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Exosomes: Generation, structure, transport, biological activity, and diagnostic application

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Cells of almost all tissues secrete to the extracellular environment a variety of vesicular structures. The most interesting vesicles are exosomes–microvesicles ranging from 30 to 100 nm in size. These vesicles contain various RNA, including mRNA, microRNA, as well as membrane and cytoplasmic proteins that can be transported in these particles to nearby and distantly located cells of various tissues using physiological fluids (blood, urine, saliva, etc.). Exosomes are necessary for normal functioning of the organism and their repertoire changes during the development of pathologies. This review presents the data on generation, secretion, and transport of exosomes, their structure and roles in normal conditions and in the process of the malignant tumor development. Prospects of the application of exosomal biomarkers for the development of early non-invasive cancer diagnosis are also discussed.
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ISSN 1990-7478, Biochemistry (Moscow), Supplement Series A: Membrane and Cell Biology, 2016, Vol. 10, No. 3, pp. 163–173. © Pleiades Publishing, Ltd., 2016.
Original Russian Text © S.N. Tamkovich, O.S. Tutanov, P.P. Laktionov, 2016, published in Biologicheskie Membrany, 2016, Vol. 33, No. 3, pp. 163–175.
Exosomes: Generation, Structure, Transport, Biological Activity,
and Diagnostic Application
S. N. Tamkovicha, b, *, O. S. Tutanova, and P. P. Laktionova
aInstitute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences,
pr. Akademika Lavrentieva 8, Novosibirsk, 630090 Russia
bNovosibirsk State University, ul. Pirogova 2, Novosibirsk, 630090 Russia
*e-mail: s.tamk@niboch.nsc.ru
Received July 28, 2015; in final form, October 24, 2015
Abstract—Cells of almost all tissues secrete to the extracellular environment a variety of vesicular structures.
The most interesting vesicles are exosomes – microvesicles ranging from 30 to 100 nm in size. These vesicles
contain various RNA, including mRNA, microRNA, as well as membrane and cytoplasmic proteins that can
be transported in these particles to nearby and distantly located cells of various tissues using physiological flu-
ids (blood, urine, saliva, etc.). Exosomes are necessary for normal functioning of the organism and their
repertoire changes during the development of pathologies. This review presents the data on generation, secre-
tion, and transport of exosomes, their structure and roles in normal conditions and in the process of the
malignant tumor development. Prospects of the application of exosomal biomarkers for the development of
early non-invasive cancer diagnosis are also discussed.
Keywords: exosomes, microvesicles, biomarkers, cell-to-cell signaling, microRNA, proteomic markers
DOI: 10.1134/S1990747816020112
INTRODUCTION
The growing interest to microvesicles – structures
ranging in size from tens of nanometers to several
microns produced by living and actively functioning
cells – started a few years ago, and a special attention
is paid to exosomes – endosomal membrane particles
of 30100 nm in size. It is known that they contain
cytosolic and membrane proteins, as well as function-
ally active ribonucleic acids (mRNA, microRNA,
rRNA, tRNA, etc.) [1–3]. It is now established that
exosomes are involved in the transportation of pro-
teins and nucleic acids and immune response regula-
tion, including the antigen presentation, transporta-
tion of infectious agents, and development of patho-
logical processes [4]. It has been recently found that
exosomes are involved in tumor progression. In par-
ticular, exosomes produced by prostate cancer cells
affect the microenvironment of the tumor and pro-
mote its growth [5], and exosomes produced by plate-
lets in lung cancer promote an active intratumoral
angiogenesis [6].
It is known that the body cells secrete exosomes
into the extracellular space, including biological fluids,
such as blood, lymph, cerebrospinal fluid, urine, and
saliva. These vesicular structures are stable for a long
time. They are carried through the blood and lymph
circulation and can be detected in the body far away
from the localization of the parent cells. Structure,
properties and biological activity of exosomes and fea-
tures of their circulation are now being studied inten-
sively. Identification of tumor cells-specific markers in
the blood circulating exosomes will be useful for elab-
oration of approaches to non-invasive diagnosis of
malignant neoplasms.
1.1. Generation and Secretion of Exosomes
Different sources use the terms microvesicles and
exosomes, as well as special names for specific vesicular
particles (microparticles, microvesicles, ectosomes,
shedding microvesicles, nanovesicles, exosomes, exo-
some-like particles, apoptotic vesicles, prominino-
somes, prostatosomes, dexosomes, texosomes, epididi-
mosomes, argosomes, archeosomes, or oncosomes).
These terms appeared due to the use of different bio-
logical materials, as well as methods and research
aims. According to the most rigorous definition, exo-
somes are extracellular membrane particles of endo-
cytic origin ranging from 30 to 100 nm emerging
during the formation of multivesicular bodies and
secreted into the extracellular space as a result of the
fusion of multivesicular bodies with the plasma mem-
brane [1]. Microvesicles are a number of extracellular
membrane particles that, in addition to exosomes,
include apoptotic blebs – membrane particles with a
diameter of 50 to 500 nm separated from the cells in
the process of apoptosis, and shedding microvesicles –
REVIEWS
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TAM KOV ICH et a l.
microparticles with a diameter of 100 to 1000 nm
secreted by various cells under physiological condi-
tions and probably playing an important role, along
with exosomes, in the cell-to-cell communication and
transport of proteins and nucleic acids [7]. The con-
tamination of exosomal preparations with microparti-
cles of similar size considerably complicates the task of
obtaining a high-quality exosome isolation protocol
and the correct interpretation of results obtained on
the total particle pool.
The paper of Pan and Johnstone [8] published in
1983 is considered to be the first scientific description
of exosomes. The authors showed that in the process
of maturation of sheep reticulocytes, transferrin recep-
tors are released with exosomes. Initially, exosomes
were considered either as specific formations peculiar
only to this type of cells or as metabolic by-products.
The discovery of the ability of antigen-presenting cells
to secrete exosomes with their own immunostimula-
tory activity changed the attitude of the scientific
world to the vesicles of this type [9]. In 2007, Valadi et
al. showed that exosomes were able to carry a wide
pool of RNA [10]. This year, it was found that in addi-
tion to mRNAs and microRNAs, other classes of
RNAs are transported by exosomes: tRNA, rRNA,
snRNA, snoRNA, piRNA, and scaRNA [3]. All this,
together with constantly renewed data on the role of
exosomes in the body under normal conditions and
during the development of pathological processes
aroused a heightened interest among scientists.
The exosome formation process can be divided into
four stages: initiation, endocytosis, formation of mul-
tivesicular bodies, and secretion [11]. In the f irst stage,
the invagination of the cell membrane sites containing
ubiquitinated surface receptors forms early endo-
somes. The fate of future multivesicular bodies is
largely determined by small GTPases of the Rab fam-
ily. Protein Rab5 that regulates the fusion of early
endosome membranes binds to the formed endosome
allowing effector proteins to start their work: phospho-
inositol-3-kinase, early endosomal antigen 1, and
rabenosyn-5. The latter form a GDP/GTP exchange
complex Rabex-5 that stabilizes the active form of
Rab5, mediates further membrane fusion, and pro-
vides a platform for the recognition of proteins con-
taining FYVE domain [12, 13]. As a result, the formed
complex binds to FYVE domain of the protein, which
is part of a protein complex ESCRT-0 (Endosomal
Sorting Complex Required for Transport). This bind-
ing leads to the assembly of other parts of ESCRT-0 on
the formed endosomal membrane, which in turn initi-
ates the assembly of ESCRT-I and ESCRT-II that fur-
ther mediates the invagination of the membrane in the
newly formed multivesicular body and the formation
of ESCRT-III responsible for the final formation of
microvesicles and their release into the cavity of the
multivesicular body [14]. In addition, there is an
ESCRT-independent way of the exosome formation,
but this mechanism is poorly understood [15]. In the
process of maturation, a multivesicular body can
either be degraded by becoming a part of lysosomes, or
processed by the Golgi complex [16] or secreted in the
form of exosomes into the extracellular space. These
processes are inhibited by other GTPases of the Rab
family that organize short and long ways of recycling
(Rab4 and Rab11) and are responsible for taking the
path of lysosomal degradation (Rab7) and transport to
the Golgi complex (Rab6) [16]. Further fate of micro-
particles – degradation or secretion – is mediated by
the action of proteins Rab27A and Rab27B, playing a
significant but not decisive role in these processes.
The inactivation of both proteins reduces secretion by
50% but does not block it in full [17], which indicates
the existence of alternative mechanisms and other fac-
tors participating in this process [16]. In particular, it
is shown that the secretion of exosomes is affected by
proteins Rab5A, Rab9A and Rab2 [17]. Furthermore,
both under normal conditions and during the develop-
ment of malignant neoplasms, the secretion of exo-
somes is increased by heat shock [18], low pH values of
the environment [19], high intracellular concentration
of calcium ions [20], loss of adhesion [21], etc. Exo-
somes are secreted into the extracellular space due to
the fusion of the formed multivesicular bodies and the
cell plasma membrane. Thus, exosomes secreted into
the extracellular environment carry on their surface
ligand-binding domains of receptors and other trans-
membrane proteins identical to the donor cell, which
allows the vesicles to enter into all interactions charac-
teristic of the parent cells (Fig. 1).
Exosomes secreted by donor cells can act both
locally, by interacting with neighboring recipient cells
(paracrine and autocrine regulation [22]), and dis-
tantly, by getting into the bloodstream and moving
through organs and tissues (endocrine regulation). The
exosomes can be transported through the body in the
flow of the extracellular medium and affect distant
cells. To date, the following mechanisms of interac-
tions of exosomes with recipient cells are described:
(1) Binding of exosomes with cells without inter-
nalization mediated by the ligand-receptor interac-
tions without membrane fusion (for example, presen-
tation of antigens [9]);
(2) Attachment/fusion of the membranes of exo-
somes and target cells resulting in the transfer of proteins
anchored in the vesicle membrane into the plasma mem-
brane (for example, binding of exosomes to endothelial
cells through the α4β1-integrin/VCAM-1 pathway
leading to the transportation of glycosylphosphatidyli-
nositol-bound proteins (acetylcholinesterase, CD55,
CD58, CD59) into the plasma membrane of recipient
cells [23]);
(3) Internalization of exosomes by target cells
through endocytosis and transcytosis leading to the
displacement of vesicle contents into the cell (for
example, dendritic cells present peptides from the
internalized exosomes on their surface [24]);
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EXOSOMES: GENERATION, STRUCTURE, TRANSPORT 165
(4) Action of exosome components after lysis in the
extracellular medium. Lack of interaction of exosomes
with membranes of target cells involves the destruc-
tion of exosomal membranes. The study of exosomes
secreted by ovarian cancer cell lines 1A9-VAS-3 (con-
trol) and 1A9-VS-1 (VEGF overexpression) con-
ducted by Taraboletti et al. [25] shows that there is a
destruction of exosomal membranes with consequent
release of the contents at acidic pH values characteristic
of the tumor microenvironment in hypoxia. This leads
to increased chemotactic effects of exosomes secreted
by 1A9-VS-1 cells. On the basis of the fact that in
hypoxia the VEGF synthesis is increased through the
activation of HIF-1, the authors assumed that high lev-
els of VEGF reflect both an increase in its synthesis at
low pH and an increase in the share of VEGF resulting
from the release of collapsing vesicles [25].
1.2. Molecular Composition of Exosomes
It is known that 1 μL of blood contains about 3 mil-
lion of exosomes. Theoretically, each exosome includes
lipids, 100 proteins and 10000 nucleotides of RNA
and DNA [26].
The lipid composition of the exosomal membrane
differs from that of the cytoplasmic membrane of a
secreting cell. In particular, Laulagnier et al. [27] have
shown that the ratio of phosphatidylcholine, phospha-
tidylethanolamines, phosphatidylinositol, phosphati-
dylserine, and sphingomyelins is more balanced in
exosomal membranes than in membranes of secreting
cells (26 : 26 : 19 : 19 : 20 and 43 : 23 : 12 : 12 : 9, respec-
tively). In addition, in the exosome membranes the
“flip-flop” transitions are more common than in
plasma membrane, and in this sense they are more
similar to the organelle membranes. This behavior of
exosomal membranes may be associated both with the
process of maturation of microvesicles and with the
lack of specific enzymes that regulate the flip-flop in
the cell membrane, such as flippase [27]. High levels
of sphingomyelin and phosphatidylinositol ensure
enhanced stability of exosomal membranes within a
physiological range of pH values of biological fluids
and protect them against proteolytic or lipolytic deg-
radation in the process of circulation [27]. It is found
that exosomal membranes contain lipid rafts enriched
with a number of proteins (tyrosine kinase Src, glyco-
sylphosphatidylinositol-containing proteins, etc.) [28].
Protein composition of exosomes is usually studied
by 2D electrophoresis, Western blotting, flow cyto-
metry, immunoelectron microscopy, and mass spec-
trometry [29]. It is noteworthy that, depending on
their origin, exosomes may carry both RNA and pro-
tein markers of secreting cells. According to the cur-
Fig. 1. Mechanism of maturation and secretion of exosomes (modified from [16]).
Ubiquitination
Early
endosome
МB
MB
Rab5
Rab4
Endosome
maturation
Rab7
Rab5
Multivesicular
body (MB)
ESCRTs
Rab27b
Slac2-b
Lysosome
Rab27a
Slp4
Endocytosis
Exosome
formation
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TAM KOV ICH et a l.
rent data, proteins of exosomes include both non-tis-
sue-specific proteins found in most of the exosomes
regardless of their origin (tetraspanins CD63, CD81
and CD9, proteins required for the transport of exo-
somes and their binding to the target cells (GTPases,
annexins, flotillin), heat shock proteins (Hsc70,
Hsp90), proteins involved in the biogenesis of mul-
tivesicular bodies (Alix, TSG101) [26]), and tissue-
specific proteins, such as MHC II exhibited on the
surface of exosomes secreted by B lymphocytes and
dendritic cells, a number of proteins specific for differ-
ent tumor cell lines, in particular, ovarian cancer
(L1CAM, CD24, EMMPRIN) [30], glioma (EGFR)
[29], breast cancer (HER2) [31], etc.
As is proposed by Mathivanan et al. [7], most fre-
quently occurring exosomal proteins can be classified
into several functional groups: enzymes (32%), in par-
ticular, the family of Rab GTPases that facilitate dock-
ing and fusion of membranes; cytoskeletal proteins
(16%), such as tubulin and actin; histocompatibility
complex proteins (8%), in particular MHC I and II;
heat shock proteins (8%), such as Hsp70, Hsp90; sig-
nal transduction proteins (4%)lactadherin and oth-
ers (Fig. 2).
The results of independent studies presented in the
database ExoCarta containing the library of lipids,
proteins, mRNAs, and microRNAs found in exo-
somes so far (www.exocarta.org) allows us to single out
25 most characteristic exosomal proteins (Table 1).
In 2014, by sequencing on the Ion Torrent PGM
platform, it was found that serum and urine exosomes
contain not only matrix RNAs and microRNAs, but
also a considerable amount of RNAs of other types,
such as tRNA, rRNA, snRNA snoRNA, piRNA and
scaRNA [3].
Most interesting are microRNAs and mRNAs
packed into exosomes. Nowadays, there are direct data
indicating that exosomes carry over 750 microRNAs
and 1500 mRNAs. Their composition in microvesicles
depends on the type of biological f luid and condition
of the body (Table 2) [3, 34, 35]. It is known that
microRNAs of exosomes are involved in the post-
transcriptional regulation of gene expression of recip-
ient cells, and the transfer of intact mRNAs by exo-
somes from donor to recipient cells can participate in
the exchange of phenotypic traits between cells, as
recipient cells receive mRNAs that are not initially
expressed in them. According to Valadi et al. [10],
human cell lines HMC-1 synthesized proteins, mRNAs
of which were received from exosomes secreted by
murine cell lines MC/9. Most typical mRNAs for
serum and urine are shown in Table 3.
According to some authors, besides RNA, exo-
somes can also transfer DNA: double-stranded
genomic DNA fragments [36] and fragmented mito-
chondrial DNA [37]. However, most researchers
believe that the presence of DNA in the composition
of exosomes is an artifact related to the contamination
of vesicle preparations by apoptotic blebs of similar size.
Indeed, a detailed overview of the exosome isolation
procedures reveals controversial issues in the studies
proposing the presence of exosomal DNA. In particu-
lar, Guescini et al. [37] found that exosomes secreted by
astrocytes and glioblastoma cells contain mitochondrial
DNA. The authors filtered the culture medium through
filters with a pore diameter of 0.22 μm, deposited the
vesicles by ultracentrifugation at 110000 g for 70 min,
washed the particles with PBS, and then isolated exo-
somes on magnetic particles carrying anti-CD9 anti-
bodies on their surface. However, since CD9 is present
not only on the surface of exosomes, but also on the
surface of apoptotic blebs [38, 39], the detected DNA
may be contained in apoptotic blebs (100–220 nm in
size) present in the vesicle preparations, rather than in
exosomes.
In 2014, Kahlert et al. [36] reported on the pres-
ence of double-stranded genomic DNA in the exoso-
mal cargo derived from the culture medium of tumor
cell lines PANC-1 and T3M-4, as well as from the
blood serum of healthy donors and patients with pan-
creatic cancer. Using Sanger sequencing, it was shown
that exosomes from the blood of oncological patients
contain mutant forms of p53 and KRAS genes of the
tumor DNA [36]. To derive exosomes, the authors
also filtered the culture medium and serum through
filters with a pore diameter of 0.22 μm and deposited
vesicles by ultracentrifugation at 150 000 g for 2 h.
Apparently, as in the previous case, the presence of
DNA in the preparations may be explained by con-
tamination with apoptotic blebs.
Indeed, the contents of exosomes reflect the cytoso-
lic composition of donor cells and it is difficult to imag-
ine the mechanism of transport of genomic/mitochon-
drial DNA to these vesicles. By treating the exosomes
of tears (up to 100 nm in size) with DNAase I, it was
found that the double-stranded genomic DNA of
Fig. 2. Functional groups of most frequently encountered
exosomal proteins.
12% 8%
12%
32%
16%
8%
8%
4%
Heat shock proteins
Tetraspanins
Enzymes
Cytoskeleton proteins
MHC proteins
Membrane proteins
Signal transduction
proteins
Other proteins
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EXOSOMES: GENERATION, STRUCTURE, TRANSPORT 167
3.9 kbp in size is localized on the outer surface of the
vesicular membrane [40]. The mechanism for the
appearance of the genomic DNA on the surface of
exosomes remains unclear. It was shown that DNA
associated with DNA-binding proteins is present on
the cell surface of primary and passaged cultures, as
well as on the surface of blood cells [41]. Taking into
account the congeniality of plasma membranes and
exosomal membranes, we can assume that exosomes
also carry DNA-binding proteins. In this case, DNA
may bind to the surface of exosomes either outside
(directly in biological fluids) or inside the cells. It can
be assumed that DNA localized on the surface of tear
exosomes comes to the endosomes from the surface of
ocular epithelial cells.
The study of the structure and composition of exo-
somes is of fundamental importance. However, cur-
rently the main focus is on the role of exosomes in the
development of malignant tumors.
1.3. Role of Exosomes in the Development
of Malignant Tumors
Cancer cells secrete exosomes affecting both the
development of the primary tumor and the distant
metastasis. It is shown that these exosomes activate
tumor growth and invasion [42], affect cell adhesion
[43], and stimulate angiogenesis [44] immunosup-
pression [45], and drug resistance [16].
Participation of exosomes in immunosuppression
is shown in many studies [22]. According to Huber
et al. [45], exosomes form the immune-privileged
conditions inside the tumor. Cancer cells produce
large amounts of exosomes carrying pro-apoptotic
molecules, such as Fas ligand and TRAIL, capable of
inducing apoptosis in activated T cells, thereby block-
ing the immune response [45, 46].
The exosomes secreted by cancer cells also contain
molecules that allow tumors to grow and metastasize
actively: ADAM10, ADAM17, and cadherin-11 [43].
The increase in the adhesion of cancer cells to the
Table 1. Most frequently encountered proteins in exosomes (according to Exocarta as of January, 2014)
Protein Identif ications
in publications
1 Heat shock protein 70 kDa 52
2CD9 50
3 Glyceraldehyde 3-phosphate dehydrogenase 48
4 Beta-actin 43
5CD63 41
6CD81 39
7Annexin A2 37
8 Enolase 1 (alpha) 36
9 Heat shock protein 90 kDa alpha (cytosolic), class A, member 1 34
10 EEF1A1 34
11 P y r uv a t e k i na s e 2 33
12 Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide 32
13 Sin t ein 32
14 Albumin 32
15 Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide 31
16 EEF2 31
17 Actin, gamma 1 31
18 Lactate dehydrogenase A 30
19 Heat shock protein 90 kDa alpha (cytosolic), class B, member 1 30
20 Aldolase A 30
21 Moesin 29
22 Annexin A5 29
23 Phosphoglycerate kinase 1 28
24 Cofilin 1 28
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endothelium and fibrinogen is also achieved by trans-
ferring integrins (such as CD41) in the composition of
exosomes secreted by platelets, which leads to an
increase in the expression of several genes involved in
angiogenesis (VEGF, IL-8, HGF) and invasion
(MT1-MMP) [16].
Webber et al. [47] have shown that a number of cul-
tures of cancer cells are able to secrete exosomes con-
taining the transforming growth factor TGF-β on their
surface. TGF-β directs the differentiation of fibro-
blasts in the pathway of myofibroblasts [47]. Exosomes
of cancer cells can carry ligands to EGF receptors
(amphiregulin, heparin-binding epidermal growth
factor, TGF-α), which then bind to the respective
receptors of target cells. The cells treated with such
exosomes have a greater invasive potential than
untreated cells [42].
Exosomes secreted by cancer cells are apparently
involved in active angiogenesis accompanying tumor
progression. Indeed, Janowska-Wieczorek et al. have
shown that exosomes secreted by cell lines of human
lung cancer (A549, CRL 2066, CRL 2062, HTB 183,
HTB 177) and Lewis carcinoma (LCC) are capable of
stimulating the expression of genes involved in angio-
genesis, such as VEGF, IL-8, and HGF [6]. Moreover,
Sheldon et al. [44] found an angiogenic factor Dll4 in
the composition of exosomes and its transport from
tumor cells U87 to endothelial recipient cells. Dll4
transport leads to changes in the cell phenotype:
human umbilical vein endothelial cells (HUVECs)
produced Dll4-containing exosomes. Introduction of
U87 cells secreting Dll4-containing exosomes into
xenograft mice led to the branching of blood vessels
and an increase in their length and size [44].
It was shown that exosomes affect chemoresistance
of malignant tumors. According to Ciravolo et al. [31],
breast cancer cell lines SKBR3 and BT474 with HER2
overexpression secrete exosomes carrying HER2 on
their surface. Secretion of these vesicles is regulated by
growth factors EGF and HRG. HER2-positive exo-
somes trap trastuzumab (a drug based on monoclonal
antibodies against HER2 and widely used for the
treatment of HER2-positive breast cancer) and thus
reduce the effectiveness of the anticancer therapy [31].
In addition to the data on the involvement of exo-
somes in the processes associated with the develop-
ment of cancer, the results showing an anti-tumor
activity of exosomes in the development of malignan-
cies were published. In particular, transport of miR-16
in the exosomes secreted by multipotent mesenchymal
stromal cells leads to a decrease in tumor angiogenesis
[48]. The presentation of antigens characteristic of
tumor in the early stages of the disease, such as HER2,
can stimulate an immune response [31], and the trans-
portation of pro-apoptotic molecules, such as Fas
ligand and TRAIL, leads to an increased apoptosis in
the tumor that may inhibit the immune response and
chemoresistance in the later stages of cancer [45, 46].
Yang et al. [2] have shown that the transfer of
miR-223 in the exosomes from IL4-induced macro-
phages in the blood of healthy donors to the recipient
cells (co-cultivated breast cancer cell lines MDA-MB-
231 and SKBR3) increases their invasion and, when
treated with an anti-miR-223 antisense oligonucleotide,
reduces the invasion (invasiveness in tested and control
samples was measured in Boyden chambers [2]).
Table 2. Characteristic exosomal microRNAs in various
biological fluids (according to [3, 32, 33])
Blood plasma Blood serum Saliva Urine
miR-22-3p miR-486 miR-22 miR-204
miR-99a-5p miR-16 miR-202 miR-30a
miR-99b-5p miR-92a miR-203 miR-10b
miR-124-3p miR-101 miR-1273-d miR-7b
miR-128 miR-107 miR-10a
miR-122 miR-26a
miR-142-3p miR-23b
miR-574 miR-99a
miR-768 miR-125
miR-21 miR-21
miR-191 miR-191
let-7a let-7a
miR-451a
miR-185
miR-205
miR-378a
miR-484
miR-145
miR-320a
miR-145
miR-223
miR-150
Table 3. Most frequently encountered mRNAs in serum
and urine exosomes (according to [3])
Blood serum Urine
LOC728819 LOC728819
PPIAL4Fv1 PPIAL4Fv1
PPIAL4Fv2 PPIAL4Fv2
TTLL2 ZNF467
FNDC5 ANO9
PRR12 CDHR2
NSMCE1 SLC39A3
CT45A4 SLC8A2
ZNF880 SMARCB1
DNAJA4 ZNF324
BIOCHEMISTRY (MOSCOW), SUPPLEMENT SERIES A: MEMBRANE AND CELL BIOLOGY Vol. 10 No. 3 2016
EXOSOMES: GENERATION, STRUCTURE, TRANSPORT 169
Apparently conflicting data on the role of exo-
somes in cancer development may be related with dif-
ferent stages of tumor growth and individual charac-
teristics of the tumor cells, as well as with insufficient
knowledge of the functions of exosomes in a healthy
body. These data require further detailed study.
1.4. Tumor Markers in Blood Exosomes
1.4.1. microRNA
It is known that microRNAs are involved in the
regulation of different physiological processes, includ-
ing cell differentiation, division, apoptosis, hemato-
poiesis, and morphogenesis of various organs [49].
The role of microRNAs in pathological processes and
the development of health disorders, including hered-
itary, viral, and neoplastic diseases, is actively studied.
By now, the panels of tissue- and tumor-specific
exosomal microRNAs, potentially significant for the
diagnosis, prognosis and evaluation of cancer therapy
effectiveness, have been identified [50–56] (Table 4).
More than 750 microRNAs contained by exosomes
are identified. The amount of microRNAs involved in
the carcinogenesis mechanisms is yet to be deter-
mined. However, it is clear that the expression profile
of tissue-specific exosomal microRNAs can be used to
develop the approaches to the early diagnosis of malig-
nant tumors.
1.4.2. Proteomic Study of Exosomes
An analysis of the proteins transported by circulat-
ing exosomes is a promising area of research of tumor-
specific markers for malignancy.
According to Pant et al., exosomal proteins repre-
sent less than 0.01% of the total plasma proteome [57].
An apparent advantage of studying the exosome pro-
teome is the possibility to remove ballast plasma pro-
teins and to increase the concentration of tumor-spe-
cific proteins, including membrane proteins.
Disadvantages include a high individual variability
of an exosomal proteome even in healthy donors, which
is noted in many papers. Comparison of proteomic pro-
files of blood plasma exosomes in 15 healthy donors
Table 4. Exosomal microRNA in biological fluids and its clinical significance in various cancer diseases
Disease Source of exosomes microRNA Clinical significance
of tumor marker
Prostate cancer Blood plasma miR-1290
miR-375
Diagnostic [50]
Breast cancer Blood serum miR-101
miR-372
miR-1373
Prognostic [51]
Ovarian cancer Ascites miR-200a
miR-200c
miR-205
Diagnostic [52]
Colorectal cancer Blood serum let-7a
miR-1229
miR-1246
miR-150
miR-21
miR-223
miR-23a
Diagnostic [53]
Lung cancer Blood serum miR-17-3p
miR-21
miR-191
miR-214
miR-146
miR-155
miR-203
miR-205
Diagnostic [54]
Blood plasma miR-378-a
miR-379
miR-139-5p
miR-100
miR-154-3p
Diagnostic [55]
170
BIOCHEMISTRY (MOSCOW), SUPPLEMENT SERIES A: MEMBRANE AND CELL BIOLOGY Vol. 10 No. 3 2016
TAM KOV ICH et a l.
showed that only ten of 109 identified proteins were
present in all samples: complement component 3
(CO3), alpha-2-macroglobulin, histidine-rich glyco-
protein (HRG), pregnancy-associated alpha-2-glyco-
protein (PZP), galectin-3-binding protein (Gal3BP),
hemoglobin (HBA), fibrinogen alpha (FIBA), alpha-
1-antichymotrypsin (AACT), clusterin (CLUS), and
ceruloplasmin (CERU) [58].
Information on the protein composition of exo-
somes is constantly updated. In particular, out of 66
proteins found in 200 9 by Looze et al. in blood plasma
exosomes, only one had been identified earlier, and all
the other proteins were detected in exosomes for the
first time [59]. In 2013, Liang et al. investigated the
exosomal proteome of cell lines OVCAR3 and
IGROV1 (ovarian cancer); 1107 of 2179 exosomal pro-
teins found by them were not represented in the Exo-
Carta database [60].
The proteome of exosomes secreted by tumor cells
may contain tumor-specific proteins: HER2 in breast
cancer cell lines [61, 62], A33 antigen in colorectal
cancer cell lines [63], and EGFR in glioma cell lines
[29]. The comparative analysis of the proteins from
exosomes secreted by cell lines OVMz and SKOV3ip
(ovarian cancer) and exosomes from ascites and blood
of patients with ovarian cancer revealed a significant
content of proteins associated with tumor develop-
ment: L1CAM, CD24, EMMPRIN [30].
In 2012, Palazzolo et al. [64] performed a pro-
teomic analysis of exosomes from the culture medium
of MDA-MB-231 cells (breast cancer) and total cell
lysate. Out of 179 identified proteins, 32 were consid-
ered characteristic of exosomes. These proteins were
divided into eight functional groups: cytoskeletal pro-
teins, programmed cell death regulators, cell cycle reg-
ulators, signaling proteins, oxidative stress response
proteins, adhesion proteins, and cell motility proteins.
Exosomal proteins included 14-3-3-ε and PDC6I, reg-
ulators of proliferation, cell cycle, and programmed cell
death. It was proposed that the transfer to non-trans-
formed recipient cells by these proteins leads to subse-
quent malignant transformation of recipient cells [64].
The results of various proteomic studies of exosomes
allow determining the candidates for the role of the
protein oncomarkers (Table 5) [61–70].
Rapidly developing proteomic studies of exosomes
extend the range of these markers and help on specify-
ing their combinations that are most valuable for diag-
nostic purposes. However, for the widespread use of
these markers in healthcare, further extensive investi-
gations are needed to substantiate the diagnostic value
of new proteomic exosomal markers.
Table 5. Promising protein tumor markers in exosomes of urine and culture media
Disease Source of exosomes Protein Reference
Breast cancer SW620
MDA-MB-231
Galectin-1
HER2
14-3-3-ε
PDC6I
[65]
[64]
Lung cancer M-BE, NCIH226Br Cathepsin D
LDHB
[66]
[67]
Colorectal cancer HT-29 Annexins
Galectin-4
Tetraspan in- 8
Carcinoembryonic antigen
Epidermal growth factor receptor
Heat shock protein 90
[63]
LM 1215 Heat s hock pro tein 70
TSG10
Bladder cancer HT1376, RT112, T24 Glyceraldehyde-3-phosphate dehydrogenase
Integrin-α3
Integrin-α6
Annexin A4
[68]
Urine Retinoic acid-induced protein 3
GsGTP binding protein, α subunit
[69]
Prostate cancer Urine Prostatic acid phosphatase
Lactotransferrin
Dipeptidyl peptidase-4
Galectin-3
Kallikrein-2
Kallikrein-11
[70]
BIOCHEMISTRY (MOSCOW), SUPPLEMENT SERIES A: MEMBRANE AND CELL BIOLOGY Vol. 10 No. 3 2016
EXOSOMES: GENERATION, STRUCTURE, TRANSPORT 171
CONCLUSIONS
Exosomes are membrane structures of 30 to 100 nm
in size produced by cells and carrying proteins, RNA,
and DNA to neighboring cells and distant organs and
tissues via body fluids (blood, urine, lymph, etc.).
The knowledge of the exosome functions and con-
tent is actively updated. Attempts have been made to
identify and systematize marker proteins and nucleic
acids transported by exosomes both in normal condi-
tions and in various malignancies.
Minimally invasive venous blood sampling is an alter-
native to traditional biopsy. That is why over the past
5 years several commercial companies (Exosome Diag-
nostics, Exosome Sciences, Caris, HansaBioMed) have
been developing diagnostic tools based on the RNA
and protein markers carried by exosomes. In 2010,
Caris Life Sciences introduced the Carisome® Pros-
tate cMV 1.0 test for the prostate cancer diagnosis and
in March 2013, HansaBioMed received a patent for
the prostate cancer diagnostic method based on the
proteomic analysis of exosomes using the Exotest Kit.
Thus, the search for proteomic and microRNA
tumor markers in the exosomes circulating in the
blood of oncological patients is a crucial task for
molecular biology, and the development of non-inva-
sive sensitive cancer diagnostic tools is in high demand
among healthcare professionals.
ACKNOWLEDGMENTS
The work was supported by the Integration project
of the National Academy of Sciences of Belarus and
the Siberian Branch of the Russian Academy of Sci-
ences Analysis of circulating blood exosomes under nor-
mal conditions and in breast cancer for 2015–2017
(project no. M15CO-025).
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Translated by E. Ivanova
... The ratio of lipids in the exosomal membrane differs slightly from the composition of these lipids in the plasma membrane. In addition, exosome membrane "flip-flop" transitions are more common than in the plasma membrane due to the lack of flippases [31]. Consequently, some EVs, such as apoptotic exosome-like vesicles [32] but also exosomes secreted by cancer cells, can expose phosphatidylserine (PS) on the surface [24,33,34]. ...
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... They are composed of a certain number of specific proteins, notably the tetraspanin family (CD81, CD82, CD9 and CD63, flotillin, TSG101, and Alix) and heat choc proteins (HSP90, HSP70, HSPA5, CCT2, and HSP60) (Théry et al., 2009;AYokoi et al., 2015;Ciardiello et al., 2016;Ha et al., 2016). Exosomal membranes are composed of sphingomyelin, cholesterol, phosphatidylinositol, ceramide, phosphatidylethanolamine, phosphatidylserine, etc. (Tamkovich et al., 2016). ...
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... EVs contain surface receptors, membrane and soluble proteins, lipids, ribonucleic acids (mRNA, micro-RNA, tRNA, rRNA, small nucleolar RNA, small circular nucleolar RNA, piRNA, scaRNA, viral RNA, Y RNA and long non-coding RNA), [186][187][188][189] and even genomic and mitochondrial DNAs. 189,190 The native cargo delivery capacity of EVs has been exploited for use as drug delivery vehicles, as they are immune-compatible, noncytotoxic and non-mutagenic compared with existing viral or cellularbased therapies. 176 EVs are highly dependent on the origin and functional status of the parent cell since they reflect their phenotypic state. ...
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The focus of this review is to provide an update on the progress of microRNAs (miRNAs) as potential biomarkers for lung cancer. miRNAs are single-stranded, small non-coding RNAs that regulate gene expression and show tissue-specific signatures. Accumulating evidence indicates that miRNA expression patterns represent the in vivo status in physiology and disease. Moreover, miRNAs are stable in serum and other clinically convenient and available tissue sources, so they are being developed as biomarkers for cancer and other diseases. Cancer is currently the primary driver of the field, but miRNA biomarkers are being developed for many other diseases such as cardiovascular and central nervous system diseases. Here, we examine the framework and scope of the miRNA landscape as it specifically relates to the translation of miRNA expression patterns/signatures into biomarkers for developing diagnostics for lung cancer. We focus on examining tumor cytosol miRNAs, fluid miRNAs, and exosome miRNAs in lung cancer, the connections among these miRNAs, and the potential of miRNA biomarkers for the development of diagnostics. In lung cancer, miRNAs have been studied in both cell populations and in the circulation. However, a major challenge is to develop biomarkers to monitor cancer development and to identify circulating miRNAs that are linked to cancer stage. Importantly, the fact that miRNAs can be successfully harvested from biological fluids allows for the development of biofluid biopsies, in which miRNAs as circulating biomarkers can be captured and analyzed ex vivo. Our hope is that these minimally invasive entities provide a window to the in vivo milieu of the patients without the need for costly, complex invasive procedures, rapidly moving miRNAs from research to the clinic.
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Exosomes are tiny vesicles (30-150 nm) constantly secreted by all healthy and abnormal cells, and found in abundance in all body fluids. These vesicles, loaded with unique RNA and protein cargo, have a wide range of biological functions, including cell-to-cell communication and signalling. As such, exosomes hold tremendous potential as biomarkers and could lead to the development of minimally invasive diagnostics and next generation therapies within the next few years. Here, we describe the strategies for isolation of exosomes from human blood serum and urine, characterization of their RNA cargo by sequencing, and present the initial data on exosome labelling and uptake tracing in a cell culture model. The value of exosomes for clinical applications is discussed with an emphasis on their potential for diagnosing and treating neurodegenerative diseases and brain cancer.
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Background: Extracellular microRNAs (miRNAs) embedded in circulating exosomes may serves as prognostic biomarkers in cancer. Objective: To identify and evaluate plasma exosomal miRNAs for prognosis in castration-resis- tant prostate cancer (CRPC). Design, setting, and participants: RNA sequencing was performed to identify candidate exosomal miRNAs associated with overall survival in a screening cohort of 23 CRPC patients. Candidate miRNAs were further evaluated for prognosis using quantitative real-time polymerase chain reaction in a follow-up cohort of 100 CRPC patients. Outcome measurements and statistical analysis: Cox regression and Kaplan-Meier survival analyses were used to evaluate survival association using candidate miRNAs along with clinical prognostic factors. Results and limitations: RNA sequencing in screening cohort generated approximately 6.80 million mappable reads per patient. Of those with normalized read counts �5, 43% were mapped to miRNAs for a total of 375 known and 57 novel miRNAs. Cox regression analysis identified an association of miR-1290, -1246, and -375 with overall survival (false discover rate < 0.05). Of those, higher levels of miR-1290 and -375 were significantly associated with poor overall survival ( p < 0.004) in the follow-up cohort. Incorporation of miR-1290/-375 into putative clinical prognos- tic factors-based models in CRPC stage significantly improved predictive performance with a time- dependent area under the curve increase from 0.66 to 0.73 ( p = 6.57 � 10�6). Conclusions: Plasma exosomal miR-1290 and miR-375 are promising prognostic biomarkers for CRPC patients. Prospective validation is needed for further evaluation of these candidate miRNAs. Patient summary: In this study, we evaluated whether small RNAs circulating in blood could be used to predict clinical outcomes in late-stage prostate cancer patients. We identified two blood- based small RNAs whose levels showed significant association with survival. Our results warrant further investigation because the noninvasive blood-based test has great potential in the management of late-stage prostate cancer.
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Objective: In patients with oral squamous cell carcinoma, a high proportion of T cells in the tumor undergo apoptosis, which correlates with Fas ligand (FasL) expression on tumor cells. The present study was done to identify mechanisms responsible for apoptosis of T cells seen in the peripheral circulation of these patients. Methods: Sera of 27 patients, normal donor sera, and supernatants of cultured normal or tumor cells were fractionated by size exclusion chromatography and ultracentrifugation to isolate microvesicles. The presence of microvesicle-associated FasL was studied by Western blots, blocking with anti-Fas reagents, and immunoelectron microscopy. Biological activities of microvesicles were tested including the ability to induce apoptosis of Jurkat and T-cell blasts. Semiquantitative analysis of FasL in microvesicles was correlated with caspase-3 activity, DNA fragmentation, cytochrome c release, loss of mitochondrial membrane potential, and TCR-ζ chain expression in lymphocytes. Results: FasL-positive (FasL+) microvesicles were detected in sera of 21 of 27 patients. Microvesicles contained 42 kDa FasL. These microvesicles induced caspase-3 cleavage, cytochrome c release, loss of mitochondrial membrane potential, and reduced TCR-ζ chain expression in target lymphocytes. Biological activity of the FasL+ microvesicles was partially blocked by ZB4 anti-Fas monoclonal antibody. Microvesicle-associated FasL levels correlated with the patients' tumor burden and nodal involvement. Conclusion: Sera of patients with active oral squamous cell carcinoma contain FasL+ microvesicles, which induce the receptor and mitochondrial apoptotic pathways in Jurkat and activated T cells.