Exosomes as nucleic acid nanocarriers

    Exosomes as nucleic acid nanocarriers Jasper G. van den Boorn, Juliane Daßler, Christoph Coch, Martin Schlee, Gunther Hartmann PII: DOI: Reference:

S0169-409X(12)00209-8 doi: 10.1016/j.addr.2012.06.011 ADR 12323

To appear in:

Advanced Drug Delivery Reviews

Received date: Accepted date:

7 March 2012 20 June 2012

Please cite this article as: Jasper G. van den Boorn, Juliane Daßler, Christoph Coch, Martin Schlee, Gunther Hartmann, Exosomes as nucleic acid nanocarriers, Advanced Drug Delivery Reviews (2012), doi: 10.1016/j.addr.2012.06.011

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Exosomes as nucleic acid nanocarriers.

Gunther Hartmann1.

Institute for Clinical Chemistry and Clinical Pharmacology, University Hospital

SC RI

1:

PT

Jasper G. van den Boorn1, Juliane Daßler1, Christoph Coch1, Martin Schlee1 and

PT

ED

MA

NU

Bonn, Bonn, Germany.

CE

Corresponding author:

Dr. Jasper G. van den Boorn

AC

University Clinic Bonn Institute for Clinical Chemistry and Clinical Pharmacology Biomedical Centre (building 344) Room 1.G-020 Sigmund-Freud-Strasse 25 53127 Bonn Germany tel: @:

+49-(0)228-287-51143 [email protected]

Keywords: Exosome, extracellular vesicle, nucleic acid, miRNA, tetraspanin, ESCRT, RISC, miRISC, targeted, in vivo delivery.

ACCEPTED MANUSCRIPT Table of contents Abstract 1.0

The exosome in brief

1.1

The good, the bad, the innocent

PT

Introduction

1.2

ESCRTing RNA: a RISCy process? Exosome delivery tools Biomodulation by nucleic acid nanocarriers

NU

Tetraspanins as a recipient determinant

SC RI

The exosome carries RNA cargo in a protein context

Conclusions

2.1 2.2 2.3 2.4 3.0 4.0

MA

References

2.0

Abstract.

ED

Exosomes are nano-sized vesicles produced naturally by many cell types. They are specifically loaded with nucleic acid cargo, dependent on the exosome-

PT

producing cell and its homeostatic state. As natural intercellular shuttles of miRNA, exosomes influence an array of developmental, physiological and

CE

pathological processes in the recipient cell or tissue to which they can be selectively targeted by their tetraspanin surface-domains. By a review of current

AC

research, we here illustrate why exosomes are ideal nanocarriers for use in the targeted in vivo delivery of nucleic acids. 1.0 Introduction The term “exosome” was coined by Rose Johnstone in the late 1970s, because the vesicles she found emerging from sheep reticulocytes structurally resembled endosomes, only these particular ones were exiting material rather than entering[1,2]. Up to 1996 exosomes were at best regarded as waste-disposal vessels or by-products of cell homeostasis. This changed with the discovery that B cells can actually release functional antigen-transferring exosomes[3]. Hereafter the exosome scope widened, over time attributing an array of functionalities to these small extracellular entities. Cells can release multiple types of membrane vesicles, like plasma-membrane-budded microvesicles,

ACCEPTED MANUSCRIPT multivesicular body-derived exosomes, golgi apparatus-derived secretory vesicles or apoptotic bodies. All of these cell-derived microvesicles can carry biologically active cargo, like proteins, mRNA and miRNA[4-6]. Nonetheless, by

PT

their small size only the exosomes evade clearance by the mononuclear phagocyte system (which clears circulating particles >100 nm in size),

SC RI

maximizing their circulation time and underlining their superiority in (systemic) intercellular communication. 1.1 The exosome in brief.

NU

Exosomes are phospholipid bilayer microvesicles 50-100 nm in size, formed intracellularly by invaginations of the multivesicular body’s limiting membrane.

MA

The multivesicular body (MVB) is a late endocytic compartment, and when fusing with the cell’s plasma membrane, its liberated internal microvesicles are referred to as exosomes (also see the graphical abstract). They appear as cup-

ED

shaped structures in transfer electron microscopy and generally surface-express markers reflective of their intracellular endosomal origin like the LAMP1,

PT

LAMP2b and ALIX-1 proteins, as well as the tetraspanins CD9, CD63 and CD81[7,8]. Many immune- and somatic cell types release exosomes

CE

constitutively or do so under certain circumstances, like cellular activation. Celltype specific markers are regularly carried, like MHC molecules on dendritic cell

AC

(DC)-derived exosomes[9], CD3 on T cell exosomes[10], and EpCAM on exosomes secreted by ovarian cancer cells[11]. Also enzymes, cytoskeletal constituents and heat-shock proteins reside in the exosome lumen, which is typically devoid of cellular organelles like mitochondria or endoplasmatic reticulum[12]. For long, the exosome was regarded as the paradigmatic ‘mini version’ of the producing cell, reflecting its status quo. Especially since the membrane orientation of the exosome is equal to that of its mother cell. In the light of this view, the function and usefulness of exosomes was heavily debated and underappreciated until recently[2]. 1.2 The good, the bad, the innocent Besides DCs, B and T cells, many more cell types have been identified to release exosomes, even platelets do so when activated[13]. Additionally, exosomes have

ACCEPTED MANUSCRIPT been found in practically all body fluids, including the cerebrospinal fluid[14]. A vesicular system which is that abundant must have significant repercussions on the host. Especially in the context of immune responses exosomes induce

PT

antigen-specific cytotoxic CD8+ T cell responses against tumors by actively transferring specific antigen from tumor cells to DC[15,16], and attract immune

SC RI

cells to the tumor site in a chemokine-dependent fashion[17]. Also, exosomes have been shown to transfer pathogen-associated molecular patterns (PAMPs) from macrophages infected with various intracellular pathogens like Mycobacterium tuberculosis, Toxoplasma gondii or Salmonella typhimurium to

NU

neighbouring uninfected macrophages, thereby stimulating the induction of protective immunity[18]. A similar process aids the antigen-specific initiation of

MA

T cell immunity against cytomegalovirus-infected cells[19]. Findings like these sparked subsequent studies to employ exosomes as inducers of anti-tumor immunity[20,21]. The beneficial role of exosomes in immune cell communication elsewhere[8,22,23].

ED

is outside the scope of this review, and the subject has been extensively reviewed

PT

In contrast, exosomes are not necessarily friendly to the host by always aiding the initiation of protective immunity. They are also major players in the

CE

malignant progression of cancer by mediating immunosupression via the induction of myeloid-derived suppressor cells[24], shuttling anti-proliferative

AC

transforming growth factor-beta1 to local lymphocytes[25] and actively killing activated T cells[26,27]. In addition they aid pre-metastatic niche conditioning which facilitates systemic tumor dissemination[28,29]. The commandeering of exosomes is a tactic also used by pathogens to their infective benefit. In the most striking example, HIV transfers to receptive CD4+ T cells via the endosomal/exosomal route in immature DCs[30]. Hereby, HIV particle infectivity increases 10-fold while host humoral immunity is evaded simultaneously. Moreover, HIV-infected cells subsequently appear to release HIV Nef-protein carrying exosomes which induce apoptosis in bystander CD4+ T cells[31]. Additionally, Epstein-Barr virus (EBV; a human gamma-herpesvirus) infected cells release exosomes containing EBV-derived miRNA sequences that repress target genes in uninfected recipient cells. These target genes include CXCL11/ITAC, a gene suppressed in EBV-related lymphomas. Moreover, while

ACCEPTED MANUSCRIPT EBV DNA was found to be restricted to the infected B cell population, the EBVderived miRNAs were found beyond, indicating distant miRNA spread via exosomes to the benefit of EBV infection progression[32]. Not only viruses, but

PT

even the smallest of pathogens have discovered the usefulness of exosomes, since also prions can hitch a ride for their infectious spread[33]. Additionally,

SC RI

exosomes have also been implicated in non-infectious diseases. For example, the deposition of beta-amyloid peptide plaques in Alzheimer’s disease has been associated with exosomes[34].

It is likely that many more pathogens misuse the exosome route. But in all

NU

these processes it is not the exosome in essence that does the benefit or harm,

MA

it’s its cargo.

2.0 The exosome carries RNA cargo in a protein context Most likely, the exosome pathway finds its evolutionary origin in embryonic

ED

patterning and development. The patterned and controlled release of exosomes in embryonic tissues is likely to play a significant role in developmental

PT

intercellular communication and tissue polarity, illustrated by the apical release of Hedgehog-related protein-containing exosomes in Caenorhabditis elegans

CE

development[35]. Moreover, when we realize that besides proteins exosomes carry functional mRNAs and miRNAs, which are involved in regulation of

AC

embryogenesis[36], a role for exosomes in tissue sculpting and uniformity emerges. The young and appealing field of exosome function in animal development is reviewed by Lakkaraju et al.[37] and Kolotuev et al.[38]. The identification of Hepatitis C virus (HCV) RNA in association with exosomes present in the blood of chronically HCV infected individuals[39], was one of the first indications that exosomes can carry RNA. In an initial attempt to quantify and identify the RNA content of exosomes, a microarray-based analysis detected a set of 1300 mRNA species (the majority very weakly expressed) in exosomes of a murine mast cell line, while 16000 genes were expressed in the host cell[40]. Interestingly, the exosomal mRNA profile did not mirror the mRNA expression pattern in the mother cell. The exosomal mRNA was intact and could be translated in vitro which produced murine proteins. Additionally, incubation of human cells with the murine exosomes mediated murine protein expression

ACCEPTED MANUSCRIPT in these human recipient cells. Importantly, the murine proteins corresponded to the exosomal murine mRNAs, while the actual proteins themselves were not found in the exosomes. This last finding was the proof that exosome-mediated

PT

tranfer of RNA from cell to cell is possible and functional. Nonetheless, a functional role and in vivo relevance for exosomal mRNA transfer remains to be

SC RI

elucidated. While the mRNA and rRNA content were very low in the exosomes, roughly 120 miRNAs were enriched in comparison to their expression level in the mother cell. In contrast to cellular miRNAs, exosomal miRNAs originated mainly from the AP3M2, DGKA, PRKA1, SKP1P and FGFR1OP genes. Examples angiogenesis,

haematopoiesis,

NU

are let-7, miR-1, miR-15, miR-16, miR-181 and miR-375, which play a role in exocytosis

and

tumorogenesis.

Exosomal

MA

enrichment of certain miRNAs in comparison to cellular miRNAs suggests a biased export.

Other strong evidence for selective miRNA sorting into exosomes comes

ED

from a recent study by Mittelbrunn et al.[41] in which the miRNA content of exosomes from T and B cells and DC was compared. They identified several

PT

miRNAs, like miR-760, miR-632, miR-654-5p and miR-671-5p to significantly accumulate in the exosomes. Others, like miR-101 and miR-32 were rather more

CE

abundant in the mother cell, and some were predominantly found in exosomes from a certain cell type, like miR-92a in DC-derived exosomes. Markedly, the

AC

miRNA expression pattern in the exosomes of these cell types was found to be more similar to one another than the actual miRNA patterns found in the distinct mother cell types. Moreover, the study established that miRNA transfer during cognate immune reactions takes place via exosomes, thereby uncovering an antigen recognition-dependent crossover of miRNAs from the T cell to the DC. A striking interaction, able to functionally modify gene expression in the DC[41]. Additionally it has been shown that glioblastoma exosomal mRNA and miRNA modulates cells in the tumor stroma to support tumorigenesis and promote tumor cell proliferation[42], a likewise process has been found in colorectal cancer cells[43]. In the latter two studies the RNA contents of exosomes from the investigated cancer cells was found to reflect the expression pattern in the mother cell. Also, the identification of distinct miRNA and mRNA species present in glioblastoma[42] and colorectal cancer[43] derived exosomes,

ACCEPTED MANUSCRIPT or the typical caveolin-1 carrying exosomes circulating in melanoma patients[44], raised the valid thought of using them as biomarkers of disease. This could aid diagnostics and therapeutic strategies in the future[45]. If

PT

exosomes are specialized RNA shuttles, how is the cargo loaded?

SC RI

2.1 ESCRTing RNA: a RISCy process?

Ubiquitinated protein cargo destined for exosomal loading typically interacts with the endosomal sorting complex required for transport (ESCRT). For miRNAs to be selectively loaded into exosomes their likewise interaction with

NU

ESCRT is a feasible thought.

Independently, two research groups described the association of argonaute

MA

(AGO)-2 and GW182, components of the RNA-induced silencing complex (RISC), with MVBs[46,47]. Interestingly, monocyte-derived exosomes were highly enriched for GW182 and contained AGO proteins. Moreover, isolation of MVB

ED

fractions indicated mature miRNAs like miR-16 and let-7a to associate with these fractions, suggesting miRNA-loaded incomplete RISC complexes (pre-miRISC) to

PT

associate with MVB membranes[46]. For them to be subsequently loaded into the lumen of a budding exosome, they possibly interact with ESCRT.

CE

The ESCRT pathway is an intricate protein machinery involved in MVB formation, vesicle budding and protein cargo sorting, among other membrane-

AC

reorganization processes[48]. The ESCRT system basically comprises four ESCRT complexes (0 through III) and the Vps4 complex. The hallmark of ESCRTmediated MVB cargo loading is the recognition of ubiquitinated proteins on the MVB membrane by ESCRT-0, a process that can associate with clathrin domains. The ESCRT-0 cargo complex then interacts with the ESCRT-I and –II complexes, the total complex of which will then associate with ESCRT-III. The latter protein is not involved in cargo recognition, but rather mediates vesicle budding after an initial cargo de-ubiquitination step. Following vesicle scission, the ESCRT-III complex disassociates from the MVB membrane utilizing energy provided by the Vps4 complex[48] (also see the graphical abstract). Indeed, GW182[46] and AGO2[49] can be found ubiquitinated when purified, however this only indicates their possible interaction with ESCRT-0. Gibbings et al.[46] then proceeded to establish that RISCs regularly associate

ACCEPTED MANUSCRIPT with ESCRT complexes by knocking down proteins essential for MVB formation, like Hrs and Alix. This significantly increased cellular GW182 content, indicating the absence of MVB formation to accumulate intracellular RISC complexes,

PT

possibly by the lack of export. Accordingly, the accumulation of MVBs and their associated RISCs enhanced RNAi efficiency, while knockdown of MVB-associated

SC RI

ESCRT complexes attenuated RNAi[46,47]. Additionally, Lee et al.[47] show that ubiquitination stimulates RISC loading, further supporting the finding that premiRISCs couple to the ESCRT complex on MVB membranes in mediating RNAi. Besides this new role for MVBs in RNAi, the recruitment of pre-miRISCs to

NU

the ESCRT complex strongly suggests that these complexes are loaded into budding exosomes, and implies a novel sorting role for the ESCRT complex. Since

MA

predominantly GW182, but not AGO2, was enriched in the exosomes, the incomplete miRISC complex is not exported into the exosome lumen. Thereby, ESCRT-associated pre-miRISC complexes unable to timely bind their mRNA

ED

target sequences, or mRNA/pre-miRISC complexes unable to timely bind AGO2, could be destined for exosomal export (also see the graphical abstract). This

PT

‘balancing act’ between ESCRT-associated pre-miRISCs, and AGO2/mRNA-target association suggests overabundant miRNA species to be destined for exosomal

CE

export. Thereby, over expression of miRNAs would enhance their exosomal transport. An effect which was indeed observed in a recent study by Kosaka et

AC

al.[50]

Since the intracellular miRNA balance could thereby directly influence the formation of MVBs, such an exosomal expulsion of overabundant miRNAs could be regarded as a regulatory process with a direct role in intercellular communication, and tissue transcriptome uniformity and -homogenization. 2.2 Exosome delivery tools The latter alights a route toward the use of exosomes for targeted miRNA delivery. Currently there are only two studies that have employed exosomes to deliver a therapeutic agent, with only one delivering a nucleic acid. In the first, the anti-inflammatory substance curcumin or the STAT3-inhibitor JSI124 effectively treated brain inflammatory diseases in mice when delivered intranasally, encapsulated in diffusion-loaded exosomes[51]. In this study the

ACCEPTED MANUSCRIPT intranasally applied mouse EL4 thymoma-derived exosomes reached the brain rather spontaneously. The second study, by Alvarez-Erviti et al.[52], was the first to use immature DC-derived exosomes in an intravenous application to target

PT

the mouse brain. Moreover, they were the first to deliver a macromolecular substance using exosomes. The DC were engineered to express the rabies viral

SC RI

glycoprotein (RVG) fused to the Lamp2b protein, ensuring exosomal surface expression of the Lamp2b-RVG fusion protein. This targeted the exosomes in vivo to neurons, microglia and oligodendrocytes. The exosomes were electroporated with a siRNA against BACE1, which is involved in beta-amyloid plaque formation

NU

in Alzheimer’s disease. Subsequently, the exosomes reduced BACE1 expression in targeted neurons in vivo. While there are certain issues to take into

MA

account[53], this approach for the very first time establishes the proof-ofconcept and feasibility of using exosomes as a delivery tool in systemic, targeted siRNA delivery in vivo.

ED

Exosomes as an siRNA delivery agent have a multitude of advantages over the typical viruses, lipid nanoparticles and polycationic delivery agents currently

PT

in use[53]. Most importantly, they deliver their cargo directly into the cytosol, bypassing the need for endosomal escape, while their inertness avoids attack

CE

and clearance in the extracellular environment.

AC

2.3 Biomodulation by nucleic acid nanocarriers The docking of pre-miRISC complexes to the ESCRT complex as a site of RNAi, the possible balance act between AGO/mRNA-target association and ESCRTmediated exosomal loading of miRNAs, and the observed transport of overexpressed miRNAs into exosomes suggests that exosomes could be generated which selectively or predominantly carry a desired miRNA cargo. MiRNAs post-transcriptionally control approximately 60% of all proteinencoding genes, and thereby influence a wide choice of cellular processes like differentiation, proliferation and apoptosis. Also, de-regulation of miRNA expression has been found in a broad panel of human diseases, ranging from cancer

(like

miR-9

disrupting

CpG

island

hypermethylation)[54]

to

neurodegenerative disease (miR-29 in Alzheimer’s disease[55], miR-7 in Parkinson for example[56]), Down’s syndrome (miR-155 and -802)[57] and

ACCEPTED MANUSCRIPT autoinflammation (miR-146a implicated in rheumatoid arhthritis)[58]. A comprehensive overview of non-coding RNA and miRNA function in human disease, was recently published by Esteller et al.[59]

PT

The ‘corrective use’ of distinct miRNA species and in particular the use of miRNA-silencing antagonists (antagomirs) poses a new therapeutic approach to

SC RI

correct pathological miRNA profiles. Impressive results have already been obtained by using antagomirs in vivo to lower blood cholesterol[60] or control the growth of transplanted neuroblastoma[61]. MiRNA or antagomir overexpression in cells that lack expression of their target sequences or lack

NU

expression of AGO proteins may optimize their packaging in exosomes. The formation of the RNAi silencing complex would be subdued and the ESCRT

MA

complex could favor exosomal loading instead of implementing RNAi. 2.4 Tetraspanins as a recipient determinant

ED

Tetraspanins are a protein family of fourfold membrane-crossing proteins, showing broad expression on virtually all cell types and involvement in many

PT

intercellular interactions. They regularly associate with one another forming clusters in tetraspanin membrane domains (TEMs) or with neighbouring

CE

transmembrane proteins like integrins[62]. Of particular interest to exosomes is their role in cellular fusion, illustrated by the observation that a sperm cell

AC

cannot fuse with a CD9 or CD81 tetraspanin-lacking oocyte[63]. Exosomes do not inconsiderately fuse with any recipient cell that happens

to be around. They display distinct tissue homing, which is likely related to their high expression of adhesion molecules like integrins and tetraspanins[64]. For example, LFA-1 appears to be a major player in the exosomal communication between T cells and DC[65,66], while B cell-derived exosomes rather display distinct target selectivity by their expression of beta1 and beta2 integrins[67]. Recently, Rana et al.[68] demonstrated that the integrin/tetraspanin expression pattern found in exosomal TEMs can influence their targeting. In a set of tetraspanin and integrin modification experiments, they show that it is likely not the actual pattern of tetraspanins that dictates exosome homing, but the association of certain tetraspanins and integrins. The exosomes from the rat pancreatic adenocarcinoma cell line BSp73ASML (ASML) were found to express

ACCEPTED MANUSCRIPT the metastasis-enhancing tetraspanins CD151 and Tspan8, and alpha6beta4 integrin among other distinct molecules. The exosomes of ASML cells which display alpha6beta4 integrin association with both CD151 and Tspan8 home to

PT

lung and lymph node stromal cells, where they support pre-metastatic niche formation[69]. Exosomal overexpression of Tspan8 in the presence of CD151

SC RI

and absence of alpha6beta4 integrin abolishes their potential to form premetastatic niches, and makes them rather home to endothelial cells and their progenitors, where they initiate angiogenesis[70]. Selective expression of the beta4

integrin

chain

on

exosomes

normally

expressing

CD151

and

NU

overexpressing Tspan8 makes them loose this capacity to home to endothelial cells, but gain capacity to target stromal cells and home to liver and lung

MA

tissue[68,71]. Finally, ASML-derived exosomes expressing CD151 but not Tspan8 or alpha6beta4 do not induce angiogenesis or prime metastatic niches[68]. Importantly, the association of Tspan8 with the alpha4 integrin chain appeared

ED

essential to mediate exosome fusion with endothelial recipient cells[70]. These results show the rather poorly studied tetraspanins, and in

PT

particular the expression pattern of adhesion molecules and their association with tetraspanins in TEMs, to be key regulators of exosome tissue homing and of

CE

subsequent membrane fusion with the recipient cell. This invites further scrutiny into the exact targeting capabilities of integrin and tetraspanin combinations. It

AC

is conceivable that in the future expression modulation of exosomal integrins and tetraspanins will allow the targeting of therapeutic exosomes during systemic application. 3.0 Conclusions Current research on RNAi and antagomirs indicates their promise for future therapies. However, the hurdle towards clinical application of nucleic acids clearly is the targeted, systemic delivery of these agents in vivo. Local delivery is not always feasible for practical reasons or simply impossible in the case of systemic or disseminated disease. The available in vivo nucleic acid delivery agents have a variety of targeting difficulties and unwanted side effects, which seriously limits their (long-term) use. Hence, there is a strong need for an immunologically inert, nano-sized delivery tool, which delivers its cargo to the

ACCEPTED MANUSCRIPT cytosol of recipient cells, and which can be specifically loaded with RNA and be targeted upon intravenous application in vivo. Exosomes have the potential to adress these needs and to revolutionize

PT

targeted delivery of nucleic acids. Current research shows that we are on the brink of unveiling the mechanisms that cells use to target their exosomes to

SC RI

certain tissues or cell populations and deciphering how exosomes are distinctly loaded with their RNA cargo. The answers to these questions could position exosomes on the front line of in vivo delivery, permitting us to ultimately use

NU

nature’s own intercellular nucleic acid carrier to the benefit of curing disease. 4.0 References

[5] [6] [7] [8] [9] [10] [11] [12]

MA

ED

[4]

PT

[3]

CE

[2]

B.T. Pan, R.M. Johnstone, Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: selective externalization of the receptor., Cell. 33 (1983) 967–978. J. COUZIN, Cell biology: The ins and outs of exosomes., Science. 308 (2005) 1862–1863. G. Raposo, H. Nijman, W. Stoorvogel, R. Leijendekker, C. Harding, C. Melief, et al., B lymphocytes secrete antigen-presenting vesicles, J. Exp. Med. 183 (1996) 1161–1172. E. Cocucci, G. Racchetti, J. Meldolesi, Shedding microvesicles: artefacts no more., Trends in Cell Biology. 19 (2009) 43–51. S.F. Mause, C. Weber, Microparticles: protagonists of a novel communication network for intercellular information exchange., Circ. Res. 107 (2010) 1047–1057. B. György, T.G. Szabó, M. Pásztói, Z. Pál, P. Misják, B. Aradi, et al., Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles., Cell. Mol. Life Sci. 68 (2011) 2667–2688. R.J. Simpson, S.S. Jensen, J.W.E. Lim, Proteomic profiling of exosomes: current perspectives., Proteomics. 8 (2008) 4083–4099. C. Théry, M. Ostrowski, E. Segura, Membrane vesicles as conveyors of immune responses, Nature Publishing Group. 9 (2009) 581–593. S. Viaud, C. Thery, S. Ploix, T. Tursz, V. Lapierre, O. Lantz, et al., Dendritic Cell-Derived Exosomes for Cancer Immunotherapy: What's Next?, Cancer Research. 70 (2010) 1281–1285. N. Blanchard, D. Lankar, F. Faure, A. Regnault, C. Dumont, G. Raposo, et al., TCR activation of human T cells induces the production of exosomes bearing the TCR/CD3/zeta complex., J. Immunol. 168 (2002) 3235–3241. S. Runz, S. Keller, C. Rupp, A. Stoeck, Y. Issa, D. Koensgen, et al., Malignant ascites-derived exosomes of ovarian carcinoma patients contain CD24 and EpCAM, Gynecologic Oncology. 107 (2007) 563–571. C. Thery, A. Regnault, J. Garin, J. Wolfers, L. Zitvogel, P. RicciardiCastagnoli, et al., Molecular characterization of dendritic cell-derived exosomes. Selective accumulation of the heat shock protein hsc73., J. Cell Biol. 147 (1999) 599–610.

AC

[1]

ACCEPTED MANUSCRIPT

[19]

[20] [21]

[22] [23] [24] [25] [26] [27]

PT

SC RI

NU

[18]

MA

[17]

ED

[16]

PT

[15]

CE

[14]

H.F. Heijnen, A.E. Schiel, R. Fijnheer, H.J. Geuze, J.J. Sixma, Activated platelets release two types of membrane vesicles: microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules., Blood. 94 (1999) 3791–3799. J.M. Street, P.E. Barran, C.L. MacKay, S. Weidt, C. Balmforth, T.S. Walsh, et al., Identification and proteomic profiling of exosomes in human cerebrospinal fluid, J Transl Med. 10 (2012) 5. J. Wolfers, A. Lozier, G. Raposo, A. Regnault, C. Thery, C. Masurier, et al., Tumor-derived exosomes are a source of shared tumor rejection antigens for CTL cross-priming., Nat Med. 7 (2001) 297–303. S. Dai, D. Wei, Z. Wu, X. Zhou, X. Wei, H. Huang, et al., Phase I Clinical Trial of Autologous Ascites-derived Exosomes Combined With GM-CSF for Colorectal Cancer, Molecular Therapy. 16 (2008) 782–790. T. Chen, J. Guo, M. Yang, X. Zhu, X. Cao, Chemokine-Containing Exosomes Are Released from Heat-Stressed Tumor Cells via Lipid Raft-Dependent Pathway and Act as Efficient Tumor Vaccine, The Journal of Immunology. 186 (2011) 2219–2228. S. Bhatnagar, K. Shinagawa, F.J. Castellino, J.S. Schorey, Exosomes released from macrophages infected with intracellular pathogens stimulate a proinflammatory response in vitro and in vivo, Blood. 110 (2007) 3234–3244. J.D. Walker, C.L. Maier, J.S. Pober, Cytomegalovirus-infected human endothelial cells can stimulate allogeneic CD4+ memory T cells by releasing antigenic exosomes., The Journal of Immunology. 182 (2009) 1548–1559. N. Chaput, J. Ta eb, N.L.E.C. Schartz, F. Andr, E. Angevin, L. Zitvogel, Exosome-based immunotherapy, Cancer Immunol Immunother. 53 (2004) 234–239. S. Viaud, S. Ploix, V. Lapierre, C. Théry, P.-H. Commere, D. Tramalloni, et al., Updated technology to produce highly immunogenic dendritic cellderived exosomes of clinical grade: a critical role of interferon-γ., J Immunother. 34 (2011) 65–75. A. Bobrie, M. Colombo, G. Raposo, C. Théry, Exosome Secretion: Molecular Mechanisms and Roles in Immune Responses, Traffic. 12 (2011) 1659–1668. M. Simons, G. Raposo, Exosomes--vesicular carriers for intercellular communication., Current Opinion in Cell Biology. 21 (2009) 575–581. R. Valenti, V. Huber, M. Iero, P. Filipazzi, G. Parmiani, L. Rivoltini, Tumorreleased microvesicles as vehicles of immunosuppression., Cancer Research. 67 (2007) 2912–2915. A. Clayton, J.P. Mitchell, J. Court, M.D. Mason, Z. Tabi, Human TumorDerived Exosomes Selectively Impair Lymphocyte Responses to Interleukin-2, Cancer Research. 67 (2007) 7458–7466. G. Andreola, L. Rivoltini, C. Castelli, V. Huber, P. Perego, P. Deho, et al., Induction of lymphocyte apoptosis by tumor cell secretion of FasLbearing microvesicles., J. Exp. Med. 195 (2002) 1303–1316. J.W. Kim, E. Wieckowski, D.D. Taylor, T.E. Reichert, S. Watkins, T.L. Whiteside, Fas ligand-positive membranous vesicles isolated from sera of patients with oral cancer induce apoptosis of activated T lymphocytes.,

AC

[13]

ACCEPTED MANUSCRIPT

[34] [35] [36] [37] [38] [39] [40]

[41]

[42]

[43]

PT

SC RI

NU

[33]

MA

[32]

ED

[31]

PT

[30]

CE

[29]

AC

[28]

Clin. Cancer Res. 11 (2005) 1010–1020. J.L. Hood, R.S. San, S.A. Wickline, Exosomes Released by Melanoma Cells Prepare Sentinel Lymph Nodes for Tumor Metastasis, Cancer Research. 71 (2011) 3792–3801. H. Peinado, S. Lavotshkin, D. Lyden, The secreted factors responsible for pre-metastatic niche formation: Old sayings and new thoughts, Seminars in Cancer Biology. 21 (2011) 139–146. R. Wiley, S. Gummuluru, Immature dendritic cell-derived exosomes can mediate HIV-1 trans infection, Proc. Natl. Acad. Sci. U.S.a. 103 (2006) 738–743. G.C.M.L.T.V.K.B.Y.C.N.J.K.A.P.B.M.P. Metka Lenassi, HIV Nef is secreted in exosomes and triggers apoptosis in bystander CD4+ T cells, Traffic. 11 (2010) 110. D.M. Pegtel, K. Cosmopoulos, D.A. Thorley-Lawson, M.A.J. van Eijndhoven, E.S. Hopmans, J.L. Lindenberg, et al., Functional delivery of viral miRNAs via exosomes, Proceedings of the National Academy of Sciences. 107 (2010) 6328–6333. B. Fevrier, D. Vilette, F. Archer, D. Loew, W. Faigle, M. Vidal, et al., Cells release prions in association with exosomes., Proc. Natl. Acad. Sci. U.S.a. 101 (2004) 9683–9688. L. Rajendran, M. Honsho, T.R. Zahn, P. Keller, K.D. Geiger, P. Verkade, et al., Alzheimer's disease beta-amyloid peptides are released in association with exosomes., Proc. Natl. Acad. Sci. U.S.a. 103 (2006) 11172–11177. S. Liegeois, The V0-ATPase mediates apical secretion of exosomes containing Hedgehog-related proteins in Caenorhabditis elegans, J. Cell Biol. 173 (2006) 949–961. A. Pauli, J.L. Rinn, A.F. Schier, Non-coding RNAs as regulators of embryogenesis., Nat Rev Genet. 12 (2011) 136–149. A. Lakkaraju, E. Rodriguez-Boulan, Itinerant exosomes: emerging roles in cell and tissue polarity, Trends in Cell Biology. 18 (2008) 199–209. I. Kolotuev, A. Apaydin, M. Labouesse, Secretion of Hedgehog-Related Peptides and WNT During Caenorhabditis elegans Development, Traffic. 10 (2009) 803–810. F. Masciopinto, C. Giovani, S. Campagnoli, L. Galli-Stampino, P. Colombatto, M. Brunetto, et al., Association of hepatitis C virus envelope proteins with exosomes., Eur. J. Immunol. 34 (2004) 2834–2842. H. Valadi, K. Ekström, A. Bossios, M. Sjöstrand, J.J. Lee, J.O. Lötvall, Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells, Nat Cell Biol. 9 (2007) 654–659. M. Mittelbrunn, C. Gutiérrez-Vázquez, C. Villarroya-Beltri, S. González, F. Sánchez-Cabo, M.Á. González, et al., Unidirectional transfer of microRNAloaded exosomes from T cells to antigen-presenting cells., Nat Commun. 2 (2011) 282. J. Skog, T. Würdinger, S. van Rijn, D.H. Meijer, L. Gainche, W.T. Curry, et al., Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers, Nat Cell Biol. 10 (2008) 1470–1476. B. Hong, J.-H. Cho, H. Kim, E.-J. Choi, S. Rho, J. Kim, et al., Colorectal cancer

ACCEPTED MANUSCRIPT

[50] [51]

[52] [53] [54] [55]

[56] [57]

[58] [59]

PT

SC RI

NU

[49]

MA

[48]

ED

[47]

PT

[46]

CE

[45]

AC

[44]

cell-derived microvesicles are enriched in cell cycle-related mRNAs that promote proliferation of endothelial cells, BMC Genomics. 10 (2009) 556. M. Logozzi, A. De Milito, L. Lugini, M. Borghi, L. Calabrò, M. Spada, et al., High Levels of Exosomes Expressing CD63 and Caveolin-1 in Plasma of Melanoma Patients, PLoS ONE. 4 (2009) e5219. M.A. Cortez, C. Bueso-Ramos, J. Ferdin, G. Lopez-Berestein, A.K. Sood, G.A. Calin, MicroRNAs in body fluids--the mix of hormones and biomarkers., Nat Rev Clin Oncol. 8 (2011) 467–477. D.J. Gibbings, C. Ciaudo, M. Erhardt, O. Voinnet, Multivesicular bodies associate with components of miRNA effector complexes and modulate miRNA activity, Nat Cell Biol. 11 (2009) 1143–1149. Y.S. Lee, S. Pressman, A.P. Andress, K. Kim, J.L. White, J.J. Cassidy, et al., Silencing by small RNAs is linked to endosomal trafficking., Nat Cell Biol. 11 (2009) 1150–1156. W.M. Henne, N.J. Buchkovich, S.D. Emr, The ESCRT Pathway, Developmental Cell. 21 (2011) 77–91. M. Matsumoto, S. Hatakeyama, K. Oyamada, Y. Oda, T. Nishimura, K.I. Nakayama, Large-scale analysis of the human ubiquitin-related proteome., Proteomics. 5 (2005) 4145–4151. N. Kosaka, H. Iguchi, Y. Yoshioka, F. Takeshita, Y. Matsuki, T. Ochiya, Secretory mechanisms and intercellular transfer of microRNAs in living cells., Journal of Biological Chemistry. 285 (2010) 17442–17452. X. Zhuang, X. Xiang, W. Grizzle, D. Sun, S. Zhang, R.C. Axtell, et al., Treatment of Brain Inflammatory Diseases by Delivering Exosome Encapsulated Anti-inflammatory Drugs From the Nasal Region to the Brain, Molecular Therapy. 19 (2011) 1769–1779. L. Alvarez-Erviti, Y. Seow, H. Yin, C. Betts, S. Lakhal, M.J.A. Wood, Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes., Nat. Biotechnol. 29 (2011) 341–345. J.G. van den Boorn, M. Schlee, C. Coch, G. Hartmann, SiRNA delivery with exosome nanoparticles, Nat. Biotechnol. 29 (2011) 325–326. L. Ma, J. Young, H. Prabhala, E. Pan, P. Mestdagh, D. Muth, et al., miR-9, a MYC/MYCN-activated microRNA, regulates E-cadherin and cancer metastasis, Nat Cell Biol. 12 (2010) 247–U52. S.S. Hébert, K. Horré, L. Nicolaï, A.S. Papadopoulou, W. Mandemakers, A.N. Silahtaroglu, et al., Loss of microRNA cluster miR-29a/b-1 in sporadic Alzheimer's disease correlates with increased BACE1/betasecretase expression., Proceedings of the National Academy of Sciences. 105 (2008) 6415–6420. S. Gehrke, Y. Imai, N. Sokol, B. Lu, Pathogenic LRRK2 negatively regulates microRNA-mediated translational repression., Nature. 466 (2010) 637– 641. D.E. Kuhn, G.J. Nuovo, A.V.J. Terry, M.M. Martin, G.E. Malana, S.E. Sansom, et al., Chromosome 21-derived MicroRNAs Provide an Etiological Basis for Aberrant Protein Expression in Human Down Syndrome Brains, Journal of Biological Chemistry. 285 (2010) 1529–1543. K.M. Pauley, S. Cha, miRNA-146a in rheumatoid arthritis: a new therapeutic strategy., Immunotherapy. 3 (2011) 829–831. M. Esteller, Non-coding RNAs in human disease., Nat Rev Genet. 12

ACCEPTED MANUSCRIPT

[66]

[67] [68] [69] [70]

[71]

PT

SC RI

NU

[65]

MA

[64]

ED

[63]

PT

[62]

CE

[61]

AC

[60]

(2011) 861–874. J. Krützfeldt, N. Rajewsky, R. Braich, K.G. Rajeev, T. Tuschl, M. Manoharan, et al., Silencing of microRNAs in vivo with 'antagomirs'., Nature. 438 (2005) 685–689. L. Fontana, M.E. Fiori, S. Albini, L. Cifaldi, S. Giovinazzi, M. Forloni, et al., Antagomir-17-5p Abolishes the Growth of Therapy-Resistant Neuroblastoma through p21 and BIM, PLoS ONE. 3 (2008) e2236. M.E. Hemler, Tetraspanin functions and associated microdomains., Nat Rev Mol Cell Biol. 6 (2005) 801–811. E. Rubinstein, A. Ziyyat, J.-P. Wolf, F. Le Naour, C. Boucheix, The molecular players of sperm-egg fusion in mammals., Semin. Cell Dev. Biol. 17 (2006) 254–263. M. Zöller, Tetraspanins: push and pull in suppressing and promoting metastasis, Nat Rev Cancer. 9 (2008) 40–55. E.N.M. Nolte-'t Hoen, S.I. Buschow, S.M. Anderton, W. Stoorvogel, M.H.M. Wauben, Activated T cells recruit exosomes secreted by dendritic cells via LFA-1, Blood. 113 (2009) 1977–1981. Y. Xie, H. Zhang, W. Li, Y. Deng, M.A. Munegowda, R. Chibbar, et al., Dendritic cells recruit T cell exosomes via exosomal LFA-1 leading to inhibition of CD8+ CTL responses through downregulation of peptide/MHC class I and Fas ligand-mediated cytotoxicity., The Journal of Immunology. 185 (2010) 5268–5278. A. Clayton, A. Turkes, S. Dewitt, R. Steadman, M.D. Mason, M.B. Hallett, Adhesion and signaling by B cell-derived exosomes: the role of integrins., The FASEB Journal. 18 (2004) 977–979. S. Rana, M. Zöller, Exosome target cell selection and the importance of exosomal tetraspanins: a hypothesis, Biochem. Soc. Trans. 39 (2011) 559–562. T. Jung, D. Castellana, P. Klingbeil, I. Cuesta Hernández, M. Vitacolonna, D.J. Orlicky, et al., CD44v6 dependence of premetastatic niche preparation by exosomes., Neoplasia. 11 (2009) 1093–1105. I. Nazarenko, S. Rana, A. Baumann, J. McAlear, A. Hellwig, M. Trendelenburg, et al., Cell surface tetraspanin Tspan8 contributes to molecular pathways of exosome-induced endothelial cell activation., Cancer Research. 70 (2010) 1668–1678. M. Herlevsen, D.-S. Schmidt, K. Miyazaki, M. Zöller, The association of the tetraspanin D6.1A with the alpha6beta4 integrin supports cell motility and liver metastasis formation., Journal of Cell Science. 116 (2003) 4373–4390.

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

MA

NU

SC RI

PT

Graphical abstract