Blockage of transferred exosome-shuttled miR-494 inhibits melanoma growth and metastasis

There is emerging evidence of bioactive material transport by exosomes in melanoma. However, the functions of exosome content underlying such cancer progression remain largely unknown. We aimed at determining whether exosome secretion contributes to cellular microRNA-494 (miR-494) loss and investigated the roles of miR-494 in melanoma progression. The exosomes from blood serum and cell culture conditioned media were separated by ultracentrifugation. A short hairpin RNA was used to silence rab27a for inhibiting exosome release. To address the functional role of exosomal miR-494, we assessed cell proliferation, migration, invasion capabilities, and cell apoptosis. Finally, subcutaneous xenograft and lung-metastasis models were constructed to determine the effect of exosomal miR-494 in vivo. Based on long noncoding RNA microarray analysis of melanocyte and melanoma-derived exosomes from the Gene Expression Omnibus database, we discovered that miR-494 was enriched in melanoma-derived exosomes. And miR-494 was increased in exosomes secreted from melanoma patients’ serum and A375 cells. Rab27a depletion reduced exosome secretion and rescued the abundance of cellular miR-494. Functional studies revealed that knockdown of rab27a and subsequent accumulation of miR-494 significantly suppressed the malignant phenotypes of melanoma cells via inducing cell apoptosis. Nude mice experiments confirmed that tumor growth and metastasis were suppressed by increasing miR-494 accumulation after rab27a depletion. In conclusion, blocking transferred exosome-shuttled miR-494 is a potential therapeutic option for melanoma.     

Microenvironmental pH Is a Key Factor for Exosome Traffic in Tumor Cells

Exosomes secreted by normal and cancer cells carry and deliver a variety of molecules. To date, mechanisms referring to tumor exosome trafficking, including release and cell-cell transmission, have not been described. To gain insight into this, exosomes purified from metastatic melanoma cell medium were labeled with a lipid fluorescent probe, R18, and analyzed by spectrofluorometry and confocal microscopy. A low pH condition is a hallmark of tumor malignancy, potentially influencing exosome release and uptake by cancer cells. Using different pH conditions as a modifier of exosome traffic, we showed (i) an increased exosome release and uptake at low pH when compared with a buffered condition and (ii) exosome uptake by melanoma cells occurred by fusion. Membrane biophysical analysis, such as fluidity and lipid composition, indicated a high rigidity and sphingomyelin/ganglioside GM3 (N-acetylneuraminylgalactosylglucosylceramide) content in exosomes released at low pH. This was likely responsible for the increased fusion efficiency. Consistent with these results, pretreatment with proton pump inhibitors led to an inhibition of exosome uptake by melanoma cells. Fusion efficiency of tumor exosomes resulted in being higher in cells of metastatic origin than in those derived from primary tumors or normal cells. Furthermore, we found that caveolin-1, a protein involved in melanoma progression, is highly delivered through exosomes released in an acidic condition. The results of our study provide the evidence that exosomes may be used as a delivery system for paracrine diffusion of tumor malignancy, in turn supporting the importance of both exosomes and tumor pH as key targets for future anti-cancer strategies.     

Neurotransmitter-Triggered Transfer of Exosomes Mediates Oligodendrocyte–Neuron Communication

Reciprocal interactions between neurons and oligodendrocytes are not only crucial for myelination, but also for long-term survival of axons. Degeneration of axons occurs in several human myelin diseases, however the molecular mechanisms of axon-glia communication maintaining axon integrity are poorly understood. Here, we describe the signal-mediated transfer of exosomes from oligodendrocytes to neurons. These endosome-derived vesicles are secreted by oligodendrocytes and carry specific protein and RNA cargo. We show that activity-dependent release of the neurotransmitter glutamate triggers oligodendroglial exosome secretion mediated by Ca²⁺ entry through oligodendroglial NMDA and AMPA receptors. In turn, neurons internalize the released exosomes by endocytosis. Injection of oligodendroglia-derived exosomes into the mouse brain results in functional retrieval of exosome cargo in neurons. Supply of cultured neurons with oligodendroglial exosomes improves neuronal viability under conditions of cell stress. These findings indicate that oligodendroglial exosomes participate in a novel mode of bidirectional neuron-glia communication contributing to neuronal integrity.     

Analysis of the RNA content of the exosomes derived from blood serum and urine and its potential as biomarkers

Exosomes are tiny vesicles (30–150 nm) constantly secreted by all healthy and abnormal cells, and found in abundance in all body fluids. These vesicles, loaded with unique RNA and protein cargo, have a wide range of biological functions, including cell-to-cell communication and signalling. As such, exosomes hold tremendous potential as biomarkers and could lead to the development of minimally invasive diagnostics and next generation therapies within the next few years. Here, we describe the strategies for isolation of exosomes from human blood serum and urine, characterization of their RNA cargo by sequencing, and present the initial data on exosome labelling and uptake tracing in a cell culture model. The value of exosomes for clinical applications is discussed with an emphasis on their potential for diagnosing and treating neurodegenerative diseases and brain cancer.     

Protein profile of exosomes from trabecular meshwork cells

To better understand the role of exosomes in the trabecular meshwork (TM), the site of intraocular pressure control, the exosome proteome from primary cultures of human TM cell monolayers was analyzed. Exosomes were purified from urine and conditioned media from primary cultures of human TM cell monolayers and subjected to a two dimensional HPLC separation and MS/MS analyses using the MudPIT strategy. Spectra were searched against a human protein database using Sequest. Protein profiles were compared to each other and the Exocarta database and the presence of specific protein markers confirmed by Western blot analyses of exosomes from aqueous humor and human TM cell strains (n=5) that were untreated, or exposed to dexamethasone and/or ionomycin. TM cell exosomes contained 108 of the 143 most represented exosome proteins in ExoCarta, including previously characterized markers such as membrane organizing and tetraspanin proteins. Several cell-specific proteins in TM exosomes were identified including myocilin, emilin-1 and neuropilin-1. All TM exosome proteins had flotation densities on sucrose gradients and release responses to ionomycin typical for exosomes. Taken together, TM exosomes have a characteristic exosome protein profile plus contain unique proteins, including the glaucoma-causing protein, myocilin; suggesting a role for exosomes in the control of intraocular pressure.     

Adiponectin is partially associated with exosomes in mouse serum

Exosomes are membrane vesicles 30–120 nm in diameter that are released by many cell types and carry a cargo of proteins, lipids, mRNA, and microRNA. Cultured adipocytes reportedly release exosomes that may play a role in cell-to-cell communication during the development of metabolic diseases. However, the characteristics and function of exosomes released from adipocytes in vivo remain to be elucidated. Clearly, adipocyte-derived exosomes could exist in the circulation and may be associated with adipocyte-specific proteins such as adipocytokines. We isolated exosomes from serum of mice by differential centrifugation and analyzed adiponectin, leptin, and resistin in the exosome fraction. Western blotting detected adiponectin but no leptin and only trace amounts of resistin in the exosome fraction. The adiponectin signal in the exosome fraction was decreased by proteinase K treatment and completely quenched by a combination of proteinase K and Triton X-100. Quantitative ELISA showed that the exosome fraction contains considerable amounts of adiponectin, but not leptin or resistin. The concentration of adiponectin in the serum and the ratio of adiponectin to total protein in the exosome fraction were lower in obese mice than in lean mice. These results suggest that a portion of adiponectin exists as a transmembrane protein in the exosomes in mouse serum. We propose adiponectin as a marker of exosomes released from adipocytes in vivo.     

Exosomes surf on filopodia to enter cells at endocytic hot spots,traffic within endosomes,and are targeted to the ER

Exosomes are nanovesicles released by virtually all cells, which act as intercellular messengers by transfer of protein, lipid, and RNA cargo. Their quantitative efficiency, routes of cell uptake, and subcellular fate within recipient cells remain elusive. We quantitatively characterize exosome cell uptake, which saturates with dose and time and reaches near 100% transduction efficiency at picomolar concentrations. Highly reminiscent of pathogenic bacteria and viruses, exosomes are recruited as single vesicles to the cell body by surfing on filopodia as well as filopodia grabbing and pulling motions to reach endocytic hot spots at the filopodial base. After internalization, exosomes shuttle within endocytic vesicles to scan the endoplasmic reticulum before being sorted into the lysosome as their final intracellular destination. Our data quantify and explain the efficiency of exosome internalization by recipient cells, establish a new parallel between exosome and virus host cell interaction, and suggest unanticipated routes of subcellular cargo delivery.     

Exosome-mediated transduction of mechanical force regulates prostate cancer migration via microRNA

Physical cues in the extracellular microenvironment regulate cancer cell metastasis. Functional microRNA (miRNA) carried by cancer derived exosomes play a critical role in extracellular communication between cells and the extracellular microenvironment. However, little is known about the role of exosomes loaded miRNAs in the mechanical force transmission between cancer cells and extracellular microenvironment. Herein, our results suggest that stiff extracellular matrix (ECM) induced exosomes promote cancer cell migration. The ECM mechanical force regulated the exosome miRNA cargo of prostate cancer cells. Exosome miRNAs regulated by the ECM mechanical force modulated cancer cell metastasis by regulating cell motility, ECM remodeling and the interaction between cancer cells and nerves. Focal adhesion kinase mediated-ECM mechanical force regulated the intracellular miRNA expression, and F-actin mediate-ECM mechanical force regulated miRNA packaging into exosomes. The above results demonstrated that the exosome miRNA cargo promoted cancer metastasis by transmitting the ECM mechanical force. The ECM mechanical force may play multiple roles in maintaining the microenvironment of cancer metastasis through the exosome miRNA cargo.     

Anticarcinogenic impact of extracellular vesicles (exosomes) from cord blood stem cells in malignant melanoma: A potential biological treatment

Incidence of Malignant Melanoma has become the 5th in the UK. To date, the major anticancer therapeutics include cell therapy, immunotherapy, gene therapy and nanotechnology-based strategies. Recently, extracellular vesicles, especially exosomes, have been highlighted for their therapeutic benefits in numerous chronic diseases. Exosomes display multifunctional properties, including inhibition of cancer cell proliferation and initiation of apoptosis. In the present in vitro study, the antitumour effect of cord blood stem cell (CBSC)-derived exosomes was confirmed by the CCK-8 assay (p < 0.05) on CHL-1 melanoma cells and improve the repair mechanism on lymphocytes from melanoma patients. Importantly, no significant effect was observed in healthy lymphocytes when treated with the exosome concentrations at 24, 48 and 72 h. Comet assay results (OTM and %Tail DNA) demonstrated that the optimal exosome concentration showed a significant impact (p < 0.05) in lymphocytes from melanoma patients whilst causing no significant DNA damage in lymphocytes of healthy volunteers was 300 μg/ml. Similarly, the Comet assay results depicted significant DNA damage in a melanoma cell line (CHL-1 cells) treated with CBSC-derived exosomes, both the cytotoxicity of CHL-1 cells treated with CBSC-derived exosomes exhibited a significant time-dependent decrease in cell survival. Sequencing analysis of CBSC exosomes showed the presence of the let-7 family of miRNAs, including let-7a-5p, let-7b-5p, let-7c-5p, let-7d-3p, let-7d-5p and two novel miRNAs. The potency of CBSC exosomes in inhibiting cancer progression in lymphocytes from melanoma patients and CHL-1 cells whilst causing no harm to the healthy lymphocytes makes it a potential candidate as an anticancer therapy.     

Reconstitution, activities, and structure of the eukaryotic RNA exosome

The RNA exosome is a multisubunit 3' to 5' exoribonuclease complex that participates in degradation and processing of cellular RNA. To determine the activities and structure of the eukaryotic exosome, we report the reconstitution of 9-subunit exosomes from yeast and human and reconstitution of 10- and 11-subunit exosomes from yeast. Comparative biochemical analysis between purified subunits and reconstituted exosomes using AU-rich, polyadenylated (poly[A]), generic, and structured RNA substrates reveals processive phosphorolytic activities for human Rrp41/Rrp45 and the 9-subunit human exosome, processive hydrolytic activities for yeast Rrp44 and the yeast 10-subunit exosome, distributive hydrolytic activities for Rrp6, and processive and distributive hydrolytic activities for the yeast 11-subunit exosome. To elucidate the architecture of a eukaryotic exosome, its conserved surfaces, and the structural basis for RNA decay, we report the X-ray structure determination for the 286 kDa nine-subunit human exosome at 3.35 A.     

Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET

Tumor-derived exosomes are emerging mediators of tumorigenesis. We explored the function of melanoma-derived exosomes in the formation of primary tumors and metastases in mice and human subjects. Exosomes from highly metastatic melanomas increased the metastatic behavior of primary tumors by permanently 'educating' bone marrow progenitors through the receptor tyrosine kinase MET. Melanoma-derived exosomes also induced vascular leakiness at pre-metastatic sites and reprogrammed bone marrow progenitors toward a pro-vasculogenic phenotype that was positive for c-Kit, the receptor tyrosine kinase Tie2 and Met. Reducing Met expression in exosomes diminished the pro-metastatic behavior of bone marrow cells. Notably, MET expression was elevated in circulating CD45(-)C-KIT(low/+)TIE2(+) bone marrow progenitors from individuals with metastatic melanoma. RAB1A, RAB5B, RAB7 and RAB27A, regulators of membrane trafficking and exosome formation, were highly expressed in melanoma cells. Rab27A RNA interference decreased exosome production, preventing bone marrow education and reducing, tumor growth and metastasis. In addition, we identified an exosome-specific melanoma signature with prognostic and therapeutic potential comprised of TYRP2, VLA-4, HSP70, an HSP90 isoform and the MET oncoprotein. Our data show that exosome production, transfer and education of bone marrow cells supports tumor growth and metastasis, has prognostic value and offers promise for new therapeutic directions in the metastatic process.     

人脐血血浆外泌体提取方法的比较北大核心CSCD

为探寻高效且稳定的提取人脐血血浆外泌体的方法,利用超高速离心法、蔗糖垫密度梯度离心法、改良超速离心法和聚乙二醇(polyethylene glycol, PEG)沉淀法提取人脐血血浆外泌体,并比较4种方法的优劣。利用透射电镜、动态光散射技术观察外泌体的形态、结构及大小;聚氰基丙烯酸正丁酯(bicinchoninic acid, BCA)法测定外泌体蛋白总量;Western blotting检测外泌体表面标志蛋白CD63、HSP70以及外泌体阴性蛋白GM130 (高尔基标志蛋白)的表达。结果表明,与提取外泌体的“金标准”,即超高速离心法相比,蔗糖垫密度梯度离心法稳定性好,获取的外泌体粒径较均一,但操作较复杂,耗时长;改良超速离心法操作较简单,纯度较高;PEG沉淀法提取的外泌体蛋白量最高,操作时间最短,但杂质较多。结果表明,4种方法均能从人脐血血浆中获取外泌体,但在操作时间、纯度、提取量等方面存在一定差异。因此,应根据实验目的和具体要求选择合适的提取人脐血血浆外泌体的方法。     

Tubular cell-derived exosomal miR-150-5p contributes to renal fibrosis following unilateral ischemia-reperfusion injury by activating fibroblast in vitro and in vivo

Unilateral ischemia reperfusion injury (UIRI) with longer ischemia time is associated with an increased risk of acute renal injury and chronic kidney disease. Exosomes can transport lipid, protein, mRNA, and miRNA to corresponding target cells and mediate intercellular information exchange. In this study, we aimed to investigate whether exosome-derived miRNA mediates epithelial-mesenchymal cell communication relevant to renal fibrosis after UIRI. The secretion of exosomes increased remarkably in the kidney after UIRI and in rat renal tubular epithelium cells (NRK-52E) after hypoxia treatment. The inhibition of exosome secretion by Rab27a knockout or GW4869 treatment ameliorates renal fibrosis following UIRI in vivo. Purified exosomes from NRK-52E cells after hypoxia treatment could activate rat kidney fibroblasts (NRK-49F). The inhibition of exosome secretion in hypoxic NRK-52E cells through Rab27a knockdown or GW4869 treatment abolished NRK-49F cell activation. Interestingly, exosomal miRNA array analysis revealed that miR-150-5p expression was increased after hypoxia compared with the control group. The inhibition of exosomal miR-150-5p abolished the ability of hypoxic NRK-52E cells to promote NRK-49F cell activation in vitro, injections of miR-150-5p enriched exosomes from hypoxic NRK-52E cells aggravated renal fibrosis following UIRI, and renal fibrosis after UIRI was alleviated by miR-150-5p-deficient exosome in vivo. Furthermore, tubular cell-derived exosomal miR-150-5p could negatively regulate the expression of suppressor of cytokine signaling 1 to activate fibroblast. Thus, our results suggest that the blockade of exosomal miR-150-5p mediated tubular epithelial cell-fibroblast communication may provide a novel therapeutic target to prevents UIRI progression to renal fibrosis.     

Heparanase Regulates Secretion,Composition, and Function of Tumor Cell-derived Exosomes

Emerging evidence indicates that exosomes play a key role in tumor-host cross-talk and that exosome secretion, composition, and functional capacity are altered as tumors progress to an aggressive phenotype. However, little is known regarding the mechanisms that regulate these changes. Heparanase is an enzyme whose expression is up-regulated as tumors become more aggressive and is associated with enhanced tumor growth, angiogenesis, and metastasis. We have discovered that in human cancer cells (myeloma, lymphoblastoid, and breast cancer), when expression of heparanase is enhanced or when tumor cells are exposed to exogenous heparanase, exosome secretion is dramatically increased. Heparanase enzyme activity is required for robust enhancement of exosome secretion because enzymatically inactive forms of heparanase, even when present in high amounts, do not dramatically increase exosome secretion. Heparanase also impacts exosome protein cargo as reflected by higher levels of syndecan-1, VEGF, and hepatocyte growth factor in exosomes secreted by heparanase-high expressing cells as compared with heparanase-low expressing cells. In functional assays, exosomes from heparanase-high cells stimulated spreading of tumor cells on fibronectin and invasion of endothelial cells through extracellular matrix better than did exosomes secreted by heparanase-low cells. These studies reveal that heparanase helps drive exosome secretion, alters exosome composition, and facilitates production of exosomes that impact both tumor and host cell behavior, thereby promoting tumor progression.     

Plasma exosomes can deliver exogenous short interfering RNA to monocytes and lymphocytes

Despite the promise of RNA interference (RNAi) and its potential, e.g. for use in cancer therapy, several technical obstacles must first be overcome. The major hurdle of RNAi-based therapeutics is to deliver nucleic acids across the cell's plasma membrane. This study demonstrates that exosome vesicles derived from humans can deliver short interfering RNA (siRNA) to human mononuclear blood cells. Exosomes are nano-sized vesicles of endocytic origin that are involved in cell-to-cell communication, i.e. antigen presentation, tolerance development and shuttle RNA (mainly mRNA and microRNA). Having tested different strategies, an optimized method (electroporation) was used to introduce siRNA into human exosomes of various origins. Plasma exosomes (exosomes from peripheral blood) were used as gene delivery vector (GDV) to transport exogenous siRNA to human blood cells. The vesicles effectively delivered the administered siRNA into monocytes and lymphocytes, causing selective gene silencing of mitogen-activated protein kinase 1. These data suggest that human exosomes can be used as a GDV to provide cells with heterologous nucleic acids such as therapeutic siRNAs.     

Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes

To realize the therapeutic potential of RNA drugs, efficient, tissue-specific and nonimmunogenic delivery technologies must be developed. Here we show that exosomes-endogenous nano-vesicles that transport RNAs and proteins-can deliver short interfering (si)RNA to the brain in mice. To reduce immunogenicity, we used self-derived dendritic cells for exosome production. Targeting was achieved by engineering the dendritic cells to express Lamp2b, an exosomal membrane protein, fused to the neuron-specific RVG peptide. Purified exosomes were loaded with exogenous siRNA by electroporation. Intravenously injected RVG-targeted exosomes delivered GAPDH siRNA specifically to neurons, microglia, oligodendrocytes in the brain, resulting in a specific gene knockdown. Pre-exposure to RVG exosomes did not attenuate knockdown, and non-specific uptake in other tissues was not observed. The therapeutic potential of exosome-mediated siRNA delivery was demonstrated by the strong mRNA (60%) and protein (62%) knockdown of BACE1, a therapeutic target in Alzheimer's disease, in wild-type mice.     

Engineered exosome-mediated messenger RNA and single-chain variable fragment delivery for human chimeric antigen receptor T-cell engineering

Background aimsMost current chimeric antigen receptor (CAR) T cells are generated by viral transduction, which induces persistent expression of CARs and may cause serious undesirable effects. Messenger RNA (mRNA)-based approaches in manufacturing CAR T cells are being developed to overcome these challenges. However, the most common method of delivering mRNA to T cells is electroporation, which can be toxic to cells.MethodsThe authors designed and engineered an exosome delivery platform using the bacteriophage MS2 system in combination with the highly expressed protein lysosome-associated membrane protein 2 isoform B on exosomes.ResultsThe authors’ delivery platform achieved specific loading and delivery of mRNA into target cells and achieved expression of specific proteins, and anti-CD3/CD28 single-chain variable fragments (scFvs) expressed outside the exosomal membrane effectively activated primary T cells in a similar way to commercial magnetic beads.ConclusionsThe delivery of CAR mRNA and anti-CD3/CD28 scFvs via designed exosomes can be used for ex vivo production of CAR T cells with cancer cell killing capacity. The authors’ results indicate the potential applications of the engineered exosome delivery platform for direct conversion of primary T cells to CAR T cells while providing a novel strategy for producing CAR T cells in vivo.     

Oncogenic and Non‐Malignant Pancreatic Exosome Cargo Reveal Distinct Expression of Oncogenic and Prognostic Factors Involved in Tumor Invasion and Metastasis

Exosomes are small extracellular membrane vesicles important in intercellular communication, with their oncogenic cargo attributed to tumor progression and pre‐metastatic niche formation. To gain an insight into key differences in oncogenic composition of exosomes, human non‐malignant epithelial and pancreatic cancer cell models and purified and characterized resultant exosome populations are utilized. Proteomic analysis reveals the selective enrichment of known exosome markers and signaling proteins in comparison to parental cells. Importantly, valuable insights into oncogenic exosomes (362 unique proteins in comparison to non‐malignant exosomes) of key metastatic regulatory factors and signaling molecules fundamental to pancreatic cancer progression (KRAS, CD44, EGFR) are provided. It is reported that oncogenic exosomes contain factors known to regulate the pre‐metastatic niche (S100A4, F3, ITGβ5, ANXA1), clinically‐relevant proteins which correlate with poor prognosis (CLDN1, MUC1) as well as protein networks involved in various cancer hallmarks including proliferation (CLU, CAV1), invasion (PODXL, ITGA3), metastasis (LAMP1, ST14) and immune surveillance escape (B2M). The presence of these factors in oncogenic exosomes offers an understanding of select differences in exosome composition during tumorigenesis, potential components as prognostic and diagnostic biomarkers in pancreatic cancer, and highlights the role of exosomes in mediating crosstalk between tumor and stromal cells.     

Free Extracellular miRNA Functionally Targets Cells by Transfecting Exosomes from Their Companion Cells

Lymph node and spleen cells of mice doubly immunized by epicutaneous and intravenous hapten application produce a suppressive component that inhibits the action of the effector T cells that mediate contact sensitivity reactions. We recently re-investigated this phenomenon in an immunological system. CD8+ T lymphocyte-derived exosomes transferred suppressive miR-150 to the effector T cells antigen-specifically due to exosome surface coat of antibody light chains made by B1a lymphocytes. Extracellular RNA (exRNA) is protected from plasma RNases by carriage in exosomes or by chaperones. Exosome transfer of functional RNA to target cells is well described, whereas the mechanism of transfer of exRNA free of exosomes remains unclear. In the current study we describe extracellular miR-150, extracted from exosomes, yet still able to mediate antigen-specific suppression. We have determined that this was due to miR-150 association with antibody-coated exosomes produced by B1a cell companions of the effector T cells, which resulted in antigen-specific suppression of their function. Thus functional cell targeting by free exRNA can proceed by transfecting companion cell exosomes that then transfer RNA cargo to the acceptor cells. This contrasts with the classical view on release of RNA-containing exosomes from the multivesicular bodies for subsequent intercellular targeting. This new alternate pathway for transfer of exRNA between cells has distinct biological and immunological significance, and since most human blood exRNA is not in exosomes may be relevant to evaluation and treatment of diseases.