Extracellular vesicles as circulating cancer biomarkers: opportunities and challenges

Extracellular vesicles as circulating cancer biomarkers: opportunities and challenges Extracellular vesicles (EVs) are small, lipid-bound particles containing nucleic acid and protein cargo which are excreted from cells under a variety of normal and pathological conditions. EVs have garnered substantial research interest in recent years, due to their potential utility as circulating biomarkers for a variety of diseases, including numerous types of cancer. The following review will discuss the current understanding of the form and function of EVs, their specific role in cancer pathogenesis and their potential for non-invasive disease diagnosis and/or monitor - ing. This review will also highlight several key issues for this field, including the importance of implementing robust and reproducible sample handling protocols, and the challenge of extracting an EV-specific biomarker signal from a complex biological background. Keywords: Exosome, Microvesicle, Extracellular vesicle, Cancer, Biomarker Introduction Overview of extracellular vesicles (EVs) Extracellular vesicles (EVs) have garnered much recent Extracellular vesicle is a general term used to describe interest due to their potential utility as circulating bio- cell-derived sub-micron membranous vesicles which are markers for cancer. EVs have been implicated in a diverse released into the extracellular space. Following release, range of physiological functions due to their capacity to EVs can enter the circulation and have been isolated convey protein and nucleic acid species from a donor cell from numerous bodily fluids including blood [ 1], urine to a recipient. Tumour-derived EVs have been demon- [2], saliva [3], ascites [1] and breast milk [4]. This review strated to carry disease-associated molecular cargo, and will consider two major EV subclasses: exosomes, which further, observed to modulate the behaviour of recipient are endosomally derived, and microvesicles (MVs, also cells towards a pro-oncogenic phenotype. The correlation referred to as ectosomes) which bud from the plasma between the tumour cell and tumour-EV proteome and membrane surface. Various other terms have been used transcriptome across multiple tumour contexts has high- to describe specific subsets of EVs, however, the general lighted the potential for tumour-EVs as candidate mark- terms ‘exosome’ and ‘microvesicle’ are the most widely ers for disease diagnosis and monitoring. This review recognised within the field. summaries the current understanding of EV form and It is generally understood that most cells release a function in the context of cancer, highlighting key and mixture of both exosomes and MVs into the extracellu- transformative works in this space. We also discuss some lar space [5, 6]. Given this, it can be difficult to reliably of the current limitations in this field, and the challenges associate physical, molecular and functional properties to address for EV biomarkers to have clinical utility. with a specific vesicle subclass. This section will provide a brief summary of the current understanding of exosome and microvesicle formation and release, as illustrated in Fig. 1. The subsequent sections will consider both vesicle classes together, using the term ‘EVs’ to denote a mixed population. *Correspondence: m.trau@uq.edu.au Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, QLD, Australia Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Lane et al. Clin Trans Med (2018) 7:14 Page 2 of 11 proteins (e.g. Rab, Annexin A2, Annexin A5) [16–18]. The mechanisms underlying exosomal cargo selection have yet to be fully elucidated, and appear to be modulated by a range of protein and lipid species. It appears that MVE formation and exosome budding is in part modulated by the endosomal complex required for transport (ESCRT) machinery, a system of five protein complexes involved in the reorganisation of cellular membranes [19–21]. Exoso- mal budding and cargo selection appears to be partially mediated by tetraspanins, a class of membrane spanning proteins [16, 22]. Certain members of the tetraspanin family, including CD9, CD63, CD81 and CD82 are used as conventional exosome markers, and are thought to be ubiquitously present on vesicles derived from various cel- lular sources [16, 22]. It is hypothesised that tetraspanin- enriched membrane microdomains within the MVE may facilitate the recruitment of specific protein cargo for inclusion in the resultant vesicles [17, 23]. There is also some evidence that exosome budding may be mediated by the presence and/or abundance of certain lipid spe- cies, although, this mechanism appears to be cell type Fig. 1 Schematic of the process of exosome and microvesicle specific. For example, Trajkovic and colleagues [24] secretion. Exosomes are endosomally derived, and bud inside an reported that in oligodendrocyte precursor cells (Oli- intermediate structure known as a multi-vesicular element (MVE). neu), inhibition of ceramide formation decreased vesicle The MVE subsequently fuses with the plasma membrane of the cell, budding, however, this effect was not observed in either releasing the contents. Microvesicles bud directly from the plasma prostate cancer (PC-3) [25] or melanoma (MNT-1) cell membrane surface, preceded by a rearrangement of the membrane lipid bilayer and the local cytoskeleton lines [20] in separate investigations. In total, it is evident that there is specific selection of exosome cargo, and that this is regulated by multiple cellular mechanisms. A more detailed review of the process of exosome biogenesis and Exosomes release has been presented by Hessvik and Llorente [26]. Endosomally-derived vesicles were first described in the early 1980’s during studies of reticulocyte maturation [7, 8]. This early work demonstrated that transferrin recep - Microvesicles tor shed from maturing reticulocytes in culture was Microvesicles (MVs) were first described in the 1960’s associated with sub-200 nm vesicular structures [7]. Elec- by the observation that platelets released lipid-rich par- tron microscopy demonstrated that these vesicles arose ticles with pro-coagulant activity from the cell surface within a larger endocytic cellular compartment termed a into their surroundings [27]. It was later discovered that multi-vesicular element (MVE) [8, 9]. This MVE was then this surface shedding, sometimes referred to as ‘ectocy- observed to fuse with the plasma membrane of the cell tosis’ [28], occurred across numerous cell types includ- and release the small vesicles to the extracellular space [8, ing monocytes [29], neutrophils [28], oligodendrocytes 9]. It was initially hypothesised that vesicle release in this [30] and tumour cells. In the late 1990’s, Heijnen and col- manner was a reticulocyte-specific mechanism to shed leagues [31] first observed that release of both microvesi - unneeded protein material during maturation [10, 11]. cles and endosomally-derived exosomes could arise from Later studies during the 1990’s and early 2000’s, however, a single cell. suggested that this phenomenon occurred across numer- Microvesicles typically carry some of the plasma mem- ous haematic and non-haematic cell types, including brane components of the cell of origin, which can include B cells [12], dendritic cells [13], epithelial cells [14] and integrins, selectins and/or tetraspanins [32]. The MV notably, tumour cells [15]. proteome, however, does not directly reflect that of the Exosomes are known to carry protein cargo specific to cell, implying that some selection of cargo occurs dur- their cell of origin, however, they also appear to carry a ing vesiculation [32]. MVs are more physically heterog- core set of constituents including cytoskeletal proteins enous than exosomes, and are reported to range in size (e.g. actin, myosin), heat shock proteins (e.g. HSP70, from 0.1 to 1 µm [33]. MVs also appear to carry a diverse HSP90), tetraspanins, and vesicular transport associated range of protein cargo, and as such, a ubiquitous set of Lane et al. Clin Trans Med (2018) 7:14 Page 3 of 11 specific MV markers have yet to be clearly defined. The some of the most important discoveries in the field of EV most commonly used marker is the lipid phosphatidyl- research is included as Fig. 2. serine with proteins including integrin-β1, flotillin-1 and tissue factor proposed as candidates [33]. The sequence Immune‑associated roles of EVs of events underlying MV release have been relatively well In the mid-1990’s it was reported that EVs secreted defined. MV release appears to be driven by an increase from antigen presenting cells (APCs) appeared to have 2+ in intracellular Ca levels which triggers a membrane immunogenic properties. An early study by Raposo rearrangement [34]. There is a simultaneous cytoskel - and colleagues [12] showed that EVs shed by B lympho- etal rearrangement, initiated by transforming protein cytes carried major histocompatibility complex class II RhoA, and culminating in MV budding [35]. The process (MHC-II) molecules on their surface, and were capable of budding and release of MVs and their potential role in of inducing antigen-specific T helper cell responses. A tumorigenesis has been reviewed in detail by Surman and subsequent investigation reported that EVs secreted from colleagues [33]. dendritic cells (DCs) contained both MHC-I and MHC- II molecules, and were similarly capable of inducing an immune response in  vivo [13]. Wolfers and colleagues Physiological roles of EVs [15] later reported that tumour cells also secrete EVs Despite initially being thought of as a mechanism for cel- bearing MHC-I molecules. These tumour-derived EVs lular waste removal, it has subsequently become appar- were shown to transfer tumour antigens to DCs, enabling ent that EVs, including exosomes and microvesicles, play a T cell specific anti-tumour response in vivo [15]. In the several important roles in normal and pathological physi- context of infection, macrophages infected with Myco- ology. EVs appear to have immunogenic properties, and bacterium bovis were demonstrated to secrete EVs which + + can be involved in antigen presentation to immune effec - could activate specific CD4 and C D8 T cell responses tor cells [12]. Further, EVs have been implicated in cell– [38]. In total, there is a substantial body of evidence to cell communication, and have been observed to transfer suggest that EVs represent an important mechanism for functional nucleic acids and proteins between cells [36]. communication between APCs and immune effector This function appears to be particularly important in a cells. disease context, and may represent a mechanism to pro- These findings stimulated interest in the potential to mote tumour growth and metastasis [37]. The following use DC-derived EVs as a component of an autologous section will discuss the current understanding of the key cancer vaccine. An early study by Zitvogel and colleagues physiological functions of EVs. A timeline describing [13] using a mouse model demonstrated that DC-derived Fig. 2 Timeline of key discoveries in extracellular vesicle research. Microvesicles were first reported in the 1960’s, and exosomes in the 1980’s. The physiological role of EVs in antigen presentation and cell–cell communication were first reported in the 1990’s and 2000’s respectively. From the late 2000’s onwards, several key works have highlighted the role of tumour EVs in promoting cancer growth and metastasis, and highlighted their potential utility as biomarkers Lane et al. Clin Trans Med (2018) 7:14 Page 4 of 11 EVs were capable of inducing an anti-tumour response lines, including the parent cell line, in  vitro [47]. This in vivo, slowing tumour growth and in some cases, com- same phenomenon was later noted in an in  vivo model pletely eradicating an established tumour. Several phase of breast cancer [48]. Further, in contrast to the immu- I human trials were subsequently run, enrolling patients nogenic capacity of DC-derived EVs, tumour-EVs appear with melanoma [39] and non-small cell lung cancer [40] to exert immunosuppressive effects [49]. Tumour-EVs respectively. In both of these trials, EVs were produced have been observed to suppress the activity of natural from patient DCs, pulsed with antigenic peptides and killer cells [50, 51] and T cells [52] and to promote the injected. These treatments appeared to promote dis - differentiation of myeloid derived suppressor cells [53, ease stabilisation in a few patients, however, the efficacy 54]. This is postulated to contribute to immune tolerance of DC-derived EVs has yet to be established for cancer of the tumour, and therefore inhibition of these tumour- immunotherapy [39–41]. The use of EVs as cancer immu - EV activities has been proposed as a therapeutic strategy notherapeutics is reviewed in depth in [41]. [53]. In total, these studies provide strong evidence that EVs are a mechanism of communication for tumour cells to promote proliferation, invasiveness and evasion of the EVs in cell–cell communication host immune system. In the mid-2000’s, it was postulated that EVs may rep- In addition to influencing the local tumour environ - resent a mechanism of cell–cell communication beyond ment, there is also evidence to suggest that EVs may be their immunogenic capacity [42]. In a landmark study, involved in initiating and/or supporting tumour metasta- Valadi and colleagues [36] demonstrated that EVs iso- sis at distant sites. It has been reported that EVs derived lated from a mouse mast cell line could transfer function- from the highly metastatic B16-F10 melanoma cell line ally intact mRNA to be translated in human mast cells. could recruit bone marrow derived cells (BMDCs) to The investigators also noted that the EVs appeared to promote the establishment of metastatic lesions [55]. A carry several species of miRNA, and hypothesised these similar mechanism was observed in a model of pancre- could also be transferred between cells [36]. These ini - atic ductal adenocarcinoma, whereby tumour-derived tial findings have since been recapitulated in numerous EVs were found to specifically interact with a subset of studies. For example, Gross et al. reported that EVs from resident liver cells, inducing fibrosis and the recruitment Drosophila melanogaster and human cell lines carry Wnt of BMDCs to this site [56]. Another recent and notewor- proteins, a key class of morphogen, which are capable of thy study examined the potential role of EVs in the phe- activating downstream signalling pathways in recipient nomenon of metastatic organotrophism across a variety cells [43]. EV-mediated communication has also been of cancer types [57]. This study implicated integrins on implicated in several other key developmental processes, the EV surface as a key factor determining the establish- including early implantation [44], angiogenesis, and ment of pre-metastatic niche sites in specific organs [57]. protection of the fetus and placenta from the maternal Taken together, these reports indicate that tumour cells immune system [45]. may employ EV-mediated communication to facilitate metastasis to distant sites. EVs in cancer Despite the diverse roles of EVs in normal physiology, arguably the most well studied form EV-mediated com- EVs as cancer biomarkers munication has been in the context of tumour growth There has been considerable interest in exploring the use and metastasis. Skog and colleagues [37] were amongst of tumour-EVs for disease diagnosis and monitoring. It is the first to explore this phenomenon, reporting that EV generally understood that EVs contain nucleic acid and from glioblastoma cells could upregulate angiogenic protein cargo representative of the secreting cell, and this behaviour in normal brain endothelial cells, via the trans- has been established across a number of tumour contexts fer of nucleic acid and protein. This phenomenon has [58]. The presence of tumour-EVs in circulating bodily since been observed across numerous tumour contexts, fluids such as blood, urine and cerebrospinal fluid means and several notable examples will be described here. In they represent a readily accessible source of biomarkers. an in  vitro model of hypoxia, Park et  al. [46] observed This suggests they may have particular utility for longi - that squamous carcinoma cells secrete proteins and tudinal disease monitoring and early detection of relapse EVs which together lead to decreased adhesiveness and [59]. It has also been reported that certain EV-associated increased angiogenic behaviour in recipient cells. In a protein and nucleic acid species may be predictive of later study, EVs secreted from a highly invasive vari- response to treatment. In total, there is a growing body ant of the HS578T (HS578Ts(i) ) breast cancer cell line of evidence that suggests EVs could represent a rich and were demonstrated to upregulate the proliferative, migra- accessible source of cancer biomarkers. tory and angiogenic potential of several recipient cell Lane et al. Clin Trans Med (2018) 7:14 Page 5 of 11 Amongst the first reports exploring the biomarker therapies in breast cancer, Cetuximab (anti-EGFR) ther- potential of tumour-EVs was a comparison of the con- apy in colon cancer [73] and Pazopanib (chemotherapy) tent of glioblastoma EVs to their cells of origin [37]. Skog in soft tissue sarcoma [74]. In these studies, exposure et al. [37] reported that EVs contained tumour-associated to EVs from resistant cells was demonstrated to disrupt RNA and protein species that were a ‘snapshot’ of the drug-associated signalling pathways in sensitive recipi- content of the secreting cell. Subsequently, the presence ents, and this is proposed to contribute to the develop- of known cancer-associated miRNA [60], mRNA [37, 61], ment of resistance. Notably, a distinct mechanism has lncRNA [62] and post-translational protein modifications also been described for Trastuzumab (anti-HER2) ther- [63] in tumour-derived EVs has been established across apy in breast cancer [75]. EV-associated HER2 appears numerous investigations. This phenomenon has been to be capable of binding this drug, thereby reducing the demonstrated in multiple cancer types, and was exempli- available concentration and diminishing the therapeutic fied by a recent study by Hurwitz and colleagues profil - effect [75]. In total, these observations have suggested ing sixty cancer cell lines. The EV proteome was found that tumour-EV biomarkers have potential prognostic to reflect the cellular proteome and transcriptome across and predictive value. all samples analysed. This was exemplified by hierarchi - cal clustering based on the EV proteomic data, where Current challenges samples were found to segregate based on the tissue Whilst tumour-EVs represent a promising class of circu- type of the originating cell [58]. The correlation between lating biomarker, it is worthwhile to note some current tumour-EV and tumour cell content is particularly valu- limitations in this field of research. One major challenge able where the ability to conduct a tissue biopsy is lim- for this field is the lack of standardisation of protocols for ited, such as in tumours of the brain and central nervous EV enrichment and characterisation. The use of disparate system. For example, studies of glioblastoma multiforme EV handling and analysis protocols means that reported have indicated that tumour-EV in the cerebrospinal fluid sample characteristics can vary between studies, and contain elevated levels of miR21 relative to healthy con- this complicates inter-study comparisons. In response to trols, and that EV-miR21 levels reflect tumour burden this, the EV-TRACK knowledgebase (http://evtra ck.org) [64, 65]. Prognostically informative tumour-EV miRNA [76] was recently established. This resource is designed signatures have similarly been identified in pancreatic to facilitate inter-study methodological comparisons and cancer [66], colorectal cancer [67] and non-small cell develop guidelines for experimental design and reporting lung cancer [68]. Similarly, in a study of Non-Hodgkin’s in EV research. lymphoma patients, Provencio and colleagues [69] iden- The following section will outline some of the com - tified that the presence of several candidate mRNAs monly used methods for EV enrichment and characteri- including C-MYC, BCL-6 and PTEN in plasma-derived sation, highlighting specific issues associated with each. EVs was predictive of progression free survival. These, We also present a brief discussion of some of the major and other reports, have suggested that tumour-EVs may challenges for translation of tumour-EV biomarkers to therefore have potential for non-invasive longitudinal the clinic. disease monitoring [70]. It has also been suggested that the nature of tumour- Isolation and enrichment of EVs EV release may provide opportunities for early disease The use of appropriate sample handling methods is of detection. Melo and colleagues [71] reported that in an particular importance for the biomarker potential of in vivo model of pancreatic cancer, the level of EVs bear- tumour-EVs to be realised. There is substantial evidence ing a candidate marker protein was elevated prior to that the method of sample handling can impact the the tumour being detectable by conventional imaging apparent physical and molecular characteristics of these techniques. Similarly, in a study of acute myloid leukae- samples [70]. Variability can be introduced by biases in mia (AML), Hornick et  al. [59] observed that AML-EVs the isolation or detection of certain EV components [77], were detected in the circulation prior to leukaemic or as contaminating artefacts which are not completely blasts appearing in the blood. There is also evidence that removed during sample processing [78]. Moreover, it has tumour-EVs may have utility in predicting response to also been suggested that more thorough reporting of EV treatment. Tumour-derived EVs have been implicated in handling and measurement protocols is warranted, in resistance to numerous therapeutics by mediating the order to facilitate inter-study comparisons and improve transfer of specific miRNA and/or protein species from the reproducibility of results [76, 79, 80]. drug-resistant to drug-sensitive cells. This phenome - The main methods used to isolate EVs are differential non has been demonstrated across several cancer types ultracentrifugation, density gradient ultracentrifuga- and therapies including Tamoxifen (anti-estrogen) [72] tion, polymer-facilitated precipitation (e.g. ExoQuick, Lane et al. Clin Trans Med (2018) 7:14 Page 6 of 11 Physical and molecular characterisation of EVs Total Exosome Isolation Kit), immunoaffinity isolation The utility of tumour-EV biomarker studies is under - and, size exclusion chromatography (SEC). The major- pinned by the ability to accurately determine sample ity of EV investigations employ one or more of these characteristics, including size distribution, concentration methods as part of an isolation workflow. Developing and the molecular contents. The nature of EV samples, an appropriate workflow is dependent on the start- however, presents some unique challenges for physi- ing material, required purity of isolates and available cal and molecular characterisation. Physically, EVs exist equipment. The relative advantages and limitations of in the sub-100  nm range and are heterogenous in size, various EV isolation protocols have been the subject which limits the applicability of conventional nanoparti- of several previous reports, and will be briefly summa- cle characterisation techniques. Further, molecular char- rised here. acterisation is complicated by the difficulty in isolating a Differential ultracentrifugation is arguably the highly pure EV population devoid of protein and nucleic ‘gold standard’ for EV isolation. This method, ini- acids from non-EV sources. The following section will tially described by Théry and colleagues [81], involves discuss some of the specific factors to consider for EV a series of sequential centrifugation steps designed characterisation. to enrich < 200  nm vesicles. Although widely used, Physical characterisation of EVs is considered an there is evidence that ultracentrifugation may induce important experimental step to verify that vesicle size vesicle aggregation [82], and further, that protein distribution and concentration are as expected for the contaminants may be co-isolated with EVs [83]. A sample. Platforms commonly employed for measure- theoretical analysis of ultracentrifugation by Livshits ment include transmission electron microscopy (TEM), and colleagues [79] also highlighted that variabil- dynamic light scattering (DLS), nanoparticle tracking ity in the pelleting efficiency of different rotors can analysis (NTA) [91], flow cytometry [92] and tunable lead to variable sample recovery using this technique. resistive pulse sensing (TRPS) [92–94]. There are several Density gradient ultracentrifugation is an extension general and platform-specific issues to consider when of this method, where samples are subjected to over- interpreting and reporting EV measurements, which will night centrifugation on a sucrose or iodixanol gradient briefly be summarised here. For a more detailed discus - [84]. This method is generally effective at separating sion of the various platforms employed for EV charac- EVs from other contaminants [85, 86], however, it is terisation, refer to an investigation by Van der Pol and laborious and may lead to sample loss [87]. Size exclu- colleagues [95]. sion chromatography (SEC) methods have also been TEM is arguably the ‘gold standard’ technique for phys- adapted for EV enrichment. Lobb and colleagues [88] ical characterisation of EVs. This technique allows direct assessed this method and found it to perform simi- visualisation of the size and morphology of single vesicles larly to density gradient ultracentrifugation in terms of with a resolution of ≤ 1  nm [95]. It has been suggested, isolate purity. The use of commercial polymer-based however, that artefacts may be introduced during sample reagents such as ExoQuick and Invitrogen Total Exo- preparation and fixing, including vesicle shrinkage [92]. some Isolation Kit expedite the isolation process and To counter this, cryo-electron microscopy (cryo-TEM) avoid high speed centrifugation, however, the purity has become widely used in EV research [96, 97]. Unlike of EVs produced by these methods is generally poor conventional TEM, cryo-TEM samples do not require [89]. Immunoaffinity based methods allow the isola- staining and fixing [96, 97]. This is thought to better tion of EVs bearing specific surface markers, enabling preserve vesicle morphology, allowing visualisation of the interrogation of EV subpopulations of interest [90]. native EV structure [98]. Both TEM and cryo-TEM are This method generally produces pure and homog- largely qualitative methods, as the number of vesicles enous yields [84, 86], however, performance is highly which can be analysed is limited [99]. Flow cytometry dependent on the antibody used for capture. is a technique conventionally used for single cell analy- Ultimately, the most appropriate EV isolation tech- sis which has been adapted for EV characterisation [100, nique will depend on the sample type, the purpose of 101]. The EV sample is focused into a narrow stream and the investigation, the downstream analyses to be per- passes through a laser beam, with the light scattering and formed and the available equipment and resources. It fluorescence profile of each vesicle individually detected is important for sample handling workflows to be indi - and recorded [101, 102]. This can be used to determine vidually evaluated and optimised with respect to isolate individual EV size and/or verify the presence of fluores - yield and quality. Increased stringency in the evaluation cently labelled molecules of interest, and has been sug- and reporting of EV isolation protocols will serve to gested as a way to interrogate the heterogeneity within increase experimental reproducibility and better facili- EV populations [100, 103]. There are, however, currently tate inter-study comparisons. Lane et al. Clin Trans Med (2018) 7:14 Page 7 of 11 several practical limitations of this technique. Critically, the EV extraction method and the RNA isolation proto- the small size and low refractive index of EVs means they col [77, 109], and inter-sample comparisons should be generally do not scatter enough light to trigger detec- conducted with regards to this. Eldh and colleagues [109] tion on a conventional flow cytometer, with only clusters observed that due to the differences between cellular and of multiple EVs and very large EVs detected [101, 102]. EV membranes that cellular RNA extraction protocols Several investigations have demonstrated successful trig- may require some modification for optimal performance. gering from fluorescence, by uniformly labelling EVs Further, Akers et  al. [64] noted that transcripts conven- with a general membrane or protein dye [103, 104]. Care tionally used for normalisation such as the housekeeping must be taken, however, to remove any unincorporated genes GAPDH and 18S rRNA may not reliably correlate dye which can give a non-specific signal [104]. Further, with EV RNA yield, and an alternate method of normali- achieving sufficiently bright immunofluorescent labelling sation should be employed. of EV-associated markers can be challenging as a single As for RNA, conventional analysis techniques are typi- EV contains a limited number of target molecules [100]. cally employed to characterise EV protein cargo. Detec- Unlike EM and flow cytometry, DLS, NTA and TRPS tion of a small number of pre-determined protein targets are all bulk measurement techniques. DLS generates is typically achieved by Western blot, using a standard size distribution information based on fluctuations in sample preparation workflow as described by Choi et al. the intensity of measured light over time due to Brown- [85]. Where characterisation of the full EV proteome is ian motion [105]. This technique enables rapid and bulk required, such as for biomarker discovery, liquid chro- sample characterisation, however, there is a propensity matography tandem–mass spectrometry (LC–MS/ of this technique to over-represent larger particles in the MS) methods have been used [110, 111]. There are sev - sample as these dominate the light scattering signal [105]. eral challenges for characterisation of EVs by LC–MS/ This must be taken into consideration when interpret - MS, notably the difficulty in depleting the non-vesicular ing measurements of physically heterogeneous samples protein components from complex samples which mask such as EVs. NTA builds a size distribution by tracking detection of less abundant EV associated proteins [112]. the Brownian motion of individual particles, and is there- This is particularly challenging when working with pro - fore less affected by outliers than DLS [91]. It is similarly tein-rich biological fluids, such as serum or plasma. For rapid, enabling the measurement of thousands of single proteomic studies, therefore, the EV isolation method is EVs over a few minutes [106]. It is worthwhile to note, critically important. A detailed discussion of the issues however, that it is difficult to determine the lower size surrounding proteomic analysis of EVs is presented by limit for EVs that are reliably detected and tracked using Abramowicz et al. [112]. this system. The limit of detection is dependent on both All of the aforementioned characterisation methods are the refractive index of the particles and the suspending performed on the total EV population, which is likely to fluid, with previous estimates for EVs ranging between 50 comprise exosomes, microvesicles and other non-vesic- and 90 nm [91, 92]. Robustly defining the limits of detec - ular components. As previously mentioned, EV isolation tion for a system is important to ensure that size and con- and characterisation techniques do not allow the user centration information are based on true, confident EV to concretely distinguish between these and so reliably detection and not confounded by system noise. TRPS is attributing physical and molecular properties to a par- a non-optical measurement technique based on the elec- ticular EV class is difficult. Further, it can be difficult to trical impedance induced by a particle as it traverses a ascertain if identified proteins and nucleic acids are true conical nanopore [105]. This system generally requires EV cargo [80]. It is important to establish that the mole- an expert user to operate, as number of user-defined cules of interest are truly contained within EVs to ensure parameters must be optimised for each measurement that they are reproducibly enriched during EV sample [94]. Instrument setup and the limit of detection varies processing, as opposed to the stochastic enrichment of between measurements, and can be empirically deter- co-isolates such as serum proteins and circulating nucleic mined as described in [94]. acids. In general, selection of the most appropriate char- Molecular characterisation of EVs is typically achieved acterisation methodologies will depend on the purpose using conventional nucleic acid and protein analysis of the investigation, as well as the equipment and exper- techniques. For evaluation of RNA, the most commonly tise available. used methods are reverse transcription PCR (RT-PCR) to detect transcripts of interest [61, 72, 107] or RNA and Translation of EV biomarkers to a clinical setting miRNA sequencing to obtain the full transcriptome [67, There are specific challenges to be addressed before the 108]. Importantly, however, the observed miRNA and potential of tumour-EV biomarkers can be realised in a mRNA profiles have been reported to be influenced by clinical setting. There are several specific issues related Lane et al. Clin Trans Med (2018) 7:14 Page 8 of 11 Authors’ contributions to the collection of circulating EVs from human subjects. REL wrote the manuscript. DK, MMH and MT reviewed the manuscript and The level of circulating EVs is known to be influenced by provided critical revisions. All authors read and approved the final manuscript. numerous factors, including the time of day when the Author details sample is collected [113] and by physical activity under- Australian Institute for Bioengineering and Nanotechnology, The Univer- taken prior to collection [114]. These factors may influ - sity of Queensland, St Lucia, QLD, Australia. The University of Queensland ence the subsequent analysis. In addition, György and Diamantina Institute, Faculty of Medicine, Translational Research Institute, The University of Queensland, Woolloongabba, QLD, Australia. QIMR-Berghofer colleagues [115] have observed that after blood collec- Medical Research Institute, Herston, QLD, Australia. School of Chemistry tion, some cells may continue to produce vesicles in vitro. and Molecular Biosciences, The University of Queensland, St Lucia, QLD, They reported that the level of artefactual vesiculation Australia. was dependent on the type of tube used for blood collec- Competing interests tion. In total, Mora and colleagues [116] point out that Darren Korbie and Matt Trau are both associated with Xing Technologies, a for routine ‘biobanking’ of EVs to be feasible that collec- spin-out company from the Trau lab at the University of Queensland, focusing on translating diagnostic technologies. tion, isolation and storage protocols would need to be thoroughly optimised and standardised. Most EV inves- Availability of data and materials tigations to date have centred on in  vitro cell line mod- Not applicable. els of disease, with limited numbers of clinical samples Consent for publication subjected to analysis. The feasibility of high throughput Not applicable. isolation of tumour-EVs from complex biological fluids Ethics approval and consent to participate has therefore yet to be demonstrated. This demonstra - Not applicable. tion, as well as continued evaluation and improvement of EV sample handling and characterisation methods, is Funding REL is the recipient of an Australian Government Research Training Program warranted to continue to progress the use of tumour-EV scholarship. Though not directly funding this work, REL, DK and MT would like biomarkers towards clinical applications. to acknowledge the National Breast Cancer Foundation (NBCF) Australia, for ongoing financial support of research activities in their laboratory. Conclusions Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- It is now apparent that EVs participate in a range of phys- lished maps and institutional affiliations. iological processes and represent an important intercellu- lar communication mechanism. There is much evidence Received: 13 March 2018 Accepted: 23 May 2018 that tumour-EVs carry tumour-associated cargo, and that they actively facilitate cancer growth. Their potential as readily accessible cancer biomarkers has been explored across a number of different contexts. There are, however, References still several issues to be addressed before tumour-EV 1. Keller S, Konig AK, Marme F, Runz S, Wolterink S, Koensgen D et al biomarkers can be considered truly feasible in a clini- (2009) Systemic presence and tumor-growth promoting effect of ovar - cal setting. Currently, there is a lack of standardisation ian carcinoma released exosomes. 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Extracellular vesicles as circulating cancer biomarkers: opportunities and challenges

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Abstract

Extracellular vesicles (EVs) are small, lipid-bound particles containing nucleic acid and protein cargo which are excreted from cells under a variety of normal and pathological conditions. EVs have garnered substantial research interest in recent years, due to their potential utility as circulating biomarkers for a variety of diseases, including numerous types of cancer. The following review will discuss the current understanding of the form and function of EVs, their specific role in cancer pathogenesis and their potential for non-invasive disease diagnosis and/or monitor - ing. This review will also highlight several key issues for this field, including the importance of implementing robust and reproducible sample handling protocols, and the challenge of extracting an EV-specific biomarker signal from a complex biological background. Keywords: Exosome, Microvesicle, Extracellular vesicle, Cancer, Biomarker Introduction Overview of extracellular vesicles (EVs) Extracellular vesicles (EVs) have garnered much recent Extracellular vesicle is a general term used to describe interest due to their potential utility as circulating bio- cell-derived sub-micron membranous vesicles which are markers for cancer. EVs have been implicated in a diverse released into the extracellular space. Following release, range of physiological functions due to their capacity to EVs can enter the circulation and have been isolated convey protein and nucleic acid species from a donor cell from numerous bodily fluids including blood [ 1], urine to a recipient. Tumour-derived EVs have been demon- [2], saliva [3], ascites [1] and breast milk [4]. This review strated to carry disease-associated molecular cargo, and will consider two major EV subclasses: exosomes, which further, observed to modulate the behaviour of recipient are endosomally derived, and microvesicles (MVs, also cells towards a pro-oncogenic phenotype. The correlation referred to as ectosomes) which bud from the plasma between the tumour cell and tumour-EV proteome and membrane surface. Various other terms have been used transcriptome across multiple tumour contexts has high- to describe specific subsets of EVs, however, the general lighted the potential for tumour-EVs as candidate mark- terms ‘exosome’ and ‘microvesicle’ are the most widely ers for disease diagnosis and monitoring. This review recognised within the field. summaries the current understanding of EV form and It is generally understood that most cells release a function in the context of cancer, highlighting key and mixture of both exosomes and MVs into the extracellu- transformative works in this space. We also discuss some lar space [5, 6]. Given this, it can be difficult to reliably of the current limitations in this field, and the challenges associate physical, molecular and functional properties to address for EV biomarkers to have clinical utility. with a specific vesicle subclass. This section will provide a brief summary of the current understanding of exosome and microvesicle formation and release, as illustrated in Fig. 1. The subsequent sections will consider both vesicle classes together, using the term ‘EVs’ to denote a mixed population. *Correspondence: m.trau@uq.edu.au Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, QLD, Australia Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Lane et al. Clin Trans Med (2018) 7:14 Page 2 of 11 proteins (e.g. Rab, Annexin A2, Annexin A5) [16–18]. The mechanisms underlying exosomal cargo selection have yet to be fully elucidated, and appear to be modulated by a range of protein and lipid species. It appears that MVE formation and exosome budding is in part modulated by the endosomal complex required for transport (ESCRT) machinery, a system of five protein complexes involved in the reorganisation of cellular membranes [19–21]. Exoso- mal budding and cargo selection appears to be partially mediated by tetraspanins, a class of membrane spanning proteins [16, 22]. Certain members of the tetraspanin family, including CD9, CD63, CD81 and CD82 are used as conventional exosome markers, and are thought to be ubiquitously present on vesicles derived from various cel- lular sources [16, 22]. It is hypothesised that tetraspanin- enriched membrane microdomains within the MVE may facilitate the recruitment of specific protein cargo for inclusion in the resultant vesicles [17, 23]. There is also some evidence that exosome budding may be mediated by the presence and/or abundance of certain lipid spe- cies, although, this mechanism appears to be cell type Fig. 1 Schematic of the process of exosome and microvesicle specific. For example, Trajkovic and colleagues [24] secretion. Exosomes are endosomally derived, and bud inside an reported that in oligodendrocyte precursor cells (Oli- intermediate structure known as a multi-vesicular element (MVE). neu), inhibition of ceramide formation decreased vesicle The MVE subsequently fuses with the plasma membrane of the cell, budding, however, this effect was not observed in either releasing the contents. Microvesicles bud directly from the plasma prostate cancer (PC-3) [25] or melanoma (MNT-1) cell membrane surface, preceded by a rearrangement of the membrane lipid bilayer and the local cytoskeleton lines [20] in separate investigations. In total, it is evident that there is specific selection of exosome cargo, and that this is regulated by multiple cellular mechanisms. A more detailed review of the process of exosome biogenesis and Exosomes release has been presented by Hessvik and Llorente [26]. Endosomally-derived vesicles were first described in the early 1980’s during studies of reticulocyte maturation [7, 8]. This early work demonstrated that transferrin recep - Microvesicles tor shed from maturing reticulocytes in culture was Microvesicles (MVs) were first described in the 1960’s associated with sub-200 nm vesicular structures [7]. Elec- by the observation that platelets released lipid-rich par- tron microscopy demonstrated that these vesicles arose ticles with pro-coagulant activity from the cell surface within a larger endocytic cellular compartment termed a into their surroundings [27]. It was later discovered that multi-vesicular element (MVE) [8, 9]. This MVE was then this surface shedding, sometimes referred to as ‘ectocy- observed to fuse with the plasma membrane of the cell tosis’ [28], occurred across numerous cell types includ- and release the small vesicles to the extracellular space [8, ing monocytes [29], neutrophils [28], oligodendrocytes 9]. It was initially hypothesised that vesicle release in this [30] and tumour cells. In the late 1990’s, Heijnen and col- manner was a reticulocyte-specific mechanism to shed leagues [31] first observed that release of both microvesi - unneeded protein material during maturation [10, 11]. cles and endosomally-derived exosomes could arise from Later studies during the 1990’s and early 2000’s, however, a single cell. suggested that this phenomenon occurred across numer- Microvesicles typically carry some of the plasma mem- ous haematic and non-haematic cell types, including brane components of the cell of origin, which can include B cells [12], dendritic cells [13], epithelial cells [14] and integrins, selectins and/or tetraspanins [32]. The MV notably, tumour cells [15]. proteome, however, does not directly reflect that of the Exosomes are known to carry protein cargo specific to cell, implying that some selection of cargo occurs dur- their cell of origin, however, they also appear to carry a ing vesiculation [32]. MVs are more physically heterog- core set of constituents including cytoskeletal proteins enous than exosomes, and are reported to range in size (e.g. actin, myosin), heat shock proteins (e.g. HSP70, from 0.1 to 1 µm [33]. MVs also appear to carry a diverse HSP90), tetraspanins, and vesicular transport associated range of protein cargo, and as such, a ubiquitous set of Lane et al. Clin Trans Med (2018) 7:14 Page 3 of 11 specific MV markers have yet to be clearly defined. The some of the most important discoveries in the field of EV most commonly used marker is the lipid phosphatidyl- research is included as Fig. 2. serine with proteins including integrin-β1, flotillin-1 and tissue factor proposed as candidates [33]. The sequence Immune‑associated roles of EVs of events underlying MV release have been relatively well In the mid-1990’s it was reported that EVs secreted defined. MV release appears to be driven by an increase from antigen presenting cells (APCs) appeared to have 2+ in intracellular Ca levels which triggers a membrane immunogenic properties. An early study by Raposo rearrangement [34]. There is a simultaneous cytoskel - and colleagues [12] showed that EVs shed by B lympho- etal rearrangement, initiated by transforming protein cytes carried major histocompatibility complex class II RhoA, and culminating in MV budding [35]. The process (MHC-II) molecules on their surface, and were capable of budding and release of MVs and their potential role in of inducing antigen-specific T helper cell responses. A tumorigenesis has been reviewed in detail by Surman and subsequent investigation reported that EVs secreted from colleagues [33]. dendritic cells (DCs) contained both MHC-I and MHC- II molecules, and were similarly capable of inducing an immune response in  vivo [13]. Wolfers and colleagues Physiological roles of EVs [15] later reported that tumour cells also secrete EVs Despite initially being thought of as a mechanism for cel- bearing MHC-I molecules. These tumour-derived EVs lular waste removal, it has subsequently become appar- were shown to transfer tumour antigens to DCs, enabling ent that EVs, including exosomes and microvesicles, play a T cell specific anti-tumour response in vivo [15]. In the several important roles in normal and pathological physi- context of infection, macrophages infected with Myco- ology. EVs appear to have immunogenic properties, and bacterium bovis were demonstrated to secrete EVs which + + can be involved in antigen presentation to immune effec - could activate specific CD4 and C D8 T cell responses tor cells [12]. Further, EVs have been implicated in cell– [38]. In total, there is a substantial body of evidence to cell communication, and have been observed to transfer suggest that EVs represent an important mechanism for functional nucleic acids and proteins between cells [36]. communication between APCs and immune effector This function appears to be particularly important in a cells. disease context, and may represent a mechanism to pro- These findings stimulated interest in the potential to mote tumour growth and metastasis [37]. The following use DC-derived EVs as a component of an autologous section will discuss the current understanding of the key cancer vaccine. An early study by Zitvogel and colleagues physiological functions of EVs. A timeline describing [13] using a mouse model demonstrated that DC-derived Fig. 2 Timeline of key discoveries in extracellular vesicle research. Microvesicles were first reported in the 1960’s, and exosomes in the 1980’s. The physiological role of EVs in antigen presentation and cell–cell communication were first reported in the 1990’s and 2000’s respectively. From the late 2000’s onwards, several key works have highlighted the role of tumour EVs in promoting cancer growth and metastasis, and highlighted their potential utility as biomarkers Lane et al. Clin Trans Med (2018) 7:14 Page 4 of 11 EVs were capable of inducing an anti-tumour response lines, including the parent cell line, in  vitro [47]. This in vivo, slowing tumour growth and in some cases, com- same phenomenon was later noted in an in  vivo model pletely eradicating an established tumour. Several phase of breast cancer [48]. Further, in contrast to the immu- I human trials were subsequently run, enrolling patients nogenic capacity of DC-derived EVs, tumour-EVs appear with melanoma [39] and non-small cell lung cancer [40] to exert immunosuppressive effects [49]. Tumour-EVs respectively. In both of these trials, EVs were produced have been observed to suppress the activity of natural from patient DCs, pulsed with antigenic peptides and killer cells [50, 51] and T cells [52] and to promote the injected. These treatments appeared to promote dis - differentiation of myeloid derived suppressor cells [53, ease stabilisation in a few patients, however, the efficacy 54]. This is postulated to contribute to immune tolerance of DC-derived EVs has yet to be established for cancer of the tumour, and therefore inhibition of these tumour- immunotherapy [39–41]. The use of EVs as cancer immu - EV activities has been proposed as a therapeutic strategy notherapeutics is reviewed in depth in [41]. [53]. In total, these studies provide strong evidence that EVs are a mechanism of communication for tumour cells to promote proliferation, invasiveness and evasion of the EVs in cell–cell communication host immune system. In the mid-2000’s, it was postulated that EVs may rep- In addition to influencing the local tumour environ - resent a mechanism of cell–cell communication beyond ment, there is also evidence to suggest that EVs may be their immunogenic capacity [42]. In a landmark study, involved in initiating and/or supporting tumour metasta- Valadi and colleagues [36] demonstrated that EVs iso- sis at distant sites. It has been reported that EVs derived lated from a mouse mast cell line could transfer function- from the highly metastatic B16-F10 melanoma cell line ally intact mRNA to be translated in human mast cells. could recruit bone marrow derived cells (BMDCs) to The investigators also noted that the EVs appeared to promote the establishment of metastatic lesions [55]. A carry several species of miRNA, and hypothesised these similar mechanism was observed in a model of pancre- could also be transferred between cells [36]. These ini - atic ductal adenocarcinoma, whereby tumour-derived tial findings have since been recapitulated in numerous EVs were found to specifically interact with a subset of studies. For example, Gross et al. reported that EVs from resident liver cells, inducing fibrosis and the recruitment Drosophila melanogaster and human cell lines carry Wnt of BMDCs to this site [56]. Another recent and notewor- proteins, a key class of morphogen, which are capable of thy study examined the potential role of EVs in the phe- activating downstream signalling pathways in recipient nomenon of metastatic organotrophism across a variety cells [43]. EV-mediated communication has also been of cancer types [57]. This study implicated integrins on implicated in several other key developmental processes, the EV surface as a key factor determining the establish- including early implantation [44], angiogenesis, and ment of pre-metastatic niche sites in specific organs [57]. protection of the fetus and placenta from the maternal Taken together, these reports indicate that tumour cells immune system [45]. may employ EV-mediated communication to facilitate metastasis to distant sites. EVs in cancer Despite the diverse roles of EVs in normal physiology, arguably the most well studied form EV-mediated com- EVs as cancer biomarkers munication has been in the context of tumour growth There has been considerable interest in exploring the use and metastasis. Skog and colleagues [37] were amongst of tumour-EVs for disease diagnosis and monitoring. It is the first to explore this phenomenon, reporting that EV generally understood that EVs contain nucleic acid and from glioblastoma cells could upregulate angiogenic protein cargo representative of the secreting cell, and this behaviour in normal brain endothelial cells, via the trans- has been established across a number of tumour contexts fer of nucleic acid and protein. This phenomenon has [58]. The presence of tumour-EVs in circulating bodily since been observed across numerous tumour contexts, fluids such as blood, urine and cerebrospinal fluid means and several notable examples will be described here. In they represent a readily accessible source of biomarkers. an in  vitro model of hypoxia, Park et  al. [46] observed This suggests they may have particular utility for longi - that squamous carcinoma cells secrete proteins and tudinal disease monitoring and early detection of relapse EVs which together lead to decreased adhesiveness and [59]. It has also been reported that certain EV-associated increased angiogenic behaviour in recipient cells. In a protein and nucleic acid species may be predictive of later study, EVs secreted from a highly invasive vari- response to treatment. In total, there is a growing body ant of the HS578T (HS578Ts(i) ) breast cancer cell line of evidence that suggests EVs could represent a rich and were demonstrated to upregulate the proliferative, migra- accessible source of cancer biomarkers. tory and angiogenic potential of several recipient cell Lane et al. Clin Trans Med (2018) 7:14 Page 5 of 11 Amongst the first reports exploring the biomarker therapies in breast cancer, Cetuximab (anti-EGFR) ther- potential of tumour-EVs was a comparison of the con- apy in colon cancer [73] and Pazopanib (chemotherapy) tent of glioblastoma EVs to their cells of origin [37]. Skog in soft tissue sarcoma [74]. In these studies, exposure et al. [37] reported that EVs contained tumour-associated to EVs from resistant cells was demonstrated to disrupt RNA and protein species that were a ‘snapshot’ of the drug-associated signalling pathways in sensitive recipi- content of the secreting cell. Subsequently, the presence ents, and this is proposed to contribute to the develop- of known cancer-associated miRNA [60], mRNA [37, 61], ment of resistance. Notably, a distinct mechanism has lncRNA [62] and post-translational protein modifications also been described for Trastuzumab (anti-HER2) ther- [63] in tumour-derived EVs has been established across apy in breast cancer [75]. EV-associated HER2 appears numerous investigations. This phenomenon has been to be capable of binding this drug, thereby reducing the demonstrated in multiple cancer types, and was exempli- available concentration and diminishing the therapeutic fied by a recent study by Hurwitz and colleagues profil - effect [75]. In total, these observations have suggested ing sixty cancer cell lines. The EV proteome was found that tumour-EV biomarkers have potential prognostic to reflect the cellular proteome and transcriptome across and predictive value. all samples analysed. This was exemplified by hierarchi - cal clustering based on the EV proteomic data, where Current challenges samples were found to segregate based on the tissue Whilst tumour-EVs represent a promising class of circu- type of the originating cell [58]. The correlation between lating biomarker, it is worthwhile to note some current tumour-EV and tumour cell content is particularly valu- limitations in this field of research. One major challenge able where the ability to conduct a tissue biopsy is lim- for this field is the lack of standardisation of protocols for ited, such as in tumours of the brain and central nervous EV enrichment and characterisation. The use of disparate system. For example, studies of glioblastoma multiforme EV handling and analysis protocols means that reported have indicated that tumour-EV in the cerebrospinal fluid sample characteristics can vary between studies, and contain elevated levels of miR21 relative to healthy con- this complicates inter-study comparisons. In response to trols, and that EV-miR21 levels reflect tumour burden this, the EV-TRACK knowledgebase (http://evtra ck.org) [64, 65]. Prognostically informative tumour-EV miRNA [76] was recently established. This resource is designed signatures have similarly been identified in pancreatic to facilitate inter-study methodological comparisons and cancer [66], colorectal cancer [67] and non-small cell develop guidelines for experimental design and reporting lung cancer [68]. Similarly, in a study of Non-Hodgkin’s in EV research. lymphoma patients, Provencio and colleagues [69] iden- The following section will outline some of the com - tified that the presence of several candidate mRNAs monly used methods for EV enrichment and characteri- including C-MYC, BCL-6 and PTEN in plasma-derived sation, highlighting specific issues associated with each. EVs was predictive of progression free survival. These, We also present a brief discussion of some of the major and other reports, have suggested that tumour-EVs may challenges for translation of tumour-EV biomarkers to therefore have potential for non-invasive longitudinal the clinic. disease monitoring [70]. It has also been suggested that the nature of tumour- Isolation and enrichment of EVs EV release may provide opportunities for early disease The use of appropriate sample handling methods is of detection. Melo and colleagues [71] reported that in an particular importance for the biomarker potential of in vivo model of pancreatic cancer, the level of EVs bear- tumour-EVs to be realised. There is substantial evidence ing a candidate marker protein was elevated prior to that the method of sample handling can impact the the tumour being detectable by conventional imaging apparent physical and molecular characteristics of these techniques. Similarly, in a study of acute myloid leukae- samples [70]. Variability can be introduced by biases in mia (AML), Hornick et  al. [59] observed that AML-EVs the isolation or detection of certain EV components [77], were detected in the circulation prior to leukaemic or as contaminating artefacts which are not completely blasts appearing in the blood. There is also evidence that removed during sample processing [78]. Moreover, it has tumour-EVs may have utility in predicting response to also been suggested that more thorough reporting of EV treatment. Tumour-derived EVs have been implicated in handling and measurement protocols is warranted, in resistance to numerous therapeutics by mediating the order to facilitate inter-study comparisons and improve transfer of specific miRNA and/or protein species from the reproducibility of results [76, 79, 80]. drug-resistant to drug-sensitive cells. This phenome - The main methods used to isolate EVs are differential non has been demonstrated across several cancer types ultracentrifugation, density gradient ultracentrifuga- and therapies including Tamoxifen (anti-estrogen) [72] tion, polymer-facilitated precipitation (e.g. ExoQuick, Lane et al. Clin Trans Med (2018) 7:14 Page 6 of 11 Physical and molecular characterisation of EVs Total Exosome Isolation Kit), immunoaffinity isolation The utility of tumour-EV biomarker studies is under - and, size exclusion chromatography (SEC). The major- pinned by the ability to accurately determine sample ity of EV investigations employ one or more of these characteristics, including size distribution, concentration methods as part of an isolation workflow. Developing and the molecular contents. The nature of EV samples, an appropriate workflow is dependent on the start- however, presents some unique challenges for physi- ing material, required purity of isolates and available cal and molecular characterisation. Physically, EVs exist equipment. The relative advantages and limitations of in the sub-100  nm range and are heterogenous in size, various EV isolation protocols have been the subject which limits the applicability of conventional nanoparti- of several previous reports, and will be briefly summa- cle characterisation techniques. Further, molecular char- rised here. acterisation is complicated by the difficulty in isolating a Differential ultracentrifugation is arguably the highly pure EV population devoid of protein and nucleic ‘gold standard’ for EV isolation. This method, ini- acids from non-EV sources. The following section will tially described by Théry and colleagues [81], involves discuss some of the specific factors to consider for EV a series of sequential centrifugation steps designed characterisation. to enrich < 200  nm vesicles. Although widely used, Physical characterisation of EVs is considered an there is evidence that ultracentrifugation may induce important experimental step to verify that vesicle size vesicle aggregation [82], and further, that protein distribution and concentration are as expected for the contaminants may be co-isolated with EVs [83]. A sample. Platforms commonly employed for measure- theoretical analysis of ultracentrifugation by Livshits ment include transmission electron microscopy (TEM), and colleagues [79] also highlighted that variabil- dynamic light scattering (DLS), nanoparticle tracking ity in the pelleting efficiency of different rotors can analysis (NTA) [91], flow cytometry [92] and tunable lead to variable sample recovery using this technique. resistive pulse sensing (TRPS) [92–94]. There are several Density gradient ultracentrifugation is an extension general and platform-specific issues to consider when of this method, where samples are subjected to over- interpreting and reporting EV measurements, which will night centrifugation on a sucrose or iodixanol gradient briefly be summarised here. For a more detailed discus - [84]. This method is generally effective at separating sion of the various platforms employed for EV charac- EVs from other contaminants [85, 86], however, it is terisation, refer to an investigation by Van der Pol and laborious and may lead to sample loss [87]. Size exclu- colleagues [95]. sion chromatography (SEC) methods have also been TEM is arguably the ‘gold standard’ technique for phys- adapted for EV enrichment. Lobb and colleagues [88] ical characterisation of EVs. This technique allows direct assessed this method and found it to perform simi- visualisation of the size and morphology of single vesicles larly to density gradient ultracentrifugation in terms of with a resolution of ≤ 1  nm [95]. It has been suggested, isolate purity. The use of commercial polymer-based however, that artefacts may be introduced during sample reagents such as ExoQuick and Invitrogen Total Exo- preparation and fixing, including vesicle shrinkage [92]. some Isolation Kit expedite the isolation process and To counter this, cryo-electron microscopy (cryo-TEM) avoid high speed centrifugation, however, the purity has become widely used in EV research [96, 97]. Unlike of EVs produced by these methods is generally poor conventional TEM, cryo-TEM samples do not require [89]. Immunoaffinity based methods allow the isola- staining and fixing [96, 97]. This is thought to better tion of EVs bearing specific surface markers, enabling preserve vesicle morphology, allowing visualisation of the interrogation of EV subpopulations of interest [90]. native EV structure [98]. Both TEM and cryo-TEM are This method generally produces pure and homog- largely qualitative methods, as the number of vesicles enous yields [84, 86], however, performance is highly which can be analysed is limited [99]. Flow cytometry dependent on the antibody used for capture. is a technique conventionally used for single cell analy- Ultimately, the most appropriate EV isolation tech- sis which has been adapted for EV characterisation [100, nique will depend on the sample type, the purpose of 101]. The EV sample is focused into a narrow stream and the investigation, the downstream analyses to be per- passes through a laser beam, with the light scattering and formed and the available equipment and resources. It fluorescence profile of each vesicle individually detected is important for sample handling workflows to be indi - and recorded [101, 102]. This can be used to determine vidually evaluated and optimised with respect to isolate individual EV size and/or verify the presence of fluores - yield and quality. Increased stringency in the evaluation cently labelled molecules of interest, and has been sug- and reporting of EV isolation protocols will serve to gested as a way to interrogate the heterogeneity within increase experimental reproducibility and better facili- EV populations [100, 103]. There are, however, currently tate inter-study comparisons. Lane et al. Clin Trans Med (2018) 7:14 Page 7 of 11 several practical limitations of this technique. Critically, the EV extraction method and the RNA isolation proto- the small size and low refractive index of EVs means they col [77, 109], and inter-sample comparisons should be generally do not scatter enough light to trigger detec- conducted with regards to this. Eldh and colleagues [109] tion on a conventional flow cytometer, with only clusters observed that due to the differences between cellular and of multiple EVs and very large EVs detected [101, 102]. EV membranes that cellular RNA extraction protocols Several investigations have demonstrated successful trig- may require some modification for optimal performance. gering from fluorescence, by uniformly labelling EVs Further, Akers et  al. [64] noted that transcripts conven- with a general membrane or protein dye [103, 104]. Care tionally used for normalisation such as the housekeeping must be taken, however, to remove any unincorporated genes GAPDH and 18S rRNA may not reliably correlate dye which can give a non-specific signal [104]. Further, with EV RNA yield, and an alternate method of normali- achieving sufficiently bright immunofluorescent labelling sation should be employed. of EV-associated markers can be challenging as a single As for RNA, conventional analysis techniques are typi- EV contains a limited number of target molecules [100]. cally employed to characterise EV protein cargo. Detec- Unlike EM and flow cytometry, DLS, NTA and TRPS tion of a small number of pre-determined protein targets are all bulk measurement techniques. DLS generates is typically achieved by Western blot, using a standard size distribution information based on fluctuations in sample preparation workflow as described by Choi et al. the intensity of measured light over time due to Brown- [85]. Where characterisation of the full EV proteome is ian motion [105]. This technique enables rapid and bulk required, such as for biomarker discovery, liquid chro- sample characterisation, however, there is a propensity matography tandem–mass spectrometry (LC–MS/ of this technique to over-represent larger particles in the MS) methods have been used [110, 111]. There are sev - sample as these dominate the light scattering signal [105]. eral challenges for characterisation of EVs by LC–MS/ This must be taken into consideration when interpret - MS, notably the difficulty in depleting the non-vesicular ing measurements of physically heterogeneous samples protein components from complex samples which mask such as EVs. NTA builds a size distribution by tracking detection of less abundant EV associated proteins [112]. the Brownian motion of individual particles, and is there- This is particularly challenging when working with pro - fore less affected by outliers than DLS [91]. It is similarly tein-rich biological fluids, such as serum or plasma. For rapid, enabling the measurement of thousands of single proteomic studies, therefore, the EV isolation method is EVs over a few minutes [106]. It is worthwhile to note, critically important. A detailed discussion of the issues however, that it is difficult to determine the lower size surrounding proteomic analysis of EVs is presented by limit for EVs that are reliably detected and tracked using Abramowicz et al. [112]. this system. The limit of detection is dependent on both All of the aforementioned characterisation methods are the refractive index of the particles and the suspending performed on the total EV population, which is likely to fluid, with previous estimates for EVs ranging between 50 comprise exosomes, microvesicles and other non-vesic- and 90 nm [91, 92]. Robustly defining the limits of detec - ular components. As previously mentioned, EV isolation tion for a system is important to ensure that size and con- and characterisation techniques do not allow the user centration information are based on true, confident EV to concretely distinguish between these and so reliably detection and not confounded by system noise. TRPS is attributing physical and molecular properties to a par- a non-optical measurement technique based on the elec- ticular EV class is difficult. Further, it can be difficult to trical impedance induced by a particle as it traverses a ascertain if identified proteins and nucleic acids are true conical nanopore [105]. This system generally requires EV cargo [80]. It is important to establish that the mole- an expert user to operate, as number of user-defined cules of interest are truly contained within EVs to ensure parameters must be optimised for each measurement that they are reproducibly enriched during EV sample [94]. Instrument setup and the limit of detection varies processing, as opposed to the stochastic enrichment of between measurements, and can be empirically deter- co-isolates such as serum proteins and circulating nucleic mined as described in [94]. acids. In general, selection of the most appropriate char- Molecular characterisation of EVs is typically achieved acterisation methodologies will depend on the purpose using conventional nucleic acid and protein analysis of the investigation, as well as the equipment and exper- techniques. For evaluation of RNA, the most commonly tise available. used methods are reverse transcription PCR (RT-PCR) to detect transcripts of interest [61, 72, 107] or RNA and Translation of EV biomarkers to a clinical setting miRNA sequencing to obtain the full transcriptome [67, There are specific challenges to be addressed before the 108]. Importantly, however, the observed miRNA and potential of tumour-EV biomarkers can be realised in a mRNA profiles have been reported to be influenced by clinical setting. There are several specific issues related Lane et al. Clin Trans Med (2018) 7:14 Page 8 of 11 Authors’ contributions to the collection of circulating EVs from human subjects. REL wrote the manuscript. DK, MMH and MT reviewed the manuscript and The level of circulating EVs is known to be influenced by provided critical revisions. All authors read and approved the final manuscript. numerous factors, including the time of day when the Author details sample is collected [113] and by physical activity under- Australian Institute for Bioengineering and Nanotechnology, The Univer- taken prior to collection [114]. These factors may influ - sity of Queensland, St Lucia, QLD, Australia. The University of Queensland ence the subsequent analysis. In addition, György and Diamantina Institute, Faculty of Medicine, Translational Research Institute, The University of Queensland, Woolloongabba, QLD, Australia. QIMR-Berghofer colleagues [115] have observed that after blood collec- Medical Research Institute, Herston, QLD, Australia. School of Chemistry tion, some cells may continue to produce vesicles in vitro. and Molecular Biosciences, The University of Queensland, St Lucia, QLD, They reported that the level of artefactual vesiculation Australia. was dependent on the type of tube used for blood collec- Competing interests tion. In total, Mora and colleagues [116] point out that Darren Korbie and Matt Trau are both associated with Xing Technologies, a for routine ‘biobanking’ of EVs to be feasible that collec- spin-out company from the Trau lab at the University of Queensland, focusing on translating diagnostic technologies. tion, isolation and storage protocols would need to be thoroughly optimised and standardised. Most EV inves- Availability of data and materials tigations to date have centred on in  vitro cell line mod- Not applicable. els of disease, with limited numbers of clinical samples Consent for publication subjected to analysis. The feasibility of high throughput Not applicable. isolation of tumour-EVs from complex biological fluids Ethics approval and consent to participate has therefore yet to be demonstrated. This demonstra - Not applicable. tion, as well as continued evaluation and improvement of EV sample handling and characterisation methods, is Funding REL is the recipient of an Australian Government Research Training Program warranted to continue to progress the use of tumour-EV scholarship. Though not directly funding this work, REL, DK and MT would like biomarkers towards clinical applications. to acknowledge the National Breast Cancer Foundation (NBCF) Australia, for ongoing financial support of research activities in their laboratory. Conclusions Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- It is now apparent that EVs participate in a range of phys- lished maps and institutional affiliations. iological processes and represent an important intercellu- lar communication mechanism. There is much evidence Received: 13 March 2018 Accepted: 23 May 2018 that tumour-EVs carry tumour-associated cargo, and that they actively facilitate cancer growth. Their potential as readily accessible cancer biomarkers has been explored across a number of different contexts. There are, however, References still several issues to be addressed before tumour-EV 1. Keller S, Konig AK, Marme F, Runz S, Wolterink S, Koensgen D et al biomarkers can be considered truly feasible in a clini- (2009) Systemic presence and tumor-growth promoting effect of ovar - cal setting. Currently, there is a lack of standardisation ian carcinoma released exosomes. 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Clinical and Translational MedicineSpringer Journals

Published: May 31, 2018

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