Extracellular Vesicles in Oncology: Progress ... - Wiley Online Library

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Extracellular Vesicles in Oncology: Progress and Pitfalls in the Methods of Isolation and Analysis Donatella Lucchetti, Andrea Fattorossi, and Alessandro Sgambato*

platelets but distinguishable from them. Wolf described this particulate as “platelet dust” which was subsequently replaced by the term “extracellular vesicles.”[1] In 1981, Trams and coworkers[2] showed that cultured normal and neoplastic cells release vesicles that could be involved in physiological processes. Few years later, Johnstone discovered that, during the in vitro maturation, reticulocytes release small vesicles and called them “exosomes.”[3] Only few articles on exosomes have been published during the years soon after their discovery but the extracellular vesicles have been recently re-discovered by cancer scientists and represent now one of the most exciting and rapidly advancing fields in cancer research (Figure 1A). The term of EVs was introduced by the International Society of Extracellular vesicles (ISEV) and their classification is based on their biogenesis. The three main classes of extracellular vesicles are exosomes, microvesicles, and apoptotic bodies. Here, we will focus on the first two classes of extracellular vesicles: microvesicles and exosomes[4] (Figure 1B). Microvesicles originate directly from the budding of the plasma membrane[5] while exosomes represent the intraluminal vesicles (ILVs) of multivesicular bodies (MVBs), which are formed during the maturation of endosomes. Some MVBs are fated for degradation, whereas others can fuse with the plasma membrane, leading to the exosome secretion.[5,6] The generation of the ILVs within MVBs involves the segregation of cargo at the endosomal membrane, the formation of inward budding vesicles and their release in the endosomal lumen containing a small portion of cytosol. A group of proteins, forming the endosomal sorting complex required for transport (ESCRT), can regulate the biogenesis of exosomes.[7,8] However, the formation of exosomes can also take place in ESCRTindependent manners.[9] All cell-derived vesicles are enclosed by a lipid bilayer but their size is different: exosomes range from 30 to 150 nm whereas microvesicles range from 100 to 1000 nm.[10] Although size is the simplest method to distinguish exosomes from microvesicles, a subpopulation of small microvesicles which originate directly by plasma membrane, called exosome-like vesicles (20–50 nm), confuses this standard classification[11] (Table 1).

The possibility to study solid tumors through the analysis of extracellular vesicles in biological fluids is one of the most exciting and rapidly advancing field in cancer research. The extracellular vesicles are tiny sacs released in both physiological and pathological conditions and can be used to monitor the evolution of several pathological states, including neoplastic diseases. Indeed, these vesicles carry biological informations and can affect the behavior of recipient cells by transferring proteins, DNA, RNA, and microRNA. In this review the authors analyze the methods to collect biological fluid samples (urine, plasma/serum, and cell supernatant), and to isolate and quantify extracellular vesicles highlighting advantages and drawbacks. Moreover, the authors provide an overview on the adoption and the advantages of the methods (such as digital PCR, next generation sequencing, reverse-phase protein microarrays, flow-cytometry, etc.) most frequently used to analyze the molecular content of extracellular vesicles. Despite the great scientific interest on this topic, there is still a great uncertainty about which is the best method for the collection, isolation, quantification, and molecular evaluation of these vesicles and a standardization is needed. The features of EVs make them ideal candidates for liquid biopsy-based biomarkers. However, the small size of EVs makes their analysis very difficult and requires multiple advanced technologies, being therefore a limitation.

1. Extracellular Vesicles: From the Beginning to Biomarkers Discovery Extracellular vesicles (EVs) are heterogeneous membranebound organelles that are shed from the surfaces of cells into the extracellular environment and are involved in extra cellular communication. The extracellular vesicles have been described for the first time by Wolf in 1967.[1] They showed that in normal plasma and serum, there was a minute particulate isolated through high-speed centrifugation originating from Dr. D. Lucchetti, Prof. A. Sgambato Institute of General Pathology Università Cattolica del Sacro Cuore Largo Francesco Vito 1, 00168 Rome, Italy E-mail: [email protected] Prof. A. Fattorossi Department of Obstetrics and Gynecology Fondazione Policlinico A. Gemelli Largo Agostino Gemelli 8, 00168 Rome, Italy

DOI: 10.1002/biot.201700716

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Donatella Lucchetti is a post-doc researcher supported by an AIRC fellowship at Catholic University of Sacred Heart in Rome, Italy. In particular, Dr. Lucchetti is interested in exploring the role of extracellular vesicles in cancer tumorigenesis and progression with particular attention to CSCs. She won an AIRC fellowship in 2016 to study the relationship among differentiation, mutational status of colorectal cancer stem cells, and extracellular vesicles release. Moreover, she was awarded a post-doctoral fellowship from the Fondazione Umberto Veronesi.

Figure 1. A) Histogram of extracellular vesicles (EVs) studies over the past 30 years. Interest in EVs has strikingly increased in the last 10 years when more than 80% of the studies on EVs have been published. The histogram was generated based on PubMed indexed EVs studies (keywords: extracellular vesicles, exosomes, or microparticles). The resulting articles were verified by reading the title and abstract to ensure that the study referred to EVs. B) Exosomes originate from multivesicular bodies (MVBs), late endosome-derived cell compartments, which bud off parts of their limiting membrane into their lumen, forming intraluminal vesicles. The exocytic MVBs fuse their membrane with the plasma membrane upon cell stimulation. Microvesicles, on the other hand, originate directly from plasma membrane.

The different origin of EVs implies that the membrane composition of microvesicles is more similar to that of the parent cell than exosomes. Both types of EVs can contain microRNA, RNA, DNA, cytokines, growth factors, and other proteins involved in cell-to-cell communication both in physiological and pathological conditions. Through this complex “trafficking,” EVs can also transport molecular elements potentially able to favor tumorigenesis.[4] The release of EVs into the extracellular space offers an opportunity to examine them in body fluids such as blood, urine, liquor, and malignant effusions. Moreover, the possibility to easily isolate EVs from biological fluids makes them potentially useful biomarkers for the clinical management of neoplastic diseases. The advantages of EVs as biomarkers and/or source of biomarkers are i) the non-invasive way to access to these bioactive vesicles; ii) EVs are easily accessible and amenable to functional and molecular analysis[12]; iii) analysis of circulating

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Andrea Fattorossi is a contract professor at Dept. of Gynecology and Obstetrics, Catholic University of Sacred Heart, Rome and is head of the Laboratory of Tumour Immunology of the same Institution. His major research projects are 1) changes in the immune profile of tumour draining lymph nodes in relationship with disease; 2) clinical grade expansion of anti-tumour cytotoxic T cells for the development of a cell therapy using anti-tumor cytotoxic T cells; 3) multicentrer Phase II and III randomized studies on vaccination in patients with ovarian cancer; and 4) microvesicles identification by advanced flow cytometry. Alessandro Sgambato is a full professor of General Pathology at Catholic University of Sacred Heart. His intense scientific interest has been devoted from the beginning to the molecular mechanisms underlying malignant cell transformation. Currently, Prof. Sgambato is focusing on the role of extracellular vesicles in the tumorigenic process.

EVs would represent a “liquid biopsy” with the advantage of not requiring the partial or total removal of tumors to get informations about their molecular genetics landscape and allowing repeated analyses over time. EVs-related biomarker studies could be divided into three groups according to the analytes tested: i) quantity, ii) protein composition, iii) nucleic acid composition depending on the variable referred to for the evaluation of their potential role as biomarkers. Despite a great scientific interest has grown on this topic, a great limit to its further development is the lack of a standardization of the methods for collection, isolation, quantification, and molecular evaluation of EVs.[13] Recently, the ISEV has established an EV-TRACK platform to guide EVs

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Table 1. Major features of extracellular membranous vesicles.

Size (diameter) Flotation density

Exosomes

Microvesicles

30–100 nm

100–1000 nm

1.13–1.19 g mL

Morphology

1

1.032–1.068 g mL

Exosome-like vesicles 20–80 nm 1

1.1–1.19 g mL

1

Cup-shaped

Cup-shaped

LBPA, low phosphatydilserine, cholesterol, ceramide, sphyngomyelin

High phosphatydilserine, cholesterol

ND

Alix, TSG101, HSC70, CD63, CD81, CD9

Integrin, selectin

Integrin, selectin

Site of origin

MVBs

Plasma membrane

Plasma membrane

Composition

miRNA, mRNA, protein, DNA, lipids

miRNA, mRNA, protein, lipids

miRNA, mRNA, protein, lipids

Lipid composition Possible protein marker

Cup-shaped

LBPA, lysobisphosphatidic acid; Alix, ALG-2 interacting protein X; TSG, tumor susceptibility 101; HSP, heat shock protein; MVB, multivescicular body; ND, not determined.

researchers in specific aspects to identify the minimal experimental requirements for the study of extracellular vesicles and to interpret and reproduce the experiments.[14] In this review, we analyze the methods to collect biological fluid samples, and to isolate and quantify EVs. Moreover, we provide an overview on the methods most frequently used to analyze the molecular content of EVs.

2. Sample Collection and Storage 2.1. Collection and Storage of Blood Samples Blood products (serum and plasma) are the most commonly used biofluids in EVs studies (69%).[15] The advantages of blood are multiple and include the homogeneity of the samples, the ease of access and the extensive archives from clinical studies. The best choice between plasma or serum samples for collection of EVs is still controversial.[16,17] A worldwide survey on techniques used for the isolation and characterization of EVs has indicated the plasma as the most commonly used blood product, with a percent of 47% versus the 22% of serum samples.[15] The use of serum is suggested when the biological question involves platelets and platelet EVs-associated nucleic acids.[13] Besides this situation, plasma samples are preferred because the plasma is “the physiological fluid” of the blood and EVs isolation would not be affected by platelets-released EVs[18] (Table 2). The standardization of pre-analytical and analytical methods for the measurement of EVs in blood samples remains a challenge. The activation of platelets is possible with physical forces associated with the blood draw procedure: to minimize this activation it is suggested to adopt a 21-gauge or larger

needles and a butterfly system.[19–21] Nevertheless, further studies are needed to define the best method of venipuncture for EVs isolation. Moreover, it is suggested to discard the first few milliliters of blood before the collection to avoid the effects of the pressure and the fibroblasts contamination.[22] Moreover, hemolyzed samples should not be used and if included in the analysis the obtained results should be used with care.[13] For the plasma collection, the choice of anticoagulants (e.g., EDTA, citrate, and heparin) remains an open question.[23] It is recommended to choose the anticoagulant on the basis of specific research questions. For example, it is not recommended to use heparin or EDTA if interested in the analysis of nucleic acids since both anticoagulants may interfere with PCR reactions. Alternatively, the downstream assay reagent may be chosen on the basis of the anticoagulants selected.[24] At present, citrate is the most commonly used anticoagulant and has been recommended by the International Society on Thrombosis and Haemostasis.[22] Additional studies are clearly needed to address the issue of the choice of the best anticoagulant to isolate EVs from plasma samples. Kalra and coworkers[24] characterized the exosomes spiked in plasma under different storage conditions and showed that samples stored at 4, 20, and 80 C, and analyzed on days 10, 30, and 90 contained the exosomal maker TSG101, indicating that exosomes are stable over a period of at least 3 months. Although TSG101 was observed in all samples, its band intensity, as assessed by WB, varied suggesting that exosomes are more stable when stored at 80 C. Moreover, platelets removal is essential because platelets release EVs during freezethaw cycles.[22] A study of Bæk and coworkers[23] showed that the main factors impacting on EVs isolation are the storage temperature, the period of time and prospective transportation before the initial

Table 2. Advantages and disadvantages of serum and plasma samples. Advantages Serum

Plasma

Disadvantages

-EVs may be slightly more abundant in serum than plasma;

-Not a physiological fluid except in wound healing;

-Possibility to use extensive archives from clinical studies

-Many serum EVs (50%) are released by platelets during coagulation

-Is a physiological fluid of the blood;

-More difficult to deplete plasmatic proteins from EVs preparations

-Platelets can be excluded from the samples

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centrifugation, and the centrifugation protocol. On the contrary, long-term storage and freeze-thawing did not appear to have a critical influence. Collectively, the available literature highlights that, in a given study, all samples must be of the same type and must be handled similarly to compare the results.

expensive EVs-depleted FBS. To our knowledge, no study has defined the conditions for the correct storage of cell supernatant before the isolation as yet. In this case, we suggest the general rule for storage and isolation conditions: ‘the sooner, the better’ preferring storage at 80 C rather than at 20 C.[34]

2.2. Collection and Storage of Urinary Samples

3. EVS Isolation Methods

A correct samples collection is fundamental for the isolation of intact EVs in urine. The first issue debated in the literature is urine collection to be done with or without a digital rectal examination (DRE) if prostatic EVs are going to be analyzed? Dijkstra and coworkers[25] showed that the DRE before the collection increases the EVs secretion into urethra. Subsequently, Hendriks confirmed that the mean levels of prostatic markers are significantly higher in urinary EVs collected after performing a DRE.[26] However, several studies have shown that it is possible to identify prostate markers in urinary EVs without the need of prostate massage. Indeed, the collection of urine without DRE simplifies the sample handing, obviates sample variability and patient discomfort associated with prostate massage, and supports the future development of a potential EVs-based urinary diagnostic test for prostate cancer.[27,28] Another important problem is the presence of large amounts of Tamm-Horsfall protein (THP) or uromodulin, which is the most abundant protein in urine under physiological conditions. THP traps EVs after the centrifugation steps thus preventing an efficient and reproducible isolation.[29–31] Fernandez-Llama and coworkers[32] showed that EVs entrapment in THP can be eliminated by chemical reduction of disulfide bonds with dithiothreitol, which is able to depolymerize the THP and Zhou and coworkers[33] showed that protease inhibitors are necessary for the optimal isolation of intact urine EVs. The same authors suggested freezing the samples at 80 C and not at 20 C to avoid the degradation of EVs. They showed that extensive vortexing after thawing resulted in 87.4 and 100% recovery of EVs in urine frozen at 20 and 80 C, respectively. Moreover, the urinary EVs-associated proteins were preserved almost completely after long-term storage at 80 C.[33]

3.1. Differential Centrifugation and Rate Zonal Centrifugation

2.3. Collection and Storage of Culture Supernatant Samples An important factor to be taken into consideration during the isolation of EVs from cell culture supernatant is the presence of an additional “artificial” EVs source, i.e., the fetal bovine serum (FBS) which is routinely added to cell cultures and is rich of EVs.[34,35] There are four options to control this potential confounding factor: i) after cell culture reaches 70–80% of confluence, the medium should be changed with basal culture medium supplemented only with glutamine and antibiotics followed by EVs collection after 48 h; ii) using the bovine serum albumin instead of the standard FBS[34–36]; iii) if cells do not survive in serum free condition, the FBS-derived EVs could be pre-depleted from FBS by using he ultracentrifugation protocol described by several authors (i.e., centrifugation of the FBS containing medium at 100 000g for 16 h and then sterilefiltering); and iv) using the commercially available but more

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Ultracentrifugation (or better defined “differential centrifugation”) is the most widely adopted EVs isolation method for all the biological fluids described above and is therefore currently considered the “gold standard” to isolate EVs.[15] However, differential centrifugation often produces unsatisfactory results such as a relatively low yield or an insufficient purity of isolated EVs[37] (Table 3). Moreover, very often, researches apply the same differential centrifugation protocols using different rotors and, perhaps most importantly, they do not consider the differences in viscosity of the various samples. Being the differential centrifugation protocols based on the different size, density, and shape of EVs this often leads to confused data. Indeed, the viscosity of the biofluids significantly affect the sedimentation coefficiency and requires an adjustment of the centrifugation protocols in order to isolate EVs with the right size. MomenHeravy and coworkers[38,39] showed that the EVs recovery is inversely correlated with the viscosity of the containing biofluid. Livshits and co-authors have developed a web-calculator to help the researcher selecting the best differential centrifugation protocols for their samples on the bases of the parameters described above (http://vesicles.niifhm.ru/).[36] In conclusion, the limitations of the differential centrifugation method are i) it is time consuming; ii) it requires a great amount of start sample for the isolation of EVs; iii) small contaminating microvesicles (exosome-like vesicles) can still be present in the preparation of exosomes; and iv) EVs can be contaminated by protein aggregates (e.g., lipoproteins), especially in EVs isolated from blood. To minimize the protein aggregates that co-sediment with EVs, the rate zonal centrifugation has been proposed and used by a subset of researchers. Rate zonal centrifugation exploits the buoyant density of EVs in gradients (sucrose, OptiPrepTM) which allows the separation of EVs with different buoyant densities; however, since vesicles with similar buoyant densities may cosediment, the problem of contaminating small microvesicles (exosome-like vesicles) and protein aggregates remains largely unsolved.[40,41] It follows from all the above that the differential centrifugation method is not suitable in a clinical setting and the identification of more standardized, cost-effective, and easyto-handle methods represents an area of intense research activity.[42]

3.2. Immunoaffinity Capture Immunoaffinity capture utilizing monoclonal antibodies to surface proteins is often adopted for the isolation of EVs,

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Table 3. Procedures for isolation/purification of EVs. Method

Isolation principle

Advantages

Differential centrifugation/rate zonal centrifugation

Sequential separation of particles based on size, density and shape of EVs

Low cost; reduced contamination risks; large sample capacity; yields large amounts of EVs

Time consuming; requires a greater amount of start sample; small contaminating microvesicles can still be present in the preparation of exosomes; contamination with protein aggregates; not suitable to be used in a hospital setting

Immunoaffinity capture-based techniques

EVs isolation based on specific interaction between membrane-bound antigens of EVs and immobilized antibodies

Is suitable when the starting material is limiting: yields large amounts of EVs

Clear knowledge of EVs markers specific for each class of EVs is needed; not all vesicles express the markers at the same level

Ultrafiltration

EVs isolation based on separation of suspended particles is primarily dependent on their size or molecular weight

Fast procedure; does not require special equipment, low equipment cost

Possibility of clogging; vesicles trapping; EVs loss due to adhesion to the membranes

Commercially available kit

Based on precipitation of EVs altering their solubility or dispersibility using agglutinating agents

Optimal recovery of EVs; suitable in a hospital setting; ease to use

Currently are the most expensive methods; aggregation and precipitation of other elements present in the suspension

and especially exosomes. The method is particularly indicated when the starting sample is small (typically 2.5 Kb), and the higher quality of EVs-DNA isolated. Moreover, it is well known that free RNA is immediately degraded and eliminated in the blood stream, while it remains stable inside the EVs. If the role of the EVs-derived genomic sequencing will be confirmed in other studies, better diagnostic and prognostic tools could be developed for personalized medicine.

5.3. Flow Cytometry Flow cytometry is an attractive method for the characterization of EVs. The measurement of small particles with flow cytometry dates back to 1979 with the measurement of light-scatter signal from individual viruses.[72] At present, although several studies have been published on the characterization of EVs by flow cytometry and attempted clinical correlations the reliability of such measures remains debatable. New generation flow cytometers (FC) e.g., Gallios and Cytoflex (Beckman Coulter), BD-fortessa (Becton Dickinson), and Apogee (Apogee Flow Systems) can discriminate EVs as small as 150 nm by side or forward scatter signals (Figure 2B). However, particle size measurement by flow cytometry is complex. The refractive index (which contributes the most to the forward and side scatter signals) of EVs is heterogeneous and is generally estimated to be considerably lower than that of the commercial beads used for instrument calibration.[73,74]. For example, the light scattering of a 500 nm polystyrene bead roughly correspond to a biological particle of about 1400 nm, thus this technique still do not permit an accurate analysis of the samples.[75] The fluorescence-trigging signal is needed for the analysis of smaller EVs: the side scatter is not sufficient for discrimination of EVs because they are smaller than the illumination wavelength.[76,77] Evaluation of EVs concentration requires the use of calibrated beads of known concentration, and an accurate volume measurement. Moreover, whereas cells expose >1 105 surface antigens that can be fluorescently labeled, an individual vesicle may bear from 10 up to 100 molecules and the immunofluorescent signals from EVs will be dimmed.[78]

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Other limitations of FC in EVs field are i) the coincidence of single versus double-stained EVs and ii) the swarm effect that leads to an underestimation of the total EVs. To prevent swarm detection and coincidence effect a dilution series on the samples being run can be usefully adopted.[79,80] However, a standardization of sample processing protocols is a necessary step for future application especially for clinical multicenter studies. Standard flow cytometer detects EVs with a diameter above 500 nm and particles smaller cannot be analyzed directly but need to be bounded with beads. In this approach, the EVs are captured on the surface of microspheres via chemical conjugation or via antibody capture, and detected using a fluorescent antibody that recognizes EVs surface markers. The analysis with beads provides information on the expression of specific targets, but it does not allow to quantify the concentration of EVs and do not provide information on individual EVs.[81] Despite all limitations, however, FC remains the fastest method to identify EVs in clinical samples and also allows multiplex fluorescence detection.

6. Progress, Pitfalls, and Potential of Extracellular Vesicles Field in Cancer Research Liquid biopsy is an important topic in cancer research thanks to recent findings on the mechanisms responsible for tumor development and to the emergence of new molecular investigation methods. Liquid biopsy allows early diagnosis and subsequent monitoring of neoplastic disease through a simple biological fluid test.[82] The aim of the liquid biopsy is to replace tumor tissue analysis with an accurate and non-invasive genetic and molecular analysis of a malignant tumor, enabling a personalized therapy. Nowadays, it is recognized that EVs are involved in cellular communication and represent a key player in many diseases. The features of EVs make them ideal candidates for liquid biopsy-based biomarkers. In fact: i) EVs are tissue-specific; ii) cargo of EVs (RNA and DNA) is protected from nucleases; and iii) EVs represent a mirror of both physiological and pathological cellular conditions. Therefore, EVs can help physician in choosing the optimal treatment for each patient in the various stages of a neoplastic disease. However, the small size of EVs makes their analysis very difficult and requires multiple advanced technologies, being therefore a limitation. Moreover, to translate the EVs studies at the clinical level, standardization of protocols will be necessary. Thus, despite EVs are the most exciting and rapidly advanced topic in cancer research we are still far from their use in clinical practice, and further studies are needed to understand and exploit all the potentials of this huge tool.

Abbreviations AFM, atomic force microscopy; DLS, dynamic light scattering; DRE, digital rectal examination; EVs, extracellular vesicles; FBS, fetal bovine serum; FC, flow cytometry; ILVs, intraluminal vesicles; MVBs, multivesicular bodies; RPPA, reverse phase protein array; TEM, trasmission electron microscopy; THP, Tamm-Horsfall protein; WB, Western blot.

Conflict of Interest The authors declare no commercial or financial conflict of interest.

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Keywords biomarkers, extracellular vesicles, liquid biopsy, oncology, standardization Received: November 22, 2017 Revised: May 28, 2018 Published online:

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