugates [46]. In galactosemia patients a shift from prevalent high
mannose to complex glycans was observed. This shift was specific to
EVs since it was not detected in the urinary Tamm-Horsfall glycoprotein
[44]. Therefore, EVs glycans constituted useful biomarkers in galacto-
semia, which is a potentially lethal hepatotoxic syndrome for new born
infants and that in the long-term causes complications related with the

Table 1
Selected glycoconjugates and glycans associated with EVs.

Molecule Biofluid/tumor cells Cancer/disease References

Prion Infected moRK13, GT1-7 cells Prion disease [41]

ADAM10 SKOV3 cells Ovarian [51]
Specific glycosignatures T-cells (Jurkat-Tat-CCR5, SupT1and H9) – [6]
Specific glycosignatures T-cells (Jurkat-Tat-CCR5, SupT1and H9) – [7]
HCT-15 and HT-29 cells Colon
SkMel-5 cells Melanoma
Breast milk –
Specific glycosignatures SKOV3 cells Ovarian [42]
H4 cells Neuroglioma
N-glycan profiles Urine Galactosemia [44]
EGFR, EGFRvIII, podoplanin SkMG3, GBM20/3, GLI36vIII, GLI36R132H, LNZ308, A172 cells and blood Glioblastoma [79]
Specific glycosignatures, LGALS3BP SKOV3, OVMz cells Ovarian [8,16]
Glycan profiles Urine – [45]
Syndecan-1,-2,-4, glypican U-87 MG and LN18 cells Glioblastoma [48]
a2,3-Linked sialic acid B cells – [73]
N-glycoproteins Urine – [43]
EWI-2 N-glycans Sk-Mel-5 cells Melanoma [58]
Highly glycosylated EMMPRIN MCF-7 cells Breast cancer [89]
CD24, EpCAM CaOV3, OVCAR3, SKOV3, OV90, OVCA429, UCI101, ES-2, TOV112D, TOV21G, UWB1.289, GG, M130, Ovarian [81,82]
ascites
GlcNAc-containing glycans Urine – [46]
Integrins Several cancer cells, tissues, blood Melanoma [65,67]
Breast
Pancreatic
O-GlcNAc SW480 and SW620 cells Colorectal [90]

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J. Costa
central nervous system and ovaries.
EVs contain heparan sulfate proteoglycans, and syndecans play a
major role in exosome biogenesis (Section 4.1) and are relevant in the
interaction between exosomes and target cells (Section 4.2).
Concerning lipids EVs also have characteristic compositions and
they are enriched in glycosphingolipids (including gangliosides GM3,
GM1, GD2), sphingomyelin, cholesterol, and phosphatidylserine [47].
On the other hand, GPI-anchored proteins are present in EVs, for
example, glypican [48].
EVs contain nucleic acids including mRNA, miRNA and DNA and,
therefore, may serve as vehicles for the transfer of genetic information
among cells and under disease conditions could be a source for the
identification of biomarkers. This topic is subject of intensive research
[1].
3.2. Separation techniques
Several populations of EVs are released from cells and have over-
lapping biophysical and biochemical properties. In view of this, the
purification of homogeneous EVs populations constitutes a challenge at
present time and several methodologies may be used [49,50]. The most
widely used approaches for EVs separation are based on differential
centrifugation, predominantly, ultracentrifugation of cell supernatants
and biological fluids. For purity improvement and separation refine-
ment of different EVs populations, ultracentrifugation in gradients of
sucrose [8,51] or iodixanol [35] can be used. More recently, the interest
in size-exclusion chromatography for the separation of EVs has
increased [52]. In addition, affinity chromatography, including immu-
noisolation with antibodies against specific EVs components [35] allow
the recovery of more homogeneous populations based on EVs surface
epitopes. In view of EVs enrichment in specific glycans, lectin affinity
chromatography is also used in their purification [49,53]. Particularly,
it constitutes the basis for the diagnostic platform ELLSA™ (Enzyme
Linked Lectin Specific Assay) to isolate disease-specific exosomes from
body fluids. Heparin-affinity in combination with ultrafiltration can
also be used for the purification of EVs released from normal and cancer
cells, and from human blood plasma [54].
4. Glycoconjugates from extracellular vesicles
4.1. Biogenesis and sorting
Syndecan HSPGs are involved in the biogenesis and cargo loading of
exosomes via the intracellular adaptor syntenin and several regulators
that include Alix, the small GTPase ARF6, lipid-modifying enzyme
PLD2 and heparanase [55]. Heparanase caused enhanced exosome
secretion and altered exosome composition and function in different
human cancer cells [56]. More recently, it was shown that heparanase
stimulated the production of exosomes with syntenin-1, syndecan and
CD63, but not CD9, CD81 or flotillin-1 [57]. The underlying mechanism
proposed that syndecan clustered upon heparanase and protease
cleavage, which resulted in enhanced formation of intraluminal vesicles
(in multivesicular endosomes), subsequently released as exosomes [55].
Other sorting mechanisms have been proposed based on the
observations that EVs are strongly glycosylated and enriched in specific
glycoproteins and glycans [6–8,16,42] (Section 3.1). Supporting this
idea was the finding that complex N-glycosylation of the glycoprotein
EWI-2 was essential for its sorting into EVs in a melanoma cell line
using the N-glycosylation inhibitor deoxymannojirymycin and by
mutating EWI-2 N-glycosylation sites [58]. Other studies reported that
kifunensin, an inhibitor of glycoprotein processing from high mannose
to complex-type N-glycans, affected the composition of EVs from
ovarian carcinoma cells [16].
Glycan-based sorting of glycoproteins into exosomes could involve
cellular lectins, such as galectins. Galectin-3 and galectin-4 were found
in EVs [7,59] and inhibition of glycosylation caused decreased levels of
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BBA - Reviews on Cancer 1868 (2017) 157–166
galectin-3 in melanoma cells [58]. Furthermore, galectin-5 has been
proposed to mediate the concentration of poly-N-lactosamine contain-
ing glycoproteins into reticulocytes [60]. It is quite known that glycan-
lectin interactions are involved in the sorting of glycoproteins in other
cellular settings [61]. Therefore, an alternative pathway of glycoprotein
sorting to EVs that would involve glycans and lectins is not too far
fetching. In this scenario glycans would interact with lectins and
promote the specific sorting into EVs possibly via glycoprotein oligo-
merization or enrichment in membrane domains. More experimental
evidence is still required in support of this hypothesis.
To proceed further in elucidating sorting mechanisms of specific
glycoconjugates into EVs, improved purification strategies to obtain
more homogeneous fractions of EVs, as well as plasma membrane and
multivesicular endosome membrane fractions will be helpful.
4.2. Interaction with target cells
EVs can interact and be internalized by cells thus constituting
vehicles of biomolecule transfer and, consequently, provide the means
for communication among cells. In disease, they are involved in the
spreading of toxic molecules from cell to cell underlying the transmis-
sion of pathogenicity, which is relevant in cancer [1,2]. On the other
hand, EVs from healthy cells or from stem cells have beneficial effects
on sick cells [62].
EVs may modify target cell and organ function. For example, EVs
from glioblastoma cells were shown to transmit mRNA, miRNA and
angiogenic proteins to brain microvascular endothelial cells in culture
[63]; microvesicles containing the oncogenic receptor EGFRvIII also
transferred the oncogenic activity to cancer cells lacking EGFRvIII [64].
Moreover, EVs play different roles in tumorigenesis, extracellular
matrix remodeling, metastases, tumor angiogenesis and tumor immu-
nity [1,5]. Very importantly, EVs were found to promote the formation
of pre-metastatic niches in distant sites with the remodeling of
extracellular matrix in different cancers [65–67] (see Section 4.3.1).
EVs are recognized by molecules at the surface of target cells and
they can be internalized by various mechanisms including clathrin-
mediated endocytosis, phagocytosis, macropinocytosis and plasma or
endosomal membrane fusion [42,68] (Fig. 2). Several proteins, in many
cases glycosylated are relevant in EVs uptake, including, tetraspanins,
integrins and other cell adhesion molecules, proteoglycans and lectins
[68].
The HSPGs constitute cell-surface receptors for EVs endocytosis.
HSPGs of the syndecan and glypican type were found to mediate the
uptake of EVs by glioblastoma cells, in a process that was dependent on
the 2-O and N-sulfation groups of the HSPGs [48]; uptake was
specifically inhibited by free heparan sulfate chains, whereas closely
related chondroitin sulfate had no effect. HSPGs were also relevant for
the uptake of EVs by hepatic stellate cells [69]. Furthermore, heparin
partially inhibited EVs uptake by bladder cancer cells [70]. HSPGs from
EVs of myeloma cell lines or myeloma patients interacted with HSPGs
from tumor cells or marrow stromal cells via fibronectin [71].
Lectins are also involved in exosome uptake. For example, galectin-
5 from exosomes of rat reticulocyte was suggested to mediate exosome
uptake by macrophages [60]. On the other hand, exosomes were taken
up by dendritic cells in a process involving the interaction between a C-
type lectin and mannose/glucosamine-rich C-type lectin receptor [72].
B-cell derived exosomes displayed sialic acid α2,3-linked on their
surface that mediated the binding to sialoadhesin (CD169, Siglec1)
from macrophages in spleen and lymph node [73], therefore, playing a
role in immune response.
Lamp2b fusion proteins displayed at the surface of exosomes have
been used as a means to improve cell targeting. The N-glycosylation
engineering of those surface proteins through the introduction of N-
glycosylation sites was shown to increase their stability having as
consequence an enhanced uptake by recipient neuroblastoma cells
[74].
J. Costa
4.3. Cancer glyco-biomarkers
EVs transport cellular material to the extracellular environment, are
capable of crossing biological barriers and are found in biological fluids
including blood, urine and cerebrospinal fluid. Therefore, they consti-
tute valuable targets for biomarker identification. Potential biomarkers
have been identified in EVs from glioblastoma, urogenital cancers
including bladder and prostate cancer, melanoma, colorectal, breast,
ovarian, pancreatic cancers and lung adenocarcinoma [75–77].
Furthermore, the enhanced levels of EVs in body fluids from cancer
patients have been proposed as potential biomarkers [78].
EVs are highly glycosylated since they are derived from the plasma
membrane or from multivesicular endosomes. In cancer, EVs carry
molecules expressed by tumor cells, including glycoconjugates, to the
extracellular environment and in this context EVs are considered as
surrogates of the originating tumor cells that are found in body fluids.
The proportions of the different molecules may be specific to EVs in
view of specific sorting events that take place during EVs biogenesis.
For example, the enrichment of specific molecules, which are used as
EVs markers (such as, glycosylated tetraspanin CD63, sialoglycoprotein
LGALS3BP) has been observed; furthermore, characteristic glycosigna-
tures of EVs relatively to total cellular membranes [7,16], or to plasma
membrane [8] also suggest glycan-based sorting to EVs possibly
involving intracellular lectins (Section 4.1). Considering that changes
in glycosylation are a hallmark of cancer (Section 2.3), glycoconjugates
from EVs and their respective glycosylation could constitute potential
biomarker targets in cancer. In this context two major aspects may be
considered: the identification of potential novel glyco-biomarkers and
the improvement of glyco-biomarkers already used in the clinics. These
issues will be discussed in the next two sections.
4.3.1. Glycoconjugates from extracellular vesicles as potential cancer
biomarkers
Recently, several studies have addressed the potential of EVs
glycoconjugates and their glycosylation as biomarkers in cancer
(Table 1). For example, in glioblastoma EVs elevated expression of
several proteins including the N-glycosylated receptors EGFR and
EGFRvIII were detected, and EVs were shown to constitute a surrogate
of primary tumor mutations and to be useful in monitoring the effects of
treatment [79]. EGFR overexpression is detected in a high number of
glioblastoma multiforme cases, and EGFRvIII, a genomic deletion
variant of EGFR, which is constitutively active and highly oncogenic
is also often expressed. Mutations in EGFR that result in deregulation of
its activity are also present in other types of cancer and several studies
describing its glycosylation and association with cancer have been
performed, for example, glycomic analysis showed that EGFR-specific
N-glycan signatures included high bisecting β1,4-GlcNAcylation and
low α2,3-sialylation relative to EGFR-negative colorectal cancer tissues
[80], but the relevance of EGFR glycosylation from EVs has been less
explored.
In ovarian cancer patients, malignant ascites-derived exosomes
contain the N- and O-glycosylated GPI-anchor CD24 and the N-
glycosylated epithelial cell adhesion molecule EpCAM that are impor-
tant for diagnostics [81,82]. CD24 is a well-established marker not only
in ovarian but also other types of cancer where it is associated with a
poor prognosis; it was also detected in some tumor stem cells [82]. Anti-
CD24 antibody SWA11 studies using A549 lung and SKOV3 ovarian
carcinoma cells in mouse showed that CD24 targeting increased
infiltration of tumor tissues with immune cells suggesting involvement
of antibody-dependent cell-mediated cytotoxicity and strong alterations
in intratumoral cytokine microenvironment; moreover, combination of
SWA11 mAb with gemcitabine treatment strongly potentiated its anti-
cancer efficacy in A549 lung cancer model [83]. CD24 glycans from
SKOV3 cells contained α2,3/6-linked sialic acid and were recognized by
Siglec-5 and P-selectin [84], but further studies are required to
elucidate in detail its glycosignatures and relevance in cancer. On the
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BBA - Reviews on Cancer 1868 (2017) 157–166
other hand, glycomics of EVs glycoproteins from SKOV3 and OVMz
ovarian cancer cells showed the presence of bisecting GlcNAc-contain-
ing-glycans and high mannose glycans [8,16]. These structures were
proposed to constitute ovarian cancer biomarkers, which was in
agreement with the upregulation of bisecting GlcNAc in tissues from
patients [27] and predominance of high mannose-type glycans in tumor
tissues region [85].
LGALS3BP is a heavily glycosylated protein with sialic acid and
complex N-glycans, that is associated with EVs from different cancer
cells [8,16,86] (EVPedia, Vesiclepedia, ExoCarta). In ovarian carcinoma
cells it was found to contain α2,3-linked sialic acid based on lectin
blotting with Maackia amurensis lectin and digestion with Streptococcus
pneumoniae sialidase [8,16,42]. LGALS3BP is an extracellular matrix
protein and binds galectin-3 as well as other glycoproteins from the
extracellular matrix including integrins; these interactions could pro-
vide the basis for its localization in EVs. LGALS3BP has been proposed
as a potential cancer biomarker, for example, in pancreatic carcinoma
[87] and prostate cancer [88].
In breast cancer a highly glycosylated form of the extracellular
matrix metalloproteinase inducer (EMMPRIN) was found as a marker
for proinvasive EVs from tumor cells. Furthermore, EMMPRIN was
present at high levels on EVs from metastatic breast cancer patients in
vivo. Anti-EMMPRIN strategies, such as EV de-N-deglycosylation with
peptide N-glycosidase F, gene knockdown, and specific blocking
peptides, inhibited EV-induced invasion [89]; therefore, EMMPRIN
glycosylation appeared relevant for tumor cell phenotype.
O-GlcNAcylation is a post-translational modification mainly of
nuclear, cytoplasmic and mitochondrial proteins but recently it has
also been found in proteins from EVs. Furthermore, O-GlcNAcylation of
many EV proteins from colorectal cancer cells was increased in
metastatic cells, thus suggesting its potential as biomarker [90]. In this
study the proteins transitional endoplasmic reticulum ATPase and
RuVB-like 1 contained O-GlcNAc and their levels were significantly
increased in EVs from metastatic cells.
A recent body of evidence has demonstrated that exosomes have the
potential to form pre-metastatic niches creating a favorable environ-
ment in target organs for metastases formation. Exosomes from highly
metastatic melanoma increased the metastatic behavior of primary
tumors and an exosome-specific melanoma signature with prognostic
and therapeutic potential, comprised of TYRP2, VLA-4 (integrin α4β1),
HSP70, an HSP90 isoform and the MET oncoprotein was found [65].
More recently, in pancreatic cancer it was described that the formation
of the pre-metastatic niche involved upregulation of macrophage
migration inhibitory factor (elevated in plasma exosomes from pan-
creatic cancer patients), which induced expression of the N-linked
glycoprotein transforming growth factor beta (TGF-beta) by Kupffer
cells, which in turn, led to the production of the N- and O-linked
glycoprotein fibronectin by hepatic stellate cells [66]. In this context,
integrins play a very relevant role; more specifically, tumor exosome
integrins determined organotropic metastasis (integrins α6β4 and α6β1
associated with lung metastasis, integrin αvβ5 associated with liver
metastasis), and clinical data from patients with breast or pancreatic
cancer indicated that exosomal integrins could be used to predict organ-
specific metastasis [67]. Integrins are N-glycosylated proteins that
exhibit glycosylation changes in cancer with implications in malig-
nancy [24], and a particularly interesting topic would be to explore
glycosylation of EVs integrins in depth.
Glycolipids from EVs have also been suggested as potential cancer
biomarkers elsewhere [47].
Summing-up, there is a recent constellation of observations that
point to EVs glycoconjugates/glycosignatures as emerging targets with
potential in the cancer biomarker field.
4.3.2. Detection of clinical glyco-biomarkers in extracellular vesicles
Several glycoconjugates already used in the clinics as cancer
biomarkers have been detected in EVs (Table 2). Selected glyco-
J. Costa BBA - Reviews on Cancer 1868 (2017) 157–166

Table 2
Selected glycoconjugate/glycan tumor markers found in EVs.
Tumor markers were extracted from https://www.cancer.gov/about-cancer/diagnosis-staging/diagnosis/tumor-markers-fact-sheet. Type of glycosylation was extracted from http://
www.uniprot.org. The identification of the targets in the EVs databases was only considered from human origin and it was not necessarily associated with the type of cancer for which the
glycoconjugate is a biomarker. Date: 2nd February 2017.

Tumor markers used in the clinics Identification in EVs

Marker Cancer Biofluid/ Uniprot Glycosylation type EVPedia Vesiclepedia ExoCarta

tumor cells entry

Alpha-fetoprotein (AFP) Liver and germ cell tumors Blood P02771 N-glycosylated + VP_174 174
Beta-human chorionic gonadotropin Choriocarcinoma, germ cell Urine, blood P0DN86 N- and O-glycosylated − VP_1082 1082
(Beta-hCG) tumors
C-kit/CD117 Gastrointestinal stromal tumor, Tumor P10721 N-glycosylated − VP_3815 3815
mucosal melanoma
CA15-3/CA27.29 (MUC1) Breast Blood P15941 N- and O-glycosylated + VP_4582 4582
CA19-9 Pancreatic, gallbladder, bile Blood − Glycan sialyl-Lewisa [94]
duct, gastric
CA-125 (MUC16) Ovary Blood Q8WXI7 N- and O-glycosylated + VP_94,025 94025
Carcinoembryonic antigen (CEA) Colorectal and others Blood P06731 N-glycosylated + VP_1048 1048
Chromogranin A (CgA) Neuroendocrine tumors Blood P10645 O-glycosylated + VP_1113 1113
Estrogen receptor (ER) Breast Tumor P03372 O-GlcNAc + VP_2099 2099
HE4 Ovary Blood Q14508 N-glycosylated + VP_10406 10406
HER2/neu Breast, gastric, gastroesophageal Tumor P04626 N-glycosylated + VP_2064 2064
junction adenocarcinoma
Prostate-specific antigen (PSA) Prostate Blood P07288 N-glycosylated + VP_354 354
Urokinase plasminogen activator (uPA), Breast Tumor P00749, O-Fucose/N- +/+ VP_5328, VP- 5328/5054
plasminogen activator inhibitor (PAI- P05121 glycosylated, N- 5054
1) glycosylated

biomarkers that are membrane bound will be discussed in the next
paragraphs.
CA15-3 and CA27.29 are epitopes from mucin 1 (MUC1) that are
used in breast cancer diagnosis. MUC1 is a type I transmembrane
protein with a heavily O- and N-glycosylated extracellular domain.
Aberrantly glycosylated MUC1 is overexpressed in most human epithe-
lial cancers, including breast cancer [18]. MUC1 was found in lipid rafts
from plasma membrane and exosomes of human breast carcinoma cells
MCF-7 [91].
Carbohydrate antigen 19-9 (CA19-9) corresponds to the sialylated
fucosylated structure sialyl-Lewisa present on glycolipids and glycopro-
teins. CA19-9 is a widely used biomarker in pancreatic cancer, although
it presents limited sensitivity and specificity. It has been tested in
combination with other biomarkers; for example, CA19-9 and CA125
have good sensitivity for detecting preclinical pancreatic cancer and
can be used to evaluate prognosis [92,93]. Recently, CA19-9 has been
detected in pancreatic cancer derived exosomes [94].
CA-125 is an ovarian cancer antigen that is screened in the serum of
patients as a biomarker. It is present in mucin 16 (MUC16), which is a
highly N- and O-glycosylated type I membrane glycoprotein with a
large extracellular domain. This biomarker alone has specificity limita-
tions but it is useful for monitoring the effectiveness of treatment and
detecting recurrence [95]. More recently, approaches involving the
glycoprofiling of MUC16 have shown promising results towards in-
creased biomarker sensitivity for ovarian cancer [96]. Furthermore, the
screening for CA-125, EpCAM and CD24 using a microfluidic approach
on the ExoSearch chip, which provides an enriched preparation of
blood plasma exosomes, showed significant diagnostic power for
ovarian cancer [97].
Carcinoembryonic antigen (CEA) belongs to the immunoglobulin
superfamily, it is heavily N-glycosylated and is bound to the plasma
membrane via a GPI-anchor. CEA is the main marker in colorectal
cancer, in prognosis and postoperative surveillance and for therapy
monitoring [98]. CEA has been detected in EVs from colorectal cancer
cells and from plasma of patients with colorectal cancer [99]. Recently,
exosomes from colorectal cancer cells, which were CEA positive, were
found to induce morphological and functional changes in colonic
mesenchymal stromal cells, possibly favoring tumor growth and
malignant progression [100].
The human epidermal growth factor receptor 2 (HER2, also known
as ErBB2 or HER2/neu) is a type 1 transmembrane glycoprotein with an
extracellular ectodomain and an intracellular domain with tyrosine
kinase activity. HER2 is overexpressed in several types of cancer,
particularly, in breast cancer [101]. Recently, it was found that cancer
cell-derived HER2 exosomes could stimulate the MAPK pathway in
monocytes with possible implications in the formation of tumor-
associated macrophages [102]. Further data suggested that HER2-
positive exosomes could decrease the efficacy of antibody Trastuzumab
therapy [103].
These glyco-biomarkers used in clinical practice usually have
sensitivity and specificity limitations when used alone. However, an
interesting aspect would be their screening in EVs, which could
potentially improve their predictive value. Of particular interest would
also be to investigate if disease-associated glycoforms are enriched in
EVs from body fluids.
5. Conclusions and future perspectives
The study of EVs has been subject of increased interest in recent
years and the recognized role of EVs in transmitting biomolecules
among cells as well as their potential to provide disease biomarkers are
particularly relevant. Several aspects currently remain a challenge, such
as purification of homogeneous populations of vesicles, regulation of
EVs release and modulation of recognition and uptake of EVs by target
cells.
Glycoconjugates and their glycosylation in EVs could emerge as
useful tools for major advances in the field. For example, a useful
approach for EVs fractionation in view of their diversity, is to explore
their characteristic glycosylation properties as the basis for affinity
purification. Concerning biogenesis, the HSPG syndecan participates in
exosome formation and cargo loading. Furthermore, characteristic
glycosignatures from EVs suggest that novel specific mechanisms,
possibly related with intracellular lectins, participate in glycoprotein
sorting into EVs.
In the context of EVs interaction with other cells, HSPGs from host
cells play a well-recognized role, and the binding partners from EVs to
HSPGs are currently an interesting topic of research. EVs have been
largely explored as therapeutic vehicles of delivery (e.g., drugs, nucleic

163
J. Costa
acids, proteins) in cancer and in neurodegenerative diseases.
Modulation of EVs surface glycosylation could be a promising strategy
for protein stabilization and to improve interactions with target cells.
EVs also hold a great potential for the identification of disease
biomarkers since stressed cells release EVs that are found in body fluids,
such as plasma, urine or cerebrospinal fluid; therefore, EVs are
considered surrogates of the originating cells. In cancer major altera-
tions in cell surface glycosylation take place, which will be reflected at
the surface of EVs. Therefore, the study of glycoconjugates and their
glycosylation from body fluids EVs constitutes a promising strategy to
identify novel cancer markers. In addition, many cancer markers
already used in the clinics are glycosylated and also found in EVs; the
investigation of these clinical biomarkers specifically in EVs may
improve their predictive value.
In conclusion, glycosylation appears to be highly relevant in the
extracellular vesicle field. Particularly in cancer it holds great promise
towards novel biomarker identification as well as improvement of
clinical biomarkers.
Transparency Document
The http://dx.doi.org/10.1016/j.bbcan.2017.03.007 associated
with this article can be found in the online version.
Acknowledgments
We thank Prof. Peter Altevogt, DKFZ, Germany, for critical reading
of the manuscript and useful suggestions. This work was supported by
Euronanomed 2 ERA-NET, project GlioEx, Fundação para a Ciência e a
Tecnologia (FCT), Portugal, grant ENMed/0001/2013; FCT/Ministério
da Educação e Ciência, grant iNOVA4Health-UID/Multi/04462/2013.
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