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Address correspondence to: Malene Jørgensen, PhD, Department of Clinical Immunology, Aalborg University Hospital, Urbansgade 32-36, DK-9000 Aalborg, Denmark.
Extracellular vesicles (EVs) are small, membrane-enclosed entities released from cells in many different biological systems. These vesicles play an important role in cellular communication by virtue of their protein, RNA, and lipid content, which can be transferred among cells. The complement of biomolecules reflects the parent cell, and their characterization may provide information about the presence of an aberrant process. Peripheral blood is a rich source of circulating EVs, which are easily accessible through a blood sample. An analysis of EVs in peripheral blood could provide access to unparalleled amounts of biomarkers of great diagnostic and prognostic value. The objectives of this review are to briefly present the current knowledge about EVs and to introduce a toolbox of selected techniques, which can be used to rapidly characterize clinically relevant properties of EVs from peripheral blood.
Methods
Several techniques exist to characterize the different features of EVs, including size, enumeration, RNA cargo, and protein phenotype. Each technique has a number of advantages and pitfalls. However, with the techniques presented in this review, a possible platform for EV characterization in a clinical setting is outlined.
Findings
Although EVs have great diagnostic and prognostic potential, a lack of standardization regarding EV analysis hampers the full use of this potential. Nevertheless, the analysis of EVs in peripheral blood has several advantages compared with traditional analyses of many soluble molecules in blood.
Implications
Overall, the use of EV analysis as a diagnostic and prognostic tool has prodigious clinical potential.
In recent years, interest in the characterization, biogenesis, and function of extracellular vesicles (EVs) has increased immensely. These membrane-derived vesicles play vital roles in a plethora of processes in several biological systems. In humans, EVs are pivotal to cellular communication for the maintenance of homeostasis and the development and progression of pathologic conditions, such as cancer. Consequently, this communication forms the basis for the use of EV analysis in a clinical setting because EVs seem to be a promising source of biomarkers of diagnostic and prognostic value. EV analysis can likely be used as one component of treatment surveillance. In addition, EVs have the potential of being used as drug therapy entities, delivering a tailored pharmacologic cargo to a specific target.
Classification of EVs
In general, EVs are a heterogeneous population of membrane-enclosed vesicles released from a variety of cells into the extracellular space in vivo and in vitro. One general feature for these vesicles is that they are enclosed by a membrane that consists of a phospholipid bilayer. However, the EVs can be divided into a number of subpopulations each with specific characteristics, including their biogenesis, size, cellular origin, protein composition, mRNA and microRNA (miRNA) content, and/or biological function. With biogenesis as a classification tool, the EVs can be divided into 3 major groups: exosomes, microvesicles (MVs), and apoptotic bodies. Many of the properties of EVs, and in particular exosomes, have been reviewed extensively elsewhere
; therefore, the following section states the overall characteristics of these 3 EV groups.
Exosomes
Exosome is the vesicle type that has been studied most intensely. They are approximately 30 to 100 nm in diameter and originate from inward budding of the limiting membrane of multivesicular bodies, which are late endosomal compartments present in the cytosol of the cell.
When the multivesicular bodies fuse with the plasma membrane, the release of the exosomes to the extracellular space is facilitated. The biogenesis of the exosomes causes the orientation of the membrane proteins to be similar to that of the plasma membrane. The exosomal membrane is enriched in cholesterol, ceramide, and sphingomyelin and exposes the phospholipid phosphatidylserine.
In addition, exosomes contain several proteins that are currently used as markers to identify exosomes. These makers are not ubiquitously expressed on all exosomes but are found in a large proportion of these vesicles. Therefore, they are generally accepted as exosomal markers. These markers include TSG101, Alix, and the tetraspanins CD9, CD63, and CD81.
Along with these hallmark proteins, the phenotype of exosomes often reflects a molecular signature of the cell from which they originate. This cell-specific signature may provide some indication about the functionality of the exosomes because some of these signature molecules could ensure the delivery of the exosomes to the correct target cell,
The composition of the exosome cargo can also be related to the potential biological function of this vesicle type. They have been known to contain proteins from both the plasma membrane and cytosol along with mRNA and the non–protein-coding miRNAs and small interfering RNAs.
The size of MVs ranges from 100 to 1000 nm. They are formed from outward budding of the plasma membrane, thus releasing the MVs directly into the extracellular space.
Hence, the membrane proteins of MVs retain the topologic features of those found in the plasma membrane. Generally, most MVs incorporate phosphatidylserine in the outer leaflet of the membrane.
This feature has frequently been used to isolate and identify MVs from biological samples along with a combination of cell-specific protein markers to determine their cellular origin.
The majority of circulating platelet-derived microparticles fail to bind annexin V, lack phospholipid-dependent procoagulant activity and demonstrate greater expression of glycoprotein Ib.
Currently, there is a less extensive list of markers to identify MVs when compared with exosomes. Nonetheless, CD40 ligand, adenosine diphosphate ribosylation factor 6, and several integrins and selectins have been proposed as MV markers.
Like exosomes, the phenotype of MVs reflects their parent cell, and the content of the vesicle cargo also includes membrane and cytosolic proteins, mRNA, and miRNAs.
They are formed from blebbing of the plasma membrane in cells undergoing apoptosis, releasing the apoptotic bodies straight into the extracellular space.
Unlike the 2 other vesicle types, apoptotic bodies are distinguished by containing organelles, DNA fragments, and histones as a part of the vesicular cargo in addition to proteins and other molecules from the cytosol of the parent cell.
an enormous amount of research has been performed to delineate all aspects of this phenomenon, however, predominantly related to exosomes and MVs. Within the field of immunology, the evidence of the immunomodulatory effects of EVs has steadily increased throughout the years. Several studies document that many of the cells of the adaptive and innate immune system release EVs, including T and B lymphocytes, dendritic cells, and mast cells.
Exosomes have in particular been implicated in several contexts of antigen presentation. T cells can be activated by major histocompatibility complex II–bearing exosomes from antigen-presenting cells,
On the transcriptional level, miRNA can also be transferred from T cells to antigen-presenting cells by exosomes, and this molecular transfer changes the gene expression of the recipient cell.
In relation to pathologic challenges of the immune system, EVs have been associated with the spread of several types of infection. Accordingly, EVs from cells infected with HIV-1, Epstein-Barr virus, hepatitis C virus, and cytomegalovirus mediate the spread of these pathogens by entering the recipient cell.
Transfer of the chemokine receptor CCR5 between cells by membrane-derived microparticles: a mechanism for cellular human immunodeficiency virus 1 infection.
Tumor-derived EVs can also play a role in transferring oncogenic activity, as was observed with the vesicular transfer of the oncogenic epidermal growth factor receptor vIII from glioma cells.
Currently, it is apparent that several aspects of the mechanisms and functional consequences of EVs have yet to be determined. Nonetheless, it is also evident from the work already performed that EVs are central to an abundance of cellular processes, including those of a pathologic nature, and consequently have great potential as targets for diagnosis, prognosis, or treatment.
Diagnostic and Prognostic Potential of EVs In Peripheral Blood
Numerous studies have established that EVs can be detected in a multitude of biological fluids, such as saliva, urine, blood, ascites, breast milk, and cerebrospinal fluid.
Because EVs are released constitutively into the bloodstream and this release increases on cellular activation, as well as in many pathologic conditions,
Elevated levels of circulating platelet microparticles, VEGF, IL-6 and RANTES in patients with gastric cancer: possible role of a metastasis predictor.
a mere enumeration of EVs may indicate the presence of an aberrant process. In many aspects of diagnostics, the use of blood samples is already implemented in the clinic because it is known that blood harbors a vast amount of biomarkers and other biologically relevant molecules. In addition, most tissues will contribute to this molecular reservoir due to dense vascularization of the body. In line with this, the analysis of EVs in peripheral blood is likely to provide an indicator of the systemic health status, which can be used in clinical settings.
Analysis of EVs in Peripheral Blood
The use of EV analysis encompasses several advantages over the traditional analyses of many soluble molecules in blood, such as hormones and cytokines. One significant advantage is the inherent protection of the EV cargo of proteins and RNA from degradation, thus rendering them intact and functional.
This has proven to be particularly significant for the use of miRNA as valuable biomarkers because most RNA in blood exists as cargo of EVs. Regarding another aspect of stability, EVs appear to have a relatively long half-life in blood.
Therefore, EVs can likely be transported from any location of the body to the bloodstream, thus making them easily accessible for analysis, compared with biopsies. This also has a significant advantage for the patient because the collection of a blood sample is a minimally invasive procedure associated with much less discomfort than a biopsy. Another advantage of EVs links to the great dynamic range of molecules present in the bloodstream. It can often become an issue to detect relevant biomarkers because these diagnostic molecules frequently constitute a small part of the total amount of molecules in a blood sample.
However, on the basis of the presence of a relatively small panel of markers, EVs can be identified, and the phenotype and cargo, holding biomarkers of otherwise undetectable amounts, can be analyzed and interpreted into clinically relevant information. Furthermore, EV size distribution, phenotype, or cargo content can seemingly change according to the progression of a disease.
Elevated levels of circulating platelet microparticles, VEGF, IL-6 and RANTES in patients with gastric cancer: possible role of a metastasis predictor.
The access to cell-specific and disease state–specific EV profiles of proteins and RNA from a blood sample renders a sophisticated fingerprint of a disease of diagnostic and prognostic value.
EV Analysis in Risk Stratification
Cases are emerging in which EV analyses of blood samples seem to be good candidates as a part of a diagnostic platform. In addition, some examples exist in which this type of analysis shows a promising prognostic potential. As previously mentioned, the number of detectable EVs in a blood sample seems to increase in several types of cancers compared with healthy individuals.
Elevated levels of circulating platelet microparticles, VEGF, IL-6 and RANTES in patients with gastric cancer: possible role of a metastasis predictor.
Elevated levels of circulating platelet microparticles, VEGF, IL-6 and RANTES in patients with gastric cancer: possible role of a metastasis predictor.
In further relation to cancer, protein and RNA biomarkers have been identified in EVs from the peripheral circulation, with the preponderance of cases that involve miRNAs found in exosomes. Accordingly, several cases reveal that either a single or a panel of differentially expressed miRNAs can significantly identify patients with cancer compared with control individuals.
The use of EV protein markers in risk stratification has also been investigated. One study found that the presence of 2 exosomal proteins in exosomes from plasma distinguished patients with ovarian cancer from controls.
In another study, EVs from patients with gastric cancer had significantly increased expression of 2 proteins, including HER2/neu, when compared with controls.
From this study, an interesting notion arises, indicating that some EV markers may not exclusively be used in diagnosis but could also indicate which patients are eligible for a specific treatment. A list of disease-related EV proteins from blood is given in Table II.
Table IISelected protein extracellular vesicle markers isolated from blood.
The characterization of EVs in peripheral blood can be based on several of their biochemical and biophysical properties. These properties include size, cargo, density, morphologic findings, lipid composition, and protein phenotype. Currently, a wide range of techniques facilitates EV analysis. Some techniques have existed for years and are being further developed to embrace the challenges of this type of analysis. Other techniques have emerged as a consequence of the increasing interest within the field. The focus of the following section is to introduce a toolbox of selected techniques that could be part of a potential platform to rapidly characterize several clinically relevant properties of EVs in peripheral blood. An overview of the steps from blood sample to output of the analysis will be presented for each technique along with advantages and pitfalls. A summary of several key features associated with the presented techniques is outlined in Figure 1.
Figure 1Overview of the techniques used to analyze extracellular vesicles (EVs) present in blood samples from a clinical and diagnostic perspective. The 3 major characterization areas are illustrated (phenotyping, size and enumeration, and RNA cargo) and the major advantages and pitfalls are outlined for each of the techniques presented in this review. abs = antibodies; cDNA = complementary DNA; LOD = limit of detection; F-NTA = fluorescence nanoparticle tracking analysis; micro-NMR = micro–nuclear magnetic resonance; MVs = microvesicles; NTA = nanoparticle tracking analysis; qRT-PCR = quantitative real-time reverse transcription–polymerase chain reaction; SIOS = scanning ion occlusion sensing.
One aspect that is a prerequisite for almost all techniques used for EV analysis is isolation of EVs from the biological fluid in which they are present. Hence, it is evident that EV isolation is essential, and with no standardized protocols yet, this is a major focus in the field of EV research.
The isolation includes numerous sequential centrifugation steps with increasing centrifugal force, thus using size and density properties of the EV subsets to separate these from other components of blood. Initially, low-speed steps (300-500g) are applied to remove whole cells.
Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes.
Nevertheless, the choice of isolation procedure may have a considerable effect on the downstream analyses of EVs from blood. In addition to isolation, the preanalytical procedures also encompass choice of anticoagulant, processing temperature, and storage before analysis. These variables are all likely to affect the dynamic material that blood constitutes, and although standardizations are in the pipeline, several recommendations have been published.
Several methods exist to characterize the protein composition of EVs, related to either a surface marker phenotype or the proteins present in the EV cargo. Although the EV phenotype is particularly important in the determination of cellular and subcellular origin, it can in combination with a protein cargo analysis also provide clues about the functionality of the EVs. The following section focuses on 4 techniques used for characterization of EV proteins because they have great potential as reliable methods used in EV analysis. Common to all 4 methods is the dependence and use of antibodies. Consequently, they all rely on the specificity and sensitivity of the applied antibodies. Currently, no proteins are known to be constitutively sorted into vesicles independently of the subcellular origin of the vesicle and the activation status of the producing cell. This lack of invariant housekeeping markers hampers the quantitative analysis of vesicles. In the 1990s, exosomes derived from B cells were discovered to contain the lysosomal membrane protein CD63,
These early studies also found that exosomal CD63 is present in a much lower amount compared with that of the producing cells. In contrast, CD81 was >10-fold up-regulated in exosomes.
The phenotyping of plasma exosomes from 7 healthy donors revealed a similar tendency, indicating that CD63 is less represented and in a heterogeneous manner in relation to CD81 and CD9.
which may have been attributed solely to its initial discovery and description. Consequently, it is important to choose the markers for EV phenotyping carefully.
Western Blotting
The analytical technique called Western blotting (WB) or immunoblotting is a widely accepted method used to detect specific protein markers in EVs. In general, WB is applied to validate the presence or absence of EV protein markers in purified samples based on the availability of specific antibodies.
Activated platelets release two types of membrane vesicles: microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules.
. A major advantage of WB is the possibility of detecting intravesicular and membrane-associated proteins with the same technology (Figure 1). The basic principle of WB
Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure.
is outlined in Figure 2A. The results obtained by WB are semiquantitative and can be correlated to the presence of a uniformly expressed protein. Multiple steps of centrifugation of the EV-containing samples are generally needed before analysis.
Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes.
In addition, the amount of EV-containing sample used for WB can vary greatly. To illustrate this, it was possible to generate 5 WBs using an EV sample of 200 µL of plasma,
whereas another study used WB analysis for the detection of CD63 in EVs and was able to accomplish this with approximately 0.2 µg of EV protein, equivalent to 108 EVs.
Because WB depends on a high protein concentration, sample preparation with ultracentrifugation is needed, which may be difficult in many clinical laboratories. Nonetheless, if the diagnostic or prognostic marker is of intravesicular origin, WB remains the only usable detection method of those currently applied.
Figure 2Outline of the workflow and timeframes for the presented techniques used for phenotyping of extracellular vesicles (EVs) from peripheral blood. (A) Western Blotting, (B) flow cytometry of microvesicles (MVs), (C) flow cytometry of exosomes, (D) micro–nuclear magnetic resonance (micro-NMR), and (E) EV Array. abs = antibodies; MNP = magnetic nanoparticles; PS = phosphatidylserine.
Flow cytometry is a powerful technique for multiparametric analysis of single biological particles and remains the most extensively used technique for enumeration and phenotyping of EVs in clinical samples.
By suspending particles in a hydrodynamically focused fluid stream passing a laser beam, this technique allows simultaneous analysis of the physical characteristics, including size and granularity, and expression of multiple antigens of up to thousands of particles per second.
Different approaches are applied when phenotyping either MVs or exosomes by flow cytometry. MVs are typically identified as particles with a forward scatter smaller than an internal standard that consists of approximately 1-µm beads.
Figure 2B illustrates how MVs can be distinguished using a blend of size-calibrated fluorescent beads. When analyzing a plasma sample, better specificity of MV detection is obtained by identifying those MVs that expose phosphatidylserine and cell-specific markers (Figure 2B). In addition, MV count per analyzed volume can easily be calculated by adding a known number of fluorescent latex beads as an internal standard. For the phenotyping of MVs by flow cytometry, use of 50 to 100 µL of platelet-free plasma is recommended.
Use of flow cytometry to phenotype exosomes and other EVs <200 nm normally depends on adsorption of these vesicles onto antibody-coated beads (Figure 2C). The beads are large enough to facilitate the detection of these small vesicles, which would otherwise fall below the lower limit of detection (Figure 2C). A multiparametric analysis of the EVs captured onto the beads can be performed using fluorescent-labeled antibodies against selected EV surface markers.
To be able to use flow cytometry for plasma exosome characterization, an isolation procedure is needed. As an example, a 3-step centrifugation isolation on 3 mL of plasma can be used as a preparatory procedure before the phenotyping.
The previous generation of flow cytometers is challenged by the small size and signals from most EVs, and several laboratories have reported that a size of 0.5 µm is the cutoff value for accurate identification.
Standardization of platelet-derived microparticle counting using calibrated beads and a Cytomics FC500 routine flow cytometer: a first step towards multicenter studies?.
However, the enhanced sensitivity of modern digital flow cytometers allows the detection of EVs in the range of approximately 200 nm to 1.0 µm21 (Figure 1).
Micro–Nuclear Magnetic Resonance
Micro–nuclear magnetic resonance (µ-NMR) is a highly sensitive and rapid analytical technique developed for profiling circulating EVs from blood samples.
The technique uses size and immunoaffinity to define an EV population and favors EVs in the range of 50 to 150 nm. The EVs are labeled with target-specific magnetic nanoparticles and detected by a hand-sized NMR system
The output of the µ-NMR technique is a MV expression of an EV protein marker, which is calculated as the decay rate of the target protein normalized to that of a selected exosomal marker, such as CD63.
The output is quantitative in a relative way across samples but cannot be assigned a definitive number of vesicles or number of marker molecules on each vesicle.
Before loading the blood sample onto the chip, a 2-step centrifugation purification is needed. The pelleted EVs can be loaded onto the µ-NMR device for analysis, and the relative contents of a single EV marker are determined within seconds. Signals are detectable to approximately 104 of EVs, which is equivalent to 0.02 ng of EV protein.
this may not be the case with EVs from all cellular systems. Hence, care should be taken to choose the appropriate exosomal marker for normalization.
EV Array
The EV array is based on the technology of protein microarray and is capable of detecting and phenotyping EVs from unpurified starting material in a high-throughput manner.
The technology is developed to perform multiplexed phenotyping of EVs in an open platform; however, it is optimized to analyze the exosomal EV subset. Protein microarrays are well accepted as powerful tools to search for antigens or antibodies in various sample types.
Micrometer-sized spots of capturing antibodies against known EV surface antigens are printed in a customized spot setup. The basic principle of the subsequent analysis is outlined in Figure 2E. The captured vesicles are detected using a cocktail of antibodies against the tetraspanins CD9, CD63 and CD81.
The antibodies are applied to ensure that all exosomes captured are detected, as well as excluding other types of EVs from being detected. The read-out is a fluorescence signal for each individual microarray spot (Figure 2E). The method is semiquantitative and gives the phenotype of EVs for 21 protein markers simultaneously. Plasma samples are analyzed directly without any further purification or isolation steps, and only 10 µL of plasma is needed. It has been established that only 2.5 × 104 exosomes is required for each microarray analysis.
The technique uses an overnight incubation to capture the EVs. Because of the incubation time and the remaining development procedure, this method takes approximately 2 days to complete, making it the slowest assay of the ones presented in this review.
Enumeration and Size
Determination of the amount and size distribution of EVs in a blood sample is greatly valuable because of the previously mentioned observation that an increased production of EVs has been detected in several pathologic conditions. In addition, knowing the size of the EVs present can be informative because it can indicate which vesicle type is the most dominant type in an unprocessed sample and provide information about the quality of an isolation procedure.
Several methods exist to enumerate EVs and determine their size. The following section focuses on 2 of these because they have great potential as fast methods used in EV analysis.
Nanoparticle Tracking Analysis
Nanoparticle tracking analysis (NTA) allows for a quick size determination and quantification of EVs in a suspension. It was first introduced in 2006 and has in recent years received significant attention within the field of EV analysis.
The technique is based on relating the Brownian motions of a particle to its size. Brownian motions are experienced by all particles in a fluid, here constituted by EVs in a suspension. These motions can be tracked when the particles are illuminated by a laser beam and scatter the light
Hence, NTA seems to offer a more sensitive enumeration than other conventional methods, such as flow cytometry. For the analysis of plasma, the high content of lipoprotein particles may pose a significant problem.
As an interesting notion, selected NTA instruments allow for the expansion to fluorescence NTA in which EVs can be labeled with antibodies conjugated to stable fluorophores and subsequently be phenotyped in addition to the enumeration and concentration determination.
Figure 3Outline of the workflow and timeframe for the presented techniques used for size determination and enumeration of extracellular vesicles (EVs). (A) Nanoparticle tracking analysis; (B) Scanning Ion Occlusion Sensing.
The scanning ion occlusion sensing (SIOS) method is a relatively new technology that facilitates a fast analysis of EV size, concentration, and biochemical composition. The technology is nonoptical, label free, and based on passing each EV through a nanopore in a membrane by means of single-molecule electrophoresis.
The basic principle is shown in Figure 3B. A transmembrane current drives the electrophoresis, and an ionic current through the nanopore channel can be measured.
In addition to the size and surface charge, the total number of EVs passing the nanopore is detected, and the read-out can be depicted as a plot of concentration versus size distribution (Figure 3B). The SIOS has a reported usability for particles in the large range of 70 nm to 10 µm and in the concentration of 105 to 1012 particles/mL.
The use of complex samples can pose some difficulties because protein aggregates cannot be separated from MVs, which is explained by their similar size (Figure 1). In contrast to NTA, SIOS does not depend on the refractive index of the particles analyzed because it is a nonoptical technique.
RNA cargo
Along with the increasing attention on EVs, it has become consistently apparent that their RNA cargo contributes to the potential diagnostic and prognostic value of these vesicles. As mentioned previously, several research groups have reported that the RNA present in serum and plasma is in fact protected from the highly active RNases when being stored inside the different kind of vesicles.
The expression of miRNAs seems to depend on the cellular function and in some cases on the cellular stage. In addition, a number of miRNAs are specific to cell type or tissue.
As previously mentioned, a rapidly expanding list of miRNAs as potential biomarkers is forming, in which a differential expression can be detected between healthy and pathologic conditions, in particular related to cancer. A selection of such EV miRNA markers is given in Table I. When focusing on the discovery of potential miRNA biomarkers, approaches such as microarray analyses and deep sequencing are useful techniques to perform a wide search. Such a search can be performed with pooled samples of a chosen disease group versus a control group. After single or multiple candidates are identified, a more targeted manner is chosen to validate and quantify these findings to reveal the true diagnostic potential.
Because the qRT-PCR technique is the most relevant as a possible clinically test to characterize the EV RNA cargo, this is the focus of the following section.
qRT-PCR
The qRT-PCR technique is based on the PCR, which is used to amplify and simultaneously quantify a targeted RNA molecule.
Relative quantification of mRNA levels in Jurkat T cells with RT-real time-PCR (RT-rt-PCR): new possibilities for the screening of anti-inflammatory and cytotoxic compounds.
For ≥1 specific sequences in a sample, qRT-PCR enables detection and quantification. The quantity can be an absolute number of copies or a relative amount when normalized to RNA input or additional reference genes. Initially, the characterization of the EV RNA cargo requires extraction of the RNA
before qRT-PCR (Figure 4). The output of the quantification is a signal that relates to the amount of RNA present in the sample. Because this has no unit, the results of the relative quantification can be compared across a number of different qRT-PCR analyses. A total of ≥1 reference genes are applied to correct for nonspecific variation, such as the differences in the quantity and quality of RNA used, which can affect the efficiency of reverse transcription and therefore the entire PCR process. In most of the reported work on EV RNA cargo analysis, a comprehensive isolation procedure is applied (Figure 4). The isolation often involves multiple centrifugation steps and filtration.
However, an optimized protocol was recently published, describing the extraction and qRT-PCR analysis of EV RNA directly from plasma and serum isolated with only a single centrifugation step at low speed and time.
Figure 4Outline of the workflow and timeframe for the analysis of extracellular vesicle (EV) RNA cargo by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). cDNA = complementary DNA.
The techniques presented in this review could all potentially be part of a platform to characterize EVs from peripheral blood and transform these data into clinically relevant information. However, to fully harness the diagnostic and prognostic potential of EVs, a number of aspects remain to be delineated. A fundamental aspect, which is imperative to address, is standardization.
The standardization relates to several areas of EV analysis and research and includes nomenclature, preanalytical conditions, and isolation procedures. This will give rise to standardized protocols and facilitate unbiased comparisons of results from different studies across laboratories. Furthermore, it will greatly aid in the development of robust clinical assays. The next challenge, before using EVs in a diagnostic manner, is to select the most optimal methods, concerning sample requirements, apparatus, and analysis accessibility. In addition, selection of the most optimal combination of target markers is essential to several of the methods. A common feature for all the methods outlined is the need for an extensive isolation procedure before EV analysis. In that regard, considerations should be taken concerning the loss of material and thereby valuable information. Therefore, the method of analysis should most optimally work on unpurified plasma or serum samples. A prerequisite for the incorporation of EV analysis into robust clinical assays is the discovery and validation of relevant diagnostic or prognostic EV targets. Currently, thousands of EV-related proteins and RNA components have been identified by proteomic and transcriptomic approaches.
Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes.
Even if cell lines are only an approximation of the in vivo conditions, they serve as useful model systems and encompass a relevant source of cell surface–expressed biomarkers and intracellular proteins and RNAs, relating to cancer in particular. These in vitro model systems are consequently valuable in the matter of identifying which proteins and potential biomarkers are conveyed to the EVs. As an example, this strategy was adopted to identify the protein Claudin as an EV-expressed biomarker for ovarian cancer. Likewise, the model systems of cell lines have a great potential to elucidate the biogenesis of EVs and their subsequent influence on the recipient cells. This finding has been reported in a study in which a mast cell line was exposed to oxidative stress and the subsequent cell-derived EVs revealed a changed RNA profile compared with those derived from cells grown under normal conditions. This study additionally clarified that the stress-derived EVs were able to affect nonstressed cells to become more resistant to the oxidative stress.
Nonetheless, the use of cell lines and their autologous production of EVs must be carefully interpreted. Cell lines are kept in a closed and controlled system, mainly as monocultures, and will therefore only receive and respond to the signals specifically applied to them. Because the cells receive no signals or EVs from their original surrounding environment, they will most likely merely autocommunicate. Therefore, cell lines will only partly reflect the authenticity of the biogenesis of the EVs. In the biomarker discovery, the use of mass spectrometry (MS) has for at least 2 decades been essential for the proteomics-driven research of EVs and is the only technique available that allows EV characterization from all common clinical biospecimens, including serum, plasma, urine, synovial fluid, ascites, and feces. Accordingly, the identification of several important EV protein markers has been facilitated by MS.
Currently, technical methods for targeted MS platforms are being applied in clinical assays and include multiple reaction monitoring and selected reaction monitoring. These methods enable MS as a high-throughput technique to perform rapid analyses of EV proteins, which could also be used in a diagnostic fashion and not exclusively in the discovery phase for EV biomarkers. Nonetheless, the development of standardizations for EV analysis along with a continued effort to unravel all aspects of EV proteins and RNA will improve the understanding of the molecular mechanisms and biological functions of EVs. Despite being in its infancy, the field of EV research has undoubtedly received an enormous interest. Consequently, numerous initiatives have been implemented in the EV society, including several databases (ExoCarta,
), conferences (International Society for Extracellular Vesicles conference on microvesiculation and disease), and a dedicated journal (Journal of Extracellular Vesicles).
The launch of Journal of Extracellular Vesicles (JEV), the official journal of the International Society for Extracellular Vesicles - about microvesicles, exosomes, ectosomes and other extracellular vesicles.
J Extracell Vesicles.2012 Apr 16; ([Epub ahead of print])
The apparent role of EVs in a vast number of biological processes, along with many of their intriguing features, forms the basis of extending EV analysis beyond basic research and into a clinical and therapeutic context. Despite being a relatively new field, the potential and versatility of EV analysis are supported by an increasing number of publications. The applications of this type of analysis include the areas of diagnostics and prognostics, as well as drug therapy, regenerative medicine, and vaccines. For the purpose of diagnosis and prognosis, the use of EVs seems particularly promising because these vesicles contain a plethora of clinically relevant molecules, such as proteins and RNA from the parent cell. Thereby, the miRNA and protein patterns, which are unique for a specific pathologic condition, can be used. The analysis of EVs could accordingly be incorporated as a screening tool and applied to confirm a diagnosis. Furthermore, EV analysis has the potential to become an element in treatment surveillance and companion diagnostics, resulting in a patient-optimized treatment. Related to therapeutics, EVs have been proposed as a new type of drug delivery system. Such a system involves engineered EVs loaded with a therapeutic cargo and expressing ligands, which target a particular tissue or cell type. The inherent protection of the cargo and tailored cellular targeting simultaneously enhance the solubility, stability, and specificity of the therapeutic agent. In another approach, the EVs constitute the therapeutic target, and the aim is to inhibit their release or to perform a systemic depletion. In terms of regenerative capabilities, EVs from stem cells have clinical potential.
The EVs accordingly constitute a cell-free approach, which mediates many of the regenerative properties from the stem cells. This circumvents several issues related to stem cell transplantations, consequently making handling and storage easier and increasing the stability. This cell-free approach has also been adapted in the field of vaccines and immunotherapy. Particularly in relation to cancer, EVs have been used as potent inducers of antitumor responses.
Exosomes as potent cell-free peptide-based vaccine, I: dendritic cell-derived exosomes transfer functional MHC class I/peptide complexes to dendritic cells.
The results of these studies indicate a promising future for the use of EV-based treatment. The abovementioned examples of the clinical use of EV analysis clearly indicate its feasibility. In line with the scope of this review, the potential of using EV analysis of a simple blood sample seems encouraging, and this is particularly pronounced in relation to diagnostics. Despite the extensive amount of information, which is possible to obtain from a single blood sample, this complexity is at the same time a challenge that complicates EV analysis. Nonetheless, advances and continued research within the field allow for the development and improvement of techniques and standard protocols, which seek to meet these challenges. The increased understanding of this may lead to a paradigm shift, particularly related to immune regulation and cellular communication in cancer. Taken together, technology and biology will inevitably pave the way for the future use of EV analysis in many clinical applications.
Conflicts of Interest
The authors have indicated that they have no conflicts of interest regarding the content of this article.
Acknowledgments
Ms. Revenfeld, Ms. Bæk, Dr. Nielsen, and Dr. Jørgensen performed the literature search, the figure creation, and wrote or contributed to the writing of the manuscript. All authors contributed to the design of the article and revised and accepted the final manuscript.
The authors gratefully acknowledge Dr. Shona Pedersen (Department of Biochemical Chemistry, Aalborg University Hospital, Aalborg, Denmark) for input and discussion of technical issues regarding size and enumeration of EVs. The authors also kindly acknowledge the editorial support provided by Dr. Lotte H. Pugholm and Dr. Evo K.L. Søndergaard (Department of Clinical Immunology, Aalborg University Hospital, Aalborg, Denmark).
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The majority of circulating platelet-derived microparticles fail to bind annexin V, lack phospholipid-dependent procoagulant activity and demonstrate greater expression of glycoprotein Ib.
Transfer of the chemokine receptor CCR5 between cells by membrane-derived microparticles: a mechanism for cellular human immunodeficiency virus 1 infection.
Elevated levels of circulating platelet microparticles, VEGF, IL-6 and RANTES in patients with gastric cancer: possible role of a metastasis predictor.
Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes.
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Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure.
Standardization of platelet-derived microparticle counting using calibrated beads and a Cytomics FC500 routine flow cytometer: a first step towards multicenter studies?.
Relative quantification of mRNA levels in Jurkat T cells with RT-real time-PCR (RT-rt-PCR): new possibilities for the screening of anti-inflammatory and cytotoxic compounds.
The launch of Journal of Extracellular Vesicles (JEV), the official journal of the International Society for Extracellular Vesicles - about microvesicles, exosomes, ectosomes and other extracellular vesicles.
J Extracell Vesicles.2012 Apr 16; ([Epub ahead of print])
Exosomes as potent cell-free peptide-based vaccine, I: dendritic cell-derived exosomes transfer functional MHC class I/peptide complexes to dendritic cells.
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