Exosomes, a subset of extracellular vesicles (EVs), function as a mode of intercellular communica-tion and molecular transfer. Exosomes facilitate the direct extracellular transfer of proteins, lipids, andmiRNA/mRNA/DNAs between cells in vitro and in vivo. The immunological activities of exosomes affectimmunoregulation mechanisms including modulating antigen presentation, immune activation, immunesuppression, immune surveillance, and intercellular communication. Besides immune cells, cancer cellssecrete immunologically active exosomes that influence both physiological and pathological processes.The observation that exosomes isolated from immune cells such as dendritic cells (DCs) modulate theimmune response has enforced the way these membranous vesicles are being considered as potentialimmunotherapeutic reagents. Indeed, tumour- and immune cell-derived exosomes have been shown tocarry tumour antigens and promote immunity, leading to eradication of established tumours by CD8+
+
T cells and CD4 T cells, as well as directly suppressing tumour growth and resistance to malignanttumour development. Further understanding of these areas of exosome biology, and especially of molec-ular mechanisms involved in immune cell targeting, interaction and manipulation, is likely to providesignificant insights into immunorecognition and therapeutic intervention. Here, we review the emergingroles of exosomes in immune regulation and the therapeutic potential in cancer.
The immune system embodies ordered biological processes andtructures that serve to recognise and respond to the surrounding
immune surveillance [1]. In both adaptive and innate immunity,several immune pro-tumour effector mechanisms are dysreg-ulated, leading to the hypothesis that inflammation in certaininstances can facilitate carcinogenesis and tumour progression by
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xtracellular environment. In the context of cancer, precancer-us and malignant cells can provoke an immune response thatbolishes transformed and/or malignant cells; a process known as
∗ Corresponding author at: La Trobe Institute for Molecular Science (LIMS), Larobe University, Bundoora, Victoria 3083, Australia. Tel.: +61 3 9479 3961.
modulating immunoregulation [2]. Further, in some cancer types,an inflammatory microenvironment restricts the immune systemfrom rejecting malignant cells, and promotes the development oftumours [3]. Throughout the tumour microenvironment, multi-
their roles in immune regulation and cancer. Semin Cell Dev Biol
ple immune cell types, including T lymphocytes, B lymphocytes,macrophages, and natural killer (NK) cells regulate tumourigen-esis depending on the tissue composition and cellular stimuli[4,5]. Recently, the role of extracellular vesicles (EVs), in particular,
present antigenic peptides (namely MHC–peptide complexes) to
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ndosome-derived exosomes, secreted by tumour cells in mod-lating the immune response has been highlighted [6–12]. Asocal mediators of intercellular communication and immunologi-al function, exosomes can enhance opsonisation, regulate antigenresentation, and induce immune activation and immune sup-ression. Further, there is increasing evidence highlighting thatxosomes may facilitate cancer progression by regulating differ-nt immune cell types towards a pro-tumourigenic environment13,159].
Exosomes are homogenous membrane vesicles (40–150 nmiameter), derived from the exocytosis of intraluminal vesicleswithin multivesicular bodies, MVBs), and released into the extra-ellular space when fused with the plasma membrane [14,15].ost cell types release exosomes through this mechanism includ-
ng haematopoietic cells, reticulocytes, B- and T-lymphocytes,endritic cells, mast cells, platelets, intestinal epithelial cells,strocytes, neurons and tumour cells [16]. Specific characteris-ics associated with exosomes include their composition (bilipidicayer), size, density (1.09–1.13 g/mL) and protein content, includ-ng endosome-derived (endosomal sorting complex required forransport proteins; ESCRTs, such as Alix and Tsg101), sorting- andrafficking-related (endosomal Rab GTPases) and cell membrane-erived (tetraspanins, CD63, CD81 and CD82) [17,18]. In addition torotein constituents, exosomes are also comprised of various lipidsnd lipid-raft-associated proteins originating either at the plasmaembrane or from early/late endosome compartments, includ-
ng cholesterol, sphinogmyelin, and flotillins [15,19,20]. Exosomesre involved in the transfer of various mRNAs and microRNAsmiRs) to neighbouring cells for translation [21,22]. Pancreatic can-er cell-derived exosomes have recently been shown to containouble-stranded DNA of mutated KRAS and p53 [23], the most fre-uent genetic mutations in human pancreatic cancer. Interactionsetween tumour-derived exosomes and recipient cells are medi-ted through direct signalling interactions via surface-expressedolecules or by transfer of exosomes and/or their cargo [24,25].
umour-derived exosomes are released both locally and into theirculation to interact with an assortment of target cells, includingther tumour, stroma, and immune cells. The precise mechanismf exosome internalisation is still unclear, however, receptor-ediated endocytosis (e.g., LFA1, TIM1 and TIM4), phagocytosis,
nd direct plasma membrane fusion have been proposed [26,27].t has been demonstrated that the low pH of the tumour microen-ironment is essential for exosome uptake by human metastaticelanoma in vitro, that is suggested to be related to elevated sta-
ility and lipid/cholesterol content of exosomal membranes in ancidic environment [28].
Exosomes adopt many distinct roles that vary depending onheir cellular origin, from modulating immune function, enhancingumour-cell invasion, and intercellular communication. In an efforto understand immune-related functions of exosomes, recent stud-es have investigated specific components of exosomes that directlyr indirectly regulate the tumour immune response [11,29–31].hese components have been shown to include peptide-boundHC class I and II, T-cell stimulatory molecules (B7.2, ICAM-1),
nd other immune molecules such as MFG-E8, FasL, galectin-9,GF-�, TNF-�, or NKG2D ligand [32–44]. Exosomes derived fromancer cells and virally infected cells have been shown to influ-nce the tumour extracellular environment [35,36,45–48]. Tumourells secrete immunologically active exosomes, capable of inhibi-ing tumour growth through anti-tumour immune responses [49]nd promotion of tumour growth by inhibiting anti-tumour immu-ity [50] or enhancing the metastatic process [35,36,45–48,51,52].
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his review will summarise the role of exosomes in immunoregu-ation, including antigen presentation, activation and suppressionf the immune response, and expression of cell surface opsonins
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and complement factors as a means of immune surveillance andimmunorecognition in the context of cancer.
2. Modulating the immune response: the role of exosomes
The immune response is the collective process of innateand adaptive immunity [53]. These responses are regulated bymany different components, including proteins, RNAs, and lipids;a process known as immunomodulation. In cancer, complexnetworks are mediated by tumour cells or pro-inflammatory cellconstituents, to facilitate immunomodulation and result in therepression of adaptive immunity against cancer cells [54–56]. Withtheir diverse, and often contrasting immune-related functions,exosomes have recently gained attention during tumourigene-sis, in particular on cancer immune surveillance and tumourescape [11], promoting tumour progression. Exosomes facilitateangiogenesis [57–60], directly suppressing cytotoxic T lympho-cytes and NK-cells’ anti-tumour responses, and inducing activationof immune suppressor cell subsets, leading to loss of tumourimmune surveillance [9]. Furthermore, the suppressor activity oftumour vesicles seems to involve induction of the generation,expansion, and suppressive function of human regulatory T cells[61,62]. The molecules expressed on exosomes involved in regu-lating the immune response include FasL, TRAIL, membrane-boundTGF-�, CD73, and galectins [36,43,44,63–67]. Tumour-derived exo-somes can also suppress immune-cell responses by inhibiting thecytotoxicity of CD8+ T cells and NK cells [63,68–71]. This inhibi-tion is mediated predominantly through NKG2D down-regulation[68–70] and TGF-� [71] on the tumour-derived exosome surface.The inhibition of cytotoxic T-cell function has also been suggestedto be induced in part by the increased T-cell ROS content mediatedby melanoma-derived exosomes [72].
In addition to their direct roles in the extracellular tumourenvironment, exosomes have been shown to be significant inthe regulation of immune responses during cancer immunother-apy [33,73–84]. Exosomes carrying MHC–peptide complexes andantigens are crucial in initiation and amplification of an immuneresponse. Pioneering work by Raposo and co-workers [33] demon-strated that B lymphoblast-derived exosomes that bear MHC classII–peptide complexes were capable of activating human and mouseantigen specific T cell clones. The presentation of these complexesto T cells identified a possible role of exosomes in the modulationof the adaptive immune response. Zitvogel and colleagues demon-strated that exosomes derived from dendritic cells (DCs) pulsedwith antigenic peptides induced potent anti-tumour CD8+ T cellresponses in murine mastocytoma P815 and mammary carcinomaTS/A tumour models, resulting in the regression of establishedtumours [78]. Presentation of MHC–peptide complexes to naïve Tlymphocytes and the priming of cytotoxic T lymphocytes (CTLs)by these exosomes were shown to selectively promote the anti-tumour immune response [78]. Further, this study highlighted thatDC maturation accompanied exosome production. These resultsand subsequent studies discussed in this review further highlightthe importance and often contrasting functional effects of exo-somes in mediating and influencing the immune surveillance.
2.1. Exosomes and their roles in antigen presentation and T cellactivation
Antigen-presenting cells (APCs), such as dendritic cells (DCs),
their roles in immune regulation and cancer. Semin Cell Dev Biol
T cells. Exosomes derived from various cell types have beenshown to play crucial roles in carrying and presenting func-tional MHC–peptide complexes to modulate antigen-specific CD8+
esponse [43] through direct presentation and cross-presentations illustrated in Fig. 1A and B. Direct presentation occurs whenHC–peptide complexes on the exosomes are directly engaged
y antigen-specific T cells leading to T cell activation. In cross-resentation, APCs acquire antigens carried by exosomes, furtherrocess these antigens and present their peptides to CD8+ Tells. Moreover, cross-presentation can occur whereby antigeniceptide–MHC complexes are together transferred onto DCs andhen presented to T cells (termed cross-dressing) [85]. In the
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ontext of exosome function, MHC–peptide complexes from exo-omes have been attributed to cross-dressing of APCs to activatello-reactive T cells [37] but yet to be confirmed as a mecha-ism as exosome-mediated T cell cross-priming [86,87]. Thery and
ig. 1. The role of exosomes in modulating the immune response: (A) tumour-derived ehown to prime CD8+ and CD4+ T cells. These exosomes express tumour antigens (e.g., as
ound antigens). These exosomes also express some receptor ligands and adhesion mondirectly in APC binding. In addition, antigen cross-presentation through MHC–peptidexosomes to induce T cell activation. It is however still debated. (B) APC-derived exosomectivation of NK cells. DC-derived exosomes, when used as potential vaccines, have beeno host DCs to prime CD8+ and CD4+ T cells, respectively. These exosomes also exhibit
n vivo. Exosomes therefore are vehicles for MHC–peptide complexes (pMHC) and that
xosomes derived from APCs loaded with specific peptides and/or antigens are also caediated activation of NK cells. (C) Exosomes have been demonstrated to have more gene
TNF-�, RANTES), cytokine/chemokine generation/secretion (CCL2), and enhancing immifferent mechanisms including attenuation of T cell-mediated killing (exosomes enrichedexosomes suppress CD3 �-chain expression by T cells, blocking NKG2D-dependent cytotells (MDSCs) (exosomes contain PGE2, TGF-�, HSP72 in tumour-derived vesicles). Thesxosomes carrying and transferring opsonins and complement factors enhance extracellodulating the inflammatory response. CD55 and CD59 on exosomes derived from imma
poptotic cells (through opsonic receptors, e.g., iC3b) for phagocytosis. Further, targetingD9, CD11a, CD54, CD81, and phosphatidylserine on exosomes, and CD11a and CD54 on
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colleagues [40] highlighted exosomes secreted by DCs, in addi-tion to inducing naïve T cell stimulation in vivo and in vitro, couldstimulate T cells in the presence of MHC class II-deficient DCs.In the absence of intact antigen, exosomal peptide–MHC (pMHC)complexes could stimulate specific T cell response in vitro in thepresence of mature DCs. Exosomes therefore mediate the trans-fer and cross-dressing of pMHC complexes between different DCpopulations. Further, Wakim and Bevan [87] showed that exo-somes derived from SIINFEKL-pulsed H-2Kb DCs failed to cross
their roles in immune regulation and cancer. Semin Cell Dev Biol
dress H-2Kbm1 DCs to induce OT-I T cell proliferation; howeverthe co-culture of the two DC population enabled cross-dressingof antigenic peptide–MHC class I complexes. This study indi-cated that exosomes are not the means by which intact antigenic,
xosomes internalised by or fused with antigen-presenting cells (APCs) have beenMHC class I- and class II-peptide complexes and as intra-exosomal and membrane-
lecules (e.g., LFA1, MFGE8, TIM1/4, tetraspanins) participating either directly or complex “cross-dressing” APCs have been shown to be potentially mediated bys can directly modulate the antigen-specific response of CD8+ and CD4+ T cells and
demonstrated to transfer functional MHC class I- and class II-peptide complexescomparable efficacy as mature DCs to stimulate antigen-specific T cell activationrequire co-stimulatory molecules expressed by recipient DCs to stimulate T cells.pable of inducing an immune response, including NKG2D ligand, IL-15/IL-15R�-ral immune stimulatory roles, including activation of pro-inflammatory mediatorsune activation through Hsp70. (D) Exosomes repress immune responses through
for CD95L, TRAIL or galectin 9, which promotes T cell apoptosis), NK cell cytotoxicityoxicity of NK cells and CD8+ T cells), and activation of myeloid-derived suppressore mechanisms facilitate immune escape, and tumour invasion and metastasis. (E)ular surveillance, apoptotic cell phagocytosis/clearance, antigen presentation, andture DCs, have been shown to attenuate the inflammatory response by opsonising
of circulating exosomes to DCs has been shown mediated by MFG-E8/lactadherin,DCs.
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4 D.W. Greening et al. / Seminars in Cell & Developmental Biology xxx (2015) xxx–xxx
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Fig. 1.
eptide-MHC class I complexes are cross-dressed onto DCs. Clearly,ith these opposing data, the issue of exosome-mediated cross-ressing of DCs requires further investigation.
Exosomes carrying native tumour antigen or functionalHC–peptide complexes have been widely investigated and
hown to play a critical role in antigen cross-presentation dur-ng cancer surveillance (Fig. 1A) [33,77–79,88]. In an in vitroystem, Wolfers et al. [77] demonstrated that dendritic cells acti-ated tumour-specific CD8+ T cells following uptake of humanelanoma exosomes and that these exosomes are enrichedith HSP70 and full-length tumour antigens. In mouse tumour
ells, these authors further demonstrated that exosomes inducedD8+ T cell cross priming and tumour rejection in vivo [77].onocytes-derived dendritic cells (MoDCs) pulsed with exosomes
rom melanoma ascites induced Mart1/Melan A-specific, HLA-A2estricted CD8+ T cell responses ex vivo [88]. Further, bulk lym-hocytes from these melanoma patients stimulated with MoDCs
oaded with ascites-derived exosomes expanded into tumourpecific cytotoxic T lymphocytes. The authors therefore pro-osed that such ascites-derived exosomes could be a suitableumour antigen source for exosome-based vaccines against cancer88].
Exosomes have also been shown to directly induce T cellctivation in the absence of APCs as they carry MHC–peptide com-lexes, sometimes even co-stimulatory molecules, that includexosomes isolated from viral infected MoDCs [89]. Utsugi-Kobukait al. [41] demonstrated that exosomes secreted from chickenvalbumin (OVA)-pulsed BmDCs, presented OVA257–264 peptideo its specific CD8+ T cell hybridomas. Interestingly, exosomesecreted by OVA257–264 peptide- or OVA protein-pulsed maturemDCs stimulated T cell hybridomas more efficiently than the
mmature counterparts likely due to higher abundance of co-timulatory molecules on the mature BmDC derived exosomes41]. In vivo, exosomes have been shown to also transfer func-ional MHC–peptide complexes to DCs leading to priming of CD8+
nd CD4+ T cells [48,90,91] (Fig. 1B). Furthermore, DC-derivedxosomes from knock-out mice lacking MHC class II–peptide com-
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lex and ICAM-1 expression were not able to prime naïve Tells directly [39]. Therefore, changes in protein composition andriming abilities of exosomes reflect maturation signals receivedy DCs.
nued ).
Although exosomes also transfer class II–peptide complexes,their capacity to directly activate CD4+ T cells is less well under-stood. It was reported that 50% of MHC class II–peptide complexesare lost on activated B cells every day including 12% loss as a resultof exosome release [38]. These exosomes were shown to directlystimulate primed, but not naïve, CD4+ T cells. Interestingly, recycledMHC II–peptide complexes were also able to directly activate CD4+
T cells [38]. Mallegol et al. [92] demonstrated in vitro that exo-somes, secreted by HLA-DR4-expressing intestinal epithelial cellline T84 and pulsed with human serum albumin peptide HSA64–76,could not directly activate HLA-DR4-restricted HSA64–76-specificCD4+ T-cell hybridoma. However, the same T-cell hybridoma wasactivated by human MoDCs loaded with these exosomes, even moreefficiently than the MoDCs loaded with the soluble antigen. In addi-tion, intestinal epithelial exosomes were shown to contain MHCclass II–peptide complexes, and tetraspanins CD9, CD81 and CD82[93] which may enhance local antigen presentation. These resultsindicate that exosomes carrying MHC II–peptide complexes maydirectly activate CD4+ T cells and MoDCs loaded with peptide-pulsed exosomes could be more efficient than MoDCs loaded withsoluble antigen, arguing that exosomes may enhance the immunesurveillance in the mucosal surface [94] and in other locations.
In transplantation, indirect allorecognition initiated by recipientDCs presenting donor allogeneic antigens is one of the mech-anisms responsible for transplant rejection [95]. Using Thy1.1congenic, TCR transgenic 1H3.1 CD4+ T cells specific for the IAb-IE�52–68 complex, Montecalvo et al. [37] demonstrated that donorDC-derived exosomes mediated the transfer of functional allo-geneic IAb-IE�52–68 complexes to Balb/c recipient DCs which inturn initiated the indirect allorecognition. The authors thereforeproposed that donor DC-derived exosomes may amplify allorecog-nition during organ transplantation [37,40]. Such studies haveraised controversy regarding the feasibility of direct presentationby exosomes, as indirect presentation might be a possibility andhas not been excluded. Some studies proved the need of indirectpresentation by DCs for exosomes stimulated T-cells [37,96], whileother studies have demonstrated direct functional presentation
their roles in immune regulation and cancer. Semin Cell Dev Biol
through exosomes themselves [39,97].Cell surface and integral membrane/adhesion proteins on
exosomes are important in mediating associated cell recog-nition, adhesion, antigen uptake, and recipient cell function
24,27,28,98–100]. Taken together, functional MHC/peptideomplexes and tumour antigens such as, Mart-1, gp100, Her2/Neund CEA present in exosomes have been shown to be a promisingnti-tumour therapeutic vaccines in clinical intervention via anti-en presentation and T cell activation [43,44,101]. Other ligands,ncluding lactadherin/milk fat globule E8 (MFGE8), tetraspaninsnd externalised phosphatidylserine, are also present on exo-omes. These ligands and adhesion molecules participate eitherirectly or indirectly in the binding of exosomes to APCs [102–104].oreover, intestinal epithelial cell-derived exosomes were shown
o contain enriched transmembrane proteins CD9, CD81, CD82nd glycoprotein A33 antigen that were capable of binding humanerum albumin (HSA). HSA bound exosomes preferentially interactith DCs, thereby enhancing antigen presentation to T cells [92].
.2. Exosomes: promoting immune responses
Exosomes have recently been shown to be involved in the pro-nflammatory response and are capable of promoting immunityFig. 1C). Exosomes derived from bacteria-infected macrophagesave been shown to be immunomodulatory and stimulateacrophages and neutrophils to secrete pro-inflammatory medi-
tors, including TNF-� and RANTES (up-regulating iNOS expres-ion) [105,106]. Further, exosomes released from macrophagesnfected with intracellular pathogens stimulate a pro-inflammatoryesponse in vitro and in vivo [106]. Pathogen-associated molecularatterns (PAMP) on these exosomes may also play critical roles
n enhancing immune surveillance [105,106]. Whilst its been wellstablished that T cells secrete exosomes, distinct exosome popu-ations have been identified from CD4+ T cells, of which one vesicleype increases upon T-cell activation depending on the level of co-timulation [90]. This suggests that T-cell activation differentiallyegulates the release of distinct exosome subpopulations.
Mature DC-derived exosomes can activate an immuneesponse through the TNF-� pathway. Exosome-derived TNF--induced epithelial cells have been demonstrated to secretero-inflammatory cytokines (MCP-1, IL-8, TNF-�, RANTES),
ndicating a key role for exosomes in immunity [107]. More-ver, NK cells incubated with tumour-derived Hsp70-positivexosomes were induced to release granzyme B that initiatedpoptosis in human pancreatic/colorectal tumours [30]. Therefore,ro-immunogenic potential was identified as a key property ofumour-derived exosomes. In response to stress the assimilation ofsp70 within the lipid bilayer has been demonstrated to modulatexosomes release into the extracellular milieu [108]. Interestingly,ega and colleagues identified TNF-� secretion by macrophages
n membranous structures that constitute Hsp70. They suggesthat stress-induced membrane translocation of Hsp70 stimulatesn immunogenic response. Qazi et al. [109] observed the enrich-ent of exosomes in bronchoalveolar lavage fluid from patientsith sarcoidosis. Bronchoalveolar lavage fluid-derived exosomes
nduced epithelial cells to produce interleukin (IL)-8, a neutrophilhemotactant, and peripheral blood monocytes to produce IFN-�nd IL-13, factors important for activating T cell responses. Itas reported that exosomes derived from synovial fibroblasts of
heumatoid arthritis individuals contained a membrane form ofNF-� [32]. Interestingly, these exosomes were demonstrated toe internalised by activated T cells, induce Akt and NF-�B pathwayctivation and therefore delay activation-induced cell death.herefore, exposing tumour cells to stress may cause derivedxosomes to be significantly more immunogenic [110,111]. Thesectivities require the active participation of DCs to process and
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ross-present delivered antigens to T cells. It is important tomphasise that the cancer exosome content, which is under thenfluence of the microenvironment, is important for these immuneunctions.
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DC-derived exosomes also induced anti-tumour responses byactivating other immune effector cells (Fig. 1B). For example, mouseBmDC-derived exosomes harbouring functional membrane boundIL-15R� (NKG2D ligand) were shown in vivo to promote IL-15R�-and NKG2D-dependent NK cell activation and proliferation, result-ing in tumour suppression [112]. Similarly, human MoDC-derivedexosomes also carried functional NKG2D ligands and are able torestore the number and NKG2D-dependent function of NK cells inexosome-based vaccine clinical trial patients [112]. This capacityof DC-derived exosomes to suppress/activate the immune sys-tem indicates that exosomes are an important component of theimmune network, however the influence of the tumour microen-vironment is an important contributing factor to these immunefunctions. Together, these studies highlight the fundamental rolesof exosomes in the initiation and amplification of pro-inflammatoryimmune responses.
2.3. Exosomes: inhibiting immune responses
A large body of evidence points towards the established role oftumour-derived exosomes in promoting a pro-tumourigenic phe-notype and facilitating immunosuppression (extensively reviewedin [113]). Several studies further show that these exosomes havethe potential to hijack mechanisms enlisted by cancer cells toinfluence the efficacy of therapeutic agents [114,115]. Therefore,dissection of tumour exosomes stimulated pathways leading toimmune suppression will provide valuable insights into identifi-cation and/or selection for cancer therapies.
Suppression of the immune response by tumour-derived exo-somes has been shown to suppress T-cell and NK cell activity,and stimulate myeloid-derived suppressor cells (MDSCs) (Fig. 1D)[12,116]. Seminal work by Poutsiaka and colleagues [117] showedthat membranous vesicles released by murine B16 melanomacells repressed the IFN-�-dependent class II expression on murinemacrophages, which may affect antigen presentation to CD4+
T cells. Moreover, many studies have also shown that tumour-derived exosomes can induce T-cell apoptosis that facilitate evasionof immune surveillance. Andreola and colleagues demonstratedthat in melanoma cells, Fas ligand (FasL) was restricted to MVBs thatcontain melanosomes [118]. These melanosome-positive MVBswere demonstrated to further release FasL-containing exosomesthat induce apoptosis in Jurkat and lymphoid cells. Therefore, neo-plastic modulation involving release of FasL-positive exosomespromotes immune escape. Exosomes expressing FasL and TNF werealso shown to regulate T-cell apoptosis in human colorectal cancer(CRC) [64]. Exosomes from OVA-specific CD8+ T cells have beenobserved to be recruited by DCs through LFA1–ICAM-1 interac-tion, leading to down-regulation of OVA MHC–peptide expression,induction of apoptosis in OVA-loaded DCs, leading to inhibition ofOVA-specific CD8+ CTL responses in murine cancer models [119].T-cell activation and fate can therefore be regulated by tumour-derived exosomes that facilitate evasion of the immune responseand tumour progression.
Liu et al. [120] showed that pre-treatment of mice with exo-somes derived from TS/A or 4T.1 murine tumour cells, led toenhanced tumour cell growth in syngeneic and nude mice. Addi-tionally, in response to tumour-derived exosomes, expression of NKcell cytotoxic molecules was diminished and IL-2 induced NK cellproliferation was repressed, contributing to tumourigenesis. Ashiruet al. [68] observed truncated MHC class I-related chain (MIC)A (allele MICA*008) in exosomes. The authors showed that exo-somes comprising truncated MICA*008 induced down-regulation
their roles in immune regulation and cancer. Semin Cell Dev Biol
of its ligand NKG2D leading to decreased NK cytotoxicity. MICAand MICB are crucial for the induction of the NK activating recep-tor NKG2D, with expression of these molecules in sera associatedwith compromised immune response facilitating tumour escape
rom immune surveillance. Clayton and colleagues reported thatxosomes isolated from various cancer cell lines and pleural effu-ions from patients with mesothelioma, mediated attenuation ofKG2D expression by NK cells and CD8+ T cells [69]. Induction ofGF-� by exosomes was also shown to mediate down-regulation ofKG2D. Hedlund et al. [121] showed that exosomes bearing NKG2D
igands were secreted by the human placenta, possibly preventingmmune targeting of the foetus. These exosomes decreased the sur-ace expression of NKG2D receptor on NK cells and CD8+ T cells ineripheral blood mononuclear cells (PBMC) from healthy donors.urther, Admyre et al. [122] showed that exosome-like vesiclesepressed IL-2 and IFN-� production in PBMCs. Together, thesetudies highlight exosome-mediated suppression of NK cells as anmportant aspect during immune evasion and neoplastic progres-ion.
Recently, it has been suggested that cooperation betweenDSCs and tumour-derived exosomes from various murine cell
ines promotes MDSC-mediated repression of T cells. Chalmin et al.7] showed that the in vivo anti-tumour efficacy of the chemother-peutic drug cyclophosphamide in various murine models wasonsistent with the involvement of exosomal Hsp72 in potenti-ting MDSC activity. Xiang et al. [123] reported that murine TS/And 4T-1 breast tumour cell-derived exosomes induced MDSC mor-hology and activity in bone marrow myeloid cells (BMMCs), with
concomitant increase in tumour development and growth. Inddition, TGF-� and prostaglandin E2 (PGE2) were found to berucial for tumour exosomes to mediate neoplastic progressionhrough MDSCs. Liu and colleagues [124] showed that MyD88,
cytoplasmic adaptor molecule that is crucial for the propaga-ion and integration of signals generated by the TLR family, ismportant for exosomes derived from metastatic lung tumour cellsn promoting IL-6, TNF-�, and chemokine CCL2 production andnduction of MDSCs. Treatment of MyD88 KO mice with suchxosomes attenuated induction of IL-6 and chemokine CCL2, andnhanced CD8+ T cell cytotoxicity, demonstrating an important roleor exosome-mediated expansion of MDSCs and tumour metasta-is. Taken together, these studies highlight the various strategiesmployed by tumour-derived exosomes in suppressing tumourmmune responses.
.4. Opsonins and complement regulating factors as a means ofmmune surveillance (immunorecognition)
Immunorecognition is a fundamental process, important fordentification and targeting of foreign elements that influencehe extracellular environment. The complement system is com-osed of abundant proteins that react with each other to opsonizeathogens and induce a series of inflammatory responses [125].layton and colleagues [126] investigated the modulation of CD55nd CD59, two glycophosphatidylinositol (GPI)-anchored comple-ent regulator proteins that prevent formation of the complementembrane attacking complex, on exosomes derived from APCs.
xosome lysis was enhanced by CD55 and CD59 blocking anti-odies, demonstrating the role of CD55 and CD59 on exosomes forheir stability in the extracellular environment. Enrichment of cellurface CD9 and MHC class II–peptide complex was also found inC-derived exosomes [44]. The transmembrane tetraspanin CD9as suggested to facilitate direct membrane fusion, circumvent-
ng the endosome-to-lysosome fusion [127]. In addition, Morellind colleagues [103] demonstrated that exosomes are targeted,nternalised and processed by recipient splenic DCs in vivo. Ligand-
ediated targeting of exosomes towards DCs resulted in their
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nternalisation and antigen presentation to CD4+ T cells. The tar-eting of exosomes to recipient DCs is postulated to involve a suitef cell surface and membrane proteins including milk fat globuleGF factor VII (MFGE8), CD11a, CD54, CD9, and CD81 [44,103].
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Recently, the role of exosomes in presenting opsonins has beendemonstrated (Fig. 1E). Opsonins are characterised as moleculesthat target antigens or pathogens for phagocytosis, or destructionthrough the action of NK cells [125]. A recent study showed thatexosomes, derived from immature DCs, attenuated the inflamma-tory response by opsonising apoptotic cells for phagocytosis [128].The authors further observed that MFGE8 protein that was releasedby immature DCs was crucial for efficient apoptotic cell clearancethrough in vivo experimental sepsis models. Mice with MFGE8 defi-ciency showed diminished survival whereas administration of bothimmature DC-derived exosomes and treatment with recombinantMFGE8 enhanced their survival. The authors suggested that opsoni-sation of apoptotic cells induced by exosomes resulted in a reducedsystemic inflammatory response during sepsis.
3. Exosomes, immune regulation and the tumourmicroenvironment
The ability to evade immune recognition, suppress immunereactivity, and permit a chronic inflammatory response is crucialduring the progression of cancer [129,130]. A significant complica-tion is that there is no clear association between the presence ofany individual adaptive or innate immune cell type and a definedoutcome in terms of malignancy or prognosis across a range ofdifferent tumours [4,5,127]. Tumour-derived exosomes promotetumour growth by inhibiting anti-tumour immune responses andstimulating tumour proliferation and metastasis. The immune sys-tem functions by a coordinated response of many cell types thatexchange information through complex communication networks.Various studies have shown that exosomes are composed ofmRNAs, miRs, DNAs, proteins and lipid components that act on tar-get cells [17]. This exchange between immune cells and other celltypes is possibly achieved by packaging RNAs and DNAs (includingsingle- and double-stranded) into exosomes and selectively tar-geted and internalised by specific cell surface motifs [13,23,131].The delivered RNA molecules are proposed to be functional, andmRNAs can be translated, while miRs target host mRNAs to modu-late translation in the recipient cell [22,59].
Tumour-derived exosomes are important in conferring inter-cellular signals to these various immune cell types to modulateimmunosurveillance in the tumour microenvironment [132,77].Various cancer cell types are described in releasing exosomes capa-ble of inducing apoptosis in activated T cells by the transfer ofFasL and TRAIL [34,133]. miRNA transported by tumour exosomesmay act like ligands by binding to Toll-like receptors and trigger aninflammatory response. In fact, oncogene miR-21 and -29a secretedfrom lung cancer cell-derived exosomes were shown to bind TLRs tomurine (TLR7) and human (TLR8), leading to TLR-mediated NF-�Bactivation and secretion of pro-metastatic inflammatory cytokinesTNF-� and IL-6 [134]. Both TLR7 and TLR8 bind to and are acti-vated by 20-nt-long ssRNAs, which represent physiological ligandsfor these receptors [135], located in intracellular endosomes. Fur-ther, exosomes derived from human colorectal and melanoma cellsimpair the differentiation of peripheral blood monocytes to func-tional dendritic cells, instead transforming them towards MDSCs[136]. This mechanism of tumour-immune system communicationis important in understanding regulators of the tumour microenvi-ronment.
Various exogenous factors associated with the tumour microen-vironment regulate the biological function of exosomes and theirrole in immunity. These factors include heat treatment, adju-
their roles in immune regulation and cancer. Semin Cell Dev Biol
vant components and neoplastic microenvironment pressure. Choet al. [137] demonstrated that tumour-derived exosomes enrichedin Hsp70 were capable of enhancing tumour immunogenicity incomparison to tumour-derived exosomes that were independent
f external stimulus. Hsp70-enriched exosomes stimulated Th1-mmune responses, characterised by elevated production of IFN-�nd IgG2a in murine models. Moreover, exosomes derived fromctivated CD8+ T cells have been shown to express bioactive FasL,as and APO2 ligands to promote activation-induced cell deathhich may be required for an immune response [91,138]. FasL-
xpressing exosomes were shown to activate ERK and NF-�B in B16urine melanoma cells, leading to increased matrix metallopro-
einase 9 (MMP-9) expression and invasive ability of Fas-resistant16 tumour cells as shown by increased lung metastasis, whichay provide a novel view for understanding tumour escape from
he immune system [139].Dai et al. [110] reported that exosomes derived from heat-
tressed carcinoembryonic antigen (CEA)-positive tumour cellsCEA+/HS) have enhanced immunogenicity. In transgenic mice,EA+/HS exosomes contained more HSP70 and MHC-I, inducedC maturation, and enhanced immune response (both in vivo inLA-A2 transgenic mice, and CEA-specific CTL response in vitro).urther, Adams and colleagues utilised tumour exosomes in con-unction with TLR3 agonist and chemotherapy in advanced ovarianancer patients [140] to induce an effective antigen-specific Tell immune response. Collectively, these and other such stud-es emphasise the role of tumour-derived exosomes in enhancingumour immunogenicity [141]. Exosomes have also further beenhown to induce anti-tumour immunity in the absence of adjuvantsr heat treatment. Graner et al. [142] demonstrated that exosomeserived from glioblastoma cells induced protective immunity andnti-tumour immune responses in syngeneic mice. In addition,roteomic assessment of exosomes from glioma-derived patientera revealed enrichment of EGFRvIII and TGF-� in compari-on to normal, patient-matched sera. This study highlights brainumour-derived exosomes are capable of inducing effector immuneesponses, allowing exosomes to escape the blood–brain-barrier tolicit potential immune response in the recipient.
. Summary and future perspectives
Exosomes function as a fundamental mode of intercellularommunication and molecular transfer. Of key interest, tumourells secrete exosomes that can both inhibit tumour growthy eliciting anti-tumour immune responses [49] and promoteumour growth by inhibiting anti-tumour immunity or enhancingngiogenesis and/or metastases [52,143]. Tumour- and DC-derivedxosomes have been shown to carry tumour antigens and pro-ote immunity [144], leading to the eradication of established
umours [145] by CD8+ T cells [102] and CD4+ T cells [146], asell as having the capacity to directly suppress tumour growth
nd increase resistance to malignant tumour development [147].hese roles are most likely important during the immune surveil-ance of early tumour development. During the later stages ofumour development, immune surveillance in patients could beess efficient or actively suppressed by various tumour-derived
echanisms including tumour exosomes. There is increasingvidence indicating that exosomes contribute and support cancerrogression by transporting oncoproteins/mRNAs/miRs/DNAs and
mmune suppressive molecules to modulate a pro-tumourigenichenotype. Understanding recipient cell function and regulationy exosomes will certainly focus on specific mechanisms of tar-eting and delivery, uptake and transfer, including modulation ofey signalling pathways in various recipient cells both in vitro and
n vivo. The recent impact of tumour exosomes on pre-metastatic
Please cite this article in press as: Greening DW, et al. Exosomes and
rgan and tissue colonisation, highlight the importance of exo-omes as a promising therapeutic avenue. Indeed, by regulatinghe availability of circulating tumour exosomes through phar-
acological intervention, it could be speculated that multiple
PRESSlopmental Biology xxx (2015) xxx–xxx 7
functions of cancer immunity could be simultaneously recoveredor amenable to control. Further, the intrinsic ability of exosomesto traverse biological barriers and to naturally transport functionalsmall RNAs/DNAs between cells, represents an exciting deliveryvehicle for the field of translational therapy. The importance oftumour-derived exosomes in tumour progression has furtherbeen highlighted by their ability to sequester tumour-reactiveAbs, inhibiting anti-tumour cytotoxicity [148] and reducingeffectiveness of Ab-based anti-cancer drug intervention [149].As tumour-specific markers, tumour-derived exosomes could beexcellent systemic candidate biomarkers, containing an abundanceof components involved in cell–cell communication and mem-brane exchange, tumour antigens, and cell-specific markers, orotherwise as evaluating responses to therapy [150,151,84]. Finally,coupling exosomes and other types of EV with nanotechnologywill most likely form the basis where novel nanoscale cancervaccines will be developed in the near future [141,152].
The propensity of exosomes to direct, promote or suppressimmune activity is contingent upon the host cells, state of thehost cells, recipient cells and the microenvironment these inter-actions take place in. The fate of (intravenously) injected EVs isstill under discussion. It has been described that the expression ofintegrins, adhesion molecules, lipids, and other molecules on EVscontribute to the attachment and fusion of the injected vesicles to“acceptor” cells [8,103,153–155]. As a first step towards defininga context-dependent exosomal response, an in-depth biophysicalcharacterisation of exosomes and appropriate biological responsesare crucial. Different EV types from different cellular origins, or eventhe activation state of the cell-derived vesicle population, will pro-duce a specific response on target cells. Recently, the regulatoryeffect of exosomes (and other EVs) from different cell origins inthe immune system has been extensively reviewed [11]. Further,the transfer of molecules between cells during cognate immunecell interactions has been reported, and recently a novel mecha-nism of transfer of proteins and small RNA between T cells andAPCs has been described, involving exchange of exosomes duringthe formation of the immunological synapse [12]. However, theirregulatory effects in vivo remain largely unknown, especially inhumans.
Nonetheless, the prospect of enlisting exosomes as immuno-therapeutic agents remains an attractive one, with the advent ofvesicle-mediated therapeutic platforms that present an efficientand targeted mode of delivery [156]. In addition to exhibitingdistinct advantages over a cell based approach [157], vesicle ther-apeutics also features the ability to cross tissue barriers (e.g. bloodbrain barrier) and exhibit non-tumourigenic potential [158]. Giventhese various potential outcomes, it is tempting to speculate thatvesicle-based therapy may be crucial during tolerance inductionin particular in organ transplantation. This review has focused onthe importance of exosomes in immunoregulation, mechanismsincluding modulating antigen presentation, immune activation,immune suppression, immune surveillance, and intercellular com-munication throughout the extracellular environment. Furtherunderstanding of these areas of exosome biology, and especiallyof molecular mechanisms involved in immune cell interactions,is likely to provide significant insights into the phenomenon oftumour-related immune suppression and therapeutic intervention,including immunorecognition during malignancy.
Acknowledgements
their roles in immune regulation and cancer. Semin Cell Dev Biol
The authors are supported, in part, by the NHMRC program grant567122 (W.C.), NHMRC program grant 487922 (R.J.S.), and NHMRCproject grant 1057741 (R.J.S.). S.K.G. and R.X are supported by LaTrobe University Postgraduate Scholarships.
[7] Chalmin F, Ladoire S, Mignot G, Vincent J, Bruchard M, Remy-Martin JP,et al. Membrane-associated Hsp72 from tumor-derived exosomes medi-ates STAT3-dependent immunosuppressive function of mouse and humanmyeloid-derived suppressor cells. J Clin Invest 2010;120:457–71.
[8] Barres C, Blanc L, Bette-Bobillo P, Andre S, Mamoun R, Gabius HJ, et al.Galectin-5 is bound onto the surface of rat reticulocyte exosomes and mod-ulates vesicle uptake by macrophages. Blood 2010;115:696–705.
[9] Zhang HG, Grizzle WE. Exosomes and cancer: a newly described pathway ofimmune suppression. Clin Cancer Res 2011;17:959–64.
[10] Whiteside TL. Immune modulation of T-cell and NK (natural killer)cell activities by TEXs (tumour-derived exosomes). Biochem Soc Trans2013;41:245–51.
[11] Robbins PD, Morelli AE. Regulation of immune responses by extracellularvesicles. Nat Rev Immunol 2014;14:195–208.
[12] Gutierrez-Vazquez C, Villarroya-Beltri C, Mittelbrunn M, Sanchez-MadridF. Transfer of extracellular vesicles during immune cell–cell interactions.Immunol Rev 2013;251:125–42.
[13] Mittelbrunn M, Gutierrez-Vazquez C, Villarroya-Beltri C, Gonzalez S, Sanchez-Cabo F, Gonzalez MA, et al. Unidirectional transfer of microRNA-loadedexosomes from T cells to antigen-presenting cells. Nat Commun 2011;2:282.
[16] Keller S, Sanderson MP, Stoeck A, Altevogt P. Exosomes: from biogenesis andsecretion to biological function. Immunol Lett 2006;107:102–8.
[17] Thery C. Exosomes: secreted vesicles and intercellular communications.F1000 Biol Rep 2011;3:15.
[18] Zoller M. Tetraspanins: push and pull in suppressing and promoting metas-tasis. Nat Rev Cancer 2009;9:40–55.
[19] Simpson RJ, Lim JW, Moritz RL, Mathivanan S. Exosomes: proteomic insightsand diagnostic potential. Expert Rev Proteomics 2009;6:267–83.
[20] Tauro BJ, Greening DW, Mathias RA, Ji H, Mathivanan S, Scott AM,et al. Comparison of ultracentrifugation, density gradient separation, andimmunoaffinity capture methods for isolating human colon cancer cell lineLIM1863-derived exosomes. Methods 2012;56:293–304.
[21] Meckes Jr DG, Shair KH, Marquitz AR, Kung CP, Edwards RH, Raab-Traub N.Human tumor virus utilizes exosomes for intercellular communication. ProcNatl Acad Sci U S A 2010;107:20370–5.
[22] Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of geneticexchange between cells. Nat Cell Biol 2007;9:654–9.
[23] Thakur BK, Zhang H, Becker A, Matei I, Huang Y, Costa-Silva B, et al. Double-stranded DNA in exosomes: a novel biomarker in cancer detection. Cell Res2014;24:766–9.
[24] Hood JL, San RS, Wickline SA. Exosomes released by melanoma cells preparesentinel lymph nodes for tumor metastasis. Cancer Res 2011;71:3792–801.
[25] El-Hefnawy T, Raja S, Kelly L, Bigbee WL, Kirkwood JM, Luketich JD, et al.Characterization of amplifiable, circulating RNA in plasma and its potentialas a tool for cancer diagnostics. Clin Chem 2004;50:564–73.
[26] Stoorvogel W, Kleijmeer MJ, Geuze HJ, Raposo G. The biogenesis and functionsof exosomes. Traffic 2002;3:321–30.
[27] Thery C, Ostrowski M, Segura E. Membrane vesicles as conveyors of immuneresponses. Nat Rev Immunol 2009;9:581–93.
[28] Parolini I, Federici C, Raggi C, Lugini L, Palleschi S, De Milito A, et al. Microen-vironmental pH is a key factor for exosome traffic in tumor cells. J Biol Chem2009;284:34211–22.
[29] Quah BJ, O’Neill HC. The immunogenicity of dendritic cell-derived exosomes.Blood Cells Mol Dis 2005;35:94–110.
[30] Gastpar R, Gehrmann M, Bausero MA, Asea A, Gross C, Schroeder JA, et al. Heatshock protein 70 surface-positive tumor exosomes stimulate migratory andcytolytic activity of natural killer cells. Cancer Res 2005;65:5238–47.
[31] Zhang B, Yin Y, Lai RC, et al. Immunotherapeutic potential of extracellularvesicles. Front Immunol 2014;5:518.
[32] Zhang HG, Liu C, Su K, Yu S, Zhang L, Zhang S, et al. A membrane form of TNF-alpha presented by exosomes delays T cell activation-induced cell death. J
Please cite this article in press as: Greening DW, et al. Exosomes and
CJ, et al. B lymphocytes secrete antigen-presenting vesicles. J Exp Med1996;183:1161–72.
PRESSlopmental Biology xxx (2015) xxx–xxx
[34] Taylor DD, Gercel-Taylor C, Lyons KS, Stanson J, Whiteside TL. T-cell apoptosisand suppression of T-cell receptor/CD3-zeta by Fas ligand-containing mem-brane vesicles shed from ovarian tumors. Clin Cancer Res 2003;9:5113–9.
[35] Keryer-Bibens C, Pioche-Durieu C, Villemant C, Souquere S, Nishi N, HirashimaM, et al. Exosomes released by EBV-infected nasopharyngeal carcinoma cellsconvey the viral latent membrane protein 1 and the immunomodulatoryprotein galectin 9. BMC Cancer 2006;6:283.
[36] Klibi J, Niki T, Riedel A, Pioche-Durieu C, Souquere S, Rubinstein E, et al.Blood diffusion and Th1-suppressive effects of galectin-9-containing exo-somes released by Epstein–Barr virus-infected nasopharyngeal carcinomacells. Blood 2009;113:1957–66.
[37] Montecalvo A, Shufesky WJ, Stolz DB, Sullivan MG, Wang Z, Divito SJ, et al. Exo-somes as a short-range mechanism to spread alloantigen between dendriticcells during T cell allorecognition. J Immunol 2008;180:3081–90.
[38] Muntasell A, Berger AC, Roche PA. T cell-induced secretion of MHC classII–peptide complexes on B cell exosomes. EMBO J 2007;26:4263–72.
[39] Segura E, Nicco C, Lombard B, Veron P, Raposo G, Batteux F, et al. ICAM-1on exosomes from mature dendritic cells is critical for efficient naive T-cellpriming. Blood 2005;106:216–23.
[40] Thery C, Duban L, Segura E, Veron P, Lantz O, Amigorena S. Indirect activa-tion of naive CD4+ T cells by dendritic cell-derived exosomes. Nat Immunol2002;3:1156–62.
[41] Utsugi-Kobukai S, Fujimaki H, Hotta C, Nakazawa M, Minami M. MHC classI-mediated exogenous antigen presentation by exosomes secreted fromimmature and mature bone marrow derived dendritic cells. Immunol Lett2003;89:125–31.
[42] Hanayama R, Tanaka M, Miwa K, Shinohara A, Iwamatsu A, Nagata S.Identification of a factor that links apoptotic cells to phagocytes. Nature2002;417:182–7.
[43] Chaput N, Thery C. Exosomes: immune properties and potential clinicalimplementations. Semin Immunopathol 2011;33:419–40.
[44] Thery C, Regnault A, Garin J, Wolfers J, Zitvogel L, Ricciardi-Castagnoli P,et al. Molecular characterization of dendritic cell-derived exosomes. Selectiveaccumulation of the heat shock protein hsc73. J Cell Biol 1999;147:599–610.
[45] Mori Y, Koike M, Moriishi E, Kawabata A, Tang H, Oyaizu H, et al. Humanherpesvirus-6 induces MVB formation, and virus egress occurs by an exoso-mal release pathway. Traffic 2008;9:1728–42.
[46] Dukers DF, Meij P, Vervoort MB, Vos W, Scheper RJ, Meijer CJ, et al. Directimmunosuppressive effects of EBV-encoded latent membrane protein 1. JImmunol 2000;165:663–70.
[47] Ceccarelli S, Visco V, Raffa S, Wakisaka N, Pagano JS, Torrisi MR. Epstein–Barrvirus latent membrane protein 1 promotes concentration in multivesicularbodies of fibroblast growth factor 2 and its release through exosomes. Int JCancer 2007;121:1494–506.
[48] Meckes Jr DG, Gunawardena HP, Dekroon RM, Heaton PR, Edwards RH, OzgurS, et al. Modulation of B-cell exosome proteins by gamma herpesvirus infec-tion. Proc Natl Acad Sci U S A 2013;110:E2925–33.
[49] Zeelenberg IS, Ostrowski M, Krumeich S, Bobrie A, Jancic C, Boissonnas A,et al. Targeting tumor antigens to secreted membrane vesicles in vivo inducesefficient antitumor immune responses. Cancer Res 2008;68:1228–35.
[50] Jang JY, Lee JK, Jeon YK, Kim CW. Exosome derived from epigallocatechingallate treated breast cancer cells suppresses tumor growth by inhibitingtumor-associated macrophage infiltration and M2 polarization. BMC Cancer2013;13:421.
[51] Ekstrom EJ, Bergenfelz C, von Bulow V, Serifler F, Carlemalm E, Jonsson G, et al.WNT5A induces release of exosomes containing pro-angiogenic and immuno-suppressive factors from malignant melanoma cells. Mol Cancer 2014;13:88.
[52] Ichim TE, Zhong Z, Kaushal S, Zheng X, Ren X, Hao X, et al. Exosomes as a tumorimmune escape mechanism: possible therapeutic implications. J Trans Med2008;6:37.
[53] Hoebe K, Janssen E, Beutler B. The interface between innate and adaptiveimmunity. Nat Immunol 2004;5:971–4.
[54] Whiteside TL. Immune suppression in cancer: effects on immune cells, mech-anisms and future therapeutic intervention. Semin Cancer Biol 2006;16:3–15.
[55] Gajewski TF, Schreiber H, Fu YX. Innate and adaptive immune cells in thetumor microenvironment. Nat Immunol 2013;14:1014–22.
[56] Vesely MD, Kershaw MH, Schreiber RD, Smyth MJ. Natural innate and adaptiveimmunity to cancer. Annu Rev Immunol 2011;29:235–71.
[57] Hood JL, Pan H, Lanza GM, Wickline SA. Paracrine induction of endotheliumby tumor exosomes. Lab Invest 2009;89:1317–28.
[58] Gesierich S, Berezovskiy I, Ryschich E, Zoller M. Systemic induction ofthe angiogenesis switch by the tetraspanin D6.1A/CO-029. Cancer Res2006;66:7083–94.
[59] Al-Nedawi K, Meehan B, Micallef J, Lhotak V, May L, Guha A, et al. Intercellulartransfer of the oncogenic receptor EGFRvIII by microvesicles derived fromtumour cells. Nat Cell Biol 2008;10:619–24.
[60] Webber J, Steadman R, Mason MD, Tabi Z, Clayton A. Cancer exosomes triggerfibroblast to myofibroblast differentiation. Cancer Res 2010;70:9621–30.
[61] Wieckowski EU, Visus C, Szajnik M, et al. Tumor-derived microvesicles pro-mote regulatory T cell expansion and induce apoptosis in tumor-reactive
their roles in immune regulation and cancer. Semin Cell Dev Biol
activated CD8+ T lymphocytes. J Immunol 2009;183:3720–30.[62] Szajnik M, Czystowska M, Szczepanski MJ, et al. Tumor-derived microvesicles
induce, expand and up-regulate biological activities of human regulatory Tcells (Treg). PLoS ONE 2010;5:e11469.
[63] Clayton A, Mitchell JP, Court J, Mason MD, Tabi Z. Human tumor-derived exo-somes selectively impair lymphocyte responses to interleukin-2. Cancer Res2007;67:7458–66.
[64] Huber V, Fais S, Iero M, Lugini L, Canese P, Squarcina P, et al. Human colorectalcancer cells induce T-cell death through release of proapoptotic microvesi-cles: role in immune escape. Gastroenterology 2005;128:1796–804.
[65] Clayton A, Al-Taei S, Webber J, Mason MD, Tabi Z. Cancer exosomes expressCD39 and CD73, which suppress T cells through adenosine production. JImmunol 2011;187:676–83.
[66] Abusamra AJ, Zhong Z, Zheng X, Li M, Ichim TE, Chin JL, et al. Tumor exo-somes expressing Fas ligand mediate CD8+ T-cell apoptosis. Blood Cells MolDis 2005;35:169–73.
[67] Schuler PJ, Saze Z, Hong CS, Muller L, Gillespie DG, Cheng D, et al. HumanCD4 CD39 regulatory T cells produce adenosine upon co-expression of sur-face CD73 or contact with CD73 exosomes or CD73 cells. Clin Exp Immunol2014;177:531–43.
[68] Ashiru O, Boutet P, Fernandez-Messina L, Aguera-Gonzalez S, Skepper JN,Vales-Gomez M, et al. Natural killer cell cytotoxicity is suppressed by expo-sure to the human NKG2D ligand MICA*008 that is shed by tumor cells inexosomes. Cancer Res 2010;70:481–9.
[69] Clayton A, Mitchell JP, Court J, Linnane S, Mason MD, Tabi Z. Humantumor-derived exosomes down-modulate NKG2D expression. J Immunol2008;180:7249–58.
[70] Clayton A, Tabi Z. Exosomes and the MICA-NKG2D system in cancer. BloodCells Mol Dis 2005;34:206–13.
[71] Szczepanski MJ, Szajnik M, Welsh A, et al. Blast-derived microvesicles insera from patients with acute myeloid leukemia suppress natural killercell function via membrane-associated transforming growth factor-beta1.Haematologica 2011;96:1302–9.
[72] Soderberg A, Barral AM, Soderstrom M, et al. Redox-signaling transmitted intrans to neighboring cells by melanoma-derived TNF-containing exosomes.Free Radic Biol Med 2007;43:90–9.
[73] Romagnoli GG, Zelante BB, Toniolo PA, et al. Dendritic cell-derived exosomesmay be a tool for cancer immunotherapy by converting tumor cells intoimmunogenic targets. Front Immunol 2014;5:692.
[74] Naslund TI, Gehrmann U, Gabrielsson S. Cancer immunotherapy with exo-somes requires B-cell activation. Oncoimmunology 2013;2:e24533.
[75] Li QL, Bu N, Yu YC, et al. Exvivo experiments of human ovarian cancer ascites-derived exosomes presented by dendritic cells derived from umbilical cordblood for immunotherapy treatment. Clin Med Oncol 2008;2:461–7.
[76] Mignot G, Roux S, Thery C, et al. Prospects for exosomes in immunotherapyof cancer. J Cell Mol Med 2006;10:376–88.
[77] Wolfers J, Lozier A, Raposo G, Regnault A, Thery C, Masurier C, et al. Tumor-derived exosomes are a source of shared tumor rejection antigens for CTLcross-priming. Nat Med 2001;7:297–303.
[78] Zitvogel L, Regnault A, Lozier A, Wolfers J, Flament C, Tenza D, et al. Eradicationof established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes. Nat Med 1998;4:594–600.
[79] Viaud S, Thery C, Ploix S, Tursz T, Lapierre V, Lantz O, et al. Dendriticcell-derived exosomes for cancer immunotherapy: what’s next? Cancer Res2010;70:1281–5.
[80] Amigorena S. Cancer immunotherapy using dendritic cell-derived exosomes.Medicina 2000;60(Suppl. 2):51–4.
[81] Andre F, Andersen M, Wolfers J, et al. Exosomes in cancer immunotherapy:preclinical data. Adv Exp Med Biol 2001;495:349–54.
[82] Chaput N, Schartz NE, Andre F, et al. Exosomes for immunotherapy of cancer.Adv Exp Med Biol 2003;532:215–21.
[83] Andre F, Escudier B, Angevin E, et al. Exosomes for cancer immunotherapy.Ann Oncol 2004;15(Suppl. 4):iv141–4.
[84] Cho JA, Yeo DJ, Son HY, Kim HW, Jung DS, Ko JK, et al. Exosomes: a newdelivery system for tumor antigens in cancer immunotherapy. Int J Cancer2005;114:613–22.
[85] Yewdell JW, Dolan BP. Immunology: cross-dressers turn on T cells. Nature2011;471:581–2.
[86] Coppieters K, Barral AM, Juedes A, Wolfe T, Rodrigo E, Thery C, et al. No sig-nificant CTL cross-priming by dendritic cell-derived exosomes during murinelymphocytic choriomeningitis virus infection. J Immunol 2009;182:2213–20.
[87] Wakim LM, Bevan MJ. Cross-dressed dendritic cells drive memory CD8+ T-cellactivation after viral infection. Nature 2011;471:629–32.
[88] Andre F, Schartz NE, Movassagh M, Flament C, Pautier P, Morice P, et al.Malignant effusions and immunogenic tumour-derived exosomes. Lancet2002;360:295–305.
[89] Admyre C, Johansson SM, Paulie S, Gabrielsson S. Direct exosome stim-ulation of peripheral human T cells detected by ELISPOT. Eur J Immunol2006;36:1772–81.
[90] van der Vlist EJ, Arkesteijn GJ, van de Lest CH, Stoorvogel W, Nolte-’t HoenEN, Wauben MH. CD4+ T cell activation promotes the differential release ofdistinct populations of nanosized vesicles. J Extracell Vesicles 2012;1.
[91] Martinez-Lorenzo MJ, Anel A, Gamen S, Monle n I, Lasierra P, Larrad L, et al.Activated human T cells release bioactive Fas ligand and APO2 ligand inmicrovesicles. J Immunol 1999;163:1274–81.
Please cite this article in press as: Greening DW, et al. Exosomes and
[92] Mallegol J, Van Niel G, Lebreton C, Lepelletier Y, Candalh C, Dugave C,et al. T84-intestinal epithelial exosomes bear MHC class II/peptide com-plexes potentiating antigen presentation by dendritic cells. Gastroenterology2007;132:1866–76.
PRESSlopmental Biology xxx (2015) xxx–xxx 9
[93] Van Niel G, Mallegol J, Bevilacqua C, Candalh C, Brugiere S, Tomaskovic-CrookE, et al. Intestinal epithelial exosomes carry MHC class II/peptides able toinform the immune system in mice. Gut 2003;52:1690–7.
[94] Rock KL, Clark K. Analysis of the role of MHC class II presentation in the stim-ulation of cytotoxic T lymphocytes by antigens targeted into the exogenousantigen-MHC class I presentation pathway. J Immunol 1996;156:3721–6.
[95] Gould DS, Auchincloss Jr H. Direct and indirect recognition: the role of MHCantigens in graft rejection. Immunol Today 1999;20:77–82.
[96] Vincent-Schneider H, Stumptner-Cuvelette P, Lankar D, et al. Exosomes bear-ing HLA-DR1 molecules need dendritic cells to efficiently stimulate specific Tcells. Int Immunol 2002;14:713–22.
[97] Peche H, Renaudin K, Beriou G, et al. Induction of tolerance by exosomesand short-term immunosuppression in a fully MHC-mismatched rat cardiacallograft model. Am J Transplant 2006;6:1541–50.
[98] Hutagalung AH, Novick PJ. Role of Rab GTPases in membrane traffic and cellphysiology. Physiol Rev 2011;91:119–49.
[99] Feng D, Zhao WL, Ye YY, Bai XC, Liu RQ, Chang LF, et al. Cellular internalizationof exosomes occurs through phagocytosis. Traffic 2010;11:675–87.
[100] Skog J, Wurdinger T, van Rijn S, Meijer DH, Gainche L, Sena-Esteves M, et al.Glioblastoma microvesicles transport RNA and proteins that promote tumourgrowth and provide diagnostic biomarkers. Nat Cell Biol 2008;10:1470–6.
[101] Delcayre A, Shu H, Le Pecq JB. Dendritic cell-derived exosomes in cancerimmunotherapy: exploiting nature’s antigen delivery pathway. Expert RevAnticancer Ther 2005;5:537–47.
[102] Hao S, Bai O, Li F, Yuan J, Laferte S, Xiang J. Mature dendritic cells pulsed withexosomes stimulate efficient cytotoxic T-lymphocyte responses and antitu-mour immunity. Immunology 2007;120:90–102.
[103] Morelli AE, Larregina AT, Shufesky WJ, Sullivan ML, Stolz DB, Papworth GD,et al. Endocytosis, intracellular sorting, and processing of exosomes by den-dritic cells. Blood 2004;104:3257–66.
[104] Miyanishi M, Tada K, Koike M, Uchiyama Y, Kitamura T, Nagata S. Identifica-tion of Tim4 as a phosphatidylserine receptor. Nature 2007;450:435–9.
[105] Bhatnagar S, Schorey JS. Exosomes released from infected macrophages con-tain Mycobacterium avium glycopeptidolipids and are proinflammatory. J BiolChem 2007;282:25779–89.
[106] Bhatnagar S, Shinagawa K, Castellino FJ, Schorey JS. Exosomes released frommacrophages infected with intracellular pathogens stimulate a proinflam-matory response in vitro and in vivo. Blood 2007;110:3234–44.
[107] Obregon C, Rothen-Rutishauser B, Gerber P, Gehr P, Nicod LP. Active uptakeof dendritic cell-derived exovesicles by epithelial cells induces the releaseof inflammatory mediators through a TNF-alpha-mediated pathway. Am JPathol 2009;175:696–705.
[108] Vega VL, Rodriguez-Silva M, Frey T, Gehrmann M, Diaz JC, Steinem C, et al.Hsp70 translocates into the plasma membrane after stress and is released intothe extracellular environment in a membrane-associated form that activatesmacrophages. J Immunol 2008;180:4299–307.
[109] Qazi KR, Torregrosa Paredes P, Dahlberg B, Grunewald J, Eklund A, GabrielssonS. Proinflammatory exosomes in bronchoalveolar lavage fluid of patients withsarcoidosis. Thorax 2010;65:1016–24.
[110] Dai S, Wan T, Wang B, Zhou X, Xiu F, Chen T, et al. More efficient inductionof HLA-A*0201-restricted and carcinoembryonic antigen (CEA)-specific CTLresponse by immunization with exosomes prepared from heat-stressed CEA-positive tumor cells. Clin Cancer Res 2005;11:7554–63.
[111] Chen W, Wang J, Shao C, et al. Efficient induction of antitumor T cell immu-nity by exosomes derived from heat-shocked lymphoma cells. Eur J Immunol2006;36:1598–607.
[112] Viaud S, Terme M, Flament C, Taieb J, Andre F, Novault S, et al. Dendritic cell-derived exosomes promote natural killer cell activation and proliferation: arole for NKG2D ligands and IL-15Ralpha. PLoS ONE 2009;4:e4942.
[113] Filipazzi P, Burdek M, Villa A, et al. Recent advances on the role of tumorexosomes in immunosuppression and disease progression. Semin Cancer Biol2012;22:342–9.
[114] Luciani F, Spada M, De Milito A, et al. Effect of proton pump inhibitor pre-treatment on resistance of solid tumors to cytotoxic drugs. J Natl Cancer Inst2004;96:1702–13.
[115] Chen KG, Valencia JC, Lai B, et al. Melanosomal sequestration of cytotoxicdrugs contributes to the intractability of malignant melanomas. Proc NatlAcad Sci U S A 2006;103:9903–7.
[116] Bobrie A, Colombo M, Raposo G, Thery C. Exosome secretion: molecular mech-anisms and roles in immune responses. Traffic 2011;12:1659–68.
[117] Poutsiaka DD, Schroder EW, Taylor DD, Levy EM, Black PH. Membrane vesiclesshed by murine melanoma cells selectively inhibit the expression of Ia antigenby macrophages. J Immunol 1985;134:138–44.
[118] Andreola G, Rivoltini L, Castelli C, Huber V, Perego P, Deho P, et al. Induction oflymphocyte apoptosis by tumor cell secretion of FasL-bearing microvesicles.J Exp Med 2002;195:1303–16.
[119] Xie Y, Zhang H, Li W, Deng Y, Munegowda MA, Chibbar R, et al. Dendritic cellsrecruit T cell exosomes via exosomal LFA-1 leading to inhibition of CD8+ CTLresponses through downregulation of peptide/MHC class I and Fas ligand-mediated cytotoxicity. J Immunol 2010;185:5268–78.
[120] Liu C, Yu S, Zinn K, Wang J, Zhang L, Jia Y, et al. Murine mammary carci-
their roles in immune regulation and cancer. Semin Cell Dev Biol
noma exosomes promote tumor growth by suppression of NK cell function. JImmunol 2006;176:1375–85.
[121] Hedlund M, Stenqvist AC, Nagaeva O, Kjellberg L, Wulff M, Baranov V, et al.Human placenta expresses and secretes NKG2D ligands via exosomes that
down-modulate the cognate receptor expression: evidence for immunosup-pressive function. J Immunol 2009;183:340–51.
[122] Admyre C, Johansson SM, Qazi KR, Filen JJ, Lahesmaa R, Norman M, et al.Exosomes with immune modulatory features are present in human breastmilk. J Immunol 2007;179:1969–78.
[123] Xiang X, Poliakov A, Liu C, Liu Y, Deng ZB, Wang J, et al. Induction of myeloid-derived suppressor cells by tumor exosomes. Int J Cancer 2009;124:2621–33.
[124] Liu Y, Xiang X, Zhuang X, Zhang S, Liu C, Cheng Z, et al. Contribution of MyD88to the tumor exosome-mediated induction of myeloid derived suppressorcells. Am J Pathol 2010;176:2490–9.
[125] Grange C, Tapparo M, Collino F, Vitillo L, Damasco C, Deregibus MC,et al. Microvesicles released from human renal cancer stem cells stimu-late angiogenesis and formation of lung premetastatic niche. Cancer Res2011;71:5346–56.
[126] Clayton A, Harris CL, Court J, Mason MD, Morgan BP. Antigen-presenting cellexosomes are protected from complement-mediated lysis by expression ofCD55 and CD59. Eur J Immunol 2003;33:522–31.
[127] van den Boorn JG, Schlee M, Coch C, Hartmann G. SiRNA delivery with exo-some nanoparticles. Nat Biotechnol 2011;29:325–6.
[128] Miksa M, Wu R, Dong W, Komura H, Amin D, Ji Y, et al. Immature dendriticcell-derived exosomes rescue septic animals via milk fat globule epidermalgrowth factor-factor VIII [corrected]. J Immunol 2009;183:5983–90.
[129] Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell2011;144:646–74.
[130] Cavallo F, De Giovanni C, Nanni P, Forni G, Lollini PL. 2011: the immunehallmarks of cancer. Cancer Immunol Immunother 2011;60:319–26.
[131] Balaj L, Lessard R, Dai L, Cho YJ, Pomeroy SL, Breakefield XO, et al. Tumourmicrovesicles contain retrotransposon elements and amplified oncogenesequences. Nat Commun 2011;2:180.
[132] Mincheva-Nilsson L, Baranov V. Cancer exosomes and NKG2Dreceptor–ligand interactions: impairing NKG2D-mediated cytotoxicityand anti-tumour immune surveillance. Semin Cancer Biol 2014;28:24–30.
[133] Kim JW, Wieckowski E, Taylor DD, Reichert TE, Watkins S, Whiteside TL.Fas ligand-positive membranous vesicles isolated from sera of patients withoral cancer induce apoptosis of activated T lymphocytes. Clin Cancer Res2005;11:1010–20.
[134] Fabbri M, Paone A, Calore F, Galli R, Gaudio E, Santhanam R, et al. MicroRNAsbind to Toll-like receptors to induce prometastatic inflammatory response.Proc Natl Acad Sci U S A 2012;109:E2110–6.
[135] Heil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C, Akira S, et al.Species-specific recognition of single-stranded RNA via toll-like receptor 7and 8. Science 2004;303:1526–9.
[136] Valenti R, Huber V, Filipazzi P, Pilla L, Sovena G, Villa A, et al. Human tumor-released microvesicles promote the differentiation of myeloid cells withtransforming growth factor-beta-mediated suppressive activity on T lympho-cytes. Cancer Res 2006;66:9290–8.
[137] Cho JA, Lee YS, Kim SH, Ko JK, Kim CW. MHC independent anti-tumor immuneresponses induced by Hsp70-enriched exosomes generate tumor regressionin murine models. Cancer Lett 2009;275:256–65.
[138] Monleon I, Martinez-Lorenzo MJ, Monteagudo L, Lasierra P, Taules M, Itur-ralde M, et al. Differential secretion of Fas ligand- or APO2 ligand/TNF-relatedapoptosis-inducing ligand-carrying microvesicles during activation-induceddeath of human T cells. J Immunol 2001;167:6736–44.
Please cite this article in press as: Greening DW, et al. Exosomes and
[139] Cai Z, Yang F, Yu L, Yu Z, Jiang L, Wang Q, et al. Activated T cell exo-somes promote tumor invasion via Fas signaling pathway. J Immunol2012;188:5954–61.
[140] Adams M, Navabi H, Croston D, Coleman S, Tabi Z, Clayton A, et al. The ratio-nale for combined chemo/immunotherapy using a Toll-like receptor 3 (TLR3)
PRESSlopmental Biology xxx (2015) xxx–xxx
agonist and tumour-derived exosomes in advanced ovarian cancer. Vaccine2005;23:2374–8.
[141] Tan A, De La Pena H, Seifalian AM. The application of exosomes as a nanoscalecancer vaccine. Int J Nanomed 2010;5:889–900.
[142] Graner MW, Alzate O, Dechkovskaia AM, Keene JD, Sampson JH, Mitchell DA,et al. Proteomic and immunologic analyses of brain tumor exosomes. FASEBJ 2009;23:1541–57.
[143] Whiteside TL. Tumour-derived exosomes or microvesicles: another mech-anism of tumour escape from the host immune system? Br J Cancer2005;92:209–11.
[144] Guo F, Chang CK, Fan HH, Nie XX, Ren YN, Liu YY, et al. Anti-tumour effects of exosomes in combination with cyclophosphamide andpolyinosinic–polycytidylic acid. J Int Med Res 2008;36:1342–53.
[145] Hao S, Bai O, Yuan J, Qureshi M, Xiang J. Dendritic cell-derived exosomesstimulate stronger CD8+ CTL responses and antitumor immunity than tumorcell-derived exosomes. Cell Mol Immunol 2006;3:205–11.
[146] Taieb J, Chaput N, Schartz N, Roux S, Novault S, Menard C, et al. Chemoim-munotherapy of tumors: cyclophosphamide synergizes with exosome basedvaccines. J Immunol 2006;176:2722–9.
[147] Hegmans JP, Hemmes A, Aerts JG, Hoogsteden HC, Lambrecht BN.Immunotherapy of murine malignant mesothelioma using tumor lysate-pulsed dendritic cells. Am J Respir Crit Care Med 2005;171:1168–77.
[148] Battke C, Ruiss R, Welsch U, Wimberger P, Lang S, Jochum S, et al. Tumourexosomes inhibit binding of tumour-reactive antibodies to tumour cells andreduce ADCC. Cancer Immunol Immunother 2011;60:639–48.
[149] Aung T, Chapuy B, Vogel D, Wenzel D, Oppermann M, Lahmann M, et al. Exo-somal evasion of humoral immunotherapy in aggressive B-cell lymphomamodulated by ATP-binding cassette transporter A3. Proc Natl Acad Sci U S A2011;108:15336–41.
[150] Hong CS, Muller L, Boyiadzis M, Whiteside TL. Plasma-derived exosomesmediate immune suppression and serve as a biomarker of response tochemotherapy in Acute Myeloid Leukaemia (AML) (TUM7P.945). J Immunol2014; 192.
[151] Hong CS, Muller L, Whiteside TL, Boyiadzis M. Plasma exosomes as mark-ers of therapeutic response in patients with acute myeloid leukemia. FrontImmunol 2014;5:160.
[152] S ELA, Mager I, Breakefield XO, Wood MJ. Extracellular vesicles: biology andemerging therapeutic opportunities. Nat Rev Drug Discov 2013;12:347–57.
[153] Clayton A, Turkes A, Dewitt S, et al. Adhesion and signaling by B cell-derivedexosomes: the role of integrins. FASEB J 2004;18:977–9.
[154] Saunderson SC, Dunn AC, Crocker PR, et al. CD169 mediates the capture ofexosomes in spleen and lymph node. Blood 2014;123:208–16.
[155] Lai CP, Mardini O, Ericsson M, et al. Dynamic biodistribution of extra-cellular vesicles in vivo using a multimodal imaging reporter. ACS Nano2014;8:483–94.
[156] Lai RC, Yeo RW, Tan KH, et al. Exosomes for drug delivery – a novel applicationfor the mesenchymal stem cell. Biotechnol Adv 2013;31:543–51.
[157] Poh KK, Sperry E, Young RG, et al. Repeated direct endomyocar-dial transplantation of allogeneic mesenchymal stem cells: safety of ahigh dose, “off-the-shelf”, cellular cardiomyoplasty strategy. Int J Cardiol2007;117:360–4.
[158] Xin H, Li Y, Cui Y, et al. Systemic administration of exosomes released frommesenchymal stromal cells promote functional recovery and neurovascular
their roles in immune regulation and cancer. Semin Cell Dev Biol
plasticity after stroke in rats. J Cereb Blood Flow Metab 2013;33:1711–5.[159] Greening DW, Gopal SK, Mathias RA, Liu L, Sheng J, Zhu HJ, Simpson RJ. Emerg-
ing roles of exosomes during epithelial–mesenchymal transition and cancerprogression. 2015. In Press. THIS SPECIAL ISSUE in Seminars in Cell & Devel-opmental Biology - YSCDB17321-12.
