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ScienceDirect Extracellular vesicles shuffling intercellular messages: for good or for bad Alessandra Lo Cicero1,2, Philip D Stahl3 and Grac¸a Raposo1,2 The release of extracellular vesicles (EVs) is a highly conserved process exploited by diverse organisms as a mode of intercellular communication. Vesicles of sizes ranging from 30 to 1000 nm, or even larger, are generated by blebbing of the plasma membrane (microvesicles) or formed in multivesicular endosomes (MVEs) to be secreted by exocytosis as exosomes. Exosomes, microvesicles and other EVs contain membrane and cytosolic components that include proteins, lipids and RNAs, a composition that differs related to their site of biogenesis. Several mechanisms are involved in vesicle formation at the plasma membrane or in endosomes, which is reflected in their heterogeneity, size and composition. EVs have significant promise for therapeutics and diagnostics and for understanding physiological and pathological processes all of which have boosted research to find modulators of their composition, secretion and targeting. Addresses 1 Institut Curie, PSL Research University, F-75248 Paris, France 2 Centre National de la Recherche Scientifique, UMR144, Structure and Membrane Compartments, Paris F-75248, France 3 Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA Corresponding author: Raposo, Grac¸a ([email protected])

Current Opinion in Cell Biology 2015, 35:69–77 This review comes from a themed issue on Cell organelles Edited by Maya Schuldiner and Wei Guo

http://dx.doi.org/10.1016/j.ceb.2015.04.013 0955-0674/# 2015 Elsevier Ltd. All rights reserved.

Introduction EVs (exosomes, prostasomes, microvesicles, ectosomes and oncosomes among others) are targeted to recipient cells where they exchange proteins, lipids and nucleic acids involved in essential cellular processes from immune regulation to neuronal communication, including tissue physiology and regeneration. Pathophysiological processes from tumor progression to pathogen transmission appear to be influenced by EVs. EVs carry a common set of components involved in their biogenesis, structure and putatively, their interaction with target cells. Unique subsets of proteins are present in EVs released by different cell types [1,2]. Discovered in the early 1980s, the term www.sciencedirect.com

exosome was used to demarcate small (30–100 nm) vesicles of endosomal origin secreted by reticulocytes [3,4]. Exosome secretion was further described in immune cells, where small vesicles stimulate immune responses leading to tumor eradication [5,6]. A clear understanding of the different types of EVs and their respective functions has been difficult to achieve as cells can release not only exosomes but vesicles generated by budding of the plasma membrane, commonly called Microvesicles (MVs) [1] (Figure 1). The isolation methods are not yet optimal to recover highly enriched samples of each vesicle subtype [7]. Efforts are currently underway to standardize purification and characterization protocols for the different types of vesicles [8]. As an example, an improved characterization of different EV subtypes has been proposed based on lipidprotein ratios and lipid properties [9]. It is also essential to develop an acceptable nomenclature in the field [10]. In this review we will summarize recent advances in understanding the biogenesis, secretion and functions of EVs with special emphasis on exosomes.

Features and composition of exosomes Electron Microscopy (EM) is the only method to visualize exosomes, as their size (30–100 nm) is under the limit of optical resolution and it remains to be demonstrated that individual exosomes can be detected by super resolution microscopy. Exosomes from cell culture media or body fluids analyzed by ‘whole mount’ electron microscopy appear as cup shaped vesicles [6]. This feature, often used to define vesicles as exosomes, is rather an artefact of fixation, contrasting and embedding procedures. Exosomes visualized by cryo microscopy in a close-to-native state appear as round vesicles with a well-defined bilayer [1,11]. However, such morphological features do not attest to the endosomal origin of exosomes as similar small sized vesicles may also originate from plasma membrane budding (Figure 1). A clue that small vesicles isolated from the cell culture media by differential ultracentrifugation at 100 000 g are likely to correspond to exosomes are the observations in situ, in cells, by electron microscopy of fusion profiles of MVEs with the plasma membrane [6] (see also Figure 1). Immunocytochemistry, performed on isolated vesicles is often ideal to confirm enrichment of components also present in ILVs of the MVEs. However, it sets a ‘high bar’ for confirmation as the process is highly dynamic and generally only few events are captured after fixation for EM procedures. The composition of exosomes from different cells is gathered in the ExoCarta database although some entries Current Opinion in Cell Biology 2015, 35:69–77

70 Cell organelles

Figure 1

Table 1

(a)

PM

Examples of common proteins that are present in vesicles with exosomal features released by different cells. The protein content was determined by mass spectrometry analysis of exosomes from dendritic cells, melanocytes, Schwann cells, neurons, intestinal epithelial cells Proteins

Classifications

(b)

PM

Antigen presentation

MHC class I, MHC class II

Adhesion molecules

Tetraspanins: CD63, CD81, CD9, CD37, CD53, CD82 Integrins: a3, a4, aM, aL, b1, b2 MFG-E8

Membrane trafficking

Annexins: I, II, IV, V, VI, VII, XI Syndecan-1 Rab 2, Rab 5c, Rab 10, Rab 7 Arf 3, Arf 6, Arf 5 Clathrin

ESCRT proteins Heat-shock proteins

Alix, Tsg101 Hsc70, Hsp90

Cytoskeletal proteins

Actin, Cofilin 1 Moesin Tubulin: a1, a2, a6, b5, b3

Enzymes

Pyruvate kinase Alpha enolase GAPDH

Signal transduction

14-3-3 j, g, e Gb1, Gi2a Syntenin-1

Lipid raft

Flotillin-1, Flotillin-2

Others

Lactadherin Elongation factor 1a Lamp2

Current Opinion in Cell Biology

Visualisation of exosomes and MVs by electron microscopy. (a) Melanocytes were processed for ultrathin cryosectionning and immunogold labelled for the melanosomal enzyme TYRP1 (Protein AGold 10). Note in the extracellular space the membrane vesicles (exosomes) with a size ranging from 50 to 100 nm close to the deformed plasma reminiscent of an exocytic fusion event (arrows). Some vesicles are labelled (arrowheads). It cannot be excluded that the larger vesicles do not correspond to MVs that result from budding from the plasma membrane. (b) Breast cancer cells were fixed and processed for conventional electron microscopy. Vesicles of very different sizes are present in the extracellular space and some (MVs) bud directly from the plasma membrane (arrows). The smaller vesicles, not in continuity with the plasma membrane may correspond to endosome derived exosomes secreted in the same area. PM: plasma membrane. Bars: 500 nm.

include EVs using different modes of isolation and characterization. Other two databases, Vesiclepedia [12] and EVpedia [13], include published compositional data (proteins, lipids and RNAs) of both exosomes and MVs but always with caveat of suboptimal isolation methods. Table 1 shows exosomal proteins compiled from mass spectrometry data, obtained by us and main collaborators, after analysis of vesicles with exosomal features released Current Opinion in Cell Biology 2015, 35:69–77

by different cell types. A major limiting factor in the characterization of extracellular vesicles has been the absence of trustworthy methods for quantifying vesicle release. A reliable method to analyze small amounts of isolated exosomes is an optimized FACS procedure based on the labelling of isolated vesicles with fluorescent probes [14,15]. As a complement, the NanoSight LM10 based on light scattering automatically tracks and sizes nanoparticles on an individual basis although it is difficult to discriminate between small aggregates and vesicles [16].

Exosomes and microvesicles carry genetic materials The identification of mRNA and miRNA in exosomes and the ability of the transferred exosomal mRNA to be translated in target cells is a major breakthrough in exosome science [17]. Further studies confirmed the presence of RNAs and short DNA sequences associated with EVs (obtained by ultracentrifugation not preceded by differential centrifugation steps) [18,19]. The approach used to characterize EV/exosome-associated miRNA among the various studies often make it unclear if the www.sciencedirect.com

Extracellular vesicles, exosomes and MVs Lo Cicero, Stahl and Raposo 71

genetic material is just adsorbed at the surface of the vesicles or included within the lumen. A more detailed study revealed that exosomes carry a large variety of small noncoding RNA species with many of these RNAs enriched relative to cellular RNA, suggesting that cells target specific RNAs for release [20]. Among the most abundant small RNAs in shuttle RNA (i.e. RNA shuttled via exosomes) were sequences derived from vault RNA (i.e. cytoplasmic ribonucleoprotein particles), Y-RNA (aka, small non-coding RNAs), and specific tRNAs (aka, transfer RNA). Many of these highly abundant small noncoding transcripts in shuttle RNA are evolutionary well-conserved and have previously been associated to gene regulatory functions [20]. The amount of miRNA per exosome is certainly low in vesicles from plasma, seminal fluid, or secreted by dendritic cells, mast cells, and ovarian cancer cells [21]. Such low amounts may question their function but given the selective targeting of miRNA sequences to exosomes [22] and the evidence supporting exosome-associated miRNA it is conceivable that even low amounts are efficient in carrying out their intended functions. Moreover, the efficacy may be compensated by the large numbers and/or specificity of exosome targeting.

The biogenesis of exosomes and microvesicles and the sorting of cargo Exosomes are found as ILVs in the lumen of MVEs formed by the invagination of the limiting membrane of endosomes, a process that involves the segregation of cargo and pinching of a vesicle into the lumen. The Endosomal Sorting Complex Responsible for Transport (ESCRTs) Complexes [23] or other components such as lipids [24] or Tetraspanins [25] appear to be involved in exosome biogenesis. Depending on the cell type and cargo sequestered the mechanisms may vary. The diversity of mechanisms correlates with the heterogeneity in size and composition of ILVs that give rise to different exosome subpopulations [26,27] that may execute different functions [28]. While the ESCRT-0 component Hrs is required for exosome formation/secretion by dendritic cells [29], a medium high-throughput shRNA screen in HeLa-CIITA cells revealed a requirement for STAM (ESCRT-0) and TSG101 (ESCRT-I) for the secretion of MHC class II positive exosomes [26]. Interestingly, while exosome secretion was decreased with depletion of STAM and TSG101, secretion was increased after inactivation of CHMP4C (ESCRT-III) and the accessory proteins ALIX and the AAA-Atpase VPS4B. Moreover, modulation of the expression of ESCRT components also influenced the size and protein content of the released vesicles [26]. It could be tempting to propose that ‘early components’ of the ESCRT machinery regulate negatively exosome secretion whereas ‘late www.sciencedirect.com

components’ do not show a consistent effect on exosome release. However, these observations should be validated in other cell systems and for additional cargoes. Alternative mechanisms of exosome biogenesis not involving ESCRTs also operate in parallel or may be predominant depending on the cell type and particular cargo. One of these pathways involves ceramide that is concentrated in Proteolipid (PLP) enriched-exosomes from oligodendroglial cells [24]. Indeed, it has been shown recently that packaging of the cellular Prion Protein (PrP(C)) is dependent on nSMase2 and therefore on ceramide production [30]. Of note, association of PrP(Sc) (the disease-form of PrP) occurs independently of nSMase2 suggesting that other mechanisms may be involved. The tetraspanin CD63 is involved in the ESCRT-independent sorting of the luminal domain of a melanocytic specific protein PMEL to ILVs [25] and is required for the biogenesis of a subpopulation of exosomes [31]. The tetraspanin CD81, by interacting with selected proteins drives their sequestration into exosomes [32]. These reports implicate a variety of molecular machineries, ESCRTs, accessory proteins (Alix, VPS4B, Syntenin), lipids, tetraspanins in exosome formation, composition and secretion each of which could be amenable to manipulation [26,32]. The ADP ribosylation factor 6 (ARF6) and its effector the Phospholipase D2 (PLD2) have been also involved in ILV/ exosome formation [33] but ARF6 has been shown to be required for the formation of large MVs that bud from the plasma membrane of breast cancer cells [34]. The requirement for TSG101 in budding of MVs from the plasma membrane [35] suggests a redundancy of mechanisms that could be used for the biogenesis of different EVs maybe via interactions of the components involved with different effectors. The selective sequestration of cytosolic proteins into ILVs could be explained by co-mingling with other components. For example, the chaperone hsc70 and its charged cationic domain may interact with the endosomal membrane [36]. RNAs present in exosomes are distinct from the cytosolic pool and appear to be selectively sequestered [20]. A specific ‘targeting’ motif (4 nt sequence (GGAG)) is required for targeting the miRNAs to T cell exosomes. This motif is bound by a sumoylated nuclear ribonucleoprotein A2B (1hnRNPA2B1), that regulates the loading of exosomal miRNAs into exosomes [37]. In macrophages, exosomal sorting of miRNA depends on the expression levels of the target mRNA expressed in the cells of interest with consequences in intercellular communication between macrophages and endothelial cells [38].

Exosome secretion Exosome secretion involves the transport of MVEs to the cell periphery, in preparation for fusion with the plasma Current Opinion in Cell Biology 2015, 35:69–77

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membrane. These MVEs may be targeted to specific sites for polarized secretion such as in immune and neuronal synapses, or as players in cell migration within epithelial sheets (reviewed in Ref [39]). Rab GTPases are involved in exosome secretion but the requirements for specific Rabs may differ depending on the cell type. A medium high throughput screen using a selected shRNA library targeting Rab proteins revealed that loss of expression of Rab27a and Rab27b resulted in a 50% reduction in exosome secretion [40]. These observations reinforced the concept that the majority of secreted exosomes originate from the endosomal network as Rab27 associates also with lysosome related organelles and secretory lysosomes [41]. The role of Rab27 in exosome secretion has been now reported in other cell types including tumor cells [42,43,44]. These studies also revealed that previously considered exosomal proteins, such as CD9 and MfGE8 are still secreted upon Rab27 loss of function indicating that they may rather originate from the plasma membrane [27]. An earlier report suggested that Rab11 was involved in exosome release by a reticulocyte cell line [45], but whether it functions directly in secretion or controls a trafficking step required for the final exocytic step in a manner similar to cytolytic granules [46] remains unknown. Still another Rab, Rab35 with its exchange factors, regulates exosome release in oligodendrocytic cells possibly by controlling the docking/tethering of endocytic vesicles with the plasma membrane [47]. As for secretory lysosomes [48], the fusion of MVEs with the plasma membrane is likely to be mediated by SNAREs [49], but further studies are required to nail down details. The R-SNARE protein Ykt6 was reported to be involved in the secretion of Wnt via exosomes from HEK293 cells [50] but evidence for its direct role in MVE fusion and exosome release is lacking.

Exosomes and microvesicles: in sickness and in health Exosomes and MVs participate in physiological and pathological processes as they deliver their content to recipient cells by docking or fusing with the plasma membrane or by endocytosis (Figure 2). These mechanisms are not mutually exclusive and may be dependent on the cell type under study as there are experimental evidences for the different modes of interaction (summarized in Ref [2]). In the immune system the function of EVs is associated with both stimulatory and tolerogenic responses [51]. By regulating immune responses or by inducing tolerance, EVs/exosomes may be exploited for therapeutic purposes [52]. For example, Foxp3+ T regulatory cells can transfer miRNA Let-7d to T helper1 cells impairing cell proliferation and IFN-g secretion, contributing to suppression of systemic disease [53]. Secreted in situ by cancer cells, exosomes may promote tumor progression [43]. For example, exosomes from Current Opinion in Cell Biology 2015, 35:69–77

metastatic melanoma can educate and mobilize bone marrow progenitors leading to pro-vasculogenic and metastatic phenotypes [44]. Exosomes can enhance cancer cell motility as ‘TIMPless’ fibroblasts, which secrete exosomes carrying the protease ADAM10, induce a conversion of stromal fibroblasts into more invasive CAFs cells [54]. Stromal cells can also release exosomes containing RNA that can activate antiviral and NOTCH3 pathways in breast cancer cells, inducing tumor growth [55]. In contrast, breast cancer cells secrete EV/exosomes enriched in miR-122 that suppresses glucose uptake in non-tumor cells in pre-metastatic niches [56]. In metastatic bladder carcinoma cell lines, secretion of exosome-associated miR-23b results in an increased cell invasion that can be suppressed upon silencing of Rab27B. A high expression of RAB27B correlates with a poor prognosis of bladder cancer [57]. EV/exosomes active in tumor progression may stimulate angiogenesis. Both endothelial cell exosomes containing miR-214, and leukaemia cell exosomes produced under hypoxic conditions stimulate angiogenesis [58,59]. Lastly, multiple myeloma cells cultured under chronic hypoxic conditions release exosomes enriched in miR-135b, which enhances endothelial tube formation probably promoting angiogenesis [60]. In the central nervous system (CNS) EV/exosomes may be involved in the exchange of pathogenic proteins like Prions, b amyloid peptide and asynuclein [61,62]. In a physiological context, many cell types in the CNS release exosomes including neurons, astrocytes and oligodendrocytes. Exosomes from cortical neurons interact preferentially with other neurons, highlighting interneuronal communication [63] and exosomes secreted by oligodendroglial cells are reported to sustain neuronal integrity [64]. In Caenorhabditis elegans EVs loaded with PKD-2 are secreted by ciliated sensory neurons that regulate reproductive behaviour in other animals uniquely representing an example of animal to animal communication [65]. Of additional interest EV/exosomes have been proposed to participate in regeneration to reduce tissue injury and enhance tissue repair. Decreased cardiomyocyte apoptosis accompanied by improved cardiac function was observed after injection of EVs from cardiac progenitor cells in infarcted hearts [66]. Analogous findings were made in rats injected with EVs from GATA-4 mesenchymal stem cells [67]. Interestingly EVs secreted by KLF2-transduced endothelial cells reduce atherosclerotic lesion formation resulting in decreased myocardial infarction an effect attributed to miRNAs [68]. EVs may facilitate nerve regeneration as Schwann cell-derived exosomes increase axonal regeneration in vitro and enhance regeneration after sciatic nerve injury in vivo [69]. Of note, EVs released from a muscle cell line can enhance neurite outgrowth and survival [70]. www.sciencedirect.com

Extracellular vesicles, exosomes and MVs Lo Cicero, Stahl and Raposo 73

Figure 2

1

Microvesicles

RECIPIENT CELL

2 Exosomes 3

ARF6 TSG101 RNAs

RNAs 4 Rab27 Rab35 Rab11

Cytosol

Cytosol

Early Endosome MVE

ESCRTs Ceramide Tetraspanins

Lysosome

SECRETING CELL MVE

Current Opinion in Cell Biology

Cells release MVs and exosomes. MVs of different sizes bud directly from the plasma membrane, whereas exosomes correspond to vesicles that are within MVEs. These vesicles are generated by invagination of the limiting membrane which results in the sequestration of a small portion of cytosol. Exosomes are released by fusion of MVEs with the plasma membrane. Other MVEs fuse with lysosomes. Membraneassociated and transmembrane proteins on vesicles are represented as triangles and rectangles, respectively. Cytosolic proteins contained within exosomes and MVs are not represented. Arrows represent suggested directions of protein and lipid transport between organelles and between MVEs and the plasma membrane for exosome secretion. Exosomes and MVs may dock at the plasma membrane of a target cell (1) or fuse directly with the plasma membrane (2) or be endocytosed (3). Once internalized, vesicles may then fuse with the limiting membrane of an endocytic organelle (4). Pathway 2 and 3 result in the delivery of proteins and RNA into the membrane or cytosol of the target cell. MVs may have similar fates. Proposed mechanisms involved in biogenesis and secretion of exosomes and MVs are also indicated and described in the main text.

Our most recent studies reveal an unexpected role for exosomes in intercellular communication in the skin. Epidermal keratinocytes secrete exosomes that contact neighbouring melanocytes to modulate pigmentation. Exosomes released by keratinocytes from different phototypes and exposed to UVB carry selected miRNAs that increase melanin production by increasing Tyrosinase activity and the expression of other melanosomal proteins essential in melanogenesis [86].

[50,72]. Exosomes secreted by fibroblasts, promote breast cancer motility and progression [71] through Wnt signalling. Wnt signalling is also involved in the growth of B-cell lymphomas where cells interact with a continuous exchange of Wnt signalling [74] via exosomes. Exosomes containing Hedgehog have been shown to migrate along specialized filopodia called cytonemes as part of the mechanism to establish gradients in tissue development [73].

Morphogens, such as Wnt [50,71,72] and Sonic Hedgehog [28,73], are transported by a heterogeneous population of exosomes in both Drosophila and human cells

Extending their function to pathogenic organisms, exosomes have been involved in inter-parasite communication as well as in parasite-host-communication [75]. As an

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Current Opinion in Cell Biology 2015, 35:69–77

74 Cell organelles

advantage for parasite growth, plasmodium falciparum transfers genetic materials to infected red blood cells [76]. These EVs show immunomodulatory properties on human primary macrophages and neutrophils, increasing the migration rate thereby yielding an advantage for the parasite [77]. EV associated miRNAs and Y RNAs are also secreted by the gastrointestinal nematode Heligmosomoides polygyrus that infects mice, manipulating hosts and insuring RNA transfer between animal species [78]. Finally, secreted vesicles from the parasite T. vaginalis, deliver their contents to host cells and modulate host cell immune responses [79]. The pharmaceutical industry has not lost notice of the extensive developments in the field of EVs. A number of start ups have arisen as EVs in biological fluids of cancer patients may serve as biomarkers to follow disease or the course of therapy [80]. Of interest, prostate cancer cells secrete exosomes with a particular miRNA signature [81] and changes in circulating miRNA levels have been associated with prostate cancer [82]. Another therapeutical application is how EVs could be exploited in the transfer of genetic material for therapeutic approaches. EVs carry miRNA, which are transferred for target treatment [83]. Exosomes could also be manipulated to express exogenous siRNA with therapeutic potential [84] or be exploited as nanoshuttles for anti-tumor drug delivery [85].

Perspectives ‘Bright horizons’ EVs are involved in virtually every aspect of human health and disease and if leading indicators are correct, the payoff is enormous. Four hurdles to progress remain; Identification — The development of optimized isolation, characterization and quantification procedures is key to standardize reporting of results and to better assign the observed functions to different types of vesicles [28]. Biogenesis and composition — the molecular details of how specific cargo is packaged at the endosome or the plasma membrane and how signalling begets release will allow researchers to better understand EV molecular cell biology. Targeting — EVs released by one cell type and selectively targeted to a second via specific (yet unknown) recognition determinants will open an entirely new field of investigation that will focus on EV physiology and pathophysiology. Diagnosis and therapeutics — Will it be possible to diagnose an ‘incipient pathology’ by reading the content of a patient’s EVs isolated from body fluids and will it be possible to target therapeutics using recombinant EVs packaged with personalized medicines. Much progress has been made but clearly further investigation is an important investment to sort out the basic science of EV biology.

Acknowledgements We thank G. van Niel, C. Delevoye and all the members of our group for stimulating discussions. We are grateful to the International Society for Current Opinion in Cell Biology 2015, 35:69–77

Extracellular Vesicles (ISEV) that gathers together up to 800 participants in an annual meeting (www.isev.org). We apologize to colleagues whose work could not be cited This work was supported by CNRS, INSERM, Institut Curie and Clarins (to G.R.), the French National Research Agency through the ‘Investments for the Future’ program (France-BioImaging, ANR-10INSB-04) Association ARC pour la Recherche Contre le Cancer and Fondation pour la Recherche Me´dicale (G.R.).

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Exosomes as Hedgehog carriers in cytoneme-mediated transport and secretion. Nat Commun 2014, 5:5649. 74. Koch R, Demant M, Aung T, Diering N, Cicholas A, Chapuy B, Wenzel D, Lahmann M, Guntsch A, Kiecke C et al.: Populational equilibrium through exosome-mediated Wnt signaling in tumor progression of diffuse large B-cell lymphoma. Blood 2014, 123:2189-2198. 75. Marcilla A, Martin-Jaular L, Trelis M, de Menezes-Neto A, Osuna A, Bernal D, Fernandez-Becerra C, Almeida IC, Del Portillo HA: Extracellular vesicles in parasitic diseases. J Extracell Vesicles 2014, 3:25040. 76. Regev-Rudzki N, Wilson DW, Carvalho TG, Sisquella X,  Coleman BM, Rug M, Bursac D, Angrisano F, Gee M, Hill AF et al.: Cell–cell communication between malaria-infected red blood cells via exosome-like vesicles. Cell 2013, 153:1120-1133. This study shows that Plasmodium falciparum-infected red blood cells release exosomes that can directly communicate between parasites within a population. Exploiting this mechanism, parasites transfer genes and proteins increasing survival and promoting differentiation in sexual forms. 77. Mantel PY, Hoang AN, Goldowitz I, Potashnikova D, Hamza B, Vorobjev I, Ghiran I, Toner M, Irimia D, Ivanov AR et al.: Malariainfected erythrocyte-derived microvesicles mediate cellular communication within the parasite population and with the host immune system. Cell Host Microbe 2013, 13:521-534. 78. Buck AH, Coakley G, Simbari F, McSorley HJ, Quintana JF, Le Bihan T, Kumar S, Abreu-Goodger C, Lear M, Harcus Y et al.: Exosomes secreted by nematode parasites transfer small RNAs to mammalian cells and modulate innate immunity. Nat Commun 2014, 5:5488.

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79. Twu O, de Miguel N, Lustig G, Stevens GC, Vashisht AA, Wohlschlegel JA, Johnson PJ: Trichomonas vaginalis exosomes deliver cargo to host cells and mediate hostratioparasite interactions. PLoS Pathog 2013, 9:e1003482. 80. Fesler A, Jiang J, Zhai H, Ju J: Circulating microRNA testing for the early diagnosis and follow-up of colorectal cancer patients. Mol Diagn Ther 2014, 18:303-308. 81. Hessvik NP, Phuyal S, Brech A, Sandvig K, Llorente A: Profiling of microRNAs in exosomes released from PC-3 prostate cancer cells. Biochim Biophys Acta 2012, 1819:1154-1163. 82. Bryant RJ, Pawlowski T, Catto JW, Marsden G, Vessella RL, Rhees B, Kuslich C, Visakorpi T, Hamdy FC: Changes in circulating microRNA levels associated with prostate cancer. Br J Cancer 2012, 106:768-774. 83. Zhao L, Liu W, Xiao J, Cao B: The role of exosomes and ‘‘exosomal shuttle microRNA" in tumorigenesis and drug resistance. Cancer Lett 2015, 356:339-346. 84. Alvarez-Erviti L, Seow Y, Yin H, Betts C, Lakhal S, Wood MJ: Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat Biotechnol 2011, 29:341-345. 85. Zocco D, Ferruzzi P, Cappello F, Kuo WP, Fais S: Extracellular vesicles as shuttles of tumor biomarkers and anti-tumor drugs. Front Oncol 2014, 4:267. 86. Lo Cicero A, Delevoye C, Gilles-Marsens F, Lowe D, Dingli F, Gue´re´ C, Andre´ N, Vie K, van Niel G, Raposo G: Exosomes released by keratinocytes modulate melanocyte pigmentation. Nat Comm 2015. In Press.

Current Opinion in Cell Biology 2015, 35:69–77