A comprehensive overview of exosomes in ovarian cancer: emerging biomarkers and therapeutic strategies
Cheng et al. Journal of Ovarian Research
A comprehensive overview of exosomes in ovarian cancer: emerging biomarkers and therapeutic strategies
Lin Cheng 0
Shuying Wu 0
Kun Zhang 0
Yun'an Qing 0
Tianmin Xu 0
0 The Second Hospital of Jilin University , Jilin Changchun 130041 , People's Republic of China
Exosomes are nanoparticles(40-100 nm) secreted by most cells in the body, which can be isolated from several types of extracellular fluids. It has been shown that exosomes play a key role in intercellular communication and in transportation of genetic information. Emerging evidence shows that exosomes are mediators of metastasis in tumour cells, stromal cells and the extracellular matrix component through the shuttling of cargo, such as proteins, lipids, RNAs, double-stranded DNAs, non-transcribed RNAs, and microRNAs. This phenomenon has been indicated in both tumourigenesis and drug resistance. In this review, we introduce new methods of exosome extraction, focusing on the emerging role of exosomes in ovarian cancer, and discuss their potential clinical applications.
Exosomes; Ovarian cancer; Protein; RNA; DNA
Exosomes were first used in 1981 to describe exfoliated
vesicles with 5′-nucleotidase activity [
]. A few years
later, Stahl’s group discovered externalized vesicles,
which were thought to discard unwanted transferrin in
maturing sheep reticulocytes [
]. At present, exosomes
specifically refer to nanoparticles(40-100 nm) and are
classified as endogenous cellular components that
originate from multivesicular bodies(MVBs), which form by
the inward budding and fission of late endosomes.
Little progress was made regarding exosomes until
Raposo’s group [
] observed that B lymphocytes stimulate
T cells proliferation by secreting exosomes containing
functional MHCI, MHCII and T cells costimulatory
molecules, which were reported to suppress tumour growth.
Although the biological functions are not well-defined,
exosomes are known to exist in almost all types of
extracellular fluids (blood, urine, amniotic fluid, saliva, ascites,
milk, seminal fluid and cerebrospinal fluid). They also
carry many bioactive molecules, which suggests that the
secretion of exosomes is a general cellular function.
Therefore, exosomes play a significant role in intercellular
communication by transferring both proteomic and
genomic materials between cells.
Evidence shows that exosomes are released more
vigorously in pathological conditions. They are released by
a variety of tumour cells, and are present in large
numbers in specimens from patients with different types of
cancer, as well as cancer cell supernatants [
derived from tumour cells were also found to express
some tumour special factors which many be implicated
in clinical applications for the diagnosis, prognosis and
potential treatment of certain cancers [
]. For example,
exosomes derived from pancreatic carcinoma which
contain elevated levels of a specific proteoglycan, may
serve as potential non-invasive diagnostic biomarkers to
detect early stages of cancer [
Ovarian cancer(OvCa) is among the most common
types of cancer and is the leading cause of death from
gynaecological malignancies in the world [
]. More than
half of OvCa patients are in an advanced stage when
they see their doctors. The low survival rate and poor
quality of life for patients with OvCa is in part due to
the lack of early diagnostic methods and high
chemoresistance rate. Therefore, it is critically emerging to to
further understand the mechanisms of OvCa
pathophysiology in order to uncover more precise clinical
applications in the diagnosis, prognosis and treatment of
Exosome research has rapidly expanded over the last
decade. For example, it has been reported that malignant
ascites-derived exosomes of OvCa might augment
tumour invasion [
]. We believe the exosomes has
strong therapeutic potential for the diagnosis and
treatment of OvCa. Therefore, it is necessary to educate
clinicians and researchers in the field of Ovca in regards to
exosomes. The main objective of this review is to
describe recent progress in exosome research, especially
therapeutically in the field of Ovca, focusing on the
potential role of exosomes as novel biomarkers, as well as
to introduce new methods of exosome extraction.
Characteristics of Exosomes
Exosomes typically show a “cup-shaped” or “saucer-like”
morphology when analysed by electron microscopy [
In addition, they can float at 1.1–1.18 g/ml in sucrose
density gradient [
]. The surface of exosomes is
characterized by the presence of multiple families of proteins,
such as tetraspanins (CD63, CD81, CD9), heat shock
proteins (Hsc70), lysosomal proteins (Lamp2b) and
fusion proteins (CD9, flotillin, Annexin) [
tetraspanins have been used as exosome markers to
distinguish them from microvesicles, apoptotic bodies
and other vesicles. However, a precise exosome-specific
biomarker has not yet been discovered [
also contain several types of bio-active molecules, such
as proteins, lipids, mRNAs, microRNAs (miRNAs), long
non-coding RNAs (lncRNAs), genomic DNA, cDNA,
and mitochondrial DNA (mtDNA) [
]. For a more
extensive discussion on the molecular cargos of exosomes,
the reader should refer to ExoCarta
(http://www.exocarta.org), an exosome database, providing the exosome
contents identified in multiple organisms [
]. The current
version contains 41,860 proteins entries, 4946 RNA entries
and 1116 lipid entries from 286 studies (Fig. 1).
Ovarian cancer-derived Exosomal cargos and their role as potential biomarkers
Compared with conventional cancer biomarkers,
exosomal cargos have characteristic features that differentiate
them from non-cancer exosomes. They have similar, and
sometimes higher, that can be specificity and sensitivity
attributed to their origin. They have excellent stability,
and are detected in all body fluids, particularly in
peripheral blood. These features make exosomes a potentially
ideal biomarker of cancer [
Many different molecular cargos identified in
OvCaderived exosomes have drawn much attention due to
their large potential in (i) early diagnosis, (ii) prognosis,
(iii) drug resistance, and (iv) targeted therapy of OvCa.
To date, over 2000 species of protein have been
identified from OvCa-derived exosomes according to
ExoCarta. Their involvement in tumour progression and
metastasis has been reported in many of these identified
proteins, including membrane proteins (Alix, TSG 101),
and tetraspanins (CD24, CD44, CD63, CD37, CD53,
CD81), as well as enzymes (phosphate isomerase,
peroxiredoxin, gelatinolytic enzymes, aldehyde reductase).
Moreover, a study by Liang et al. illustrated that
OvCaderived exosome proteins were highly enriched in signal
pathways associated with carcinogenesis. They found
that a subset of proteins overexpressed in ovarian cancer
tissue were present in the exosomal protein list,
including epithelial cell surface antigen (EpCAM), proliferation
cell nuclear antigen (PCNA), tubulin beta-3 chain
(TUBB3), epidermal growth factor receptor (EGFR),
apolipoprotein E (APOE), claudin 3 (CLDN3), fatty acid
synthase (FASN), ERBB2, and L1CAM (CD171). These
protein may also be a source of diagnostic markers and
targets for therapeutic methods for Ovca [
them, EpCAM has been extensively studied and, is used
as a biomarker or prognostic factor of many cancers. For
example, Huang et al. demonstrated an intricate
relationship between EpCAM-regulated transcription and
altered biophysical properties of cells that promote EMT
in advanced endometrial cancer [
]. However, there are
drawbacks to the use of EpCAM as an OVCA
biomarker. For example, Shen’s group compared the
characteristics of exosomes derived from human ovarian
epithelial cells (HOSEPiC) and three OvCa cell lines
(OVCAR3, IGROV1, and ES-2), and found that the
labeled rates by anti-EpCAM antibodies were 16.4, 23.7,
15.7, and 18.5%, respectively suggesting that EpCAM
may not be an appropriate marker for detecting early
stages of OvCa [
]. The negative outcome may have
been due to the fact that EpCAM can be cleaved from
exosomes via serum metalloproteinase [
]. While some
articles suggest an outlet for combining EpCAM with
CD24 to detect Ovca-derived circulation exosomes, large
scale clinical trials is needed to verify this hypothesis. In
terms of the protein CLDN3, Morin’s group found that
CLDN3 was not likely to represent a useful biomarker
compared to CLDN4, which had a sensitivity of 51%
(32/63) and specificity of 98% (49/50) for the detection
of OvCa [
Exosomal proteins also have the potential to serve as
tumour staging and prognostic markers for response to
treatment of ovarian cancer. Marta Szajnik et al. found
that plasma from OvCa patients contained higher levels
of exosomal proteins compared to plasma from patients
with benign tumors or NCs (healthy controls).
Furthermore, the exosomal protein content was significantly
higher in advanced stages than that of early stages. In
further studies, they found that TGF-β1 and MAGE3/6
could distinguish OvCa patients from those with benign
tumours and NC. Moreover, the exosomal protein levels
variably changed and were correlated with chemotherapy
Early detection of the resistance to platinum-based
therapy is critical for improving the treatment of Ovca.
Increased expression of annexin A3 is a mechanism for
platinum resistance in ovarian cancer, which is
associated with exocytosis and the release of exosomes [
For therapy potential, the exosomal ADAM15
ectodomain effectively inhibits cancer progression by blocking
the integrin-mediated MEK/ERK signalling pathway,
providing insight into the functional significance of
exosomes that generate tumor-inhibitory factors [
MicroRNAs (miRNA), small (22–25 nucleotides in
length) noncoding RNAs, inhibit gene expression
posttranscriptionally by binding to their 3′untranslated
region, leading to the suppression of protein expression or
]. Several studies have accounted for
theirThey are involvinvolvemented in carcinogenesis, the
cell cycle, apoptosis, proliferation, invasion, metastasis,
and chemoresistance [
For instance, the levels of exosomal miR-200b and
miR-200c were found to be higher in patients with FIGO
stage III–IV, including lymph node metastasis compared
to patients with FIGO stages I–II, suggesting that these
microRNAs may be involved in tumour progression
]. Exosomal miR-21-3p can also contribute to
cisplatin resistance by potentially targeting the NAV3 gene.
MiR-21 in exosomes and tissue lysates isolated from
cancer-associated adipocytes (CAAs) and fibroblasts
(CAFs) are transferred from CAAs or CAFs to cancer
cells, where they suppress ovarian cancer apoptosis and
confer chemoresistance by binding to the direct novel
target, APAF1 [
Furthermore, miR-222-3p is enriched in OvCa-derived
exosomes, and can be transferred to macrophages to
induce a tumour-associated macrophage (TAM)-like
phenotype with SOCS3/STAT3 pathway involvement,
potentially facilitating the progression of cancer [
From a prognostic standpoint, the exosomal miRNAs
miR-21, miR-103, miR-141, miR-203, miR-205, miR-214,
miR-373, and miR-200a-c are associated with a poorer
prognosis. For more information on exosomal miRNAs
in OvCa, readers may refer to Table 1.
Although exosomes have been proposed as vehicles
for microRNA (miRNA)-based intercellular
communication and sources of miRNA biomarkers, and providers of
information on aberrant signalling pathways, some
disputes remain. By studying cancer-associated extracellular
miRNAs in patient blood samples, John R. Chevillet et
al. found that exosome fractions contained a small
minority of the miRNA content of plasma. Their data
suggests that most individual exosomes in standard
preparations do not carry biologically significant
numbers of miRNAs and are, therefore, unlikely to be
functional independently vehicles for miRNA-based
With the exception of proteins and miRNAs, several
bio-active molecules such as phosphatidyl-serine (PS),
DNAs, glycans, and glycoprotein [
], have been
reported to play an important role in exosome
internalization, signal recognition, and novel vaccines. Early
evidence suggests that uptake of OvCa
derivedexosomes by NK cells requires PS at the exosomal
surface, but the presence of PS is not sufficient [
latest study by Jayanthi Lea et al. also provides proof of
concept data supporting the high diagnostic power of PS
detection in the blood of women with suspected ovarian
]. Exosomes were found to contain
complex glycans of the di-, tri-, and tetraantennary type
with or without proximal fucose, as well as high levels of
mannose glycans. The sialoglycoprotein
galectin-3binding protein (LGALS3BP) was found to be strongly
enriched in exosomes, making it a potential marker for
]. Scientist have highlighted the
translational value of exosomal DNA (exoDNA) in
tumourderived exosomes due to its potential usefulness as a
circulating biomarker for the early detection of cancer and
metastasis. ExoDNA represents the entire genome and
reflects the mutational status of parental tumour cells
providing the following advantages: (i) its protection,
and, thus inherent stability within exosomes [
enriched tumour-derived exosomes found in complex
plasma samples [
Roles in ovarian cancer progression and metastasis
Unlike most solid tumours, OvCa rarely disseminates
through vasculature, but has a high propensity to
metastasize within the peritoneum. This allows tumour
cells to directly encounter human peritoneal mesothelial
cells (HPMC) in the initial step of metastasis. In this
seemingly “Pandora’s box”, exosomes appear to be a new
and powerful signal mediator by cleaning the mesothelial
barrier for improved cancer cell invasion [
instance, OvCa derived exosomes contain gelatinolytic
enzymes, the L1 adhesion molecule (CD171) and other
cell adhesion molecules . Koji Nakamura et al.
observed that HPMCs underwent a change in cellular
morphology to a mesenchymal, spindle phenotype when
they internalized OvCa-derived exosomes [
formation of malignant ascites is often observed in advanced
OvCa patients. Several studies revealed that malignant
ascites-derived exosomes contain multiple cargos, such as
L1CAM, CD24, ADAM10, Claudin-4 and EMMPRIN
which play a critical role in tumour progression [
In recent years, the tumour microenvironment,
including stromal cells, endothelial cells, infiltrating immune
cells, and the extracellular matrix [
] has been
recognized as having a critical role in OvCa metastasis.
Exosomes appear to be a novel and significant signalling
factor in the tumour microenvironment. In OvCa, high
LIN28A expressing OvCa cells derived from exosomes
induce EMT-related (epithelial to mesenchymale
transition) gene expression, invasion and migration when
taken up by non-metastatic target cells [
]. In regards
to immune cells, malignant ascites-derived exosomes
may induce apoptosis of the precursors of DCs
(peripheral blood lymphocytes) and PBMCs (dendritic cells)
], and may contain immunosuppressive factors, such
as TGF-β1 and IL-10, which indicate that exosomes may
be involved in the support of immune evasion in OvCa
]. Further study by Alireza Labani-Motlagh et al.
revealed two cytotoxic pathways of importance for
anticancer immunity: the NKG2D receptor-ligand pathway
and the DNAM-1-PVR/nectin-2 pathway [
addition, exosomes may induce adipose-derived
mesenchymal stem cells (ADSCs) to acquire a
tumoursupporting myofibroblast phenotype and functionality.
They also may activate macrophages to possess a
tumour-associated macrophage (TAM)-like phenotype,
which could facilitate the progression of cancer [
More recently, it was reported that CAA
(cancer-associated adipocytes) and CAF (cancer-associated fibroblasts)
derived exosomes could increase chemoresistance by
transporting microRNAs to surrounding cancer cells
. Miharu Kobayashi et al. found that significantly less
let-7 family miRNA was expressed in high invasive cells
(SKOV-3), but was highly expressed in exosomes.
Within a tumour, high invasive tumour cell derived
exosomes signal low invasive tumor cells and low invasive
tumour cell derived-exosomes can signal high invasive
tumour cells to increase invasion of recipient cells [
This suggests that the exosomes plays an important role
in the tumour microenvironment and OvCa metastasis.
Roles in ovarian cancer therapy
Exosomes are known to contribute to
immunosuppression and tumour immunity escapes [
]. A previous
BM-MSCs, SR4987 [
BM-MSCs, Placenta and Glioma
cord derived MSCs [
Adipose tissue -derived Hepatocellular
study indicated that depletion of peritoneal macrophages
by clodronate could reduce ovarian tumour progression
in vivo [
]. Inspired by the idea that macrophages may
serve as attractive targets for therapeutic intervention,
Yuan Hu et al. observed that exosomes derived from
TWEAK-stimulated macrophages (TMs) could be
internalized by OvCa cells and inhibit metastasis through the
shuttling of miR-7, subsequently leading to the
downregulation of EGFR/AKT/ERK1/2 signalling pathways [
Some believe that exosomes contain cell surface cancer
antigens, suggesting a potential for therapeutic
approaches in cancer vaccination [
]. More recently, a
plateau phenomenon has been reported, suggesting that
release of exosomes is regulated by a feedback
mechanism regardless of tissue specificity. This phenomenon
indicates that this feedback mechanism can be inhibited
by various exosomes, providing a potential therapeutic
approach to control the release of exosomes from OvCa
Exosome-facilitated drug delivery
Exosomes contain the following advantages when it
comes to their potential as cell-based therapeutic
products: (i) Therapeutic biological materials, such as
], miRNAs [
] and siRNAs [
could be loaded into exosomes. For example, a recent
study showed that human adipose mesenchymal stem
cell-derived exosomal-miRNAs are critical factors for
inducing antiproliferation signalling to A2780 and SKOV3
ovarian cancer cells ; (ii) Exosomes are taken up by
acceptor cells, through which cellular processes can be
]; (iii) Exosomes possess a high
histocompatibility and do not induce immunological rejection. To
achieve cell-specific targeting drug delivery, several
studies have tested donor cells, loading methods and
theraputic cargos of exosomes (Table 2).
Methods of exosome extraction
There are various validated methods for exosome
extraction, including ultracentrifugation, ultrafiltration,
chromatography, polymer-based precipitation and affinity
capture on antibody-coupled magnetic beads, which
have been described exhaustively by Antes’s group [
In this section, we introduce new methods of exosome
extraction (Fig. 2).
Microfluidic technology has been previously shown to
have unique advantages in genomics, proteomic analysis
and quantitative biology. This method can also be used
to separate exosomes from several body fluids. Mohsen
] categorized the microfluidic systems
developed for the detection/characterization exosomes into
six groups: (i) electrochemical [
potential , (iii) mechanical [
], (v) optical, and (vi) non-optical based.
Silica nanostructured platform for affinity capture
Similar to other affinity capture methods, the silica
nanotechnology platform selectively captures specific
exosomes based upon certain surface markers. This
approach employs silica nanosprings, made of silica glass
with unique physical and chemical properties which
consist of a wide gravimetric surface area tethered to Si
wafer substrates, creating a broad space for affinity
], as shown in Fig. 3 of how this works. Norton’s
group also reported that the biotin binding capacity for
the avidin-nanosprings was 4-fold greater than
commercially available streptavidin-coated silica beads under
identical conditions [
]. This method overcomes the
shortcomings of conventional immuno-affinity
purification, and makes large scale EV purification possible.
Aptasensor based on DNA-capped single-walled carbon Nanotube
Recently, Chinese scientists demonstrated a visible and
simple method for the detection of exosomes by
integrating DNA-capped single-walled carbon nanotubes
]. This new method consists of two main elements:
The first is s-SWCNTs, a tubular nanomaterial rich in
carboxyl groups and water solubility. The other is
aptamers, specific to CD63 and absorbed onto the surface of
s-SWCNTs, which can efficiently catalyze
H2O2mediated oxidation of 3, 3′, 5, 5′-tetramethylbenzidine
(TMB) and lead to a change from colourless to blue in
solution. Therefore, the colour of the solution represents
the quantity of exosomes and can be observed by the
naked eye or monitored by spectrometry. Moreover, this
proposed colorimetric aptasensor is universally
applicable for the detection of other targets by simply
changing the aptamer.
To date, microfluidic techniques show higher recovery
and purity of exosomes compared to conventional
methods. However, improving the throughput of
onchip isolation technologies while retaining their high
particle sorting sensitivity remains an ongoing
challenge in the field.
Thermo-acoustophoresis adds a temperature dimension
to conventional methods, integrating a concurrent
application of piezoelectric and thermoelectric effects on a
single-stream flow in a microchannel to help with the
separation and identification of extracellular vesicles,
such as exosomes and microvesicles. As vesicles in a
predetermined temperature pass through the ultrasonic
radiation field, they possess a stiffness-dependent force,
leading to their migration towards either the node or the
anti-nodes, which is determined by the acoustic contrast
factor(Φ). For a system formed by two different vesicles
with distinct membrane stiffness values, the change in Φ
brings out a temperature “window” in which opposite Φ
signs exist. As ultrasonic standing waves are able to
accommodate subtle differences, if the preinstall
temperature is set to that window, vesicles are then
separated at efficiencies exceeding 95% [
A lipid nanoprobe (LNP) system for the rapid isolation
of extracellular vesicles (EVs) including exosomes has
been reported recently. The approach includes a
labelled lipid bilayer with biotin-tagged 1, and
(ethylene glycol) (DSPE–PEG). The labelled EVs are
collected by NeutrAvidin (NA)-coated magnetic
submicrometre particles (MMPs), which shortens the
isolation procedure from hours to 15 min and does
not require large and expensive equipment [
method is also highly flexible and can be adopted for
analyses of various downstream bioactive substances,
such as DNA, RNA and proteins.
Conclusion and future perspective
Exosomes play a crucial role in multiple
pathophysiological procedures, such as inflammatory response,
immunoregulation, tumorgenesis, tumor invasion and
metastasis. Many new findings and new hypotheses
suggest the need for further research, as exosomes have
great diagnostics and therapeutic potential, yet many
questions remain. New methods have been explored to
isolate exosomes efficiently and purely, and future
studies will need to make comprehensive comparisons of
these methods. Multi-Omics, such as the
highthroughput expression analysis technique, will reveal
critical molecules and mechanisms for the packaging of
Exosomes have shown immense potential in the early
diagnosis, drug selection, prognostic evaluation, and
target therapies involved in OvCa. Nonetheless, limitations
of reported studies include cell lines and a sample size.
Therefore, there is a critical need for multiple,
largescale clinical studies regarding the involvement of
exosomes in OvCa.
ADSC: Adipose-derived mesenchymal stem cells; CAAs: Cancer-associated
adipocytes; CAFs: Cancer-associated fibroblasts; EMT: Epithelial to
mesenchymale transition; EVs: Extracellular vesicles; exoDNA: Exosomal DNA;
HPMC: Human peritoneal mesothelial cells; lncRNAs: Long non-coding RNAs;
LNP: Lipid nanoprobe; miRNAs: microRNAs; MVBs: Multivesicular bodies;
OvCa: Ovarian cancer; PS: Phosphatidyl-serine; TAM: Tumor-associated
This study was supported by grants from Jilin Province Development and
Reform Commission Funds(2014G073,2016C046–2) and Jilin Province
Department of Education Fund 2016(488).
Availability of data and materials
LC and YAQ drafted the manuscript. SYW and KZ revised the manuscript.
TMX designed the topic and revised the manuscript. All authors read and
approved the final manuscript.
Ethics approval and consent to participate
Consent for publication
I confirm that all authors of the manuscript have agreed to its content and
the order of authors listed in the manuscript has been approved by all of us.
The authors declare that they have no competing interests.
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