Towards routine detection of extracellular vesicles in clinical samples
Master thesis
1
Malgorzata Krawczyk-Durka
Supervisors:
Dr. Roy van der Meel,
Dr. Raymond M. Schiffelers
Department of Clinical Chemistry and Haematology,
University Medical Center Utrecht,
Utrecht, The Netherlands
Table of contents
Abstract ......................................................................................................................................... 4
Introduction .................................................................................................................................. 5
Extracellular vesicle classification ................................................................................................. 5
Functions of extracellular vesicles ................................................................................................ 7
Extracellular vesicles as therapeutic targets and drug delivery systems ...................................... 8
Application of extracellular vesicles in clinical diagnostics ........................................................... 9
Standardization of protocols ....................................................................................................... 10
Detection techniques .................................................................................................................. 12
Conclusions and future perspectives .......................................................................................... 15
2
Acknowledgments ....................................................................................................................... 16
References................................................................................................................................... 17
3
Abstract
Extracellular vesicles (EVs) are cell-derived particles secreted by both eukaryotic and
prokaryotic cells, as well as in the majority, if not all, body fluids. Such vesicles, initially
described as “platelet dust”, have been extensively investigated in the last few decades. Based
on their biogenesis, four main types of EVs can be distinguished: exosomes, microvesicles,
retrovirus-like particles (RLPs) and apoptotic bodies. EVs are key mediators of intercellular
communication and involved in fundamental biological processes such as blood coagulation,
tissue regeneration, stem cell maintenance and immune response modulation. Furthermore,
they have been implicated in the pathology underlying several diseases. So far, EVs have been
linked to cancer development, infectious and neurodegenerative diseases as well as the spread
of viruses and other pathogens. These vesicles are therefore gaining much attention as
potential therapeutic targets and source of clinically relevant and noninvasive biomarkers that
enable monitoring of physiology and diagnosis of several diseases. Despite the growing
interest in EVs, a number of challenges persist regarding the application of EVs for clinical
diagnostics. Standardized protocols for sample collection, analysis and processing are essential
for study-to-study comparison. So far, a number of techniques to analyze EVs are utilized in
mostly research laboratories. Depending on the information required, there are several
detection options including EV number and size distribution, molecular surface markers and
RNA content. Here, the focus is on recent findings in understanding EV biology, functions,
detection methods as well as their potential in clinical diagnostics.
Abbreviations
AAV (adeno-associated viruses), ALT (alanine aminotransferase), BBB (blood-brain barrier),
CCR5 (C-C chemokine receptor type 5), CTAD (sodium citrate containing theophylline,
adenosine and dipyridamol), DC (dendritic cell), DILI (drug-induced liver injury), DMR
(diagnostic magnetic resonance), EBV (Epstein-Barr virus) ELISA (enzyme-linked
immunosorbent assay), ESCRTs (endosomal sorting complexes required for transport), EV
(extracellular vesicle), FSC (forward scatter), GBM (glioblastoma multiforme), IL-8 (interleukin
8), I/R (ischemia/reperfusion), ISEV (International Society for Extracellular Vesicles), ILV
(intraluminal vesicle), ISTH (International Society on Thrombosis and Haemostasis), μNMR
(micro nuclear magnetic resonance), MCS (Multipotent stem cells), MMP (metalloproteinase
proteins), MNP (magnetic nanoparticle), MVB (multivesicular body), NK (natural killer), NMR
(nuclear magnetic resonance), NTA (nanoparticle tracking analysis), PS (phosphatydyloserine)
RLP (retrovirus- like particle) ROS (reactive oxygen species), qPCR (qualitative polymerase
chain reaction), RPS (resistive pulse sensing), SSC (side scatter), VEGF (vascular endothelial
growth factor)
4
Introduction
Cells can communicate via secretion of soluble factors or by direct interaction.
Recently, intercellular communication through extracellular vesicles (EVs) has gained
considerable attention.1–3 Such EVs were initially discovered in 1946 by Chargaff and West.4
Their studies suggested that cell free plasma contains a subcellular factor that promotes
clotting of blood. Twenty years later in 1967, Wolf et al. described EVs as “platelet dust”; a
fraction of plasma that possesses coagulant properties upon storage of blood platelets.5 These
vesicles were characterized by a size between 20 and 50 nm and a buoyant density of 1.020 to
1.025 g/ml.5 Since then, vesicles have been isolated from both eukaryotic and prokaryotic cells
as well as from body fluids such as blood and urine.1,2 In this review, the focus is on recent
findings regarding biology and function of EVs as well as their application as biomarkers for
clinical diagnostics.
Extracellular vesicle classification
Important criteria of EV classification are based on their cellular origin, function or
biogenesis.2 Nevertheless, there is still no consensus about classification of EVs and their
nomenclature is still unclear. Moreover, characterization of vesicles poses several limitations
like difficulties with isolation and heterogeneity in size, shape and density.1 Recently, four main
types of EVs based on their biogenesis (Figure 1) have been distinguished which include
microvesicles, exosomes, retrovirus-like particles and apoptotic bodies.1,2 (Table1)
Type of vesicles
Characteristics
Origin
Size
Density
Surface markers
Plasma membrane
50-1000 nm
Undefined
ARF6, VCAMP3
Endolysosomal
pathway and plasma
membrane
40-120 nm
1.13-1.19 g/mL
CD63, CD9
Retrovirus-like
particles
Plasma membrane
(Gag protein
interactions)
90-100 nm
1.13-1.16 g/mL
Gag
Apoptotic
bodies
Plasma membrane
50-5000 nm
1.16-1.28 g/mL
TSP, C3b, high
level of PS
Microvesicles
Exosomes
Table 1: Classification of extracellular vesicles.
5
Microvesicles are generated by budding and subsequent fission of the plasma
membrane into the extracellular space as a result of dynamic interactions between
phospholipid redistribution and cytoskeletal protein contraction.6 The diameter of these
vesicles is between 50-1,000 nm and their buoyant density is still undefined. It is believed that
microvesicles are generally larger than exosomes. Size ranges overlap between these two
types of vesicles and the distinction is usually based on their biogenesis.6
Exosomes are formed via direct budding of the plasma membrane or via the
endolysosomal pathway. In the latter case, plasma membrane is budding into multivesicular
bodies (MVBs) in the cytosol. This leads to formation of intraluminal vesicles (ILVs) within
MVBs. Next, the membrane of MVBs fuses with the plasma membrane and ILVs are released,
which are secreted as exosomes into the extracellular space.1 An important step in the
generation of ILVs includes reorganization of endosomal membrane proteins such as CD9 and
CD63 into tetraspanin enriched microdomains. Subsequently, endosomal sorting complexes
required for transport (ESCRTs) are incorporated into the budding site. ESCRTI and II promote
membrane budding and ESCRT III is crucial for completion of budding. ESCRTIII is recruited to
the site of ESCRTI and II via Alix, a protein that simultaneously binds to the TSG101 component
of the ESCRT-I complex.6,7 Exosomes are usually smaller and more homogenous than
microvesicles. They are approximately 40-120 nm in size and their density ranges between
1.13 and 1.19 g/mL.8 Such vesicles display cup-shaped morphology when analyzed by electron
microscopy.1,2 Furthermore, exosomes contain tetraspanins (CD9, CD63) and specific proteins
such us Alix and TSG101, which are often used as identification markers.3,6 Importantly, beside
specific markers, both microvesicles and exosomes contain cytoplasmic and membrane
proteins, RNA and receptors.2
Retrovirus-like particles (RLPs) are 90-100 nm in diameter and resemble retroviral
vesicles.9 RLPs are non-infectious because they do not have the complete set of genes required
for completion of the full viral cycle. These vesicles are formed by direct budding of the plasma
membrane.10 Nevertheless, the mechanism beyond this differs from exosome and microvesicle
formation. It is thought that interactions of retroviral proteins such as Gag with other elements
of the plasma membrane and cytoskeletal molecules play a pivotal role in RLP formation.11
Therefore, Gag protein might be used as a marker for retrovirus-like particles.6
The last group of EVs are termed apoptotic bodies. Their size range is between 50 nm
to 5 m and their buoyant density is from 1.16 to 1.28 g/mL.12 Apoptotic bodies are generated
when cells undergo apoptosis and they contain cell organelles and nuclear fractions.13 Such
vesicles are phagocytosed and rapidly cleared by macrophages as a respond to specific
signaling molecules. Interactions between recognition receptors on the phagocytes and
changes in the composition of cell membrane mediate this clearance.6 There are few
membrane alterations involved in this process. Translocation of phosphatidylserines (PS) to
the outer surface initiates binding of Annexin V, a molecule that is identified by phagocytes.
Another membrane change includes oxidation of surface molecules. This leads to formation of
binding sites for thrombospondin or C3b protein, which are recognized by phagocyte
receptors. Thrombospondin, C3b and Annexin V are well-characterized markers of apoptotic
bodies.6
6
Figure 1: Biogenesis of the various types of extracellular vesicles.6
Functions of extracellular vesicles
EVs are involved in a variety of fundamental cellular and biological processes.14 As
carriers of proteins, lipids and nucleic acids, EVs play important roles in intracellular signaling
by changing the composition and function of recipient cells. For example, it has been shown
that tumor-derived exosomes transfer MET, a receptor tyrosine kinase, that instruct bone
marrow progenitor cells to promote tumor growth and metastasis.15
EVs contribute to maintenance of cellular homeostasis by waste management.26,27
Vesicles containing cellular waste are secreted from the cell into the intracellular space and are
eliminated by phagocytotic cells. It has been suggested that EVs containing redundant
substances expose PS in order to enhance their clearance by phagocytosis.18 Pathological
conditions regarding waste removal by EVs may be linked to damaged phagocytes or a
deficiency in danger signal recognition.14 Furthermore, it has been suggested that EVs can
protect cells from extracellular and intracellular stress. Cells incubated with C5b-9 complex, a
kind of external stress, secrete vesicles containing this complex in order to protect cells. In
turn, caspase 3, one of the key enzymes in apoptosis, is not detectable in releasing cells but is
present in different EVs. Therefore secretion of caspase 3 protects cells from dangerous
accumulation of a form of internal stress.1,19
EVs have been also linked to stem cell maintenance.20 Such stem cell-derived vesicles
play a key role in tissue regeneration of the tissue injury.21 Furthermore, EVs participate in cell
phenotype modulation. It has been demonstrated that such vesicles can shift the phenotype of
hematopoietic stem cells towards a liver cell phenotype.22 In addition, bone marrow cell
transcriptome can be converted into a lung phenotype in vivo.23
By transporting ligands and receptors expressed on their surface EVs can modulate the
immune system.24 For example, antigen presenting exosomes derived from dendritic cells (DC)
induced T-cell mediated immune responses.25 Furthermore, EVs can suppress immune
7
responses by several mechanisms like suppression of natural killer (NK) and CD8+ cell activity,
and by inhibition of monocyte differentiation into DCs and their maturation.2,26–28
EVs released from endothelial cells display both pro- and anti-angiogenic properties.
Such vesicles can contain metalloproteinase proteins (MMP-2 and MMP-9) that support matrix
degradation and formation of new blood vessels in expanding tissues.29 Additionally, it has
been demonstrated that after subcutaneous injection of platelet-derived EVs, development of
endothelial capillaries in mice occur. This is likely due to vascular endothelial growth factor
(VEGF) that is released upon activation of platelets.30 Nevertheless, induction of angiogenesis
by EVs may also promote tumor angiogenesis and metastasis. Janowska-Wieczorek et al.
showed that EVs that bind to tumor cells support expression of metalloproteinases, VEGF,
hepatocyte growth factor and interleukin-8 (IL-8).31 EVs, especially exosomes, secreted by
tumor cells are involved in tumor angiogenesis. For example, human primary glioblastoma cellderived EVs transmit various mRNA and miRNAs that promote tumor development and
angiogenesis.32 Although EVs display mainly pro-angiogenic properties, such vesicles can also
inhibit this process by stimulating the production of reactive oxygen species (ROS).33
EVs are involved in the spread of large variety of viruses and pathogens.2 For example,
such vesicles have been shown to transport CC chemokine receptor 5 (CCR5) responsible for
viral cell entry and these vesicles can therefore promote HIV-1 infection.34 Furthermore, EVs
are implicated in spreading Epstein-Barr virus (EBV) by transferring viral microRNAs.35 Other
EVs have been shown to transport prions and other toxic proteins such us amyloid-β-peptides
and α-synuclein. These proteins can contribute to the progression of neurodegenerative
disorders like Alzheimer’s and Parkinson’s disease, respectively.36,37
Extracellular vesicles as therapeutic targets and drug delivery systems
EVs have been linked not only to regulation of many physiological processes like stem
cell maintenance, tissue repair, immune responses and blood coagulation but also to the
pathology of several diseases. As mentioned before, EVs play a role in cancer progression, the
spread of viruses and pathogenic agents like HIV-1, amyloid-β-derived peptides associated
with Alzheimer’s disease and α-synuclein linked to Parkinson’s disease. Therefore EVs have
unquestionable therapeutic potential. Several therapeutic strategies are currently under
investigation.
Recently, EVs have been investigated as potential therapeutic agents for tissue
regeneration, modulation of immune responses and immunotherapy.38 Multipotent stem cells
(MSCs), derived from the bone marrow or peripheral blood, secrete EVs, particularly
exosomes, that play a key role in regenerative medicine.39 It has been demonstrated that
application of MSCs leads to beneficial outcome, so-called paracrine effect in myocardial
infarction. Subsequently, it has been shown in various animal models of myocardial ischaemiareperfusion injury that conditioned media from hypoxic MSCs can enhance heart function and
limit the extend of infarct.40–42 Since then, MSC-based therapy have been implemented in
treatment of several diseases such as cardiovascular disease, graft versus host disease, Crohn’s
8
disease and acute kidney injury.2 EVs are also involved in the activation of the immune system
and increase of antigen presentation by generating the release of pro-inflammatory cytokines
thereby enhancing the secretion of tumor necrosis factor and increased NK cell activity.2
Therefore EVs have been used in anticancer vaccines and elimination of infections.1,2
EVs are natural transporters and intracellular delivery vehicles for various proteins and
nucleic acids and are subsequently an attractive tool for the delivery of pharmaceutical
proteins and genes. A unique feature of EVs that makes them an interesting tool in drug
delivery is their ability to cross biological barriers like the blood-brain barrier (BBB). For
example, DC-derived EVs, genetically modified to express rabies viral glycoprotein, have been
efficiently employed to deliver siRNA into the brain of mice in vivo. Moreover, the inhibition of
more than 50% of expression and production of BACE1, a therapeutic target in Alzheimer’s
disease, was observed.43 Recently, the utility of EV for gene therapy has been also investigated.
For instance, Maguire et al. demonstrated that adeno-associated viruses (AAVs) encapsulated
in EVs displaying viral capsid proteins were more efficacious than free AAVs for the delivery of
genetic material into targeted cells.44
Application of extracellular vesicles in clinical diagnostics
The identification of cell-derived vesicles isolated from body fluids suggests that EVs
are a promising source of clinically relevant and noninvasive biomarkers that enable
monitoring of physiology and diagnosis of several diseases.45 For example, exosomes isolated
from the blood of patients with glioma usually contain EGFRvIII mRNA that leads to the
expression of proangiogenic protein IL-8.32 Thus, detection of EGFRvIII mRNA in exosomes may
provide important information on tumor presence, progression and prognosis. As mentioned
before, EVs are present not only in blood but also in other body fluids. Vesicles isolated from
urine were shown to contain prostate-specific antigen and prostate-specific membrane
antigen associated with prostate cancer.46 Furthermore, exosomes from cerebrospinal fluid of
patients with Alzheimer’s disease may provide information on disease progression.47
Recently, the potential of urinary exosomes as diagnostic molecules in renal diseases
has been investigated.48 It has been shown that these exosomes contain proteins from
different parts of the nephron, e.g. podocin from glomerular podocytes; megalin, cubilin,
aquaporin-1, and type IV carbonic anhydrase from proximal tubules, and aquaporin-2 from the
collecting duct.49 Such urinary exosomes can reflect acute kidney injury. For instance,
aquaporin-1, found in exosomes of patients after renal allograft transplantation, allows
detection and monitoring of renal cellular states after ischemia/reperfusion(I/R)-induced injury
and subsequent regeneration as well as prediction of acute kidney injury.50 So far, 34 proteins
identified in urinary exosomes have been linked in various kidney diseases like autosomal
dominant polycystic kidney disease type 1, autosomal dominant and recessive nephrogenic
diabetes, antenatal Bartter syndrome type 1 and Gitelman’s syndrome.51
In another study, EVs were investigated as potential source of new biomarkers of
9
diseases and toxicities, including drug-induced liver injury (DILI).52 It has been shown that
hepatocytes generate EVs enriched in molecules reflecting drug hepatotoxicity that are
subsequently released to circulation.52 Xi Yang et al. elegantly reviewed recent advances in
utilizing EVs to discover new DILI biomarkers.52 Conventional serum biomarkers (alanine
aminotransferase (ALT), aspartate aminotransferase, alkaline phosphatase, and bilirubin)
possess several disadvantages such us low organ specificity, short half-life and poor reflection
of liver functions that limit their application. It has been shown that circulating EVs contain
liver-specific mRNAs and miRNAs, of which levels can be significantly increased in response to
DILI. Moreover, the expression level of mRNAs and miRNAs within EVs may indicate the drug
involved in hepatotoxicity and provides more information than traditional serum biomarkers
(e.g. ALT).53–55
The large variety of EV content requires several platforms of testing such as protein
typing assays, microarray assays, and RNA sequencing. Unarguably, biomarker development
platforms are gaining more attention. Nevertheless, the applicability of EVs in clinical
diagnostics needs further investigation.
Standardization of protocols
Despite the extensive studies during the past decade on EVs for biomarker
development, a number of challenges still persist. A key issue in translating EVs to suitable
clinical biomarkers is to standardize protocols for sample collection, analysis and processing.
Such standardized protocols are essential to be able to compare results obtained from
different laboratories. Though EVs are present in most, if not all body fluids, vesicles isolated
from the blood have been the most intensively investigated so far. Recently, the International
Society for Thrombosis and Haemostasis (ISTH)56 and the International Society for Extracellular
Research (ISEV)57 have described guidelines and recommendations regarding standardization
of sample collection and handling. Here, the discussion is focused on several important issues
regarding EV analysis in blood samples (Figure 2).
10
Figure 2: Essential standardization issues for EVs analysis from blood samples
Collection of blood samples involves a number of factors than can influence analysis. It
has been demonstrated that the endogenous circadian system has an effect on platelet
activation and thus on EV release.58 Furthermore, physical forces associated with blood
collection procedures can influence the number of platelet-derived EVs. Therefore, using the
same needle gauge, preferably 21-gauge or larger, gentle handling and removal of the first few
milliliters of collected blood is recommended.56,57 The choice of anticoagulant is another
important aspect in standardization of sample collection. Nevertheless there is no consensus
regarding this issue.57 Despite the fact that both the ISTH and ISEV discourage the use of
heparin-based coagulants, Jayachandran et al. have recommended the use of heparin tubes
for accurate EV counts.56,57,59 EDTA, CTAD (sodium citrate containing theophylline, adenosine
and dipyridamol) and ACD (acid-citrate-dextrose) are the most common examples of
alternative anticoagulants. Nevertheless, lowering of EV counts in blood by blocking activation
and promoting association of EVs to platelets was observed.57 In turn, György et al. have
recommended ACD tubes for the assessment of plasma EVs as ACD inhibits in vitro vesiculation
and does not interfere with protein or RNA analysis.60
Sample processing is a key issue in standardization of protocols. For many years
differential ultracentrifugation has been a gold standard for EV isolation and purification from
preclinical samples. Nevertheless, recent studies suggest that centrifugation can have a robust
influence on EV analysis and unlikely isolates only a single type of vesicle.1,61
In order to obtain plasma from blood samples, centrifugation of blood samples at
room temperature for 15 min. at 2500 x g and processing every sample with the same settings
11
are recommended by the ISTH.56
Regarding sample analysis, standardization of calibration methods is essential to
ensure study-to-study comparability. The ISTH recommends the use of 0.5-m and 0.9 m
Megamix polystyrene beads used for calibration of platelet-derived EVs measured with wide
angle (1-19) flow cytometers.56,62 Interestingly, recent studies reported promising findings
that synthetic lipid vesicles display comparable refractive index compare to EVs.63
Nevertheless, there is still no consensus regarding which calibration method is the best.64,65
Detection techniques
Several techniques have been employed or developed in recent years to detect and/or
characterize EVs in research settings. These techniques can be applied to detect EV number
and size distribution, their molecular surface markers and/or RNA content (Table 2).
Nevertheless, due to the lack of sensitive and standardized methods, a number of techniques
are not suitable for routine clinical application. Therefore, multiparameter, standardized and
high-throughput methods are required to analyze EVs from patient samples. 66
Flow cytometry is one of the most common techniques used to detect the number and
size distribution of EVs in clinical samples. With this technique vesicles are going through a
laser beam in a hydrodynamically focused fluid stream. One detector is placed in line with the
laser beam, and measures the forward-scattered light (FSC). Next, detectors measure the sidescattered light (SSC) and fluorescence intensity perpendicular to the beam. Light scattering
generates a signal that a vesicle is present.67,68 For large vesicles (>300 nm) this technique
appeared to be a reproducible method for high-throughput, multiparameter analysis.
Nevertheless, the majority of EVs are in a size range of 50-150 nm and therefore their
detection is performed with low efficiency and do not reflect the standard population.68
Heterogeneity of vesicles in body fluids has a great impact on accurate analysis. It has been
found that EV detection by flow cytometry relies on two mechanisms: detection of single, large
vesicles and swarm detection where a number of relatively small vesicles are simultaneously
illuminated by laser beam and counted as a single signal. Due to swarm detection 1000-fold
underestimation of concentrations by flow cytometry can occur.67,66,69 Recently, a novel
fluorescence based, high-resolution flow cytometric method has been developed for
quantitative and qualitative analysis of EVs.66
Nanoparticle tracking analysis (NTA) measures EV size and concentration. In this
method vesicles are illuminated by a focused laser beam and scatter light or show
fluorescence. Next, a microscope determines the position of each vesicle. For each single
vesicle, Brownian movements are tracked and the mean squared velocity is calculated.70 Beads
of known concentration are used to calibrate the system in order to get absolute size
distribution of EVs in suspension. NTA appears to be a convenient and fast method to detect
EVs ranging from 50nm to 1 m in diameter. Moreover, after labeling with a single quantum
dot detection of even smaller vesicles is possible.71 NTA quantitative measurements are less
precise in heterogeneous samples and the numbers of parameters that can be analyzed
12
simultaneously are limited. Furthermore, NTA generates a large amount of video data with
every measurement that requires enormous storage capacity and complex data
processing.66,70,72
Resistive pulse sensing (RPS) is another method used for the quantification of EVs
ranging in diameter between 50 nm and 10m.70,73 RPS is composed of two liquid cells divided
by a membrane containing a single pore, where an ionic current flows through. EVs passing
through a pore are detected as a transient change in ionic current flow that is approximately
proportional to the volume of EVs. A major drawback of measuring clinical samples with RPS is
pore clogging by particles larger than the pore such as small cells, apoptotic bodies and various
aggregates. Moreover, calibration beads can form aggregates shortly after dilution in PBS that
can change the size of the pore. In addition, a measurement takes considerable time ranging
from 30 min. to 1 hour per sample. Despite the fact that RPS is an accurate method to
determine size and concentration of vesicles, it seems that practical limitations make this
technique difficult to apply in routine clinical settings.70,73–75
Analysis of vesicles can be also be performed by specialized techniques that detect EVs
by the presence of specific markers on their surface. Diagnostic magnetic resonance (DMR) is
relatively new method that enables rapid and accurate EV analysis in clinical samples. This
integrated system includes microfluidics and micro nuclear magnetic resonance (NMR). A
Microfluidic chip can isolate vesicles directly from the blood. Next, EVs are labeled with targetspecific magnetic particles and detected by NMR.76–78 Importantly, the entire assay is
performed within the chip and no external isolation steps such as ultracentrifugation are
required. In addition, this method only requires a small volume of blood (<200 L) and the
measurement takes less than 30 min.77 Recently, this new analytical platform has been applied
to identify erythrocyte-derived vesicles as a biomarker for monitoring blood product quality.77
Moreover, this technique has been also used to detect glioblastome multiforme (GBM) vesicles
in order to monitor and predict patient responses to therapies.78 This method can be expanded
by monitoring circulating EVs in a variety of other diseases.
Immunoassays like enzyme-linked immunosorbent assays (ELISA) are conventional
methods to analyze protein in samples. Recently, technological advancements like automated
plate handling and detection enable relatively high-throughput measurement in 96-well or
384-well plates. Nevertheless, these methods usually require large amounts of EVs and involve
time-consuming isolation processes that significantly limits their utility in clinical settings.77,79
Protein microarrays are commonly used to detect antigens or antibodies in different
types of samples. Recently, the so-called Extracellular Vesicle Array that uses antigenic
capturing of EVs by protein microarray for phenotyping and enumeration of EVs has been
developed. A microarray print with spots of 21 various antibodies was used to capture
exosomes from samples. Next, these vesicles were detected with a cocktail of biotynylated
antibodies against tetraspanins CD9, CD63 and CD81 followed by fluorescence-labelled
streptavidin.80 By using this sensitive method, Jorgensen et al. determined the presence and
distribution of circulating exosomes (positive for tetraspanins) in the plasma of seven healthy
donors. The phenotyping of these vesicles showed that only the expression level of two
13
tetraspanins, CD9 and CD81, was roughly equal, whereas the expression level of CD63 was
significantly lower.80 Therefore, it opened a discussion whether CD63 is a suitable exosomal
marker. EV Array is a high-throughput method capable to detect and phenotype small EVs
directly from biological samples. Importantly, the performance of this array is economic and
does not require a large amount of sample as well as time-consuming procedures.80 In contrast
to NMR, the detection of several exosomal antigens at one time is possible.
Analysis of EV RNA content can be performed to characterize and understand vesiclemediated biological functions and mechanisms. Microarrays or real time quantitative
polymerase chain reaction (qPCR) are widely applied in order to investigate the RNA content
(mRNA, miRNA, long non-coding RNA) of EVs obtained from cell culture systems and isolated
from bodily fluids.81 Importantly, these useful and simple methods do not require specialized
sequencing equipment or complex bioinformatics approaches. However, microarrays cannot
be used for the detection of unknown miRNAs and other RNAs.82 Recently, Noerholm et al.
used a microarrays platform to show that mRNA profiles of serum-derived EVs contents differs
between glioblastoma patients and healthy individuals.83 Another method to analyze EVs by
their RNA content is deep sequencing of human plasma-derived exosomal RNA. So far, several
technologies have been developed for deep sequencing. Usually, proprietary methods
available for deep sequencing are expensive and require a qualified person to operate them.
Recently smaller platforms for routine RNA analysis have become available.81 Deep sequencing
of blood-derived exosomal RNAs as described by Huang et al. can be used as a potential
biomarker discovery tool.82
Characterization
Technique
Sensitivity/R
esolution
Highthroughput
potential
Experstise
required
References
EV number/size
distribution
Conventional Flow
cytometry
>300nm
Medium/high
Medium/high
67,68
Fluorescence based
cytometry
>50nm
Medium/high
High
66,69
Nanoparticle tracking
analysis
>50nm
Low
High
70–72
Resistive pulse
sensing
>50nm
Low
High
70,73,74
Diagnostic magnetic
resonance
30-150nm
High
Medium/high
76–78
30-100nm
High
Medium/high
80
30-100nm
Low
High
81,82
EV surface markers
EV RNA content
Protein Microarray
Deep sequencing
14
RNA Microarray
30-100nm
Medium/high
High
83
Table 2: EV detection techniques for clinical samples
Conclusions and future perspectives
EVs, extensively investigated in the last few decades, are involved in fundamental
biological processes and in the pathology of several diseases. Unarguably, such vesicles have a
great therapeutic potential and offer a source of clinically relevant and non-invasive
biomarkers that enable monitoring of physiology and diagnosis of various diseases as well
toxicity. However, to enable clinical utility of heterogeneous EVs, standardized,
multiparameter and high-throughput analysis methods are required.
The biological content of EVs is rather unexplored, moreover the distinction between
several types of EVs and their isolation is still a challenge. Nevertheless, recent technological
improvement of detection devices allows distinguishing vesicles smaller than 100 nm and the
subsequent analysis of their surface markers or content. These advances in EV detection
display a promising trend, offering valuable platforms for EV analysis in clinical diagnostic
settings. The knowledge of drawbacks of existing techniques could contribute to the design of
a perfect tool that allow physical characteristic of EVs and monitoring their biological content
as well as high-throughput analysis and reasonable costs of investigation.
To better understand the biological effects of EVs and further exploit this knowledge
into biomarker discovery tool, the analyzing methods of the content of vesicles by
transcriptome, miRNA and proteome should be further improved allowing high-throughput
analysis. In the near future, EV may be powerful clinical tools for new therapies by engineering
the vesicles containing certain level of expression of mRNA, miRNA, or proteins in the vesicles,
but also for large source of clinically relevant biomarkers.84
Realization of the full potential of EVs especially as biomarkers in clinical settings still
remains a challenge. It basically depends on standardized and high-throughput methods of
their detection. Therefore, issues regarding standardization of measurements and samples
collection should be further investigated to advance the application of EVs as reliable
diagnostic and prognostic tools.
15
Acknowledgments
I would like to especially thank my supervisor- Dr. Roy van der Meel. Firstly, for a great chance
to write my master thesis on this topic. I have never regretted that I accepted “the challenge”
to write this review during my holidays in Poland. Thanks to that, for the first time in my life I
can enjoy the fact of being a co-author of scientific publication! Secondly, I would like to
express my gratitude for a very constructive, open and friendly supervision.
Finally, I would like to thank Dr. Raymond M. Schiffelers for the opportunity to write my thesis
in his group.
16
References
1.
Van der Pol, E., Böing, A. N., Harrison, P., Sturk, A. & Nieuwland, R. Classification,
functions, and clinical relevance of extracellular vesicles. Pharmacol. Rev. 64,
676–705 (2012).
2.
EL Andaloussi, S., Mäger, I., Breakefield, X. O. & Wood, M. J. a. Extracellular
vesicles: biology and emerging therapeutic opportunities. Nat. Rev. Drug Discov.
12, 347–57 (2013).
3.
Kooijmans, S. a a, Vader, P., van Dommelen, S. M., van Solinge, W. W. &
Schiffelers, R. M. Exosome mimetics: a novel class of drug delivery systems. Int.
J. Nanomedicine 7, 1525–41 (2012).
4.
Chargaff, E. & West, R. THE BIOLOGICAL SIGNIFICANCE OF THE
THROMBOPLASTIC PROTEIN OF BLOOD. J. Biol. Chem. 166, 189–197 (1946).
5.
Wolf, P. The Nature and Significance of Platelet Products in Human Plasma. Br. J.
Haematol. 13, 269–288 (1967).
6.
Akers, J. C., Gonda, D., Kim, R., Carter, B. S. & Chen, C. C. Biogenesis of
extracellular vesicles (EV): exosomes, microvesicles, retrovirus-like vesicles, and
apoptotic bodies. J. Neurooncol. 113, 1–11 (2013).
7.
McCullough, J., Fisher, R. D., Whitby, F. G., Sundquist, W. I. & Hill, C. P. ALIXCHMP4 interactions in the human ESCRT pathway. Proc. Natl. Acad. Sci. U. S. A.
105, 7687–7691 (2008).
8.
Simons, M. & Raposo, G. Exosomes--vesicular carriers for intercellular
communication. Curr. Opin. Cell Biol. 21, 575–581 (2009).
9.
Bronson, D. L., Fraley, E. E., Fogh, J. & Kalter, S. S. Induction of retrovirus
particles in human testicular tumor (Tera-1) cell cultures: an electron
microscopic study. J. Natl. Cancer Inst. 63, 337–339 (1979).
10.
Bieda, K., Hoffmann, A. & Boller, K. Phenotypic heterogeneity of human
endogenous retrovirus particles produced by teratocarcinoma cell lines. J. Gen.
Virol. 82, 591–596 (2001).
11.
Pincetic, A. & Leis, J. The Mechanism of Budding of Retroviruses from Cell
Membranes. Adv. Virol. 2009, 1–9 (2009).
12.
Hristov, M., Erl, W., Linder, S. & Weber, P. C. Apoptotic bodies from endothelial
cells enhance the number and initiate the differentiation of human endothelial
progenitor cells in vitro. Blood 104, 2761–2766 (2004).
13.
Taylor, R. C., Cullen, S. P. & Martin, S. J. Apoptosis: controlled demolition at the
cellular level. Nat. Rev. Mol. Cell Biol. 9, 231–241 (2008).
17
14.
Yuana, Y., Sturk, A. & Nieuwland, R. Extracellular vesicles in physiological and
pathological conditions. Blood Rev. 27, 31–9 (2013).
15.
Peinado, H. et al. Melanoma exosomes educate bone marrow progenitor cells
toward a pro-metastatic phenotype through MET. Nat. Med. 18, 883–891
(2012).
16.
Martinez, M. C. & Andriantsitohaina, R. Microparticles in angiogenesis:
therapeutic potential. Circ. Res. 109, 110–119 (2011).
17.
Aharon, A. & Brenner, B. Microparticles, thrombosis and cancer. Best Pract. Res.
Clin. Haematol. 22, 61–69 (2009).
18.
Davila, M. et al. Tissue factor-bearing microparticles derived from tumor cells:
impact on coagulation activation. J. Thromb. Haemost. 6, 1517–1524 (2008).
19.
Abid Hussein, M. N. et al. Cell-derived microparticles contain caspase 3 in vitro
and in vivo. J. Thromb. Haemost. 3, 888–896 (2005).
20.
Ratajczak, J. et al. Embryonic stem cell-derived microvesicles reprogram
hematopoietic progenitors: evidence for horizontal transfer of mRNA and
protein delivery. Leuk. Off. J. Leuk. Soc. Am. Leuk. Res. Fund, U.K 20, 847–856
(2006).
21.
Camussi, G. et al. Exosome/microvesicle-mediated epigenetic reprogramming of
cells. Am. J. Cancer Res. 1, 98–110 (2011).
22.
Jang, Y.-Y., Collector, M. I., Baylin, S. B., Diehl, A. M. & Sharkis, S. J.
Hematopoietic stem cells convert into liver cells within days without fusion. Nat.
Cell Biol. 6, 532–539 (2004).
23.
Aliotta, J. M. et al. Alteration of marrow cell gene expression, protein
production, and engraftment into lung by lung-derived microvesicles: a novel
mechanism for phenotype modulation. Stem Cells 25, 2245–2256 (2007).
24.
Théry, C., Ostrowski, M. & Segura, E. Membrane vesicles as conveyors of
immune responses. Nat. Rev. Immunol. 9, 581–593 (2009).
25.
Thery, C. et al. Indirect activation of naive CD4+ T cells by dendritic cell-derived
exosomes. Nat Immunol 3, 1156–1162 (2002).
26.
Liu, C. et al. Murine mammary carcinoma exosomes promote tumor growth by
suppression of NK cell function. J. Immunol. 176, 1375–1385 (2006).
27.
Yu, S. et al. Tumor exosomes inhibit differentiation of bone marrow dendritic
cells. J. Immunol. 178, 6867–6875 (2007).
28.
Eken, C. et al. Polymorphonuclear neutrophil-derived ectosomes interfere with
the maturation of monocyte-derived dendritic cells. J. Immunol. 180, 817–824
(2008).
18
29.
Taraboletti, G. et al. Shedding of the matrix metalloproteinases MMP-2, MMP-9,
and MT1-MMP as membrane vesicle-associated components by endothelial
cells. Am. J. Pathol. 160, 673–680 (2002).
30.
Brill, A., Dashevsky, O., Rivo, J., Gozal, Y. & Varon, D. Platelet-derived
microparticles induce angiogenesis and stimulate post-ischemic
revascularization. Cardiovasc. Res. 67, 30–38 (2005).
31.
Janowska-Wieczorek, A. et al. Microvesicles derived from activated platelets
induce metastasis and angiogenesis in lung cancer. Int. J. Cancer 113, 752–760
(2005).
32.
Skog, J. et al. Glioblastoma microvesicles transport RNA and proteins that
promote tumour growth and provide diagnostic biomarkers. Nat. Cell Biol. 10,
1470–1476 (2008).
33.
Burger, J. A. & Kipps, T. J. CXCR4: a key receptor in the crosstalk between tumor
cells and their microenvironment. Blood 107, 1761–1767 (2006).
34.
Mack, M. et al. Transfer of the chemokine receptor CCR5 between cells by
membrane-derived microparticles: a mechanism for cellular human
immunodeficiency virus 1 infection. Nat. Med. 6, 769–775 (2000).
35.
Pegtel, D. M. et al. Functional delivery of viral miRNAs via exosomes. Proc. Natl.
Acad. Sci. U. S. A. 107, 6328–6333 (2010).
36.
Bellingham, S. A., Guo, B. B., Coleman, B. M. & Hill, A. F. Exosomes: Vehicles for
the Transfer of Toxic Proteins Associated with Neurodegenerative Diseases?
Front. Physiol. 3, (2012).
37.
Emmanouilidou, E. et al. Cell-produced alpha-synuclein is secreted in a calciumdependent manner by exosomes and impacts neuronal survival. J. Neurosci. 30,
6838–6851 (2010).
38.
Chaput, N. & Théry, C. Exosomes: immune properties and potential clinical
implementations. Semin. Immunopathol. 33, 419–440 (2011).
39.
Ratajczak, M. Z. et al. Pivotal role of paracrine effects in stem cell therapies in
regenerative medicine: can we translate stem cell-secreted paracrine factors
and microvesicles into better therapeutic strategies? Leukemia 26, 1166–1173
(2012).
40.
Gnecchi, M. et al. Paracrine action accounts for marked protection of ischemic
heart by Akt-modified mesenchymal stem cells. Nat. Med. 11, 367–368 (2005).
41.
Gnecchi, M., Zhang, Z., Ni, A. & Dzau, V. J. Paracrine mechanisms in adult stem
cell signaling and therapy. Circ. Res. 103, 1204–1219 (2008).
42.
Gnecchi, M. et al. Evidence supporting paracrine hypothesis for Akt-modified
mesenchymal stem cell-mediated cardiac protection and functional
improvement. FASEB J. 20, 661–669 (2006).
19
43.
Alvarez-Erviti, L. et al. Delivery of siRNA to the mouse brain by systemic injection
of targeted exosomes. Nat. Biotechnol. 29, 341–345 (2011).
44.
Maguire, C. A. et al. Microvesicle-associated AAV Vector as a Novel Gene
Delivery System. Mol. Ther. 20, 960–971 (2012).
45.
Gonda, D. D. et al. Neuro-oncologic applications of exosomes, microvesicles, and
other nano-sized extracellular particles. Neurosurgery 72, 501–10 (2013).
46.
Nilsson, R. J. A. et al. Blood platelets contain tumor-derived RNA biomarkers.
Blood 118, 3680–3683 (2011).
47.
Saman, S. et al. Exosome-associated Tau Is Secreted in Tauopathy Models and Is
Selectively Phosphorylated in Cerebrospinal Fluid in Early Alzheimer Disease. J.
Biol. Chem. 287, 3842–3849 (2012).
48.
Borges, F. T., Reis, L. a & Schor, N. Extracellular vesicles: structure, function, and
potential clinical uses in renal diseases. Braz. J. Med. Biol. Res. 46, 824–30
(2013).
49.
Pisitkun, T., Shen, R.-F. & Knepper, M. A. Identification and proteomic profiling
of exosomes in human urine. Proc. Natl. Acad. Sci. U. S. A. 101, 13368–13373
(2004).
50.
Sonoda, H. et al. Decreased abundance of urinary exosomal aquaporin-1 in renal
ischemia-reperfusion injury. Am. J. Physiol. Renal Physiol. 297, F1006–F1016
(2009).
51.
Miranda, K. C. et al. Nucleic acids within urinary exosomes/microvesicles are
potential biomarkers for renal disease. Kidney Int. 78, 191–199 (2010).
52.
Yang, X., Weng, Z., Mendrick, D. L. & Shi, Q. Circulating extracellular vesicles as a
potential source of new biomarkers of drug induced liver injury. Toxicol. Lett.
225, 401–406 (2014).
53.
Shi, Q., Yang, X. & Mendrick, D. L. Hopes and challenges in using miRNAs as
translational biomarkers for drug-induced liver injury. Biomark. Med. 7, 307–15
(2013).
54.
Shi, Q., Hong, H., Senior, J. & Tong, W. Biomarkers for drug-induced liver injury.
Expert Rev. Gastroenterol. Hepatol. 4, 225–234 (2010).
55.
Wang, K. et al. Circulating microRNAs, potential biomarkers for drug induced
liver injury. Proc. Natl. Acad. Sci. 106, 4402–4407 (2009).
56.
Lacroix, R. et al. Standardization of pre-analytical variables in plasma
microparticle determination: results of the International Society on Thrombosis
and Haemostasis SSC Collaborative workshop. J. Thromb. Haemost. 1190–1193
(2013). doi:10.1111/jth.12207
57.
Witwer, K. W. et al. Standardization of sample collection, isolation and analysis
methods in extracellular vesicle research. J. Extracell. vesicles 2, 1–25 (2013).
20
58.
Scheer, F. A. J. L. et al. The Human Endogenous Circadian System Causes
Greatest Platelet Activation during the Biological Morning Independent of
Behaviors. PLoS One 6, e24549 (2011).
59.
Jayachandran, M., Miller, V. M., Heit, J. A. & Owen, W. G. Methodology for
isolation, identification and characterization of microvesicles in peripheral
blood. J. Immunol. Methods 375, 207–214 (2012).
60.
György, B. et al. Improved circulating microparticle analysis in acid-citrate
dextrose (ACD) anticoagulant tube. Thromb. Res. 133, 285–92 (2014).
61.
Vlassov, A. V., Magdaleno, S., Setterquist, R. & Conrad, R. Exosomes: Current
knowledge of their composition, biological functions, and diagnostic and
therapeutic potentials. Biochim. Biophys. Acta - Gen. Subj. 1820, 940–948
(2012).
62.
Lacroix, R. et al. Standardization of platelet-derived microparticle enumeration
by flow cytometry with calibrated beads: results of the International Society on
Thrombosis and Haemostasis SSC Collaborative workshop. J. Thromb. Haemost.
8, 2571–4 (2010).
63.
Chandler, W. L., Yeung, W. & Tait, J. F. A new microparticle size calibration
standard for use in measuring smaller microparticles using a new flow
cytometer. J. Thromb. Haemost. 9, 1216–24 (2011).
64.
Robert, S., Poncelet, P., Lacroix, R., Raoult, D. & Dignat-George, F. More on:
calibration for the measurement of microparticles: value of calibrated
polystyrene beads for flow cytometry-based sizing of biological microparticles. J.
Thromb. Haemost. 9, 1676–8; author reply 1681–2 (2011).
65.
Mullier, F., Bailly, N., Chatelain, C., Dogné, J. M. & Chatelain, B. More on:
calibration for the measurement of microparticles: needs, interests, and
limitations of calibrated polystyrene beads for flow cytometry-based
quantification of biological microparticles. J. Thromb. Haemost. 9, 1679–81;
author reply 1681–2 (2011).
66.
Nolte-’t Hoen, E. N. M. et al. Quantitative and qualitative flow cytometric
analysis of nanosized cell-derived membrane vesicles. Nanomedicine 8, 712–20
(2012).
67.
Van der Pol, E., van Gemert, M. J. C., Sturk, a, Nieuwland, R. & van Leeuwen, T.
G. Single vs. swarm detection of microparticles and exosomes by flow
cytometry. J. Thromb. Haemost. 10, 919–30 (2012).
68.
Van der Pol, E. et al. Optical and non-optical methods for detection and
characterization of microparticles and exosomes. J. Thromb. Haemost. 8, 2596–
607 (2010).
69.
Van der Vlist, E. J., Nolte-’t Hoen, E. N. M., Stoorvogel, W., Arkesteijn, G. J. a &
Wauben, M. H. M. Fluorescent labeling of nano-sized vesicles released by cells
21
and subsequent quantitative and qualitative analysis by high-resolution flow
cytometry. Nat. Protoc. 7, 1311–26 (2012).
70.
Van der Pol, E., Coumans, F., Varga, Z., Krumrey, M. & Nieuwland, R. Innovation
in detection of microparticles and exosomes. J. Thromb. Haemost. 11 Suppl 1,
36–45 (2013).
71.
Dragovic, R. A. et al. Sizing and phenotyping of cellular vesicles using
Nanoparticle Tracking Analysis. Nanomedicine Nanotechnology, Biol. Med. 7,
780–788 (2011).
72.
Gardiner, C., Ferreira, Y. J., Dragovic, R. A., Redman, C. W. G. & Sargent, I. L.
analysis. 1, 1–11 (2013).
73.
Vogel, R. et al. Quantitative sizing of nano/microparticles with a tunable
elastomeric pore sensor. Anal. Chem. 83, 3499–3506 (2011).
74.
De Vrij, J. et al. Quantification of nanosized extracellular membrane vesicles
with scanning ion occlusion sensing. Nanomedicine (Lond). (2013).
doi:10.2217/nnm.12.173
75.
Roberts, G. S. et al. Tunable pores for measuring concentrations of synthetic and
biological nanoparticle dispersions. Biosens. Bioelectron. 31, 17–25 (2012).
76.
Issadore, D. et al. Miniature magnetic resonance system for point-of-care
diagnostics. Lab Chip 11, 2282–2287 (2011).
77.
Rho, J. et al. Magnetic Nanosensor for Detection and Pro fi ling of ErythrocyteDerived. 11227–11233 (2013).
78.
Shao, H. et al. Protein typing of circulating microvesicles allows real-time
monitoring of glioblastoma therapy. Nat. Med. 18, 1835–40 (2012).
79.
Chen, C. et al. Microfluidic isolation and transcriptome analysis of serum
microvesicles. Lab Chip 10, 505–511 (2010).
80.
Jørgensen, M. et al. Extracellular Vesicle (EV) Array: microarray capturing of
exosomes and other extracellular vesicles for multiplexed phenotyping. J.
Extracell. vesicles 2, 1–9 (2013).
81.
Hill, A. F. et al. ISEV position paper: extracellular vesicle RNA analysis and
bioinformatics. J. Extracell. vesicles 2, 1–8 (2013).
82.
Huang, X. et al. Characterization of human plasma-derived exosomal RNAs by
deep sequencing. BMC Genomics 14, 319 (2013).
83.
Noerholm, M. et al. RNA expression patterns in serum microvesicles from
patients with glioblastoma multiforme and controls. BMC Cancer 12, 22 (2012).
84.
Gaceb, A., Martinez, M. C. & Andriantsitohaina, R. Extracellular vesicles: New
players in cardiovascular diseases. Int. J. Biochem. Cell Biol. (2014).
doi:10.1016/j.biocel.2014.01.018
22
23