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1.  Exosome removal as a therapeutic adjuvant in cancer 
Exosome secretion is a notable feature of malignancy owing to the roles of these nanoparticles in cancer growth, immune suppression, tumor angiogenesis and therapeutic resistance. Exosomes are 30–100 nm membrane vesicles released by many cells types during normal physiological processes. Tumors aberrantly secrete large quantities of exosomes that transport oncoproteins and immune suppressive molecules to support tumor growth and metastasis. The role of exosomes in intercellular signaling is exemplified by human epidermal growth factor receptor type 2 (HER2) over-expressing breast cancer, where exosomes with the HER2 oncoprotein stimulate tumor growth and interfere with the activity of the therapeutic antibody Herceptin®. Since numerous observations from experimental model systems point toward an important clinical impact of exosomes in cancer, several pharmacological strategies have been proposed for targeting their malignant activities. We also propose a novel device strategy involving extracorporeal hemofiltration of exosomes from the entire circulatory system using an affinity plasmapheresis platform known as the Aethlon ADAPT™ (adaptive dialysis-like affinity platform technology) system, which would overcome the risks of toxicity and drug interactions posed by pharmacological approaches. This technology allows affinity agents, including exosome-binding lectins and antibodies, to be immobilized in the outer-capillary space of plasma filtration membranes that integrate into existing kidney dialysis systems. Device therapies that evolve from this platform allow rapid extracorporeal capture and selective retention of target particles < 200 nm from the entire circulatory system. This strategy is supported by clinical experience in hepatitis C virus-infected patients using an ADAPT™ device, the Hemopurifier®, to reduce the systemic load of virions having similar sizes and glycosylated surfaces as cancer exosomes. This review discusses the possible therapeutic approaches for targeting immune suppressive exosomes in cancer patients, and the anticipated significance of these strategies for reversing immune dysfunction and improving responses to standard of care treatments.
doi:10.1186/1479-5876-10-134
PMCID: PMC3441244  PMID: 22738135
Exosomes; Cancer; Immune suppression; Metastasis; Affinity agents; Plasmapheresis cartridges; Dialysis; Lectin; Antibodies
2.  Paracrine Induction of Endothelium by Tumor Exosomes 
Cancers utilize a nanoscale messenger system known as exosomes to communicate with surrounding tissues and immune cells. However, the functional relationship between tumor exosomes, endothelial signaling, angiogenesis, and metastasis is poorly understood. Herein, we describe a standardized approach for defining the angiogenic potential of isolated exosomes. We created a powerful technique to rapidly and efficiently isolate and track exosomes for study using dynamic light scattering in conjunction with fluorescent exosome labeling. With these methods, melanoma exosomes were observed to interact with and influence endothelial tubule morphology as well as move between endothelial tubule cells by means of tunneling nanotube structures. Melanoma exosomes also were observed to rapidly stimulate the production of endothelial spheroids and endothelial sprouts in a dose-dependent manner. In concert, tumor exosomes simultaneously elicited paracrine endothelial signaling by regulation of certain inflammatory cytokines. These data suggest that, tumor exosomes can promote endothelial angiogenic responses, which could contribute to tumor metastatic potential.
doi:10.1038/labinvest.2009.94
PMCID: PMC3316485  PMID: 19786948
exosomes; angiogenesis; tumor; cancer; endothelial; 3D assay; cytokines
3.  Exosomes and Cancer: A Newly Described Pathway of Immune Suppression 
Exosomes are small (30 to 100 nm) membrane-bound particles that are released from normal, diseased, and neoplastic cells and are present in blood and other bodily fluids. Exosomes contain a variety of molecules including signal peptides, mRNA, microRNA, and lipids. Exosomes can function to export from cells unneeded endogenous molecules and therapeutic drugs. When exosomes are taken up by specific cells, they may act locally to provide autocrine or paracrine signals or, at a distance, as a newly described nanoparticle-based endocrine system. Specifically, mRNA transferred to cells by exosomes can result in the production of new proteins. In cancer, signals via exosomes affect the immune system by inhibiting the functions of T cells and normal killer (NK) cells and by inhibiting the differentiation of precursors to mature antigen-presenting cells. Also, exosomes increase the number and/or activity of immune suppressor cells, including myeloid-derived suppressor cells, T-regulatory cells, and CD14+, HLA-DR–/low cells. The effects of exosomes on the development and progression of cancers, with an emphasis on suppression of immune surveillance, is described. Also discussed are potential uses of exosomes clinically, in the development of vaccines, in targeting tumors, and in diagnosis and/or early detection.
doi:10.1158/1078-0432.CCR-10-1489
PMCID: PMC3155407  PMID: 21224375
4.  Tumour-derived exosomes and their role in cancer-associated T-cell signalling defects 
British Journal of Cancer  2005;92(2):305-311.
Dendritic and lymphoid ‘exosomes' regulate immune activation. Tumours release membranous material mimicking these ‘exosomes,' resulting in deletion of reactive lymphocytes. Tumour-derived ‘exosomes' have recently been explored as vaccines, without analysis of their immunologic consequences. This investigation examines the composition of tumour-derived ‘exosomes' and their effects on T lymphocytes. Membranous materials were isolated from ascites of ovarian cancer patients (n=6) and Western immunoblotting was performed for markers associated with ‘exosomes.' Using cultured T cells, ‘exosomes' were evaluated for suppression of CD3-ζ and JAK 3 expressions and induction of apoptosis, measured by DNA fragmentation. ‘Exosome' components mediating suppression of CD3-ζ were isolated by continuous eluting electrophoresis and examined by Western immunoblotting. ‘Exosomes' were shown to be identical with previously characterised shed membrane vesicles by protein staining and TSG101 expression. ‘Exosomes' expressed class I MHC, placental alkaline phosphatase, B23/nucleophosmin, and FasL. ‘Exosomes' suppressed expression of T-cell activation signalling components, CD3-ζ and JAK 3 and induced apoptosis. CD3-ζ suppression was mediated by two components: 26 and 42 kDa. Only the 42 kDa component reacted with anti-FasL antibody. These results indicate that, while ‘exosomes' express tumour antigens, leading to their proposed utility as tumour vaccines, they also can suppress T-cell signalling molecules and induce apoptosis.
doi:10.1038/sj.bjc.6602316
PMCID: PMC2361848  PMID: 15655551
exosomes; activation signaling; membrane vesicles; T lymphocytes; ovarian cancer
5.  Amphiregulin exosomes increase cancer cell invasion 
Current biology : CB  2011;21(9):779-786.
SUMMARY
Autocrine, paracrine and juxtacrine are recognized modes of action for mammalian EGFR ligands that include EGF, TGF-α (TGFα), amphiregulin (AREG), heparin-binding EGF-like growth factor (HB-EGF), betacellulin, epiregulin and epigen. We identify a new mode of EGFR ligand signaling via exosomes. Human breast and colorectal cancer cells release exosomes containing full-length, signaling-competent EGFR ligands. Exosomes isolated from MDCK cells expressing individual full-length EGFR ligands displayed differential activities; AREG exosomes increased invasiveness of recipient breast cancer cells four-fold over TGFα or HB-EGF exosomes and five-fold over equivalent amounts of recombinant AREG. Exosomal AREG displayed significantly greater membrane stability than TGFα or HB-EGF. An average of 24 AREG molecules are packaged within an individual exosome, and AREG exosomes are rapidly internalized by recipient cells. Whether the composition and behavior of exosomes differ between non-transformed and transformed cells is unknown. We show that exosomes from DLD-1 colon cancer cells with a mutant KRAS allele exhibited both higher AREG levels and greater invasive potential than exosomes from isogenically matched, non-transformed cells in which mutant KRAS was eliminated by homologous recombination. We speculate that EGFR ligand signaling via exosomes may contribute to diverse cancer phenomena such as field effect and priming the metastatic niche.
doi:10.1016/j.cub.2011.03.043
PMCID: PMC3417320  PMID: 21514161
Exosomes; EGF Receptor; amphiregulin; TGF-α; HB-EGF
6.  Exosomes Communicate Protective Messages during Oxidative Stress; Possible Role of Exosomal Shuttle RNA 
PLoS ONE  2010;5(12):e15353.
Background
Exosomes are small extracellular nanovesicles of endocytic origin that mediate different signals between cells, by surface interactions and by shuttling functional RNA from one cell to another. Exosomes are released by many cells including mast cells, dendritic cells, macrophages, epithelial cells and tumour cells. Exosomes differ compared to their donor cells, not only in size, but also in their RNA, protein and lipid composition.
Methodology/Principal Findings
In this study, we show that exosomes, released by mouse mast cells exposed to oxidative stress, differ in their mRNA content. Also, we show that these exosomes can influence the response of other cells to oxidative stress by providing recipient cells with a resistance against oxidative stress, observed as an attenuated loss of cell viability. Furthermore, Affymetrix microarray analysis revealed that the exosomal mRNA content not only differs between exosomes and donor cells, but also between exosomes derived from cells grown under different conditions; oxidative stress and normal conditions. Finally, we also show that exposure to UV-light affects the biological functions associated with exosomes released under oxidative stress.
Conclusions/Significance
These results argue that the exosomal shuttle of RNA is involved in cell-to-cell communication, by influencing the response of recipient cells to an external stress stimulus.
doi:10.1371/journal.pone.0015353
PMCID: PMC3003701  PMID: 21179422
7.  Exosomes Mediate the Cytoprotective Action of Mesenchymal Stromal Cells on Hypoxia-Induced Pulmonary Hypertension 
Circulation  2012;126(22):2601-2611.
Background
Hypoxia induces an inflammatory response in the lung manifested by alternative activation of macrophages with elevation of pro-inflammatory mediators that are critical for the later development of hypoxic pulmonary hypertension (HPH). Mesenchymal stromal cell (MSC) transplantation inhibits lung inflammation, vascular remodeling and right heart failure, and reverses HPH in experimental models of disease. In this study, we aimed to investigate the paracrine mechanisms by which MSCs are protective in HPH.
Methods and Results
We fractionated mouse MSC-conditioned media to identify the biologically-active component affecting in vivo hypoxic signaling and determined that exosomes, secreted membrane microvesicles, suppressed the hypoxic pulmonary influx of macrophages and the induction of pro-inflammatory and pro-proliferative mediators, including monocyte chemoattractant protein-1 and hypoxia-inducible mitogenic factor, in the murine model of HPH. Intravenous delivery of MSC-derived exosomes (MEX) inhibited vascular remodeling and HPH, whereas MEX-depleted media or fibroblast-derived exosomes had no effect. MEX suppressed the hypoxic activation of signal transducer and activator of transcription 3 (STAT3) and the upregulation of the miR-17 superfamily of microRNA clusters, whereas it increased lung levels of miR-204, a key microRNA whose expression is decreased in human PH. MEX produced by human umbilical cord MSCs inhibited STAT3 signaling in isolated human pulmonary artery endothelial cells demonstrating a direct effect of MEX on hypoxic vascular cells.
Conclusions
This study indicates that MEX exert a pleiotropic protective effect on the lung and inhibit PH through suppression of hyperproliferative pathways, including STAT-3 mediated signaling induced by hypoxia.
doi:10.1161/CIRCULATIONAHA.112.114173
PMCID: PMC3979353  PMID: 23114789
hypertension; pulmonary; inflammation; hypoxia; signal transduction
8.  Ovarian cancer cell invasiveness is associated with discordant exosomal sequestration of Let-7 miRNA and miR-200 
Background
The role of exosomes in the pathogenesis and metastatic spread of cancer remains to be fully elucidated. Recent studies support the hypothesis that the release of exosomes from cells modifies local extracellular conditions to promote cell growth and neovascularisation. In addition, exosomes may modify the phenotype of parent and/or target cell. For example, sequestration of signaling mediators into exosomes may reduce their intracellular bioavailability to the parent cell thereby altering cell phenotype and metastatic potential. The fusion of released exosomes with target cell and delivery may also modify cell function and activity. In this study, to further elucidate the role of exosomes in ovarian cancer, the release of exosomes from two ovarian cancer cell lines of different invasive capacity and their miRNA content of exosomes were compared. The hypothesis to be tested was that ovarian cancer cell invasiveness is associated with altered release of exosomes and discordant exosomal sequestration of miRNA.
Methods
High (SKOV-3) and low (OVCAR-3) invasive ovarian cancer cell lines were used to characterize their exosome release. SKOV-3 and OVCAR-3 cells were cultured (DMEM, 20% exosome-free FBS) under an atmosphere of 8% O2 for 24 hours. Cell-conditioned media were collected and exosomes were isolated by differential and buoyant density centrifugation and characterised by Western blot (CD63 and CD9). Exosomal microRNA (let-7a-f and miR-200a-c) content was established by real-time PCR.
Results
Exosomes were identified with by the presence of typical cup-shaped spherical vesicle and the expression of exosome markers: CD63, CD9. SKOV-3 cells released 2.7-fold more exosomes (1.22 ± 0.11 μg/106 cells) compared to OVCAR-3 (0.44 ± 0.05 μg/106 cells). The let-7 family miRNA transcripts were identified in both ovarian cancer cell lines and their exosomes. The let-7 family transcripts were more abundant in OVCAR-3 cell than SKOV-3 cells. In contrast, let-7 family transcripts were more abundant in exosomes from SKOV-3 than OVCAR-3. miR-200 family transcripts were only identified in OVCAR-3 cells and their exosomes.
Conclusions
The data obtained in this study are consistent with the hypothesis that the releases of exosomes varies significantly between ovarian cancer cell lines and correlates with their invasive potential.
doi:10.1186/1479-5876-12-4
PMCID: PMC3896684  PMID: 24393345
Ovarian cancer; Exosomes; microRNA; Biomarkers; Invasion
9.  Release of Luminal Exosomes Contributes to TLR4-Mediated Epithelial Antimicrobial Defense 
PLoS Pathogens  2013;9(4):e1003261.
Exosomes are membranous nanovesicles released by most cell types from multi-vesicular endosomes. They are speculated to transfer molecules to neighboring or distant cells and modulate many physiological and pathological procedures. Exosomes released from the gastrointestinal epithelium to the basolateral side have been implicated in antigen presentation. Here, we report that luminal release of exosomes from the biliary and intestinal epithelium is increased following infection by the protozoan parasite Cryptosporidium parvum. Release of exosomes involves activation of TLR4/IKK2 signaling through promoting the SNAP23-associated vesicular exocytotic process. Downregulation of let-7 family miRNAs by activation of TLR4 signaling increases SNAP23 expression, coordinating exosome release in response to C. parvum infection. Intriguingly, exosomes carry antimicrobial peptides of epithelial cell origin, including cathelicidin-37 and beta-defensin 2. Activation of TLR4 signaling enhances exosomal shuttle of epithelial antimicrobial peptides. Exposure of C. parvum sporozoites to released exosomes decreases their viability and infectivity both in vitro and ex vivo. Direct binding to the C. parvum sporozoite surface is required for the anti-C. parvum activity of released exosomes. Biliary epithelial cells also increase exosomal release and display exosome-associated anti-C. parvum activity following LPS stimulation. Our data indicate that TLR4 signaling regulates luminal exosome release and shuttling of antimicrobial peptides from the gastrointestinal epithelium, revealing a new arm of mucosal immunity relevant to antimicrobial defense.
Author Summary
Exosomes are secreted membranous nanovesicles produced by a variety of cells. Exosomes shuttle various molecules to transfer them to neighboring or distant cells, and have been implicated as mediators in cell-cell communications to modulate physiological and pathological procedures. Here, we report that luminal release of exosomal vesicles is an important component of Toll-like receptor 4 (TLR4)-associated gastrointestinal epithelial defense against infection by Cryptosporidium parvum, an obligate intracellular protozoan that infects gastrointestinal epithelial cells. Activation of TLR4 signaling in host epithelial cells following C. parvum infection promotes luminal release of epithelial exosomes and exosomal shuttling of antimicrobial peptides from the epithelium. By direct binding to the C. parvum surface, exosomal vesicles reveal anti-C. parvum activity. Activation of TLR4 signaling in epithelial cells after LPS stimulation also increases exosomal release and exosome-associated anti-C. parvum activity. Therefore, we speculate that TLR4-mediated exosome release may be relevant to innate mucosal immunity in general, representing a new target for therapeutic intervention for infectious diseases at the mucosal surface.
doi:10.1371/journal.ppat.1003261
PMCID: PMC3617097  PMID: 23592986
10.  The Roles of Tumor-Derived Exosomes in Cancer Pathogenesis 
Exosomes are endosome-derived, 30–100 nm small membrane vesicles released by most cell types including tumor cells. They are enriched in a selective repertoire of proteins and nucleic acids from parental cells and are thought to be actively involved in conferring intercellular signals. Tumor-derived exosomes have been viewed as a source of tumor antigens that can be used to induce antitumor immune responses. However, tumor-derived exosomes also have been found to possess immunosuppressive properties and are able to facilitate tumor growth, metastasis, and the development of drug resistance. These different effects of tumor-derived exosomes contribute to the pathogenesis of cancer. This review will discuss the roles of tumor-derived exosomes in cancer pathogenesis, therapy, and diagnostics.
doi:10.1155/2011/842849
PMCID: PMC3235485  PMID: 22190973
11.  Role of Exosomes Released by Dendritic Cells and/or by Tumor Targets: Regulation of NK Cell Plasticity 
Exosomes are endosomal-derived nanovesicles released by normal and tumor cells, which transfer functionally active proteins, lipids, and nucleic acids between cells. They are important mediators of intercellular communication and act on the adjacent stroma as well as in the periphery. Recently, exosomes have been recognized to play a pathophysiological role in various diseases such as cancer or infectious diseases. Tumor cell-derived exosomes (Tex) have been shown to act as tumor promotors by educating non-malignant cells to provide a tumor supporting microenvironment, which helps to circumvent immune detection by the host and supports metastasis. However, Tex with anti-tumor, immune-activating properties were also described reflecting the complexity of exosomes. Here, we assess the role of extracellular microvesicles/exosomes as messengers affecting NK cell function in health and disease and discuss the molecular basis for the differential impact of exosomes on NK cell activity. The molecular composition/load of exosomes and the mechanisms regulating their release remain unclear and need to be further analyzed to facilitate the development of new treatment options targeting the exosomal machinery.
doi:10.3389/fimmu.2014.00091
PMCID: PMC3945280  PMID: 24639679
exosomes; NK cell regulation; tumor derived vesicles; dendritic cell-derived exosomes; microvesicles
12.  Exosome Uptake Depends on ERK1/2-Heat Shock Protein 27 Signaling and Lipid Raft-mediated Endocytosis Negatively Regulated by Caveolin-1* 
The Journal of Biological Chemistry  2013;288(24):17713-17724.
Background: Exosome vesicles can transfer molecular information previously shown to stimulate tumor development; however, the mechanism of exosome uptake is unknown.
Results: Mammalian cells internalize exosomes through lipid raft-mediated endocytosis negatively regulated by caveolin-1.
Conclusion: Our findings provide novel insights into cellular uptake of exosomes.
Significance: Our data provide potential strategies for how the exosome uptake pathway may be targeted.
The role of exosomes in cancer can be inferred from the observation that they transfer tumor cell derived genetic material and signaling proteins, resulting in e.g. increased tumor angiogenesis and metastasis. However, the membrane transport mechanisms and the signaling events involved in the uptake of these virus-like particles remain ill-defined. We now report that internalization of exosomes derived from glioblastoma (GBM) cells involves nonclassical, lipid raft-dependent endocytosis. Importantly, we show that the lipid raft-associated protein caveolin-1 (CAV1), in analogy with its previously described role in virus uptake, negatively regulates the uptake of exosomes. We find that exosomes induce the phosphorylation of several downstream targets known to associate with lipid rafts as signaling and sorting platforms, such as extracellular signal-regulated kinase-1/2 (ERK1/2) and heat shock protein 27 (HSP27). Interestingly, exosome uptake appears dependent on unperturbed ERK1/2-HSP27 signaling, and ERK1/2 phosphorylation is under negative influence by CAV1 during internalization of exosomes. These findings significantly advance our general understanding of exosome-mediated uptake and offer potential strategies for how this pathway may be targeted through modulation of CAV1 expression and ERK1/2 signaling.
doi:10.1074/jbc.M112.445403
PMCID: PMC3682571  PMID: 23653359
Caveolin; Endocytosis; Exosomes; Glioblastoma; Vesicles; Microvesicles
13.  Stromal-cell and cancer-cell exosomes leading the metastatic exodus for the promised niche 
Exosomes are thought to play an important role in metastasis. Luga and colleagues have described the production of exosomes by stromal cells such as cancer-associated fibroblasts that are taken up by breast cancer cells and are then loaded with Wnt 11, which is associated with stimulation of the invasiveness and metastasis of the breast cancer cells. Previous studies have shown that exosomes produced by breast cancer cells are taken up by stromal fibroblasts and other stromal cells, suggesting that exosomes are agents of cross-talk between cancer and stromal cells to stimulate metastasis. Imaging of exosomes by labeling with fluorescent proteins will enlighten the process by which exosomes enhance metastasis, including premetastatic niche formation.
doi:10.1186/bcr3426
PMCID: PMC3707067  PMID: 23806092
14.  Exosomes in Cancer Microenvironment and Beyond: have we Overlooked these Extracellular Messengers? 
Cancer Microenvironment  2012;5(3):323-332.
Cancer is a complex organ whose behavior is not only influenced by genetic and epigenetic changes in cancer cells but also by stromal cells, local extracellular matrix and specific tissue architecture. Intercellular communications within the cancer microenvironment are critical to coordinate the assembly of multiple cell types for an amalgamated form and function of a cancer. Exosomes are small membrane vesicles with an endosome origin that are released by cells into the extracellular environment. They carry a cargo of proteins, lipids, and nucleic acids and transfer their cargo to recipient cells and altering the recipient cells’ biochemical composition, signaling pathways, and gene regulation. Exosomes can thus serve as extracellular messengers mediating cell-cell communication. Both cancer cells and stromal cells release exosomes not only into the cancer microenvironment but also into the circulation. In this review, we summarize the research done so far on cancer-derived exosomes and assess their roles as extracellular messengers facilitating cancer progression and metastasis.
doi:10.1007/s12307-012-0110-2
PMCID: PMC3460057  PMID: 22585423
Exosome; Cancer microenvironment; Extracellular messenger; Stroma
15.  Immune modulation of T-cell and NK (natural killer) cell activities by TEXs (tumour-derived exosomes) 
Biochemical Society transactions  2013;41(1):245-251.
Body fluids of cancer patients contain TEXs (tumour-derived exosomes). Tumours release large quantities of TEXs, and the protein content of exosome or MV (microvesicle) fractions isolated from patients’ sera is high. TEXs down-regulate functions of immune cells, thus promoting tumour progression. We isolated TEXs from tumour cell supernatants and sera of patients with solid tumours or AML (acute myelogenous leukaemia). The molecular profile of TEXs was distinct from that of circulating exosomes derived from normal cells. TEXs were co-incubated with activated T-cells, conventional CD4 +CD25neg T-cells or CD56 +CD16 + NK (natural killer) cells respectively. TEXs down-regulated CD3ζ and JAK3 (Janus kinase 3) expression in primary activated T-cells and mediated Fas/FasL (Fas ligand)-driven apoptosis of CD8 + T-cells. TEXs promoted CD4 +CD25neg T-cell proliferation and their conversion into CD4 +CD25hiFOXP3 + (FOXP3 is forkhead box P3) Treg cells (regulatory T-cells), which also expressed IL-10 (interleukin 10), TGFβ1 (transforming growth factor β1), CTLA-4 (cytotoxic T-lymphocyte antigen 4), GrB (granzyme B)/perforin and effectively mediated suppression. Neutralizing antibodies specific for TGFβ1 and/or IL-10 inhibited the ability of TEXs to expand Treg cells. TEXs obtained at diagnosis from AML patients’ sera were positive for blast-associated markers CD33, CD34, CD117 and TGFβ1, and they decreased cytotoxic activity of NK cells isolated from NC (normal control) donors, induced Smad phosphorylation and down-regulated NKG2D receptor expression. Correlations between the TEX molecular profile or TEX protein levels and clinical data in cancer patients suggest that TEX-mediated effects on immune cells are prognostically important. In contrast with exosomes released by normal cells, TEXs have immunosuppressive properties and are involved in regulating peripheral tolerance in patients with cancer.
doi:10.1042/BST20120265
PMCID: PMC3721347  PMID: 23356291
immune suppression; natural killer cell (NK cell); T-cell; tumour-derived exosome (TEX); tumour escape
16.  BM mesenchymal stromal cell–derived exosomes facilitate multiple myeloma progression 
The Journal of Clinical Investigation  2013;123(4):1542-1555.
BM mesenchymal stromal cells (BM-MSCs) support multiple myeloma (MM) cell growth, but little is known about the putative mechanisms by which the BM microenvironment plays an oncogenic role in this disease. Cell-cell communication is mediated by exosomes. In this study, we showed that MM BM-MSCs release exosomes that are transferred to MM cells, thereby resulting in modulation of tumor growth in vivo. Exosomal microRNA (miR) content differed between MM and normal BM-MSCs, with a lower content of the tumor suppressor miR-15a. In addition, MM BM-MSC–derived exosomes had higher levels of oncogenic proteins, cytokines, and adhesion molecules compared with exosomes from the cells of origin. Importantly, whereas MM BM-MSC–derived exosomes promoted MM tumor growth, normal BM-MSC exosomes inhibited the growth of MM cells. In summary, these in vitro and in vivo studies demonstrated that exosome transfer from BM-MSCs to clonal plasma cells represents a previously undescribed and unique mechanism that highlights the contribution of BM-MSCs to MM disease progression.
doi:10.1172/JCI66517
PMCID: PMC3613927  PMID: 23454749
17.  Proteome Profiling of Neuroblastoma-Derived Exosomes Reveal the Expression of Proteins Potentially Involved in Tumor Progression 
PLoS ONE  2013;8(9):e75054.
Neuroblastoma (NB) is the most common extracranial solid tumor in childhood, with grim prognosis in a half of patients. Exosomes are nanometer-sized membrane vesicles derived from the multivesicular bodies (MVBs) of the endocytic pathway and released by normal and neoplastic cells. Tumor-derived exosomes have been shown in different model systems to carry molecules that promote cancer growth and dissemination. In this respect, we have here performed the first characterization and proteomic analysis of exosomes isolated from human NB cell lines by filtration and ultracentrifugation. Electron microscopy demonstrated that NB-derived exosomes exhibited the characteristic cup-shaped morphology. Dynamic light scattering studies showed a bell-shaped curve and a polydispersity factor consistent with those of exosomes. Zeta potential values suggested a good nanoparticle stability. We performed proteomic analysis of NB-derived exosomes by two dimension liquid chromatography separation and mass spectrometry analyses using the multidimensional protein identification technology strategy. We found that the large majority of the proteins identified in NB derived exosomes are present in Exocarta database including tetraspanins, fibronectin, heat shock proteins, MVB proteins, cytoskeleton-related proteins, prominin-1 (CD133), basigin (CD147) and B7-H3 (CD276). Expression of the CD9, CD63 and CD81 tetraspanins, fibronectin, CD133, CD147 and CD276 was validated by flow cytometry. Noteworthy, flow cytometric analysis showed that NB-derived exosomes expressed the GD2 disialoganglioside, the most specific marker of NB. In conclusion, this study shows that NB-derived exosomes express a discrete set of molecules involved in defense response, cell differentiation, cell proliferation and regulation of other important biological process. Thus, NB-derived exosomes may play an important role in the modulation of tumor microenvironment and represent potential tumor biomarkers.
doi:10.1371/journal.pone.0075054
PMCID: PMC3777909  PMID: 24069378
18.  Exosome Release and Low pH Belong to a Framework of Resistance of Human Melanoma Cells to Cisplatin 
PLoS ONE  2014;9(2):e88193.
Intrinsic resistance to cytotoxic drugs has been a main issue in cancer therapy for decades. Microenvironmental acidity is a simple while highly efficient mechanism of chemoresistance, exploited through impairment of drug delivery. The latter is achieved by extracellular protonation and/or sequestration into acidic vesicles. This study investigates the importance of extracellular acidosis and nanovesicle (exosome) release in the resistance of human tumour cell to cisplatin (CisPt); in parallel to proton pump inhibitors (PPI) ability of interfering with these tumour cell features. The results showed that CisPt uptake by human tumour cells was markedly impaired by low pH conditions. Moreover, exosomes purified from supernatants of these cell cultures contained various amounts of CisPt, which correlated to the pH conditions of the culture medium. HPLC-Q-ICP-MS analysis revealed that exosome purified from tumour cell culture supernatants contained CisPt in its native form. PPI pre-treatment increased cellular uptake of CisPt, as compared to untreated cells, in an acidic-depend manner. Furthermore, it induced a clear inhibition of exosome release by tumour cells. Human tumours obtained from xenografts pretreated with PPI contained more CisPt as compared to tumours from xenografts treated with CisPt alone. Further analysis showed that in vivo PPI treatment induced a clear reduction in the plasmatic levels of tumour-derived exosomes which also contained lower level of CisPt. Altogether, these findings point to the identification of a double mechanism that human malignant melanoma use in resisting to a dreadful cellular poison such as cisplatin. This framework of resistance includes both low pH-dependent extracellular sequestration and an exosome-mediated elimination. Both mechanisms are markedly impaired by proton pump inhibition, leading to an increased CisPt-dependent cytotoxicity.
doi:10.1371/journal.pone.0088193
PMCID: PMC3916404  PMID: 24516610
19.  Exosomes Derived from Mesenchymal Stem Cells Suppress Angiogenesis by Down-Regulating VEGF Expression in Breast Cancer Cells 
PLoS ONE  2013;8(12):e84256.
Exosomes are small membrane vesicles released by a variety of cell types. Exosomes contain genetic materials, such as mRNAs and microRNAs (miRNAs), implying that they may play a pivotal role in cell-to-cell communication. Mesenchymal stem cells (MSCs), which potentially differentiate into multiple cell types, can migrate to the tumor sites and have been reported to exert complex effects on tumor progression. To elucidate the role of MSCs within the tumor microenvironment, previous studies have suggested various mechanisms such as immune modulation and secreted factors of MSCs. However, the paracrine effects of MSC-derived exosomes on the tumor microenvironment remain to be explored. The hypothesis of this study was that MSC-derived exosomes might reprogram tumor behavior by transferring their molecular contents. To test this hypothesis, exosomes from MSCs were isolated and characterized. MSC-derived exosomes exhibited different protein and RNA profiles compared with their donor cells and these vesicles could be internalized by breast cancer cells. The results demonstrated that MSC-derived exosomes significantly down-regulated the expression of vascular endothelial growth factor (VEGF) in tumor cells, which lead to inhibition of angiogenesis in vitro and in vivo. Additionally, miR-16, a miRNA known to target VEGF, was enriched in MSC-derived exosomes and it was partially responsible for the anti-angiogenic effect of MSC-derived exosomes. The collective results suggest that MSC-derived exosomes may serve as a significant mediator of cell-to-cell communication within the tumor microenvironment and suppress angiogenesis by transferring anti-angiogenic molecules.
doi:10.1371/journal.pone.0084256
PMCID: PMC3877259  PMID: 24391924
20.  ExoCarta 2012: database of exosomal proteins, RNA and lipids 
Nucleic Acids Research  2011;40(D1):D1241-D1244.
Exosomes are membraneous nanovesicles of endocytic origin released by most cell types from diverse organisms; they play a critical role in cell–cell communication. ExoCarta (http://www.exocarta.org) is a manually curated database of exosomal proteins, RNA and lipids. The database catalogs information from both published and unpublished exosomal studies. The mode of exosomal purification and characterization, the biophysical and molecular properties are listed in the database aiding biomedical scientists in assessing the quality of the exosomal preparation and the corresponding data obtained. Currently, ExoCarta (Version 3.1) contains information on 11 261 protein entries, 2375 mRNA entries and 764 miRNA entries that were obtained from 134 exosomal studies. In addition to the data update, as a new feature, lipids identified in exosomes are added to ExoCarta. We believe that this free web-based community resource will aid researchers in identifying molecular signatures (proteins/RNA/lipids) that are specific to certain tissue/cell type derived exosomes and trigger new exosomal studies.
doi:10.1093/nar/gkr828
PMCID: PMC3245025  PMID: 21989406
21.  Neutral Sphingomyelinase 2 (nSMase2)-dependent Exosomal Transfer of Angiogenic MicroRNAs Regulate Cancer Cell Metastasis* 
The Journal of Biological Chemistry  2013;288(15):10849-10859.
Background: Contribution of exosomal microRNAs to cancer metastasis remains unknown.
Results: Exosomal angiogenic microRNAs secreted by metastatic cancer cells promote the metastasis through the activation of endothelial cells.
Conclusion: Horizontal transfer of exosomal miRNAs from cancer cells can dictate the microenviromental niche for the benefit of the cancer cell.
Significance: This is the first to connect cancer metastasis to the exosomal microRNA in vivo.
The release of humoral factors between cancer cells and the microenvironmental cells is critical for metastasis; however, the roles of secreted miRNAs in non-cell autonomous cancer progression against microenvironmental cells remain largely unknown. Here, we demonstrate that the neutral sphyngomyelinase 2 (nSMase2) regulates exosomal microRNA (miRNA) secretion and promotes angiogenesis within the tumor microenvironment as well as metastasis. We demonstrate a requirement for nSMase2-mediated cancer cell exosomal miRNAs in the regulation of metastasis through the induction of angiogenesis in inoculated tumors. In addition, miR-210, released by metastatic cancer cells, was shown to transport to endothelial cells and suppress the expression of specific target genes, which resulted in enhanced angiogenesis. These findings suggest that the horizontal transfer of exosomal miRNAs from cancer cells can dictate the microenviromental niche for the benefit of the cancer cell, like “on demand system” for cancer cells.
doi:10.1074/jbc.M112.446831
PMCID: PMC3624465  PMID: 23439645
Angiogenesis; Cell Biology; Cell-cell Interaction; Exosomes; Metastasis; MicroRNA; Tumor Microenvironment; Extracellular Vesicles; Cell-cell Communication; miR-210
22.  Exosomes released by K562 chronic myeloid leukemia cells promote angiogenesis in a src-dependent fashion 
Angiogenesis  2011;15(1):33-45.
Exosomes, microvesicles of endocytic origin released by normal and tumor cells, play an important role in cell-to-cell communication. Angiogenesis has been shown to regulate progression of chronic myeloid leukemia (CML). The mechanism through which this happens has not been elucidated. We isolated and characterized exosomes from K562 CML cells and evaluated their effects on human umbilical endothelial cells (HUVECs). Fluorescent-labeled exosomes were internalized by HUVECs during tubular differentiation on Matrigel. Exosome localization was perinuclear early in differentiation, moving peripherally in cells undergoing elongation and connection. Exosomes move within and between nanotubular structures connecting the remodeling endothelial cells. They stimulated angiotube formation over a serum/growth factor-limited medium control, doubling total cumulative tube length (P = 0.003). Treatment of K562 cells with two clinically active tyrosine kinase inhibitors, imatinib and dasatinib, reduced their total exosome release (P <0.009); equivalent concentrations of drug-treated exosomes induced a similar extent of tubular differentiation. However, dasatinib treatment of HUVECs markedly inhibited HUVEC response to drug control CML exosomes (P <0.002). In an in vivo mouse Matrigel plug model angiogenesis was induced by K562 exosomes and abrogated by oral dasatinib treatment (P <0.01). K562 exosomes induced dasatinib-sensitive Src phosphorylation and activation of downstream Src pathway proteins in HUVECs. Imatinib was minimally active against exosome stimulation of HUVEC cell differentiation and signaling. Thus, CML cell-derived exosomes induce angiogenic activity in HUVEC cells. The inhibitory effect of dasatinib on exosome production and vascular differentiation and signaling reveals a key role for Src in both the leukemia and its microenvironment.
doi:10.1007/s10456-011-9241-1
PMCID: PMC3595015  PMID: 22203239
Exosomes; Nanotubes; Chronic myeloid leukemia; Endothelial cells; Tyrosine kinase inhibitors
23.  Detachment of Breast Tumor Cells Induces Rapid Secretion of Exosomes Which Subsequently Mediate Cellular Adhesion and Spreading 
PLoS ONE  2011;6(9):e24234.
Exosomes are nano-vesicles secreted by a wide range of mammalian cell types. These vesicles are abundant in serum and other extracellular fluids and contain a large repertoire of proteins, mRNA and microRNA. Exosomes have been implicated in cell to cell communication, the transfer of infectious agents, and neurodegenerative diseases as well as tumor progression. However, the precise mechanisms by which they are internalized and/or secreted remain poorly understood. In order to follow their release and uptake in breast tumor cells in real time, cell-derived exosomes were tagged with green fluorescent protein (GFP)-CD63 while human serum exosomes were rhodamine isothiocynate-labeled. We show that detachment of adherent cells from various substrata induces a rapid and substantial secretion of exosomes, which then concentrate on the cell surfaces and mediate adhesion to various extracellular matrix proteins. We also demonstrate that disruption of lipid rafts with methyl-beta-cyclodextrin (MβCD) inhibits the internalization of exosomes and that annexins are essential for the exosomal uptake mechanisms. Taken together, these data suggest that cellular detachment is accompanied by significant release of exosomes while cellular adhesion and spreading are enhanced by rapid uptake and disposition of exosomes on the cell surface.
doi:10.1371/journal.pone.0024234
PMCID: PMC3167827  PMID: 21915303
24.  Plasma-Derived Exosomal Survivin, a Plausible Biomarker for Early Detection of Prostate Cancer 
PLoS ONE  2012;7(10):e46737.
Background
Survivin is expressed in prostate cancer (PCa), and its downregulation sensitizes PCa cells to chemotherapeutic agents in vitro and in vivo. Small membrane-bound vesicles called exosomes, secreted from the endosomal membrane compartment, contain RNA and protein that they readily transport via exosome internalization into recipient cells. Recent progress has shown that tumor-derived exosomes play multiple roles in tumor growth and metastasis and may produce these functions via immune escape, tumor invasion and angiogenesis. Furthermore, exosome analysis may provide novel biomarkers to diagnose or monitor PCa treatment.
Methods
Exosomes were purified from the plasma and serum from 39 PCa patients, 20 BPH patients, 8 prostate cancer recurrent and 16 healthy controls using ultracentrifugation and their quantities and qualities were quantified and visualized from both the plasma and the purified exosomes using ELISA and Western blotting, respectively.
Results
Survivin was significantly increased in the tumor-derived samples, compared to those from BPH and controls with virtually no difference in the quantity of Survivin detected in exosomes collected from newly diagnosed patients exhibiting low (six) or high (nine) Gleason scores. Exosome Survivin levels were also higher in patients that had relapsed on chemotherapy compared to controls.
Conclusions
These studies demonstrate that Survivin exists in plasma exosomes from both normal, BPH and PCa subjects. The relative amounts of exosomal Survivin in PCa plasma was significantly higher than in those with pre-inflammatory BPH and control plasma. This differential expression of exosomal Survivin was seen with both newly diagnosed and advanced PCa subjects with high or low-grade cancers. Analysis of plasma exosomal Survivin levels may offer a convenient tool for diagnosing or monitoring PCa and may, as it is elevated in low as well as high Gleason scored samples, be used for early detection.
doi:10.1371/journal.pone.0046737
PMCID: PMC3473028  PMID: 23091600
25.  Functional prostate-specific membrane antigen is enriched in exosomes from prostate cancer cells 
International Journal of Oncology  2014;44(3):918-922.
Developing simple and effective approaches to detect tumor markers will be critical for early diagnosis or prognostic evaluation of prostate cancer treatment. Prostate-specific membrane antigen (PSMA) has been validated as an important tumor marker for prostate cancer progression including angiogenesis and metastasis. As a type II membrane protein, PSMA can be constitutively internalized from the cell surface into endosomes. Early endosomes can fuse with multivesicular bodies (MVB) to form and secrete exosomes (40–100 nm) into the extracellular environment. Herein, we tested whether some of the endosomal PSMA could be transferred to exosomes as an extracellular resource for PSMA. Using PSMA-positive LNCaP cells, the secreted exosomes were collected and isolated from the cultured media. The vesicular structures of exosomes were identified by electron microscopy, and exosomal marker protein CD9 and tumor susceptibility gene (TSG 101) were confirmed by western blot analysis. Our present data demonstrate that PSMA can be enriched in exosomes, exhibiting a higher content of glycosylation and partial proteolysis in comparison to cellular PSMA. An in vitro enzyme assay further confirmed that exosomal PSMA retains functional enzymatic activity. Therefore, our data may suggest a new role for PSMA in prostate cancer progression, and provide opportunities for developing non-invasive approaches for diagnosis or prognosis of prostate cancer.
doi:10.3892/ijo.2014.2256
PMCID: PMC3928468  PMID: 24424840
prostate-specific membrane antigen; exosome; biomarker; glycosylation; prostate cancer

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