Myoferlin is a novel exosomal protein and functional regulator of cancer-derived exosomes.

PubMed

Blomme, Arnaud; Fahmy, Karim; Peulen, Olivier; Costanza, Brunella; Fontaine, Marie; Struman, Ingrid; Baiwir, Dominique; de Pauw, Edwin; Thiry, Marc; Bellahcène, Akeila; Castronovo, Vincent; Turtoi, Andrei

2016-12-13

Exosomes are communication mediators participating in the intercellular exchange of proteins, metabolites and nucleic acids. Recent studies have demonstrated that exosomes are characterized by a unique proteomic composition that is distinct from the cellular one. The mechanisms responsible for determining the proteome content of the exosomes remain however obscure. In the current study we employ ultrastructural approach to validate a novel exosomal protein myoferlin. This is a multiple C2-domain containing protein, known for its conserved physiological function in endocytosis and vesicle fusion biology. Emerging studies demonstrate that myoferlin is frequently overexpressed in cancer, where it promotes cancer cell migration and invasion. Our data expand these findings by showing that myoferlin is a general component of cancer cell derived exosomes from different breast and pancreatic cancer cell lines. Using proteomic analysis, we demonstrate for the first time that myoferlin depletion in cancer cells leads to a significantly modulated exosomal protein load. Such myoferlin-depleted exosomes were also functionally deficient as shown by their reduced capacity to transfer nucleic acids to human endothelial cells (HUVEC). Beyond this, myoferlin-depleted cancer exosomes also had a significantly reduced ability to induce migration and proliferation of HUVEC. The present study highlights myoferlin as a new functional player in exosome biology, calling for novel strategies to target this emerging oncogene in human cancer.

The Biodistribution and Immune Suppressive Effects of Breast Cancer-Derived Exosomes.

PubMed

Wen, Shu Wen; Sceneay, Jaclyn; Lima, Luize Goncalves; Wong, Christina S F; Becker, Melanie; Krumeich, Sophie; Lobb, Richard J; Castillo, Vanessa; Wong, Ke Ni; Ellis, Sarah; Parker, Belinda S; Möller, Andreas

2016-12-01

Small membranous secretions from tumor cells, termed exosomes, contribute significantly to intercellular communication and subsequent reprogramming of the tumor microenvironment. Here, we use optical imaging to determine that exogenously administered fluorescently labeled exosomes derived from highly metastatic murine breast cancer cells distributed predominantly to the lung of syngeneic mice, a frequent site of breast cancer metastasis. At the sites of accumulation, exosomes were taken up by CD45 + bone marrow-derived cells. Subsequent long-term conditioning of naïve mice with exosomes from highly metastatic breast cancer cells revealed the accumulation of myeloid-derived suppressor cells in the lung and liver. This favorable immune suppressive microenvironment was capable of promoting metastatic colonization in the lung and liver, an effect not observed from exosomes derived from nonmetastatic cells and liposome control vesicles. Furthermore, we determined that breast cancer exosomes directly suppressed T-cell proliferation and inhibited NK cell cytotoxicity, and hence likely suppressed the anticancer immune response in premetastatic organs. Together, our findings provide novel insight into the tissue-specific outcomes of breast cancer-derived exosome accumulation and their contribution to immune suppression and promotion of metastases. Cancer Res; 76(23); 6816-27. ©2016 AACR. ©2016 American Association for Cancer Research.

The chemokine receptor CCR1 is identified in mast cell-derived exosomes

PubMed Central

Liang, Yuting; Qiao, Longwei; Peng, Xia; Cui, Zelin; Yin, Yue; Liao, Huanjin; Jiang, Min; Li, Li

2018-01-01

Mast cells are important effector cells of the immune system, and mast cell-derived exosomes carrying RNAs play a role in immune regulation. However, the molecular function of mast cell-derived exosomes is currently unknown, and here, we identify differentially expressed genes (DEGs) in mast cells and exosomes. We isolated mast cells derived exosomes through differential centrifugation and screened the DEGs from mast cell-derived exosomes, using the GSE25330 array dataset downloaded from the Gene Expression Omnibus database. Biochemical pathways were analyzed by Gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway on the online tool DAVID. DEGs-associated protein-protein interaction networks (PPIs) were constructed using the STRING database and Cytoscape software. The genes identified from these bioinformatics analyses were verified by qRT-PCR and Western blot in mast cells and exosomes. We identified 2121 DEGs (843 up and 1278 down-regulated genes) in HMC-1 cell-derived exosomes and HMC-1 cells. The up-regulated DEGs were classified into two significant modules. The chemokine receptor CCR1 was screened as a hub gene and enriched in cytokine-mediated signaling pathway in module one. Seven genes, including CCR1, CD9, KIT, TGFBR1, TLR9, TPSAB1 and TPSB2 were screened and validated through qRT-PCR analysis. We have achieved a comprehensive view of the pivotal genes and pathways in mast cells and exosomes and identified CCR1 as a hub gene in mast cell-derived exosomes. Our results provide novel clues with respect to the biological processes through which mast cell-derived exosomes modulate immune responses. PMID:29511430

The chemokine receptor CCR1 is identified in mast cell-derived exosomes.

PubMed

Liang, Yuting; Qiao, Longwei; Peng, Xia; Cui, Zelin; Yin, Yue; Liao, Huanjin; Jiang, Min; Li, Li

2018-01-01

Mast cells are important effector cells of the immune system, and mast cell-derived exosomes carrying RNAs play a role in immune regulation. However, the molecular function of mast cell-derived exosomes is currently unknown, and here, we identify differentially expressed genes (DEGs) in mast cells and exosomes. We isolated mast cells derived exosomes through differential centrifugation and screened the DEGs from mast cell-derived exosomes, using the GSE25330 array dataset downloaded from the Gene Expression Omnibus database. Biochemical pathways were analyzed by Gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway on the online tool DAVID. DEGs-associated protein-protein interaction networks (PPIs) were constructed using the STRING database and Cytoscape software. The genes identified from these bioinformatics analyses were verified by qRT-PCR and Western blot in mast cells and exosomes. We identified 2121 DEGs (843 up and 1278 down-regulated genes) in HMC-1 cell-derived exosomes and HMC-1 cells. The up-regulated DEGs were classified into two significant modules. The chemokine receptor CCR1 was screened as a hub gene and enriched in cytokine-mediated signaling pathway in module one. Seven genes, including CCR1, CD9, KIT, TGFBR1, TLR9, TPSAB1 and TPSB2 were screened and validated through qRT-PCR analysis. We have achieved a comprehensive view of the pivotal genes and pathways in mast cells and exosomes and identified CCR1 as a hub gene in mast cell-derived exosomes. Our results provide novel clues with respect to the biological processes through which mast cell-derived exosomes modulate immune responses.

Role of stem cell derived exosomes in tumor biology.

PubMed

Sharma, Aman

2018-03-15

Exosomes are nano-scale messengers loaded with bio-molecular cargo of RNA, DNA, and Proteins. As a master regulator of cellular signaling, stem cell (both normal, and cancer stem cells) secreted exosome orchestrate various autocrine and paracrine functions which alter tumor micro-environment, growth and progression. Exosomes secreted by one of the two important stem cell phenotypes in cancers a) Mesenchymal stem cells, and b) Cancer stem cells not only promote cancerous growth but also impart therapy resistance in cancer cells. In tumors, normal or mesenchymal stem cell (MSCs) derived exosomes (MSC-exo) modulate tumor hallmarks by delivering unique miRNA species to neighboring cells and help in tumor progression. Apart from regulating tumor cell fate, MSC-exo are also capable of inducing physiological processes, for example, angiogenesis, metastasis and so forth. Similarly, cancer stem cells (CSCs) derived exosomes (CSC-exo) contain stemness-specific proteins, self-renewal promoting regulatory miRNAs, and survival factors. CSC-exo specific cargo maintains tumor heterogeneity and alters tumor progression. In this review we critically discuss the importance of stem cell specific exosomes in tumor cell signaling pathways with their role in tumor biology. © 2017 UICC.

Gastric cancer-derived exosomes promote peritoneal metastasis by destroying the mesothelial barrier.

PubMed

Deng, Guang; Qu, Jinglei; Zhang, Ye; Che, Xiaofang; Cheng, Yu; Fan, Yibo; Zhang, Simeng; Na, Di; Liu, Yunpeng; Qu, Xiujuan

2017-07-01

An intact mesothelium serves as a protective barrier to inhibit peritoneal carcinomatosis. Cancer-derived exosomes can mediate directional tumor metastasis; however, little is known about whether gastric cancer-derived exosomes will destroy the mesothelial barrier and promote peritoneal dissemination. Here, we demonstrate that gastric cancer-derived exosomes facilitate peritoneal metastasis by causing mesothelial barrier disruption and peritoneal fibrosis. Injury of peritoneal mesothelial cells elicited by gastric cancer-derived exosomes is through concurrent apoptosis and mesothelial-to-mesenchymal transition (MMT). Additionally, upregulation of p-ERK in peritoneal mesothelial cells is primarily responsible for the MMT while contributing little to apoptosis. Together, these data support the concept that exosomes play a crucial role in remodeling the premetastatic microenvironment and identify a novel mechanism for peritoneal metastasis of gastric carcinoma. © 2017 Federation of European Biochemical Societies.

Therapeutic Potential of Mesenchymal Stem Cell-Derived Exosomes in the Treatment of Eye Diseases.

PubMed

Harrell, C Randall; Simovic Markovic, Bojana; Fellabaum, Crissy; Arsenijevic, Aleksandar; Djonov, Valentin; Arsenijevic, Nebojsa; Volarevic, Vladislav

2018-05-18

Mesenchymal stem cells (MSCs) were, due to their immunomodulatory and pro-angiogenic characteristics, extensively explored as new therapeutic agents in cell-based therapy of uveitis, glaucoma, retinal and ocular surface diseases.Since it was recently revealed that exosomes play an important role in biological functions of MSCs, herewith we summarized current knowledge about the morphology, structure, phenotype and functional characteristics of MSC-derived exosomes emphasizing their therapeutic potential in the treatment of eye diseases.MSC-derived exosomes were as efficient as transplanted MSCs in limiting the extent of eye injury and inflammation. Immediately after intravitreal injection, MSC-derived exosomes, due to nano-dimension, diffused rapidly throughout the retina and significantly attenuated retinal damage and inflammation. MSC-derived exosomes successfully delivered trophic and immunomodulatory factors to the inner retina and efficiently promoted survival and neuritogenesis of injured retinal ganglion cells. MSC-derived exosomes efficiently suppressed migration of inflammatory cells, attenuated detrimental Th1 and Th17 cell-driven immune response and ameliorated experimental autoimmune uveitis. MSC-derived exosomes were able to fuse with the lysosomes within corneal cells, enabling delivering of MSC-derived active β-glucuronidase and consequent catabolism of accumulated glycosaminoglycans, indicating their therapeutic potential in the treatment of Mucopolysaccharidosis VII (Sly Syndrome). Importantly, beneficent effects were noticed only in animals that received MSC-derived exosomes and were not seen after therapy with fibroblasts-derived exosomes confirming specific therapeutic potential of MSCs and their products in the treatment of eye diseases.In conclusion, MSC-derived exosomes represent potentially new therapeutic agents in the therapy of degenerative and inflammatory ocular diseases.

The potential of tumor-derived exosomes for noninvasive cancer monitoring

PubMed Central

Whiteside, Theresa L.

2016-01-01

Tumor-derived exosomes (TEXs) are emerging as a new type of cancer biomarker. TEXs are membrane-bound, virus-size vesicles of endocytic origin present in all body fluids of cancer patients. Based on the expanding albeit incomplete knowledge of their biogenesis, secretion by tumor cells and cancer cell-specific molecular and genetic contents, TEXs are viewed as promising, clinically-relevant surrogates of cancer progression and response to therapy. Preliminary proteomic, genetic and functional profiling of tumor cell-derived or cancer plasma-derived exosomes confirms their unique characteristics. Alterations in protein or nucleic acid profiles of exosomes in plasma of cancer patients responding to therapies appear to correlate with clinical endpoints. However, methods for TEX isolation and separation from the bulk of human plasma-derived exosomes are not yet established and their role as biomarkers remains to be confirmed. Further development and validation of TEXs as noninvasive, liquid equivalents of tumor biopsies are necessary to move this effort forward. PMID:26289602

The potential of tumor-derived exosomes for noninvasive cancer monitoring.

PubMed

Whiteside, Theresa L

2015-01-01

Tumor-derived exosomes (TEX) are emerging as a new type of cancer biomarker. TEX are membrane-bound, virus-size vesicles of endocytic origin present in all body fluids of cancer patients. Based on the expanding albeit incomplete knowledge of their biogenesis, secretion by tumor cells and cancer cell-specific molecular and genetic contents, TEX are viewed as promising, clinically-relevant surrogates of cancer progression and response to therapy. Preliminary proteomic, genetic and functional profiling of tumor cell-derived or cancer plasma-derived exosomes confirms their unique characteristics. Alterations in protein or nucleic acid profiles of exosomes in plasma of cancer patients responding to therapies appear to correlate with clinical endpoints. However, methods for TEX isolation and separation from the bulk of human plasma-derived exosomes are not yet established and their role as biomarkers remains to be confirmed. Further development and validation of TEX as noninvasive, liquid equivalents of tumor biopsies are necessary to move this effort forward.

The functions and clinical applications of tumor-derived exosomes

PubMed Central

Shao, Yingkuan; Shen, Yanwei; Chen, Ting; Xu, Fei; Chen, Xuewen; Zheng, Shu

2016-01-01

Exosomes are extracellular vesicles with diameters ranging from 30 to 150 nm. They can be secreted by all cell types and transfer information in the form of their contents, which include proteins, lipids and nucleic acids, to other cells throughout the body. They have roles in normal physiological processes as well as in disease development. Here, we review recent findings regarding tumor-derived exosomes, including methods for their extraction and preservation. We also describe the actions of exosomes in tumorigenesis. The exosomal antigen-presenting effect during antitumor immune responses and its suppressive function in immune tolerance are discussed. Finally, we describe the potential application of exosomes to cancer therapy and liquid biopsy. PMID:27517627

Exosomes Derived From Pancreatic Stellate Cells: MicroRNA Signature and Effects on Pancreatic Cancer Cells.

PubMed

Takikawa, Tetsuya; Masamune, Atsushi; Yoshida, Naoki; Hamada, Shin; Kogure, Takayuki; Shimosegawa, Tooru

2017-01-01

Pancreatic stellate cells (PSCs) interact with pancreatic cancer cells in the tumor microenvironment. Cell constituents including microRNAs may be exported from cells within membranous nanovesicles termed exosomes. Exosomes might play a pivotal role in intercellular communication. This study aimed to clarify the microRNA signature of PSC-derived exosomes and their effects on pancreatic cancer cells. Exosomes were prepared from the conditioned medium of immortalized human PSCs. MicroRNAs were prepared from the exosomes and their source PSCs, and the microRNA expression profiles were compared by microarray. The effects of PSC-derived exosomes on proliferation, migration, and the mRNA expression profiles were examined in pancreatic cancer cells. Pancreatic stellate cell-derived exosomes contained a variety of microRNAs including miR-21-5p. Several microRNAs such as miR-451a were enriched in exosomes compared to their source PSCs. Pancreatic stellate cell-derived exosomes stimulated the proliferation, migration and expression of mRNAs for chemokine (C - X - C motif) ligands 1 and 2 in pancreatic cancer cells. The stimulation of proliferation, migration, and chemokine gene expression by the conditioned medium of PSCs was suppressed by GW4869, an exosome inhibitor. We clarified the microRNA expression profile in PSC-derived exosomes. Pancreatic stellate cell-derived exosomes might play a role in the interactions between PSCs and pancreatic cancer cells.

Exosome release of ADAM15 and the functional implications of human macrophage-derived ADAM15 exosomes.

PubMed

Lee, Hee Doo; Koo, Bon-Hun; Kim, Yeon Hyang; Jeon, Ok-Hee; Kim, Doo-Sik

2012-07-01

A disintegrin and metalloproteinase 15 (ADAM15), the only ADAM protein containing an Arg-Gly-Asp (RGD) motif in its disintegrin-like domain, is a widely expressed membrane protein that is involved in tumor progression and suppression. However, the underlying mechanism of ADAM15-mediated tumor suppression is not clearly understood. This study demonstrates that ADAM15 is released as an exosomal component, and ADAM15 exosomes exert tumor suppressive activities. We found that exosomal ADAM15 release is stimulated by phorbol 12-myristate 13-acetate, a typical protein kinase C activator, in various tumor cell types, and this results in a corresponding decrease in plasma membrane-associated ADAM15. Exosomes rich in ADAM15 display enhanced binding affinity for integrin αvβ3 in an RGD-dependent manner and suppress vitronectin- and fibronectin-induced cell adhesion, growth, and migration, as well as in vivo tumor growth. Exosomal ADAM15 is released from human macrophages, and macrophage-derived ADAM15 exosomes have tumor inhibitory effects. This work suggests a primary role of ADAM15 for exosome-mediated tumor suppression, as well as functional significance of exosomal ADAM protein in antitumor immunity.

Potential of cancer cell-derived exosomes in clinical application: a review of recent research advances.

PubMed

Sun, Yu; Liu, Jing

2014-06-01

Exosomes are 30- to 100-nm, membrane-bound vesicles that are released by most types of cells, including tumor cells. Exosomes contain a great variety of bioactive molecules, including signal peptides, microRNA, lipids, and DNA. In cancer, tumor cells aberrantly secrete large quantities of exosomes to transport paracrine signals or to contribute to tumor-environment interaction at a distance. The goal of this review was to discuss the recent advances on the mechanism of cancer-derived exosomes in tumor regulation. Pertinent articles and abstracts were identified through searches of PubMed for literature published from 1983 to December 2013. Search terms included exosome, tumor, cancer, diagnosis, and therapy. All of the exposed evidence points to communication between cancer cells and their surroundings, either mediated by cancer cell-derived exosomes or by stromal cell-derived exosomes. This communication probably supports tumor proliferation, motility, invasion, angiogenesis, and premetastatic niche preparation. In addition, recent research implies that cancer cell-derived exosomes play a suppressive role in cancer-directed immune response. The biomarkers detected in bodily fluid-derived exosomes imply a potential for exosomes in cancer diagnosis. Also, exosomes could be used as a vehicle to selectively deliver therapeutic nucleic-acid drugs or conventional drugs for tumor therapy. The tolerability and feasibility of cancer exosomes in diagnosis and therapy need to be further evaluated. Copyright © 2014 Elsevier HS Journals, Inc. All rights reserved.

Circulating exosomes and exosomal microRNAs as biomarkers in gastrointestinal cancer.

PubMed

Nedaeinia, R; Manian, M; Jazayeri, M H; Ranjbar, M; Salehi, R; Sharifi, M; Mohaghegh, F; Goli, M; Jahednia, S H; Avan, A; Ghayour-Mobarhan, M

2017-02-01

The most important biological function of exosomes is their possible use as biomarkers in clinical diagnosis. Compared with biomarkers identified in conventional specimens such as serum or urine, exosomal biomarkers provide the highest amount of sensitivity and specificity, which can be attributed to their excellent stability. Exosomes, which harbor different types of proteins, nucleic acids and lipids, are present in almost all bodily fluids. The molecular constituents of exosomes, especially exosomal proteins and microRNAs (miRNAs), are promising as biomarkers in clinical diagnosis. This discovery that exosomes also contain messenger RNAs and miRNAs shows that they could be carriers of genetic information. Although the majority of RNAs found in exosomes are degraded RNA fragments with a length of <200 nucleotides, some full-length RNAs might be present that may affect protein production in the recipient cell. In addition, exosomal miRNAs have been found to be associated with certain diseases. Several studies have pointed out miRNA contents of circulating exosomes that are similar to those of originating cancer cells. In this review, the recent advances in circulating exosomal miRNAs as biomarkers in gastrointestinal cancers are discussed. These studies indicated that miRNAs can be detected in exosomes isolated from body fluids such as saliva, which suggests potential advantages of using exosomal miRNAs as noninvasive novel biomarkers.

Proinflammatory role of epithelial cell-derived exosomes in allergic airway inflammation.

PubMed

Kulshreshtha, Ankur; Ahmad, Tanveer; Agrawal, Anurag; Ghosh, Balaram

2013-04-01

Exosomes are nanovesicles involved in intercellular communication. Their roles in various diseases are often contextual, depending on the cell type producing them. Although few studies hint toward the proinflammatory role of bronchoalveolar lavage fluid-derived exosomes in asthmatic progression, the cell types in lungs associated with exosome-mediated crosstalk and their resultant effects remain unexplored. It is well established that exosome-mediated cellular communication can influence disease phenotypes. This study explores exosome-mediated cellular crosstalk between structural and immune cells in asthma pathogenesis. Exosomes were isolated and detected from bronchoalveolar lavage fluid of control and asthmatic mice and were quantified by using a bead-based assay. Involvement of epithelial cells and macrophages were established by using immunohistochemical techniques in lung tissue sections. The role of IL-13 in exosome production was ascertained by using various in vitro and in vivo techniques. Exosome secretion was blocked in in vitro and in vivo settings by using a chemical inhibitor, and the effects on various asthmatic features were studied. Using combinatorial in vitro and in vivo approaches, we found that exosome secretion and production of exosome-associated proteins are higher in lungs of asthmatic mice compared with that seen in sham mice. Asthma is marked by enhanced secretion of exosomes by epithelial cells, but not macrophages, under the influence of IL-13. These epithelial cell exosomes induce proliferation and chemotaxis of undifferentiated macrophages. On the other hand, GW4869, which inhibited exosome production, resulted in a reduced population of proliferating monocytes and alleviation of various asthmatic features. Under the influence of IL-13, epithelial cell-derived exosomes can induce enhanced proliferation and chemotaxis of undifferentiated macrophages in the lungs during asthmatic inflammatory conditions. Copyright © 2013 American

Tumor microenvironment derived exosomes pleiotropically modulate cancer cell metabolism.

PubMed

Zhao, Hongyun; Yang, Lifeng; Baddour, Joelle; Achreja, Abhinav; Bernard, Vincent; Moss, Tyler; Marini, Juan C; Tudawe, Thavisha; Seviour, Elena G; San Lucas, F Anthony; Alvarez, Hector; Gupta, Sonal; Maiti, Sourindra N; Cooper, Laurence; Peehl, Donna; Ram, Prahlad T; Maitra, Anirban; Nagrath, Deepak

2016-02-27

Cancer-associated fibroblasts (CAFs) are a major cellular component of tumor microenvironment in most solid cancers. Altered cellular metabolism is a hallmark of cancer, and much of the published literature has focused on neoplastic cell-autonomous processes for these adaptations. We demonstrate that exosomes secreted by patient-derived CAFs can strikingly reprogram the metabolic machinery following their uptake by cancer cells. We find that CAF-derived exosomes (CDEs) inhibit mitochondrial oxidative phosphorylation, thereby increasing glycolysis and glutamine-dependent reductive carboxylation in cancer cells. Through 13C-labeled isotope labeling experiments we elucidate that exosomes supply amino acids to nutrient-deprived cancer cells in a mechanism similar to macropinocytosis, albeit without the previously described dependence on oncogenic-Kras signaling. Using intra-exosomal metabolomics, we provide compelling evidence that CDEs contain intact metabolites, including amino acids, lipids, and TCA-cycle intermediates that are avidly utilized by cancer cells for central carbon metabolism and promoting tumor growth under nutrient deprivation or nutrient stressed conditions.

Exosomes Derived from HIV-1-infected Cells Contain Trans-activation Response Element RNA*

PubMed Central

Narayanan, Aarthi; Iordanskiy, Sergey; Das, Ravi; Van Duyne, Rachel; Santos, Steven; Jaworski, Elizabeth; Guendel, Irene; Sampey, Gavin; Dalby, Elizabeth; Iglesias-Ussel, Maria; Popratiloff, Anastas; Hakami, Ramin; Kehn-Hall, Kylene; Young, Mary; Subra, Caroline; Gilbert, Caroline; Bailey, Charles; Romerio, Fabio; Kashanchi, Fatah

2013-01-01

Exosomes are nano-sized vesicles produced by healthy and virus-infected cells. Exosomes derived from infected cells have been shown to contain viral microRNAs (miRNAs). HIV-1 encodes its own miRNAs that regulate viral and host gene expression. The most abundant HIV-1-derived miRNA, first reported by us and later by others using deep sequencing, is the trans-activation response element (TAR) miRNA. In this study, we demonstrate the presence of TAR RNA in exosomes from cell culture supernatants of HIV-1-infected cells and patient sera. TAR miRNA was not in Ago2 complexes outside the exosomes but enclosed within the exosomes. We detected the host miRNA machinery proteins Dicer and Drosha in exosomes from infected cells. We report that transport of TAR RNA from the nucleus into exosomes is a CRM1 (chromosome region maintenance 1)-dependent active process. Prior exposure of naive cells to exosomes from infected cells increased susceptibility of the recipient cells to HIV-1 infection. Exosomal TAR RNA down-regulated apoptosis by lowering Bim and Cdk9 proteins in recipient cells. We found 104–106 copies/ml TAR RNA in exosomes derived from infected culture supernatants and 103 copies/ml TAR RNA in the serum exosomes of highly active antiretroviral therapy-treated patients or long term nonprogressors. Taken together, our experiments demonstrated that HIV-1-infected cells produced exosomes that are uniquely characterized by their proteomic and RNA profiles that may contribute to disease pathology in AIDS. PMID:23661700

Value of Osteoblast-Derived Exosomes in Bone Diseases.

PubMed

Ge, Min; Wu, Yingzhi; Ke, Ronghu; Cai, Tianyi; Yang, Junyi; Mu, Xiongzheng

2017-06-01

The authors' purpose is to reveal the value of osteoblast-derived exosomes in bone diseases. Microvesicles from supernatants of mouse Mc3t3 were isolated by ultracentrifugation and then the authors presented the protein profile by proteomics analysis. The authors detected a total number of 1536 proteins by mass spectrometry and found 172 proteins overlap with bone database. The Ingenuity Pathway Analysis shows network of "Skeletal and Muscular System Development and Function, Developmental Disorder, Hereditary Disorder" and pathway about osteogenesis. EFNB1 and transforming growth factor beta receptor 3 in the network, LRP6, bone morphogenetic protein receptor type-1, and SMURF1 in the pathway seemed to be valuable in the exosome research of related bone disease. The authors' study unveiled the content of osteoblast-derived exosome and discussed valuable protein in it which might provide novel prospective in bone diseases research.

Systemic T Cells Immunosuppression of Glioma Stem Cell-Derived Exosomes Is Mediated by Monocytic Myeloid-Derived Suppressor Cells

PubMed Central

Domenis, Rossana; Cesselli, Daniela; Toffoletto, Barbara; Bourkoula, Evgenia; Caponnetto, Federica; Manini, Ivana; Beltrami, Antonio Paolo; Ius, Tamara; Skrap, Miran; Di Loreto, Carla

2017-01-01

A major contributing factor to glioma development and progression is its ability to evade the immune system. Nano-meter sized vesicles, exosomes, secreted by glioma-stem cells (GSC) can act as mediators of intercellular communication to promote tumor immune escape. Here, we investigated the immunomodulatory properties of GCS-derived exosomes on different peripheral immune cell populations. Healthy donor peripheral blood mononuclear cells (PBMCs) stimulated with anti-CD3, anti-CD28 and IL-2, were treated with GSC-derived exosomes. Phenotypic characterization, cell proliferation, Th1/Th2 cytokine secretion and intracellular cytokine production were analysed by distinguishing among effector T cells, regulatory T cells and monocytes. In unfractionated PBMCs, GSC-derived exosomes inhibited T cell activation (CD25 and CD69 expression), proliferation and Th1 cytokine production, and did not affect cell viability or regulatory T-cell suppression ability. Furthermore, exosomes were able to enhance proliferation of purified CD4+ T cells. In PBMCs culture, glioma-derived exosomes directly promoted IL-10 and arginase-1 production and downregulation of HLA-DR by unstimulated CD14+ monocytic cells, that displayed an immunophenotype resembling that of monocytic myeloid-derived suppressor cells (Mo-MDSCs). Importantly, the removal of CD14+ monocytic cell fraction from PBMCs restored T-cell proliferation. The same results were observed with exosomes purified from plasma of glioblastoma patients. Our results indicate that glioma-derived exosomes suppress T-cell immune response by acting on monocyte maturation rather than on direct interaction with T cells. Selective targeting of Mo-MDSC to treat glioma should be considered with regard to how immune cells allow the acquirement of effector functions and therefore counteracting tumor progression. PMID:28107450

Systemic T Cells Immunosuppression of Glioma Stem Cell-Derived Exosomes Is Mediated by Monocytic Myeloid-Derived Suppressor Cells.

PubMed

Domenis, Rossana; Cesselli, Daniela; Toffoletto, Barbara; Bourkoula, Evgenia; Caponnetto, Federica; Manini, Ivana; Beltrami, Antonio Paolo; Ius, Tamara; Skrap, Miran; Di Loreto, Carla; Gri, Giorgia

2017-01-01

A major contributing factor to glioma development and progression is its ability to evade the immune system. Nano-meter sized vesicles, exosomes, secreted by glioma-stem cells (GSC) can act as mediators of intercellular communication to promote tumor immune escape. Here, we investigated the immunomodulatory properties of GCS-derived exosomes on different peripheral immune cell populations. Healthy donor peripheral blood mononuclear cells (PBMCs) stimulated with anti-CD3, anti-CD28 and IL-2, were treated with GSC-derived exosomes. Phenotypic characterization, cell proliferation, Th1/Th2 cytokine secretion and intracellular cytokine production were analysed by distinguishing among effector T cells, regulatory T cells and monocytes. In unfractionated PBMCs, GSC-derived exosomes inhibited T cell activation (CD25 and CD69 expression), proliferation and Th1 cytokine production, and did not affect cell viability or regulatory T-cell suppression ability. Furthermore, exosomes were able to enhance proliferation of purified CD4+ T cells. In PBMCs culture, glioma-derived exosomes directly promoted IL-10 and arginase-1 production and downregulation of HLA-DR by unstimulated CD14+ monocytic cells, that displayed an immunophenotype resembling that of monocytic myeloid-derived suppressor cells (Mo-MDSCs). Importantly, the removal of CD14+ monocytic cell fraction from PBMCs restored T-cell proliferation. The same results were observed with exosomes purified from plasma of glioblastoma patients. Our results indicate that glioma-derived exosomes suppress T-cell immune response by acting on monocyte maturation rather than on direct interaction with T cells. Selective targeting of Mo-MDSC to treat glioma should be considered with regard to how immune cells allow the acquirement of effector functions and therefore counteracting tumor progression.

Exosomes derived from high-glucose-stimulated Schwann cells promote development of diabetic peripheral neuropathy.

PubMed

Jia, Longfei; Chopp, Michael; Wang, Lei; Lu, Xuerong; Szalad, Alexandra; Zhang, Zheng Gang

2018-06-22

Schwann cells actively interact with axons of dorsal root ganglia (DRG) neurons. Exosomes mediate intercellular communication by transferring their biomaterials, including microRNAs (miRs) into recipient cells. We hypothesized that exosomes derived from Schwann cells stimulated by high glucose (HG) exosomes accelerate development of diabetic peripheral neuropathy and that exosomal cargo miRs contribute to this process. We found that HG exosomes contained high levels of miR-28, -31a, and -130a compared to exosomes derived from non-HG-stimulated Schwann cells. In vitro, treatment of distal axons with HG exosomes resulted in reduction of axonal growth, which was associated with elevation of miR-28, -31a, and -130a and reduction of their target proteins of DNA methyltransferase-3α, NUMB (an endocytic adaptor protein), synaptosome associated protein 25, and growth-associated protein-43 in axons. In vivo, administration of HG exosomes to sciatic nerves of diabetic db/db mice at 7 wk of age promoted occurrence of peripheral neuropathy characterized by impairment of nerve conduction velocity and induction of mechanic and thermal hypoesthesia, which was associated with substantial decreases in intraepidermal nerve fibers. Our findings demonstrate a functional role of exosomes derived from HG-stimulated Schwann cells in mediating development of diabetic peripheral neuropathy.-Jia, L., Chopp, M., Wang, L., Lu, X., Szalad, A., Zhang, Z. G. Exosomes derived from high-glucose-stimulated Schwann cells promote development of diabetic peripheral neuropathy.

Tumor microenvironment derived exosomes pleiotropically modulate cancer cell metabolism

PubMed Central

Zhao, Hongyun; Yang, Lifeng; Baddour, Joelle; Achreja, Abhinav; Bernard, Vincent; Moss, Tyler; Marini, Juan C; Tudawe, Thavisha; Seviour, Elena G; San Lucas, F Anthony; Alvarez, Hector; Gupta, Sonal; Maiti, Sourindra N; Cooper, Laurence; Peehl, Donna; Ram, Prahlad T; Maitra, Anirban; Nagrath, Deepak

2016-01-01

Cancer-associated fibroblasts (CAFs) are a major cellular component of tumor microenvironment in most solid cancers. Altered cellular metabolism is a hallmark of cancer, and much of the published literature has focused on neoplastic cell-autonomous processes for these adaptations. We demonstrate that exosomes secreted by patient-derived CAFs can strikingly reprogram the metabolic machinery following their uptake by cancer cells. We find that CAF-derived exosomes (CDEs) inhibit mitochondrial oxidative phosphorylation, thereby increasing glycolysis and glutamine-dependent reductive carboxylation in cancer cells. Through 13C-labeled isotope labeling experiments we elucidate that exosomes supply amino acids to nutrient-deprived cancer cells in a mechanism similar to macropinocytosis, albeit without the previously described dependence on oncogenic-Kras signaling. Using intra-exosomal metabolomics, we provide compelling evidence that CDEs contain intact metabolites, including amino acids, lipids, and TCA-cycle intermediates that are avidly utilized by cancer cells for central carbon metabolism and promoting tumor growth under nutrient deprivation or nutrient stressed conditions. DOI: http://dx.doi.org/10.7554/eLife.10250.001 PMID:26920219

Exosomes derived from human macrophages suppress endothelial cell migration by controlling integrin trafficking.

PubMed

Lee, Hee Doo; Kim, Yeon Hyang; Kim, Doo-Sik

2014-04-01

Integrin trafficking, including internalization, recycling, and lysosomal degradation, is crucial for the regulation of cellular functions. Exosomes, nano-sized extracellular vesicles, are believed to play important roles in intercellular communications. This study demonstrates that exosomes released from human macrophages negatively regulate endothelial cell migration through control of integrin trafficking. Macrophage-derived exosomes promote internalization of integrin β1 in primary HUVECs. The internalized integrin β1 persistently accumulates in the perinuclear region and is not recycled back to the plasma membrane. Experimental results indicate that macrophage-derived exosomes stimulate trafficking of internalized integrin β1 to lysosomal compartments with a corresponding decrease in the integrin destined for recycling endosomes, resulting in proteolytic degradation of the integrin. Moreover, ubiquitination of HUVEC integrin β1 is enhanced by the exosomes, and exosome-mediated integrin degradation is blocked by bafilomycin A, a lysosomal degradation inhibitor. Macrophage-derived exosomes were also shown to effectively suppress collagen-induced activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway and HUVEC migration, which are both dependent on integrin β1. These observations provide new insight into the functional significance of exosomes in the regulation of integrin trafficking. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Pancreatic Cancer-Derived Exosomes Cause Paraneoplastic β-cell Dysfunction.

PubMed

Javeed, Naureen; Sagar, Gunisha; Dutta, Shamit K; Smyrk, Thomas C; Lau, Julie S; Bhattacharya, Santanu; Truty, Mark; Petersen, Gloria M; Kaufman, Randal J; Chari, Suresh T; Mukhopadhyay, Debabrata

2015-04-01

Pancreatic cancer frequently causes diabetes. We recently proposed adrenomedullin as a candidate mediator of pancreatic β-cell dysfunction in pancreatic cancer. How pancreatic cancer-derived adrenomedullin reaches β cells remote from the cancer to induce β-cell dysfunction is unknown. We tested a novel hypothesis that pancreatic cancer sheds adrenomedullin-containing exosomes into circulation, which are transported to β cells and impair insulin secretion. We characterized exosomes from conditioned media of pancreatic cancer cell lines (n = 5) and portal/peripheral venous blood of patients with pancreatic cancer (n = 20). Western blot analysis showed the presence of adrenomedullin in pancreatic cancer-exosomes. We determined the effect of adrenomedullin-containing pancreatic cancer exosomes on insulin secretion from INS-1 β cells and human islets, and demonstrated the mechanism of exosome internalization into β cells. We studied the interaction between β-cell adrenomedullin receptors and adrenomedullin present in pancreatic cancer-exosomes. In addition, the effect of adrenomedullin on endoplasmic reticulum (ER) stress response genes and reactive oxygen/nitrogen species generation in β cells was shown. Exosomes were found to be the predominant extracellular vesicles secreted by pancreatic cancer into culture media and patient plasma. Pancreatic cancer-exosomes contained adrenomedullin and CA19-9, readily entered β cells through caveolin-mediated endocytosis or macropinocytosis, and inhibited insulin secretion. Adrenomedullin in pancreatic cancer exosomes interacted with its receptor on β cells. Adrenomedullin receptor blockade abrogated the inhibitory effect of exosomes on insulin secretion. β cells exposed to adrenomedullin or pancreatic cancer exosomes showed upregulation of ER stress genes and increased reactive oxygen/nitrogen species. Pancreatic cancer causes paraneoplastic β-cell dysfunction by shedding adrenomedullin(+)/CA19-9(+) exosomes into

Exosomes of human placenta-derived mesenchymal stem cells stimulate angiogenesis.

PubMed

Komaki, Motohiro; Numata, Yuri; Morioka, Chikako; Honda, Izumi; Tooi, Masayuki; Yokoyama, Naoki; Ayame, Hirohito; Iwasaki, Kengo; Taki, Atsuko; Oshima, Noriko; Morita, Ikuo

2017-10-03

The therapeutic potential of mesenchymal stem cells (MSCs) may be attributed partly to humoral factors such as growth factors, cytokines, and chemokines. Human term placental tissue-derived MSCs (PlaMSCs), or conditioned medium left over from cultures of these cells, have been reported to enhance angiogenesis. Recently, the exosome, which can transport a diverse suite of macromolecules, has gained attention as a novel intercellular communication tool. However, the potential role of the exosome in PlaMSC therapeutic action is not well understood. The purpose of this study was to evaluate PlaMSC-derived exosome angiogenesis promotion in vitro and in vivo. MSCs were isolated from human term placental tissue by enzymatic digestion. Conditioned medium was collected after 48-h incubation in serum-free medium (PlaMSC-CM). Angiogenic factors present in PlaMSC-CM were screened by a growth factor array. Exosomes were prepared by ultracentrifugation of PlaMSC-CM, and confirmed by transmission electron microscopy, dynamic light scattering, and western blot analyses. The proangiogenic activity of PlaMSC-derived exosomes (PlaMSC-exo) was assessed using an endothelial tube formation assay, a cell migration assay, and reverse transcription-PCR analysis. The in-vivo angiogenic activity of PlaMSC-exo was evaluated using a murine auricle ischemic injury model. PlaMSC-CM contained both angiogenic and angiostatic factors, which enhanced endothelial tube formation. PlaMSC-exo were incorporated into endothelial cells; these exosomes stimulated both endothelial tube formation and migration, and enhanced angiogenesis-related gene expression. Laser Doppler blood flow analysis showed that PlaMSC-exo infusion also enhanced angiogenesis in an in-vivo murine auricle ischemic injury model. PlaMSC-exo enhanced angiogenesis in vitro and in vivo, suggesting that exosomes play a role in the proangiogenic activity of PlaMSCs. PlaMSC-exo may be a novel therapeutic approach for treating ischemic

Cardiac progenitor-derived exosomes protect ischemic myocardium from acute ischemia/reperfusion injury

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Lijuan; Cardiovascular Disease, Internal Medicine, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH 45267; Wang, Yingjie

Highlights: ► Cardiac progenitor-derived (CPC) Exosomes protect H9C2 from apoptosis in vitro. ► CPC-exosomes protect cardiomyoyctes from MI/R induced apoptosis in vivo. ► CPC-exosomes were taken up by H9C2 with high efficiency using PKH26 labeling. ► miR-451, one of GATA4-responsive miRNA cluster, is enriched in CPC-exosomes. -- Abstract: Background: Cardiac progenitors (CPC) mediate cardioprotection via paracrine effects. To date, most of studies focused on secreted paracrine proteins. Here we investigated the CPC-derived-exosomes on protecting myocardium from acute ischemia/reperfusion (MI/R) injury. Methods and results: CPC were isolated from mouse heart using two-step protocol. Exosomes were purified from conditional medium, and confirmedmore » by electron micrograph and Western blot using CD63 as a marker. qRT-PCR shows that CPC-exosomes have high level expression of GATA4-responsive-miR-451. Exosomes were ex vivo labeled with PKH26, We observed exosomes can be uptaken by H9C2 cardiomyoblasts with high efficiency after 12 h incubation. CPC-exosomes protect H9C2 from oxidative stress by inhibiting caspase 3/7 activation invitro. In vivo delivery of CPC-exosomes in an acute mouse myocardial ischemia/reperfusion model inhibited cardiomyocyte apoptosis by about 53% in comparison with PBS control (p < 0.05). Conclusion: Our results suggest, for the first time, the CPC-exosomes can be used as a therapeutic vehicle for cardioprotection, and highlights a new perspective for using non-cell exosomes for cardiac disease.« less

Gold-carbon dots for the intracellular imaging of cancer-derived exosomes.

PubMed

Jiang, Xiaoyue; Zong, Shenfei; Chen, Chen; Zhang, Yizhi; Wang, Zhuyuan; Cui, Yiping

2018-04-27

As a novel fluorescent nanomaterial, gold-carbon quantum dots (GCDs) possess high biocompatibility and can be easily synthesized by a microwave-assisted method. Owing to their small sizes and unique optical properties, GCDs can be applied to imaging of biological targets, such as cells, exosomes and other organelles. In this study, GCDs were used for fluorescence imaging of exosomes. Tumor-specific antibodies are attached to the GCDs, forming exosome specific nanoprobes. The nanoprobes can label exosomes via immuno-reactions and thus facilitate fluorescent imaging of exosomes. When incubated with live cells, exosomes labeled with the nanoprobes can be taken up by the cells. The intracellular experiments confirmed that the majority of exosomes were endocytosed by cells and transported to lysosomes. The manner by which exosomes were taken up and the intracellular distribution of exosomes are unaffected by the GCDs. The experimental results successfully demonstrated that the presented nanoprobe can be used to study the intrinsic intracellular behavior of tumor derived exosomes. We believe that the GCDs based nanoprobe holds a great promise in the study of exosome related cellular events, such as cancer metastasis.

Gold-carbon dots for the intracellular imaging of cancer-derived exosomes

NASA Astrophysics Data System (ADS)

Jiang, Xiaoyue; Zong, Shenfei; Chen, Chen; Zhang, Yizhi; Wang, Zhuyuan; Cui, Yiping

2018-04-01

As a novel fluorescent nanomaterial, gold-carbon quantum dots (GCDs) possess high biocompatibility and can be easily synthesized by a microwave-assisted method. Owing to their small sizes and unique optical properties, GCDs can be applied to imaging of biological targets, such as cells, exosomes and other organelles. In this study, GCDs were used for fluorescence imaging of exosomes. Tumor-specific antibodies are attached to the GCDs, forming exosome specific nanoprobes. The nanoprobes can label exosomes via immuno-reactions and thus facilitate fluorescent imaging of exosomes. When incubated with live cells, exosomes labeled with the nanoprobes can be taken up by the cells. The intracellular experiments confirmed that the majority of exosomes were endocytosed by cells and transported to lysosomes. The manner by which exosomes were taken up and the intracellular distribution of exosomes are unaffected by the GCDs. The experimental results successfully demonstrated that the presented nanoprobe can be used to study the intrinsic intracellular behavior of tumor derived exosomes. We believe that the GCDs based nanoprobe holds a great promise in the study of exosome related cellular events, such as cancer metastasis.

Exosomes derived from gastric cancer cells activate NF-κB pathway in macrophages to promote cancer progression.

PubMed

Wu, Lijun; Zhang, Xu; Zhang, Bin; Shi, Hui; Yuan, Xiao; Sun, Yaoxiang; Pan, Zhaoji; Qian, Hui; Xu, Wenrong

2016-09-01

Exosomes are nano-sized membrane vesicles secreted by both normal and cancer cells. Emerging evidence indicates that cancer cells derived exosomes contribute to cancer progression through the modulation of tumor microenvironment. However, the effects of exosomes derived from gastric cancer cells on macrophages are not well understood. In this study, we investigated the biological role of gastric cancer cells derived exosomes in the activation of macrophages. We demonstrated that gastric cancer cells derived exosomes activated macrophages to express increased levels of proinflammatory factors, which in turn promoted tumor cell proliferation and migration. In addition, gastric cancer cells derived exosomes remarkably upregulated the phosphorylation of NF-κB in macrophages. Inhibiting the activation of NF-κB reversed the upregulation of proinflammatory factors in macrophages and blocked their promoting effects on gastric cancer cells. Moreover, we found that gastric cancer cells derived exosomes could also activate macrophages from human peripheral blood monocytes through the activation of NF-κB. In conclusion, our results suggest that gastric cancer cells derived exosomes stimulate the activation of NF-κB pathway in macrophages to promote cancer progression, which provides a potential therapeutic approach for gastric cancer by interfering with the interaction between exosomes and macrophages in tumor microenvironment.

The Complete Exosome Workflow Solution: From Isolation to Characterization of RNA Cargo

PubMed Central

Schageman, Jeoffrey; Li, Mu; Barta, Tim; Lea, Kristi; Gu, Jian; Magdaleno, Susan; Setterquist, Robert; Vlassov, Alexander V.

2013-01-01

Exosomes are small (30–150 nm) vesicles containing unique RNA and protein cargo, secreted by all cell types in culture. They are also found in abundance in body fluids including blood, saliva, and urine. At the moment, the mechanism of exosome formation, the makeup of the cargo, biological pathways, and resulting functions are incompletely understood. One of their most intriguing roles is intercellular communication—exosomes function as the messengers, delivering various effector or signaling macromolecules between specific cells. There is an exponentially growing need to dissect structure and the function of exosomes and utilize them for development of minimally invasive diagnostics and therapeutics. Critical to further our understanding of exosomes is the development of reagents, tools, and protocols for their isolation, characterization, and analysis of their RNA and protein contents. Here we describe a complete exosome workflow solution, starting from fast and efficient extraction of exosomes from cell culture media and serum to isolation of RNA followed by characterization of exosomal RNA content using qRT-PCR and next-generation sequencing techniques. Effectiveness of this workflow is exemplified by analysis of the RNA content of exosomes derived from HeLa cell culture media and human serum, using Ion Torrent PGM as a sequencing platform. PMID:24205503

Exosomes derived from pancreatic cancer cells induce activation and profibrogenic activities in pancreatic stellate cells.

PubMed

Masamune, Atsushi; Yoshida, Naoki; Hamada, Shin; Takikawa, Tetsuya; Nabeshima, Tatsuhide; Shimosegawa, Tooru

2018-01-01

Pancreatic cancer cells (PCCs) interact with pancreatic stellate cells (PSCs), which play a pivotal role in pancreatic fibrogenesis, to develop the cancer-conditioned tumor microenvironment. Exosomes are membrane-enclosed nanovesicles, and have been increasingly recognized as important mediators of cell-to-cell communications. The aim of this study was to clarify the effects of PCC-derived exosomes on cell functions in PSCs. Exosomes were isolated from the conditioned medium of Panc-1 and SUIT-2 PCCs. Human primary PSCs were treated with PCC-derived exosomes. PCC-derived exosomes stimulated the proliferation, migration, activation of ERK and Akt, the mRNA expression of α-smooth muscle actin (ACTA2) and fibrosis-related genes, and procollagen type I C-peptide production in PSCs. Ingenuity pathway analysis of the microarray data identified transforming growth factor β1 and tumor necrosis factor as top upstream regulators. PCCs increased the expression of miR-1246 and miR-1290, abundantly contained in PCC-derived exosomes, in PSCs. Overexpression of miR-1290 induced the expression of ACTA2 and fibrosis-related genes in PSCs. In conclusion, PCC-derived exosomes stimulate activation and profibrogenic activities in PSCs. Exosome-mediated interactions between PSCs and PCCs might play a role in the development of the tumor microenvironment. Copyright © 2017 Elsevier Inc. All rights reserved.

Cisplatin-resistant lung cancer cell-derived exosomes increase cisplatin resistance of recipient cells in exosomal miR-100-5p-dependent manner.

PubMed

Qin, Xiaobing; Yu, Shaorong; Zhou, Leilei; Shi, Meiqi; Hu, Yong; Xu, Xiaoyue; Shen, Bo; Liu, Siwen; Yan, Dali; Feng, Jifeng

2017-01-01

Exosomes derived from lung cancer cells confer cisplatin (DDP) resistance to other cancer cells. However, the underlying mechanism is still unknown. A549 resistance to DDP (A549/DDP) was established. Microarray was used to analyze microRNA (miRNA) expression profiles of A549 cells, A549/DDP cells, A549 exosomes, and A549/DDP exosomes. There was a strong correlation of miRNA profiles between exosomes and their maternal cells. A total of 11 miRNAs were significantly upregulated both in A549/DDP cells compared with A549 cells and in exosomes derived from A549/DDP cells in contrast to exosomes from A549 cells. A total of 31 downregulated miRNAs were also observed. miR-100-5p was the most prominent decreased miRNA in DDP-resistant exosomes compared with the corresponding sensitive ones. Downregulated miR-100-5p was proved to be involved in DDP resistance in A549 cells, and mammalian target of rapamycin (mTOR) expression was reverse regulated by miR-100-5p. Exosomes confer recipient cells' resistance to DDP in an exosomal miR-100-5p-dependent manner with mTOR as its potential target both in vitro and in vivo. Exosomes from DDP-resistant lung cancer cells A549 can alter other lung cancer cells' sensitivity to DDP in exosomal miR-100-5p-dependent manner. Our study provides new insights into the molecular mechanism of DDP resistance in lung cancer.

Exosomes Derived from Mesenchymal Stromal Cells Promote Axonal Growth of Cortical Neurons.

PubMed

Zhang, Yi; Chopp, Michael; Liu, Xian Shuang; Katakowski, Mark; Wang, Xinli; Tian, Xinchu; Wu, David; Zhang, Zheng Gang

2017-05-01

Treatment of brain injury with exosomes derived from mesenchymal stromal cells (MSCs) enhances neurite growth. However, the direct effect of exosomes on axonal growth and molecular mechanisms underlying exosome-enhanced neurite growth are not known. Using primary cortical neurons cultured in a microfluidic device, we found that MSC-exosomes promoted axonal growth, whereas attenuation of argonaut 2 protein, one of the primary microRNA (miRNA) machinery proteins, in MSC-exosomes abolished their effect on axonal growth. Both neuronal cell bodies and axons internalized MSC-exosomes, which was blocked by botulinum neurotoxins (BoNTs) that cleave proteins of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Moreover, tailored MSC-exosomes carrying elevated miR-17-92 cluster further enhanced axonal growth compared to native MSC-exosomes. Quantitative RT-PCR and Western blot analysis showed that the tailored MSC-exosomes increased levels of individual members of this cluster and activated the PTEN/mTOR signaling pathway in recipient neurons, respectively. Together, our data demonstrate that native MSC-exosomes promote axonal growth while the tailored MSC-exosomes can further boost this effect and that tailored exosomes can deliver their selective cargo miRNAs into and activate their target signals in recipient neurons. Neuronal internalization of MSC-exosomes is mediated by the SNARE complex. This study reveals molecular mechanisms that contribute to MSC-exosome-promoted axonal growth, which provides a potential therapeutic strategy to enhance axonal growth.

Characterization of exosomes derived from ovarian cancer cells and normal ovarian epithelial cells by nanoparticle tracking analysis.

PubMed

Zhang, Wei; Peng, Peng; Kuang, Yun; Yang, Jiaxin; Cao, Dongyan; You, Yan; Shen, Keng

2016-03-01

Cellular exosomes are involved in many disease processes and have the potential to be used for diagnosis and treatment. In this study, we compared the characteristics of exosomes derived from human ovarian epithelial cells (HOSEPiC) and three epithelial ovarian cancer cell lines (OVCAR3, IGROV1, and ES-2) to investigate the differences between exosomes originating from normal and malignant cells. Two established colloid-chemical methodologies, electron microscopy (EM) and dynamic light scattering (DLS), and a relatively new method, nanoparticle tracking analysis (NTA), were used to measure the size and size distribution of exosomes. The concentration and epithelial cellular adhesion molecule (EpCAM) expression of exosomes were measured by NTA. Quantum dots were conjugated with anti-EpCAM to label exosomes, and the labeled exosomes were detected by NTA in fluorescent mode. The normal-cell-derived exosomes were significantly larger than those derived from malignant cells, and exosomes were successfully labeled using anti-EpCAM-conjugated quantum dots. Exosomes from different cell lines may vary in size, and exosomes might be considered as potential diagnosis biomarkers. NTA can be considered a useful, efficient, and objective method for the study of different exosomes and their unique properties in ovarian cancer.

Brain-derived exosomes from dementia with Lewy bodies propagate α-synuclein pathology.

PubMed

Ngolab, Jennifer; Trinh, Ivy; Rockenstein, Edward; Mante, Michael; Florio, Jazmin; Trejo, Margarita; Masliah, Deborah; Adame, Anthony; Masliah, Eliezer; Rissman, Robert A

2017-06-09

Proteins implicated in neurodegenerative conditions such as Alzheimer's disease (AD) and Dementia with Lewy Bodies (DLB) have been identified in bodily fluids encased in extracellular vesicles called exosomes. Whether exosomes found in DLB patients can transmit pathology is not clear. In this study, exosomes were successfully harvested through ultracentrifugation from brain tissue from DLB and AD patients as well as non-diseased brain tissue. Exosomes extracted from brains diagnosed with either AD or DLB contained aggregate-prone proteins. Furthermore, injection of brain-derived exosomes from DLB patients into the brains of wild type mice induced α-synuclein (α-syn) aggregation. As assessed through immunofluorescent double labeling, α-syn aggregation was observed in MAP2 + , Rab5 + neurons. Using a neuronal cell line, we also identified intracellular α-syn aggregation mediated by exosomes is dependent on recipient cell endocytosis. Together, these data suggest that exosomes from DLB patients are sufficient for seeding and propagating α-syn aggregation in vivo.

Pathogenic Role of Exosomes in Epstein-Barr Virus (EBV)-Associated Cancers

PubMed Central

Teow, Sin-Yeang; Liew, Kitson; Khoo, Alan Soo-Beng; Peh, Suat-Cheng

2017-01-01

Exosomes are 40- to 100-nm membrane-bound small vesicles that carry a great variety of cellular cargoes including proteins, DNA, messenger RNAs (mRNAs), and microRNAs (miRNAs). These nanovesicles are detected in various biological fluids such as serum, urine, saliva, and seminal fluids. Exosomes serve as key mediators in intercellular communication by facilitating the transfer and exchange of cellular components from cells to cells. They contain various pathogenic factors whereby their adverse effects have been implicated in multiple viral infections and cancers. Interestingly, accumulating evidences showed that exosomes derived from tumour viruses or oncoviruses, exacerbate virus-associated cancers by remodelling the tumour microenvironment. In this review, we summarize the contributing factors of Epstein-Barr virus (EBV) products-containing exosomes in viral pathogenesis and their potential implications in EBV-driven malignancies. Understanding the biological role of these exosomes in the disease would undoubtedly boost the development of a more comprehensive strategy to combat EBV-associated cancers and to better predict the therapeutic outcomes. Furthermore, we also highlight the potentials and challenges of EBV products-containing exosomes being employed as diagnostic markers and therapeutic targets for EBV-related cancers. Since these aspects are rather underexplored, we attempt to underline interesting areas that warrant further investigations in the future. PMID:29104494

Cardiac progenitor cell‑derived exosomes promote H9C2 cell growth via Akt/mTOR activation.

PubMed

Li, Shentang; Jiang, Jie; Yang, Zuocheng; Li, Zhuoying; Ma, Xing; Li, Xin

2018-05-21

Exosomes are cell‑derived vesicles released from a variety of mammalian cells that are involved in cell‑to‑cell signalling. It has been reported that cardiac progenitor cells (CPCs) derived from an adult heart are one of the most promising stem cell types for cardioprotection and repair. The mammalian target of rapamycin (mTOR) signalling pathway is a pivotal regulator in CPCs, therefore, CPC‑derived exosomes were used in the present study to investigate whether it can promote H9C2 cell growth through the protein kinase B (PKB, or Akt)/mTOR signalling pathway. The CPCs were isolated from Sprague‑Dawley hearts. Following treatment with a specific medium, the exosomes were purified and identified by electron micrograph and western blot assays, using CD63 and CD81 as markers. The methyl‑thiazolyl‑tetrazolium and 5‑ethynyl‑2'‑deoxyuridine methods were used to detect H9C2 cell growth. The expression of Akt and mTOR were detected by western blot analysis following treatment with 200 or 400 µg/ml of exosomes for 24 or 48 h, respectively. It was found that, compared with higher concentrations of exosomes, prolonging the duration of exposure promoted cell growth. Accordingly, CPC‑derived exosomes stimulated the expression of Akt to a marked degree; groups treated with exosomes for 48 h showed higher expression of Akt than those treated for 24 h at the same concentration. mTOR was also stimulated by CPC‑derived exosomes. The activation of mTOR increased in accordance with the treatment time at an exosome concentration of 200 µg/ml and decreased with treatment time at an exosome concentration of 400 µg/ml. In conclusion, the present study demonstrated that CPC‑derived exosomes promoted H9C2 cell growth via the activation of Akt/mTOR in a time‑dependent manner at a relatively low exosome concentration, which may provide a novel therapy for cardiovascular disease.

Exosome-based tumor antigens-adjuvant co-delivery utilizing genetically engineered tumor cell-derived exosomes with immunostimulatory CpG DNA.

PubMed

Morishita, Masaki; Takahashi, Yuki; Matsumoto, Akihiro; Nishikawa, Makiya; Takakura, Yoshinobu

2016-12-01

For cancer immunotherapy via tumor antigen vaccination in combination with an adjuvant, major challenges include the identification of a particular tumor antigen and efficient delivery of the antigen as well as adjuvant to antigen-presenting cells. In this study, we proposed an efficient exosome-based tumor antigens-adjuvant co-delivery system using genetically engineered tumor cell-derived exosomes containing endogenous tumor antigens and immunostimulatory CpG DNA. Murine melanoma B16BL6 cells were transfected with a plasmid vector encoding a fusion streptavidin (SAV; a protein that binds to biotin with high affinity)-lactadherin (LA; an exosome-tropic protein) protein, yielding genetically engineered SAV-LA-expressing exosomes (SAV-exo). SAV-exo were combined with biotinylated CpG DNA to prepare CpG DNA-modified exosomes (CpG-SAV-exo). Fluorescent microscopic observation revealed the successful modification of exosomes with CpG DNA by SAV-biotin interaction. CpG-SAV-exo showed efficient and simultaneous delivery of exosomes with CpG DNA to murine dendritic DC2.4 cells in culture. Treatment with CpG-SAV-exo effectively activated DC2.4 cells and enhanced tumor antigen presentation capacity. Immunization with CpG-SAV-exo exhibited stronger in vivo antitumor effects in B16BL6 tumor-bearing mice than simple co-administration of exosomes and CpG DNA. Thus, genetically engineered CpG-SAV-exo is an effective exosome-based tumor antigens-adjuvant co-delivery system that will be useful for cancer immunotherapy. Copyright © 2016 Elsevier Ltd. All rights reserved.

Bovine milk-derived exosomes for drug delivery

PubMed Central

Gupta, Ramesh C.

2015-01-01

Exosomes are biological nanovesicles that are involved in cell-cell communication via the functionally-active cargo (such as miRNA, mRNA, DNA and proteins). Because of their nanosize, exosomes are explored as nanodevices for the development of new therapeutic applications. However, bulk, safe and cost-effective production of exosomes is not available. Here, we show that bovine milk can serve as a scalable source of exosomes that can act as a carrier for chemotherapeutic/chemopreventive agents. Drug-loaded exosomes showed significantly higher efficacy compared to free drug in cell culture studies and against lung tumor xenografts in vivo. Moreover, tumor targeting ligands such as folate increased cancer-cell targeting of the exosomes resulting in enhanced tumor reduction. Milk exosomes exhibited cross-species tolerance with no adverse immune and inflammatory response. Thus, we show the versatility of milk exosomes with respect to the cargo it can carry and ability to achieve tumor targetability. This is the first report to identify a biocompatible and cost-effective means of exosomes to enhance oral bioavailability, improve efficacy and safety of drugs. PMID:26604130

Human fibrocyte-derived exosomes accelerate wound healing in genetically diabetic mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Geiger, Adolf, E-mail: ageiger@dreirosen-pharma.com; Walker, Audrey, E-mail: awalker@dreirosen-pharma.com; Nissen, Erwin, E-mail: enissen@dreirosen-pharma.com

Diabetic ulcers represent a substantial societal and healthcare burden worldwide and scarcely respond to current treatment strategies. This study was addressed to evaluate the therapeutic potential of exosomes secreted by human circulating fibrocytes, a population of mesenchymal progenitors involved in normal wound healing via paracrine signaling. The exosomes released from cells sequentially stimulated with platelet-derived growth factor-BB and transforming growth factor-β1, in the presence of fibroblast growth factor 2, did not show potential immunogenicity. These exosomes exhibited in-vitro proangiogenic properties, activated diabetic dermal fibroblasts, induced the migration and proliferation of diabetic keratinocytes, and accelerated wound closure in diabetic mice in vivo.more » Important components of the exosomal cargo were heat shock protein-90α, total and activated signal transducer and activator of transcription 3, proangiogenic (miR-126, miR-130a, miR-132) and anti-inflammatory (miR124a, miR-125b) microRNAs, and a microRNA regulating collagen deposition (miR-21). This proof-of-concept study demonstrates the feasibility of the use of fibrocytes-derived exosomes for the treatment of diabetic ulcers. - Highlights: • Fibrocytes have shown potent wound healing properties in vitro and in vivo. • Their clinical use is precluded by low numbers and antigen-presenting function. • We isolated exosomes with no immunogenicity potential from human fibrocytes. • Their cargo included microRNAs and proteins that are known healing promoters. • They accelerated wound closure in diabetic mice in a dose-dependent manner.« less

Dendritic Cells Pulsed with Leukemia Cell-Derived Exosomes More Efficiently Induce Antileukemic Immunities

PubMed Central

Wei, Wei; Shen, Chang; Deng, Xiaohui; Chen, Linjun; Ma, Liyuan; Hao, Siguo

2014-01-01

Dendritic cells (DCs) and tumor cell-derived exosomes have been used to develop antitumor vaccines. However, the biological properties and antileukemic effects of leukemia cell-derived exosomes (LEXs) are not well described. In this study, the biological properties and induction of antileukemic immunity of LEXs were investigated using transmission electron microscopy, western blot analysis, cytotoxicity assays, and animal studies. Similar to other tumor cells, leukemia cells release exosomes. Exosomes derived from K562 leukemia cells (LEXK562) are membrane-bound vesicles with diameters of approximately 50–100 μm and harbor adhesion molecules (e.g., intercellular adhesion molecule-1) and immunologically associated molecules (e.g., heat shock protein 70). In cytotoxicity assays and animal studies, LEXs-pulsed DCs induced an antileukemic cytotoxic T-lymphocyte immune response and antileukemic immunity more effectively than did LEXs and non-pulsed DCs (P<0.05). Therefore, LEXs may harbor antigens and immunological molecules associated with leukemia cells. As such, LEX-based vaccines may be a promising strategy for prolonging disease-free survival in patients with leukemia after chemotherapy or hematopoietic stem cell transplantation. PMID:24622345

Intercellular Communication by Exosome-Derived microRNAs in Cancer

PubMed Central

Hannafon, Bethany N.; Ding, Wei-Qun

2013-01-01

The development of human cancers is a multistep process in which normal cells acquire characteristics that ultimately lead to their conversion into cancer cells. Many obstacles must be overcome for this process to occur; of these obstacles, is the ability to survive an inhospitable microenvironment. It is recognized that the intercommunication between tumor cells and their surrounding microenvironment is essential to overcoming this obstacle and for the tumor to progress, metastasize and establish itself at distant sites. Exosomes are membrane-derived vesicles that have recently been recognized as important mediators of intercellular communication, as they carry lipids, proteins, mRNAs and microRNAs that can be transferred to a recipient cell via fusion of the exosome with the target cell membrane. In the context of cancer cells, this process entails the transfer of cancer-promoting cellular contents to surrounding cells within the tumor microenvironment or into the circulation to act at distant sites, thereby enabling cancer progression. In this process, the transfer of exosomal microRNAs to a recipient cell where they can regulate target gene expression is of particular interest, both in understanding the basic biology of cancer progression and for the development of therapeutic approaches. This review discusses the exosome-mediated intercellular communication via microRNAs within the tumor microenvironment in human cancers, with a particular focus on breast cancer exosomes. PMID:23839094

Exosomes derived from human mesenchymal stem cells confer drug resistance in gastric cancer

PubMed Central

Ji, Runbi; Zhang, Bin; Zhang, Xu; Xue, Jianguo; Yuan, Xiao; Yan, Yongmin; Wang, Mei; Zhu, Wei; Qian, Hui; Xu, Wenrong

2015-01-01

Mesenchymal stem cells (MSCs) play an important role in chemoresistance. Exosomes have been reported to modify cellular phenotype and function by mediating cell-cell communication. In this study, we aimed to investigate whether exosomes derived from MSCs (MSC-exosomes) are involved in mediating the resistance to chemotherapy in gastric cancer and to explore the underlying molecular mechanism. We found that MSC-exosomes significantly induced the resistance of gastric cancer cells to 5-fluorouracil both in vivo and ex vivo. MSC-exosomes antagonized 5-fluorouracil-induced apoptosis and enhanced the expression of multi-drug resistance associated proteins, including MDR, MRP and LRP. Mechanistically, MSC-exosomes triggered the activation of calcium/calmodulin-dependent protein kinases (CaM-Ks) and Raf/MEK/ERK kinase cascade in gastric cancer cells. Blocking the CaM-Ks/Raf/MEK/ERK pathway inhibited the promoting role of MSC-exosomes in chemoresistance. Collectively, MSC-exosomes could induce drug resistance in gastric cancer cells by activating CaM-Ks/Raf/MEK/ERK pathway. Our findings suggest that MSC-exosomes have profound effects on modifying gastric cancer cells in the development of drug resistance. Targeting the interaction between MSC-exosomes and cancer cells may help improve the efficacy of chemotherapy in gastric cancer. PMID:26091251

Exosomes derived from human mesenchymal stem cells confer drug resistance in gastric cancer.

PubMed

Ji, Runbi; Zhang, Bin; Zhang, Xu; Xue, Jianguo; Yuan, Xiao; Yan, Yongmin; Wang, Mei; Zhu, Wei; Qian, Hui; Xu, Wenrong

2015-08-03

Mesenchymal stem cells (MSCs) play an important role in chemoresistance. Exosomes have been reported to modify cellular phenotype and function by mediating cell-cell communication. In this study, we aimed to investigate whether exosomes derived from MSCs (MSC-exosomes) are involved in mediating the resistance to chemotherapy in gastric cancer and to explore the underlying molecular mechanism. We found that MSC-exosomes significantly induced the resistance of gastric cancer cells to 5-fluorouracil both in vivo and ex vivo. MSC-exosomes antagonized 5-fluorouracil-induced apoptosis and enhanced the expression of multi-drug resistance associated proteins, including MDR, MRP and LRP. Mechanistically, MSC-exosomes triggered the activation of calcium/calmodulin-dependent protein kinases (CaM-Ks) and Raf/MEK/ERK kinase cascade in gastric cancer cells. Blocking the CaM-Ks/Raf/MEK/ERK pathway inhibited the promoting role of MSC-exosomes in chemoresistance. Collectively, MSC-exosomes could induce drug resistance in gastric cancer cells by activating CaM-Ks/Raf/MEK/ERK pathway. Our findings suggest that MSC-exosomes have profound effects on modifying gastric cancer cells in the development of drug resistance. Targeting the interaction between MSC-exosomes and cancer cells may help improve the efficacy of chemotherapy in gastric cancer.

Phenotypic modulation of auto-reactive cells by insertion of tolerogenic molecules via MSC-derived exosomes.

PubMed

Mokarizadeh, Aram; Delirezh, Nowruz; Morshedi, Ahhmad; Mosayebi, Ghasem; Farshid, Amir-Abbas; Dalir-Naghadeh, Bahram

2012-01-01

Auto-reactive cells-mediated immune responses are responsible for the current tissue damages during autoimmunity. Accordingly, functional modulation of auto-reactive cells has been a pivotal aim in many of recent studies. In the current study, we investigated the possibility for insertion of regulatory molecules onto auto-reactive cells through exosomal nano-shuttles as a novel approach for phenotype modification of auto-reactive cells. The exosomes were isolated from supernatant of mesenchymal stem cells culture. Resultant exosomes co-cultured with lymphocytes were harvested from established EAE mice in the presence of antigenic MOG35-55 peptide. After 24 hr, insertion of exosomal tolerogenic molecules (PD-L1, TGF-β, galectin-1) onto auto-reactive cells were explored through flow cytometry. The potency of exosomal inserted membrane molecules to modulate phenotype of auto-reactive lymphocytes was assessed upon ELISA test for their-derived cytokines IFN-γ and IL-17. Incorporation of exosomal molecules into lymohocytes' membrane was confirmed by flow cytometric analyses for surface levels of mentioned molecules. Additionally, the decreased secretion of IFN-γ and IL-17 were detected in exosome pre-treated lymphocytes upon stimulation with MOG peptide. Mesenchymal stem cells -derived exosomes showed to be efficient organelles for insertion of bioactive tolerogenic molecules onto auto-reactive cells and modulation of their phenotypes.

Fermentation Results in Quantitative Changes in Milk-Derived Exosomes and Different Effects on Cell Growth and Survival.

PubMed

Yu, Siran; Zhao, Zhehao; Sun, Liming; Li, Ping

2017-02-15

The discovery of microRNAs encapsulated in milk-derived exosomes has revealed stability under extreme conditions reflecting the protection of membranes. We attempted to determine the variations in nanoparticles derived from milk after fermentation, and provide evidence to determine the effects of these exosomes on cells with potential bioactivity. Using scanning electron microscopy and dynamic light scattering, we compared the morphology and particle size distribution of exosomes from yogurt fermented with three different combinations of strains with those from raw milk. The protein content of the exosome was significantly reduced in fermented milk. The cycle threshold showed that the expression of miR-29b and miR-21 was relatively high in raw milk, indicating a loss of microRNA after fermentation. Milk-derived exosomes could promote cell growth and activate the mitogen-activated protein kinase pathway. These findings demonstrated biological functions in milk exosomes and provided new insight into the nutrient composition of dairy products.

Endothelial cell-derived exosomes protect SH-SY5Y nerve cells against ischemia/reperfusion injury.

PubMed

Xiao, Bing; Chai, Yi; Lv, Shigang; Ye, Minhua; Wu, Miaojing; Xie, Liyuan; Fan, Yanghua; Zhu, Xingen; Gao, Ziyun

2017-10-01

Cerebral ischemia is a leading cause of death and disability. A previous study indicated that remote ischemic postconditioning (RIP) in the treatment of cerebral ischemia reduces ischemia/reperfusion (I/R) injury. However, the underlying mechanism is not well understood. In the present study, the authors hypothesized that the protective effect of RIP on neurological damage is mediated by exosomes that are released by endothelial cells in femoral arteries. To test this, right middle cerebral artery occlusion/reperfusion with RIP was performed in rats. In addition, an I/R injury cell model was tested that included human umbilical vein endothelial cells (HUVECs) and SH-SY5Y cells. Both the in vivo and in vitro models were examined for injury. Markers of exosomes (CD63, HSP70 and TSG101) were assessed by immunohistochemistry, western blot analysis and flow cytometry. Exosomes were extracted from both animal serum and HUVEC culture medium and identified by electron microscopy. They investigated the role of endothelial cell-derived exosomes in the proliferation, apoptosis, cell cycle, migration and invasion of I/R-injured SH-SY5Y cells. In addition, apoptosis-related molecules caspase-3, Bax and Bcl-2 were detected. RIP was determined to increase the number of exosomes and the expression levels of CD63, HSP70 and TSG101 in plasma, but not in brain hippocampal tissue. The size of exosomes released after I/R in HUVECs was similar to the size of exosomes released in rats subjected to RIP. Endothelial cell-derived exosomes partly suppressed the I/R-induced cell cycle arrest and apoptosis, and inhibited cell proliferation, migration and invasion in SH-SY5Y nerve cells. Endothelial cell-derived exosomes directly protect nerve cells against I/R injury, and are responsible for the protective role of RIP in I/R.

Merging colloidal nanoplasmonics and surface plasmon resonance spectroscopy for enhanced profiling of multiple myeloma-derived exosomes.

PubMed

Di Noto, Giuseppe; Bugatti, Antonella; Zendrini, Andrea; Mazzoldi, Elena Laura; Montanelli, Alessandro; Caimi, Luigi; Rusnati, Marco; Ricotta, Doris; Bergese, Paolo

2016-03-15

A novel approach for sorting exosomes from multiple myeloma (MM), monoclonal gammopathy of undetermined significance (MGUS) and healthy individuals is presented. The method is based on the combination of colloidal gold nanoplasmonics and surface plasmon resonance (SPR) biosensing and probes distinctive colloidal properties of MM-derived exosomes, such as molar concentration and cell membrane binding preferences. It allowed to discover that MM patients produce about four folds more exosomes than MGUS and healthy individuals. In addition, it showed that among the analyzed exosomes, only the MM-derived ones bind heparin - a structural analog of heparan sulfate proteoglycans known to mediate exosome endocytosis - with an apparent dissociation constant (Kd) equal to about 1 nM, indicating a high affinity binding. This plasmonic method complements the classical biochemical profiling approach to exosomes, expanding the MM biomarker panel and adding biosensors to the toolbox to diagnose MM. It may find applications for other diseases and has wider interest for fundamental and translational research involving exosomes. Copyright © 2015 Elsevier B.V. All rights reserved.

Morphological and molecular features of oral fluid-derived exosomes: oral cancer patients versus healthy individuals.

PubMed

Zlotogorski-Hurvitz, Ayelet; Dayan, Dan; Chaushu, Gavriel; Salo, Tuula; Vered, Marilena

2016-01-01

Oral cancer (OC) patients are at high risk to develop recurrent disease or secondary primary cancers with no available biomarkers to detect these events until a visible lesion is readily present and diagnosed by biopsy. Exosomes secreted by cancer cells are involved in tumor growth, invasion and metastasis. We aimed to determine morphological and molecular differences between oral fluid (OF)-derived exosomes of OC patients and those isolated from healthy individuals (HI). OF from OC patients (n = 36) and HI (n = 25) was initially assessed by nanoparticle tracking analysis (NTA). Following ultracentrifugation, exosomal pellets of OC patients and HI were morphologically examined by transmission electron microscopy and atomic force microscopy (AFM). Enzyme-linked immunosorbent assay (ELISA) and western blotting (WB) were used to analyze the expression of exosomal markers--CD9, CD81 and CD63. NTA showed that OC samples of OF had a significantly higher concentration of nanoparticles/ml (p = 0.01) and modal nanoparticle size (p = 0.002) compared to HI. The difference in size was structurally highlighted by AFM three-dimensional images applied on exosomal pellets. ELISA and WB showed differential expression of exosomal markers in OC exosomes compared to HI: lower expression of CD81 and CD9 in contrast to a higher expression of CD63 (~53 kDa). OF-derived exosomes from OC patients differ both morphologically and molecularly from exosomes present in HI. This study is a baseline that provides a starting point for finding exosomal biomarkers for early detection of malignant changes in high-risk patients without overt clinical signs/lesions.

NK Cell-derived Exosomes From NK Cells Previously Exposed to Neuroblastoma Cells Augment the Antitumor Activity of Cytokine-activated NK Cells.

PubMed

Shoae-Hassani, Alireza; Hamidieh, Amir Ali; Behfar, Maryam; Mohseni, Rashin; Mortazavi-Tabatabaei, Seyed A; Asgharzadeh, Shahab

2017-09-01

Immune cell-derived exosomes can increase immunity against tumors. In contrast, tumor-derived exosomes can reduce the immunity and can change the tumor microenvironment to further develop and provide metastasis. These effects take place by an alteration in the innate and adaptive immune cell functions. In this experiment, we studied the natural killer (NK) cells' effectiveness on tumor cells after expansion and thereafter incubated it with exosomes. The exosomes were derived from 2 populations of NK cells: (1) naive NK cells and, (2) NK cells previously exposed to neuroblastoma (NB) cells. Moreover, we have studied the NB-derived exosomes on NK cell function. The molecular load of the characterized exosomes (by means of nanoparticle-tracking analysis, flow cytometry, scanning electron microscopy, and western blot) from NK cells exposed to the NB cell revealed their expression of natural killer cell receptors in addition to CD56, NKG2D, and KIR2DL2 receptors. These exosomes were used to treat NK cells and thereafter administered to NB tumor cells both in vitro and in vivo. Our results showed some kind of NK cells' education by the exosomes. This education from NK cells previously exposed to NB cell-derived exosomes caused efficient and greater cytotoxicity against NB tumors, but NB-derived exosomes act as tumor promoters by providing a tumor supporting niche. Hence, this method of preparing the exosomes has a dramatic effect on activation of anti-NK cells against NB cells.

NK cell-released exosomes

PubMed Central

Fais, Stefano

2013-01-01

We have recently reported that human natural killer (NK) cells release exosomes that express both NK-cell markers and cytotoxic molecules. Similar results were obtained with circulating exosomes from human healthy donors. Both NK-cell derived and circulating exosomes exerted a full functional activity and killed both tumor and activated immune cells. These findings indicate that NK-cell derived exosomes might constitute a new promising therapeutic tool. PMID:23482694

Protective Effect of Intravitreal Administration of Exosomes Derived from Mesenchymal Stem Cells on Retinal Ischemia.

PubMed

Moisseiev, Elad; Anderson, Johnathon D; Oltjen, Sharon; Goswami, Mayank; Zawadzki, Robert J; Nolta, Jan A; Park, Susanna S

2017-10-01

Exosomes derived from human mesenchymal stem cells (hMSCs) cultured under hypoxic conditions contain proteins and growth factors that promote angiogenesis. This study investigated the effect of intravitreal administration of these exosomes on retinal ischemia using a murine model. Oxygen-induced retinopathy (OIR) was induced by exposing one-week-old male C57BL/6J mice to 5 days of 75% hyperoxic conditioning, and returning to room air. After hyperoxic conditioning, the right eye of each mouse was injected intravitreally with 1 µl saline or exosomes derived from hMSCs and compared to control mice of the same age raised in room air without OIR injected intravitreally with saline. Two weeks post-injection, fluorescein angiography (FA) and phase-variance optical coherence tomography angiography (pvOCTA) were used to assess retinal perfusion. Retinal thickness was determined by OCT. The extent of retinal neovascularization was quantitated histologically by counting vascular nuclei on the retinal surface. Among eyes with OIR, intravitreal exosome treatment partially preserved retinal vascular flow in vivo and reduced associated retinal thinning; retinal thickness on OCT was 111.1 ± 7.4µm with saline versus 132.1 ± 11.6µm with exosome, p < 0.001. Retinal neovascularization among OIR eyes was reduced with exosome treatment when compared to saline-treated eyes (7.75 ± 3.68 versus 2.68 ± 1.35 neovascular nuclei per section, p < 0.0001). No immunogenicity or ocular/systemic adverse effect was associated with intravitreal exosome treatment. Intravitreal administration of exosomes derived from hMSCs was well tolerated without immunosuppression and decreased the severity of retinal ischemia in this murine model. This appealing novel non-cellular therapeutic approach warrants further exploration.

Mesenchymal Stem Cell-Derived Exosomes: Immunomodulatory Evaluation in an Antigen-Induced Synovitis Porcine Model

PubMed Central

Casado, Javier G.; Blázquez, Rebeca; Vela, Francisco Javier; Álvarez, Verónica; Tarazona, Raquel; Sánchez-Margallo, Francisco Miguel

2017-01-01

Synovitis is an inflammatory process associated with pain, disability, and discomfort, which is usually treated with anti-inflammatory drugs or biological agents. Mesenchymal stem cells (MSCs) have been also successfully used in the treatment of inflammatory-related diseases such as synovitis or arthritis. In the last years, the exosomes derived from MSCs have become a promising tool for the treatment of inflammatory-related diseases and their therapeutic effect is thought to be mediated (at least in part) by their immunomodulatory potential. In this work, we aimed to evaluate the anti-inflammatory effect of these exosomes in an antigen-induced synovitis animal model. To our knowledge, this is the first report where exosomes derived from MSCs have been evaluated in an animal model of synovitis. Our results demonstrated a decrease of synovial lymphocytes together with a downregulation of TNF-α transcripts in those exosome-treated joints. These results support the immunomodulatory effect of these exosomes and point out that they may represent a promising therapeutic option for the treatment of synovitis. PMID:28377922

[Exosomes and Immune Cells].

PubMed

Seo, Naohiro

2017-05-01

In addition to the cytokines and cytotoxic granules, exosomes have been known as the intercellular communicator and cytotoxic missile of immune cells for the past decade. It has been well known that mature dendritic cell(DC)-derived exosomes participate in the T cell and natural killer(NK)cell activation, while immature DCs secrete tolerogenic exosomes for regulatory T(Treg)cell generation. Treg cell-derived EVs act as a suppressor against pathogenic type-1 T helper(Th1)cell responses. CD8+ T cells produce tumoricidal exosomes for preventing tumor invasion and metastasis transiently after T cell receptor(TCR)-mediated stimulation. Thus, immune cells produce functional exosomes in the activation state- and/or differentiation stage-dependent manner. In this review, the role of immune cell-derived exosomes will be introduced, focusing mainly on immune reaction against tumor.

Exosomes derived from human embryonic mesenchymal stem cells promote osteochondral regeneration.

PubMed

Zhang, S; Chu, W C; Lai, R C; Lim, S K; Hui, J H P; Toh, W S

2016-12-01

Clinical and animal studies have demonstrated the efficacy of mesenchymal stem cell (MSC) therapies in cartilage repair. As the efficacy of many MSC-based therapies has been attributed to paracrine secretion, particularly extracellular vesicles/exosomes, we determine here if weekly intra-articular injections of human embryonic MSC-derived exosomes would repair and regenerate osteochondral defects in a rat model. In this study, osteochondral defects were created on the trochlear grooves of both distal femurs in 12 adult rats. In each animal, one defect was treated with 100 μg exosomes and the contralateral defect treated with phosphate buffered saline (PBS). Intra-articular injections of exosomes or PBS were administered after surgery and thereafter weekly for a period of 12 weeks. Three unoperated age-matched animals served as native controls. Analyses were performed by histology, immunohistochemistry, and scoring at 6 and 12 weeks after surgery. Generally, exosome-treated defects showed enhanced gross appearance and improved histological scores than the contralateral PBS-treated defects. By 12 weeks, exosome-treated defects displayed complete restoration of cartilage and subchondral bone with characteristic features including a hyaline cartilage with good surface regularity, complete bonding to adjacent cartilage, and extracellular matrix deposition that closely resemble that of age-matched unoperated control. In contrast, there were only fibrous repair tissues found in the contralateral PBS-treated defects. This study demonstrates for the first time the efficacy of human embryonic MSC exosomes in cartilage repair, and the utility of MSC exosomes as a ready-to-use and 'cell-free' therapeutic alternative to cell-based MSC therapy. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Monitoring multiple myeloma by idiotype-specific peptide binders of tumor-derived exosomes.

PubMed

Iaccino, Enrico; Mimmi, Selena; Dattilo, Vincenzo; Marino, Fabiola; Candeloro, Patrizio; Di Loria, Antonio; Marimpietri, Danilo; Pisano, Antonio; Albano, Francesco; Vecchio, Eleonora; Ceglia, Simona; Golino, Gaetanina; Lupia, Antonio; Fiume, Giuseppe; Quinto, Ileana; Scala, Giuseppe

2017-10-13

Tumor-derived exosomes (TDEs) play a pivotal role in tumor establishment and progression, and are emerging biomarkers for tumor diagnosis in personalized medicine. To date, there is a lack of efficient technology platforms for exosome isolation and characterization. Multiple myeloma (MM) is an incurable B-cell malignancy due to the rapid development of drug-resistance. MM-released exosomes express the immunoglobulin B-cell receptor (Ig-BCR) of the tumor B-cells, which can be targeted by Idiotype-binding peptides (Id-peptides). In this study, we analyzed the production of MM-released exosomes in the murine 5T33MM multiple myeloma model as biomarkers of tumor growth. To this end, we selected Id-peptides by screening a phage display library using as bait the Ig-BCR expressed by 5T33MM cells. By FACS, the FITC-conjugated Id-peptides detected the MM-released exosomes in the serum of 5T33MM-engrafted mice, levels of which are correlated with tumor progression at an earlier time point compared to serum paraprotein. These results indicate that Id-peptide-based recognition of MM-released exosomes may represent a very sensitive diagnostic approach for clinical evaluation of disease progression.

Osteoclast-derived exosomal miR-214-3p inhibits osteoblastic bone formation

PubMed Central

Li, Defang; Liu, Jin; Guo, Baosheng; Liang, Chao; Dang, Lei; Lu, Cheng; He, Xiaojuan; Cheung, Hilda Yeuk-Siu; Xu, Liang; Lu, Changwei; He, Bing; Liu, Biao; Shaikh, Atik Badshah; Li, Fangfei; Wang, Luyao; Yang, Zhijun; Au, Doris Wai-Ting; Peng, Songlin; Zhang, Zongkang; Zhang, Bao-Ting; Pan, Xiaohua; Qian, Airong; Shang, Peng; Xiao, Lianbo; Jiang, Baohong; Wong, Chris Kong-Chu; Xu, Jiake; Bian, Zhaoxiang; Liang, Zicai; Guo, De-an; Zhu, Hailong; Tan, Weihong; Lu, Aiping; Zhang, Ge

2016-01-01

Emerging evidence indicates that osteoclasts direct osteoblastic bone formation. MicroRNAs (miRNAs) have a crucial role in regulating osteoclast and osteoblast function. However, whether miRNAs mediate osteoclast-directed osteoblastic bone formation is mostly unknown. Here, we show that increased osteoclastic miR-214-3p associates with both elevated serum exosomal miR-214-3p and reduced bone formation in elderly women with fractures and in ovariectomized (OVX) mice. Osteoclast-specific miR-214-3p knock-in mice have elevated serum exosomal miR-214-3p and reduced bone formation that is rescued by osteoclast-targeted antagomir-214-3p treatment. We further demonstrate that osteoclast-derived exosomal miR-214-3p is transferred to osteoblasts to inhibit osteoblast activity in vitro and reduce bone formation in vivo. Moreover, osteoclast-targeted miR-214-3p inhibition promotes bone formation in ageing OVX mice. Collectively, our results suggest that osteoclast-derived exosomal miR-214-3p transfers to osteoblasts to inhibit bone formation. Inhibition of miR-214-3p in osteoclasts may be a strategy for treating skeletal disorders involving a reduction in bone formation. PMID:26947250

Analysis of the miRNA Profiles of Melanoma Exosomes Derived Under Normoxic and Hypoxic Culture Conditions.

PubMed

Wozniak, Michal; Peczek, Lukasz; Czernek, Liliana; Düchler, Markus

2017-12-01

MicroRNAs (miRNAs) transported in melanoma-derived exosomes function as intercellular messengers supporting tumor survival and progression. Hypoxia increases melanoma phenotypic plasticity, drug resistance, and metastasis. We determined the miRNA profiles in exosomes derived from melanoma cells grown under hypoxic and normoxic conditions by microarray analyses and reverse transcription-polymerase chain reaction (RT-PCR) in order to analyze the potential influence of vesicle-transported miRNAs on cancer-related pathways and transcriptional programs. Despite phenotypical differences of the four cell lines used, their exosomes shared the majority of miRNAs. The levels of three miRNAs were higher in normoxic exosomes, whereas 15 miRNAs were significantly more abundant under hypoxic conditions. Pathway analysis pointed at several cellular processes contributing to proliferation, drug resistance, and modification of the tumor microenvironment, including immunosuppression. The miRNA-expression profiles of exosomes from patient-derived melanoma cells are modified by oxygen concentration and reflect the phenotypic changes of melanoma cells under different growth conditions. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Exosomes derived from hypoxic epithelial ovarian cancer deliver microRNA-940 to induce macrophage M2 polarization.

PubMed

Chen, Xin; Ying, Xiang; Wang, Xinjing; Wu, Xiaoli; Zhu, Qinyi; Wang, Xipeng

2017-07-01

Hypoxia is a common feature of solid tumors. It is closely related to tumor progression. Exosomal microRNAs derived from cancers are considered to be mediators between cancer cells and the tumor microenvironment. In addition, the number of tumor-associated macrophages (TAMs) in the tumor microenvironment has also been demonstrated to correlate with tumor development. However, the relationship between tumor-secreted exosomes and TAM polarization under hypoxic conditions during tumor progression is not clear. Herein, we demonstrated that hypoxia induces the high expression of microRNA-940 (miR‑940) in exosomes derived from epithelial ovarian cancer (EOC). We also found that miR‑940 is highly expressed in exosomes isolated from ascites of EOC patients. Moreover, the overexpression of miR‑940 in macrophages delivered by exosomes stimulated M2 phenotype polarization, while the M2 subtype macrophages promoted EOC proliferation and migration. These results highlight the function of hypoxia in enhancing the high level of expression of miR‑940 in tumor exosomes taken up by macrophages. We also showed that the tumor-promoting function of miR‑940 is mediated by TAM polarization in EOC. These findings show that tumor-derived exosomal miR‑940 induced by hypoxia plays an important role in stimulating TAM polarization in the progression of EOC.

Human endothelial progenitor cells-derived exosomes accelerate cutaneous wound healing in diabetic rats by promoting endothelial function.

PubMed

Li, Xiaocong; Jiang, Chunyu; Zhao, Jungong

2016-08-01

Wound healing is deeply dependent on neovascularization to restore blood flow. The neovascularization of endothelial progenitor cells (EPCs) through paracrine secretion has been reported in various tissue repair models. Exosomes, key components of cell paracrine mechanism, have been rarely reported in wound healing. Exosomes were isolated from the media of EPCs obtained from human umbilical cord blood. Diabetic rats wound model was established and treated with exosomes. The in vitro effects of exosomes on the proliferation, migration and angiogenic tubule formation of endothelial cells were investigated. We revealed that human umbilical cord blood EPCs derived exosomes transplantation could accelerate cutaneous wound healing in diabetic rats. We also showed that exosomes enhanced the proliferation, migration and tube formation of vascular endothelial cells in vitro. Furthermore, we found that endothelial cells stimulated with these exosomes would increase expression of angiogenesis-related molecules, including FGF-1, VEGFA, VEGFR-2, ANG-1, E-selectin, CXCL-16, eNOS and IL-8. Taken together, our findings indicated that EPCs-derived exosomes facilitate wound healing by positively modulating vascular endothelial cells function. Copyright © 2016 Elsevier Inc. All rights reserved.

Exosomal cancer immunotherapy is independent of MHC molecules on exosomes.

PubMed

Hiltbrunner, Stefanie; Larssen, Pia; Eldh, Maria; Martinez-Bravo, Maria-Jose; Wagner, Arnika K; Karlsson, Mikael C I; Gabrielsson, Susanne

2016-06-21

Peptide-loaded exosomes are promising cancer treatment vehicles; however, moderate T cell responses in human clinical trials indicate a need to further understand exosome-induced immunity. We previously demonstrated that antigen-loaded exosomes carry whole protein antigens and require B cells for inducing antigen-specific T cells. Therefore, we investigated the relative importance of exosomal major histocompatibility complex (MHC) class I for the induction of antigen-specific T cell responses and tumour protection. We show that ovalbumin-loaded dendritic cell-derived exosomes from MHCI-/- mice induce antigen-specific T cells at the same magnitude as wild type exosomes. Furthermore, exosomes lacking MHC class I, as well as exosomes with both MHC class I and II mismatch, induced tumour infiltrating T cells and increased overall survival to the same extent as syngeneic exosomes in B16 melanoma. In conclusion, T cell responses are independent of exosomal MHC/peptide complexes if whole antigen is present. This establishes the prospective of using impersonalised exosomes, and will greatly increase the feasibility of designing exosome-based vaccines or therapeutic approaches in humans.

Exosomes Derived from Dendritic Cells Treated with Schistosoma japonicum Soluble Egg Antigen Attenuate DSS-Induced Colitis

PubMed Central

Wang, Lifu; Yu, Zilong; Wan, Shuo; Wu, Feng; Chen, Wei; Zhang, Beibei; Lin, Datao; Liu, Jiahua; Xie, Hui; Sun, Xi; Wu, Zhongdao

2017-01-01

Exosomes are 30–150 nm small membrane vesicles that are released into the extracellular medium via cells that function as a mode of intercellular communication. Dendritic cell (DC)-derived exosomes modulate immune responses and prevent the development of autoimmune diseases. Moreover, Schistosoma japonicum eggs show modulatory effects in a mouse model of colitis. Therefore, we hypothesized that exosomes derived from DCs treated with S. japonicum soluble eggs antigen (SEA; SEA-treated DC exosomes) would be useful for treating inflammatory bowel disease (IBD). Exosomes were purified from the supernatant of DCs treated or untreated with SEA and identified via transmission electron microscopy, western blotting and NanoSight. Acute colitis was induced via the administration of dextran sulfate sodium (DSS) in drinking water (5.0%, wt/vol). Treatment with exosomes was conducted via intraperitoneal injection (i.p.; 50 μg per mouse) from day 0 to day 6. Clinical scores were calculated based on weight loss, stool type, and bleeding. Colon length was measured as an indirect marker of inflammation, and colon macroscopic characteristics were determined. Body weight loss and the disease activity index of DSS-induced colitis mice decreased significantly following treatment with SEA-treated DC exosomes. Moreover, the colon lengths of SEA-treated DC exosomes treated colitis mice improved, and their mean colon macroscopic scores decreased. In addition, histologic examinations and histological scores showed that SEA-treated DC exosomes prevented colon damage in acute DSS-induced colitis mice. These results indicate that SEA-treated DC exosomes attenuate the severity of acute DSS-induced colitis mice more effectively than DC exosomes. The current work suggests that SEA-treated DC exosomes may be useful as a new approach to treat IBD. PMID:28959207

Possibility of Exosome-Based Therapeutics and Challenges in Production of Exosomes Eligible for Therapeutic Application.

PubMed

Yamashita, Takuma; Takahashi, Yuki; Takakura, Yoshinobu

2018-01-01

Exosomes are cell-derived vesicles with a diameter 30-120 nm. Exosomes contain endogenous proteins and nucleic acids; delivery of these molecules to exosome-recipient cells causes biological effects. Exosomes derived from some types of cells such as mesenchymal stem cells and dendritic cells have therapeutic potential and may be biocompatible and efficient agents against various disorders such as organ injury. However, there are many challenges for the development of exosome-based therapeutics. In particular, producing exosomal formulations is the major barrier for therapeutic application because of their heterogeneity and low productivity. Development and optimization of producing methods, including methods for isolation and storage of exosome formulations, are required for realizing exosome-based therapeutics. In addition, improvement of therapeutic potential and delivery efficiency of exosomes are important for their therapeutic application. In this review, we summarize current knowledge about therapeutic application of exosomes and discuss some challenges in their successful use.

Embryonic Stem Cell-Derived Exosomes Promote Endogenous Repair Mechanisms and Enhance Cardiac Function Following Myocardial Infarction

PubMed Central

Khan, Mohsin; Nickoloff, Emily; Abramova, Tatiana; Johnson, Jennifer; Verma, Suresh Kumar; Krishnamurthy, Prasanna; Mackie, Alexander Roy; Vaughan, Erin; Garikipati, Venkata Naga Srikanth; Benedict, Cynthia; Ramirez, Veronica; Lambers, Erin; Ito, Aiko; Gao, Erhe; Misener, Sol; Luongo, Timothy; Elrod, John; Qin, Gangjian; Houser, Steven R; Koch, Walter J; Kishore, Raj

2015-01-01

Rationale Embryonic stem cells (ESCs) hold great promise for cardiac regeneration but are susceptible to various concerns. Recently, salutary effects of stem cells have been connected to exosome secretion. ESCs have the ability to produce exosomes however their effect in the context of the heart is unknown. Objective Determine the effect of ESC-derived exosome for the repair of ischemic myocardium and whether c-kit+ CPCs function can be enhanced with ESC exosomes Methods and Results This study demonstrates that mouse ESC derived exosomes (mES Ex) possess ability to augment function in infarcted hearts. mES Ex enhanced neovascularization, cardiomyocyte survival and reduced fibrosis post infarction consistent with resurgence of cardiac proliferative response. Importantly, mES Ex augmented cardiac progenitor cell (CPC) survival, proliferation and cardiac commitment concurrent with increased c-kit+ CPCs in vivo 8 weeks after in vivo transfer along with formation of bonafide new cardiomyocytes in the ischemic heart. miRNA array revealed significant enrichment of miR290–295 cluster and particularly miR-294 in ESC exosomes. The underlying basis for the beneficial effect of mES Ex was tied to delivery of ESC specific miR-294 to CPCs promoting increased survival, cell cycle progression and proliferation. Conclusions mES Ex provide a novel cell free system that utilizes the immense regenerative power of ES cells while avoiding the risks associated with direct ES or ES derived cell transplantation and risk of teratomas. ESC exosomes possess cardiac regeneration ability and modulate both cardiomyocyte and CPC based repair programs in the heart. PMID:25904597

Adipocyte-derived Exosomal miRNAs: A Novel Mechanism for Obesity-Related Disease

PubMed Central

Ferrante, Sarah C; Nadler, Evan P; Pillai, Dinesh K; Hubal, Monica J; Wang, Zuyi; Wang, Justin M; Gordish-Dressman, Heather; Koeck, Emily; Sevilla, Samantha; Wiles, Andrew A; Freishtat, Robert J

2014-01-01

Background Obesity is frequently complicated by comorbid conditions, yet how excess adipose contributes is poorly understood. Although adipocytes in obese individuals induce systemic inflammation via secreted cytokines, another potential mediator has recently been identified (i.e. adipocyte-derived exosomes). We hypothesized that adipocyte-derived exosomes contain mediators capable of activating end-organ inflammatory and fibrotic signaling pathways. Methods We developed techniques to quantify and characterize exosomes shed by adipocytes from 7 obese (age: 12–17.5 years, BMI: 33–50 kg/m2) and 5 lean (age: 11–19 years, BMI: 22–25 kg/m2) subjects. Results Abundant exosomal miRNAs, but no mRNAs, were detected. Comparison of obese vs. lean visceral adipose donors detected 55 differentially-expressed miRNAs (p<0.05; fold change≥|1.2|). qRT-PCR confirmed downregulation of miR-148b (ratio = 0.2 [95% confidence interval = 0.1, 0.6]) and miR-4269 (0.3 [0.1, 0.8]), and upregulation of miR-23b (6.2 [2.2, 17.8]) and miR-4429 (3.8 [1.1 to 13.4]). Pathways analysis identified TGF-β signaling and Wnt/ β-catenin signaling among the top canonical pathways expected to be altered with visceral adiposity based on projected mRNA targets for the 55 differentially expressed miRNAs. A select mRNA target was validated in vitro. Conclusion These data show that visceral adipocytes shed exosomal-mediators predicted to regulate key end-organ inflammatory and fibrotic signaling pathways. PMID:25518011

Exosomes derived from mesenchymal non-small cell lung cancer cells promote chemoresistance.

PubMed

Lobb, Richard J; van Amerongen, Rosa; Wiegmans, Adrian; Ham, Sunyoung; Larsen, Jill E; Möller, Andreas

2017-08-01

Non-small cell lung cancer (NSCLC) is the most common lung cancer type and the most common cause of mortality in lung cancer patients. NSCLC is often associated with resistance to chemotherapeutics and together with rapid metastatic spread, results in limited treatment options and poor patient survival. NSCLCs are heterogeneous, and consist of epithelial and mesenchymal NSCLC cells. Mesenchymal NSCLC cells are thought to be responsible for the chemoresistance phenotype, but if and how this phenotype can be transferred to other NSCLC cells is currently not known. We hypothesised that small extracellular vesicles, exosomes, secreted by mesenchymal NSCLC cells could potentially transfer the chemoresistance phenotype to surrounding epithelial NSCLC cells. To explore this possibility, we used a unique human bronchial epithelial cell (HBEC) model in which the parental cells were transformed from an epithelial to mesenchymal phenotype by introducing oncogenic alterations common in NSCLC. We found that exosomes derived from the oncogenically transformed, mesenchymal HBECs could transfer chemoresistance to the parental, epithelial HBECs and increase ZEB1 mRNA, a master EMT transcription factor, in the recipient cells. Additionally, we demonstrate that exosomes from mesenchymal, but not epithelial HBECs contain the ZEB1 mRNA, thereby providing a potential mechanism for the induction of a mesenchymal phenotype in recipient cells. Together, this work demonstrates for the first time that exosomes derived from mesenchymal, oncogenically transformed lung cells can transfer chemoresistance and mesenchymal phenotypes to recipient cells, likely via the transfer of ZEB1 mRNA in exosomes. © 2017 UICC.

Examination of the Specificity of Tumor Cell Derived Exosomes with Tumor Cells In Vitro

PubMed Central

Smyth, Tyson J.; Redzic, Jasmina S.; Graner, Michael W.; Anchordoquy, Thomas J.

2016-01-01

Small endogenous vesicles called exosomes are beginning to be explored as drug delivery vehicles. The in vivo targets of exosomes are poorly understood; however, they are believed to be important in cell-to-cell communication and may play a prominent role in cancer metastasis. We aimed to elucidate whether cancer derived exosomes can be used as drug delivery vehicles that innately target tumors over normal tissue. Our in vitro results suggest that while there is some specificity towards cancer cells over “immortalized” cells, it is unclear if the difference is sufficient to achieve precise in vivo targeting. Additionally, we found that exosomes associate with their cellular targets to a significantly greater extent (> 10-fold) than liposomes of a similar size. Studies on the association of liposomes mimicking the unique lipid content of exosomes revealed that the lipid composition contributes significantly to cellular adherence/internalization. Cleavage of exosome surface proteins yielded exosomes exhibiting reduced association with their cellular targets, demonstrating the importance of proteins in binding/internalization. Furthermore, although acidic conditions are known to augment the metastatic potential of tumors, we found that cells cultured at low pH released exosomes with significantly less potential for cellular association than cells cultured at physiological pH. PMID:25102470

Exosomes derived from tumor cells genetically modified to express Mycobacterium tuberculosis antigen: a novel vaccine for cancer therapy.

PubMed

Koyama, Yoshiyuki; Ito, Tomoko; Hasegawa, Aya; Eriguchi, Masazumi; Inaba, Toshio; Ushigusa, Takahiro; Sugiura, Kikuya

2016-11-01

To examine the potential of exosomes derived from the tumor cells, which had been genetically modified to express a Mycobacterium tuberculosis antigen, as a cancer vaccine aimed at overcoming the weak immunogenicity of tumor antigens. We transfected B16 melanoma cells with a plasmid encoding the M. tuberculosis antigen, early secretory antigenic target-6 (ESAT-6). The secreted exosomes bearing both tumor-associated antigens and the pathogenic antigen (or their epitopes) were collected. When the exosomes were injected into foot pads of mice, they significantly (p < 0.05) evoked cellular immunity against both ESAT-6, and B16 tumor cells. Intra-tumoral injection of the exosomes significantly suppressed (p < 0.001) tumor growth in syngeneic B16 tumor-bearing mice, while the exosomes derived from the non-transfected B16 cells showed no effect on tumor growth, although both exosomes should have similar tumor antigens. Exosomes bearing both tumor antigens and the M. tuberculosis antigen (or their epitopes) have a high potential as a candidate for cancer vaccine to overcome the immune escape by tumor cells.

Exosomes: From Garbage Bins to Promising Therapeutic Targets

PubMed Central

H. Rashed, Mohammed; Bayraktar, Emine; K. Helal, Gouda; Abd-Ellah, Mohamed F.; Amero, Paola; Chavez-Reyes, Arturo; Rodriguez-Aguayo, Cristian

2017-01-01

Intercellular communication via cell-released vesicles is a very important process for both normal and tumor cells. Cell communication may involve exosomes, small vesicles of endocytic origin that are released by all types of cells and are found in abundance in body fluids, including blood, saliva, urine, and breast milk. Exosomes have been shown to carry lipids, proteins, mRNAs, non-coding RNAs, and even DNA out of cells. They are more than simply molecular garbage bins, however, in that the molecules they carry can be taken up by other cells. Thus, exosomes transfer biological information to neighboring cells and through this cell-to-cell communication are involved not only in physiological functions such as cell-to-cell communication, but also in the pathogenesis of some diseases, including tumors and neurodegenerative conditions. Our increasing understanding of why cells release exosomes and their role in intercellular communication has revealed the very complex and sophisticated contribution of exosomes to health and disease. The aim of this review is to reveal the emerging roles of exosomes in normal and pathological conditions and describe the controversial biological role of exosomes, as it is now understood, in carcinogenesis. We also summarize what is known about exosome biogenesis, composition, functions, and pathways and discuss the potential clinical applications of exosomes, especially as biomarkers and novel therapeutic agents. PMID:28257101

[Multilateral Strategies Utilizing Exosomes for Cancer Therapy].

PubMed

Nishida-Aoki, Nao; Ochiya, Takahiro

2017-05-01

Exosomes are nano-sized extracellular vesicles which transfer their components such as RNA, DNA, and proteins from one cell to another cell. The components are released to the cytoplasm of the recipient cells, having an effect on the cells. Cancerderived exosomes promote cancer progression, invasion, gain of drug resistance, and metastasis. Recently, according to their characteristics, it is expected to apply exosomes to cancer therapies, such as utilizing exosomes as drug delivery systems(DDS) for anticancer drugs and as cancer vaccines to enhance immunity to cancer cells. More, as the cancer-derived exosomes have cancer-promoting effects on multiple stages, inhibiting the function of the cancer-derived exosomes would be helpful to cancer therapies by suppressing cancer progression. DDS and cancer vaccines utilizing exosomes are now undergoing clinical studies, although DDS is suffering from loading efficiency. Treatments by inhibiting the functions of cancer-derived exosomes have still only few reports at experimental levels. Recently, we showed in a mouse model that disruption of cancer-derived exosomes by antibodies could suppress lung metastasis of the human breast cancer cells. Exosomes will provide us the multiple strategies to fight with cancer, which can be applied to cancers from many organs. It is important to confirm safety and overcome technical problems to bring exosomes in practical use.

Exosomes derived from B16F0 melanoma cells alter the transcriptome of cytotoxic T cells that impacts mitochondrial respiration.

PubMed

Bland, Cassidy L; Byrne-Hoffman, Christina N; Fernandez, Audry; Rellick, Stephanie L; Deng, Wentao; Klinke, David J

2018-03-01

While recent clinical studies demonstrate the promise of cancer immunotherapy, a barrier for broadening the clinical benefit is identifying how tumors locally suppress cytotoxic immunity. As an emerging mode of intercellular communication, exosomes secreted by malignant cells can deliver a complex payload of coding and noncoding RNA to cells within the tumor microenvironment. Here, we quantified the RNA payload within tumor-derived exosomes and the resulting dynamic transcriptomic response to cytotoxic T cells upon exosome delivery to better understand how tumor-derived exosomes can alter immune cell function. Exosomes derived from B16F0 melanoma cells were enriched for a subset of coding and noncoding RNAs that did not reflect the abundance in the parental cell. Upon exosome delivery, RNAseq revealed the dynamic changes in the transcriptome of CTLL2 cytotoxic T cells. In analyzing transiently coexpressed gene clusters, pathway enrichment suggested that the B16F0 exosomal payload altered mitochondrial respiration, which was confirmed independently, and upregulated genes associated with the Notch signaling pathway. Interestingly, exosomal miRNA appeared to have no systematic effect on downregulating target mRNA levels. Gene expression data are available in the GEO database under the accession SuperSeries number GSE102951. © 2018 Federation of European Biochemical Societies.

Mesenchymal stem cell-derived exosomes have altered microRNA profiles and induce osteogenic differentiation depending on the stage of differentiation

PubMed Central

Wang, Xiaoqin; Omar, Omar; Vazirisani, Forugh; Thomsen, Peter

2018-01-01

Human mesenchymal stem cell (hMSC)-derived exosomes have shown regenerative effects, but their role in osteogenesis and the underlying mechanism are yet to be determined. In this study, we examined the time-course secretion of exosomes by hMSCs during the entire process of osteogenic differentiation. Exosomes derived from hMSCs in various stages of osteogenic differentiation committed homotypic cells to differentiate towards osteogenic lineage, but only exosomes from late stages of osteogenic differentiation induced extracellular matrix mineralisation. Exosomes from expansion and early and late stages of osteogenic differentiation were internalised by a subpopulation of hMSCs. MicroRNA profiling revealed a set of differentially expressed exosomal microRNAs from the late stage of osteogenic differentiation, which were osteogenesis related. Target prediction demonstrated that these microRNAs enriched pathways involved in regulation of osteogenic differentiation and general mechanisms how exosomes exert their functions, such as “Wnt signalling pathway” and “endocytosis”. Taken together, the results show that MSCs secrete exosomes with different biological properties depending on differentiation stage of their parent cells. The exosomal cargo transferred from MSCs in the late stage of differentiation induces osteogenic differentiation and mineralisation. Moreover, it is suggested that the regulatory effect on osteogenesis by exosomes is at least partly exerted by exosomal microRNA. PMID:29447276

Melanoma cell-derived exosomes promote epithelial-mesenchymal transition in primary melanocytes through paracrine/autocrine signaling in the tumor microenvironment

PubMed Central

Xiao, Deyi; Barry, Samantha; Kmetz, Daniel; Egger, Michael; Pan, Jianmin; Rai, Shesh N; Qu, Jifu; McMasters, Kelly M.; Hao, Hongying

2016-01-01

The tumor microenvironment is abundant with exosomes that are secreted by the cancer cells themselves. Exosomes are nanosized, organelle-like membranous structures that are increasingly being recognized as major contributors in the progression of malignant neoplasms. A critical element in melanoma progression is its propensity to metastasize, but little is known about how melanoma cell-derived exosomes modulate the microenvironment to optimize conditions for tumor progression and metastasis. Here, we provide evidence that melanoma cell-derived exosomes promote phenotype switching in primary melanocytes through paracrine/autocrine signaling. We found that the mitogen-activated protein kinase (MAPK) signaling pathway was activated during the exosome-mediated epithelial-to-mesenchymal transition (EMT)-resembling process, which promotes metastasis. Let-7i, an miRNA modulator of EMT, was also involved in this process. We further defined two other miRNA modulators of EMT (miR-191 and let-7a) in serum exosomes for differentiating stage I melanoma patients from non-melanoma subjects. These results provide the first strong molecular evidence that melanoma cell-derived exosomes promote the EMT-resembling process in the tumor microenvironment. Thus, novel strategies targeting EMT and modulating the tumor microenvironment may emerge as important approaches for the treatment of metastatic melanoma. PMID:27063098

Plasma-derived exosomes contribute to inflammation via the TLR9-NF-κB pathway in chronic heart failure patients.

PubMed

Ye, Wei; Tang, Xiaojun; Yang, Zhengquan; Liu, Chu; Zhang, Xin; Jin, Jing; Lyu, Jianxin

2017-07-01

Exosomes are small vesicles that contain proteins, DNA and RNA, and play an important role in inflammation; however, the underlying mechanism remains unclear. In the present study, we found increased plasma-derived exosomes in chronic heart failure patients compared with healthy controls. Further, our data demonstrated that plasma-derived exosomes carried mtDNA, and triggered an inflammatory response via the TLR9-NF-κB pathway, as well, the inflammatory effect was closely related to exosomal mtDNA copy number. However, the effect could be blocked by chloroquine (CQ), a TLR9 inhibitor. These findings reveal a new mechanism of exosome-induced inflammation, and provide a new perspective for intervention and treatment of inflammation-related diseases, such as chronic heart failure. Copyright © 2017 Elsevier Ltd. All rights reserved.

Periostin is identified as a putative metastatic marker in breast cancer-derived exosomes

PubMed Central

Vardaki, Ioulia; Ceder, Sophia; Rutishauser, Dorothea; Baltatzis, George; Foukakis, Theodoros; Panaretakis, Theocharis

2016-01-01

Breast cancer (BrCa) is the most frequent cancer type in women and a leading cause of cancer related deaths in the world. Despite the decrease in mortality due to better diagnostics and palliative care, there is a lack of prognostic markers of metastasis. Recently, the exploitation of liquid biopsies and in particular of the extracellular vesicles has shown promise in the identification of such prognostic markers. In this study we compared the proteomic content of exosomes derived from metastatic and non-metastatic human (MCF7 and MDA-MB-231) and mouse (67NR and 4T1) cell lines. We found significant differences not only in the amount of secreted exosomes but most importantly in the protein content of exosomes secreted from metastatic versus non-metastatic ones. We identified periostin as a protein that is enriched in exosomes secreted by metastatic cells and validated its presence in a pilot cohort of breast cancer patient samples with localized disease or lymph node (LN) metastasis. PMID:27589561

Periostin is identified as a putative metastatic marker in breast cancer-derived exosomes.

PubMed

Vardaki, Ioulia; Ceder, Sophia; Rutishauser, Dorothea; Baltatzis, George; Foukakis, Theodoros; Panaretakis, Theocharis

2016-11-15

Breast cancer (BrCa) is the most frequent cancer type in women and a leading cause of cancer related deaths in the world. Despite the decrease in mortality due to better diagnostics and palliative care, there is a lack of prognostic markers of metastasis. Recently, the exploitation of liquid biopsies and in particular of the extracellular vesicles has shown promise in the identification of such prognostic markers. In this study we compared the proteomic content of exosomes derived from metastatic and non-metastatic human (MCF7 and MDA-MB-231) and mouse (67NR and 4T1) cell lines. We found significant differences not only in the amount of secreted exosomes but most importantly in the protein content of exosomes secreted from metastatic versus non-metastatic ones. We identified periostin as a protein that is enriched in exosomes secreted by metastatic cells and validated its presence in a pilot cohort of breast cancer patient samples with localized disease or lymph node (LN) metastasis.

Osteoclast-derived microRNA-containing exosomes selectively inhibit osteoblast activity

PubMed Central

Sun, Weijia; Zhao, Chenyang; Li, Yuheng; Wang, Liang; Nie, Guangjun; Peng, Jiang; Wang, Aiyuan; Zhang, Pengfei; Tian, Weiming; Li, Qi; Song, Jinping; Wang, Cheng; Xu, Xiaolong; Tian, Yanhua; Zhao, Dingsheng; Xu, Zi; Zhong, Guohui; Han, Bingxing; Ling, Shukuan; Chang, Yan-Zhong; Li, Yingxian

2016-01-01

MicroRNAs have an important role in bone homeostasis. However, the detailed mechanism of microRNA-mediated intercellular communication between bone cells remains elusive. Here, we report that osteoclasts secrete microRNA-enriched exosomes, by which miR-214 is transferred into osteoblasts to inhibit their function. In a coculture system, inhibition of exosome formation and secretion prevented miR-214 transportation. Exosomes specifically recognized osteoblasts through the interaction between ephrinA2 and EphA2. In osteoclast-specific miR-214 transgenic mice, exosomes were secreted into the serum, and miR-214 and ephrinA2 levels were elevated. Therefore, these exosomes have an inhibitory role in osteoblast activity. miR-214 and ephrinA2 levels in serum exosomes from osteoporotic patients and mice were upregulated substantially. These exosomes may significantly inhibit osteoblast activity. Inhibition of exosome secretion via Rab27a small interfering RNA prevented ovariectomized-induced osteoblast dysfunction in vivo. Taken together, these findings suggest that exosome-mediated transfer of microRNA plays an important role in the regulation of osteoblast activity. Circulating miR-214 in exosomes not only represents a biomarker for bone loss but could selectively regulate osteoblast function. PMID:27462462

Triple SILAC quantitative proteomic analysis reveals differential abundance of cell signaling proteins between normal and lung cancer-derived exosomes.

PubMed

Clark, David J; Fondrie, William E; Yang, Austin; Mao, Li

2016-02-05

Exosomes are 30-100 nm sized membrane vesicles released by cells into the extracellular space that mediate intercellular communication via transfer of proteins and other biological molecules. To better understand the role of these microvesicles in lung carcinogenesis, we employed a Triple SILAC quantitative proteomic strategy to examine the differential protein abundance between exosomes derived from an immortalized normal bronchial epithelial cell line and two non-small cell lung cancer (NSCLC) cell lines harboring distinct activating mutations in the cell signaling molecules: Kirsten rat sarcoma viral oncogene homolog (KRAS) or epidermal growth factor receptor (EGFR). In total, we were able to quantify 721 exosomal proteins derived from the three cell lines. Proteins associated with signal transduction, including EGFR, GRB2 and SRC, were enriched in NSCLC exosomes, and could actively regulate cell proliferation in recipient cells. This study's investigation of the NSCLC exosomal proteome has identified enriched protein cargo that can contribute to lung cancer progression, which may have potential clinical implications in biomarker development for patients with NSCLC. The high mortality associated with lung cancer is a result of late-stage diagnosis of the disease. Current screening techniques used for early detection of lung cancer lack the specificity for accurate diagnosis. Exosomes are nano-sized extracellular vesicles, and the increased abundance of select protein cargo in exosomes derived from cancer cells may be used for diagnostic purposes. In this paper, we applied quantitative proteomic analysis to elucidate abundance differences in exosomal protein cargo between two NSCLC cell lines with distinctive oncogene mutations and an immortalized normal bronchial epithelial cell line. This study revealed proteins associated with cell adhesion, the extracellular matrix, and a variety of signaling molecules were enriched in NSCLC exosomes. The present data reveals

Therapeutic potential of CAR-T cell-derived exosomes: a cell-free modality for targeted cancer therapy.

PubMed

Tang, Xiang-Jun; Sun, Xu-Yong; Huang, Kuan-Ming; Zhang, Li; Yang, Zhuo-Shun; Zou, Dan-Dan; Wang, Bin; Warnock, Garth L; Dai, Long-Jun; Luo, Jie

2015-12-29

Chimeric antigen receptor (CAR)-based T-cell adoptive immunotherapy is a distinctively promising therapy for cancer. The engineering of CARs into T cells provides T cells with tumor-targeting capabilities and intensifies their cytotoxic activity through stimulated cell expansion and enhanced cytokine production. As a novel and potent therapeutic modality, there exists some uncontrollable processes which are the potential sources of adverse events. As an extension of this impactful modality, CAR-T cell-derived exosomes may substitute CAR-T cells to act as ultimate attackers, thereby overcoming some limitations. Exosomes retain most characteristics of parent cells and play an essential role in intercellular communications via transmitting their cargo to recipient cells. The application of CAR-T cell-derived exosomes will make this cell-based therapy more clinically controllable as it also provides a cell-free platform to diversify anticancer mediators, which responds effectively to the complexity and volatility of cancer. It is believed that the appropriate application of both cellular and exosomal platforms will make this effective treatment more practicable.

Bioinspired Cell-Derived Nanovesicles versus Exosomes as Drug Delivery Systems: a Cost-Effective Alternative.

PubMed

Goh, Wei Jiang; Zou, Shui; Ong, Wei Yi; Torta, Federico; Alexandra, Alvarez Fernandez; Schiffelers, Raymond M; Storm, Gert; Wang, Jiong-Wei; Czarny, Bertrand; Pastorin, Giorgia

2017-10-30

Cell Derived Nanovesicles (CDNs) have been developed from the rapidly expanding field of exosomes, representing a class of bioinspired Drug Delivery Systems (DDS). However, translation to clinical applications is limited by the low yield and multi-step approach in isolating naturally secreted exosomes. Here, we show the first demonstration of a simple and rapid production method of CDNs using spin cups via a cell shearing approach, which offers clear advantages in terms of yield and cost-effectiveness over both traditional exosomes isolation, and also existing CDNs fabrication techniques. The CDNs obtained were of a higher protein yield and showed similarities in terms of physical characterization, protein and lipid analysis to both exosomes and CDNs previously reported in the literature. In addition, we investigated the mechanisms of cellular uptake of CDNs in vitro and their biodistribution in an in vivo mouse tumour model. Colocalization of the CDNs at the tumour site in a cancer mouse model was demonstrated, highlighting the potential for CDNs as anti-cancer strategy. Taken together, the results suggest that CDNs could provide a cost-effective alternative to exosomes as an ideal drug nanocarrier.

Toxoplasma gondii Antigen-Pulsed-Dendritic Cell-Derived Exosomes Induce a Protective Immune Response against T. gondii Infection

PubMed Central

Aline, Fleur; Bout, Daniel; Amigorena, Sébastian; Roingeard, Philippe; Dimier-Poisson, Isabelle

2004-01-01

It was previously demonstrated that immunizing mice with spleen dendritic cells (DCs) that had been pulsed ex vivo with Toxoplasma gondii antigens triggers a systemic Th1-biased specific immune response and induces protection against infection. T. gondii can cause severe sequelae in the fetuses of mothers who acquire the infection during pregnancy, as well as life-threatening neuropathy in immunocompromised patients, in particular those with AIDS. Here, we investigate the efficacy of a novel cell-free vaccine composed of DC exosomes, which are secreted antigen-presenting vesicles that express functional major histocompatibility complex class I and II and T-cell-costimulatory molecules. They have already been shown to induce potent antitumor immune responses. We investigated the potential of DC2.4 cell line-derived exosomes to induce protective immunity against toxoplasmosis. Our data show that most adoptively transferred T. gondii-pulsed DC-derived exosomes were transferred to the spleen, elicited a strong systemic Th1-modulated Toxoplasma-specific immune response in vivo, and conferred good protection against infection. These findings support the possibility that DC-derived exosomes can be used for T. gondii immunoprophylaxis and for immunoprophylaxis against many other pathogens. PMID:15213158

Cancer Cell-derived Exosomes Induce Mitogen-activated Protein Kinase-dependent Monocyte Survival by Transport of Functional Receptor Tyrosine Kinases*

PubMed Central

Song, Xiao; Ding, Yanping; Liu, Gang; Yang, Xiao; Zhao, Ruifang; Zhang, Yinlong; Zhao, Xiao; Anderson, Gregory J.; Nie, Guangjun

2016-01-01

Tumor-associated macrophages (TAM) play pivotal roles in cancer initiation and progression. Monocytes, the precursors of TAMs, normally undergo spontaneous apoptosis within 2 days, but can subsist in the inflammatory tumor microenvironment for continuous survival and generation of sufficient TAMs. The mechanisms underlying tumor-driving monocyte survival remain obscure. Here we report that cancer cell-derived exosomes were crucial mediators for monocyte survival in the inflammatory niche. Analysis of the survival-promoting molecules in monocytes revealed that cancer cell-derived exosomes activated Ras and extracellular signal-regulated kinases in the mitogen-activated protein kinase (MAPK) pathway, resulting in the prevention of caspase cleavage. Phosphorylated receptor tyrosine kinases (RTKs), such as phosphorylated epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2), were abundantly expressed in cancer cell-derived exosomes. Knock-out of EGFR or/and HER-2, or alternatively, inhibitors against their phosphorylation significantly disturbed the exosome-mediated activation of the MAPK pathway, inhibition of caspase cleavage, and increase in survival rate in monocytes. Moreover, the deprived survival-stimulating activity of exosomes due to null expression of EGFR and HER-2 could be restored by activation of another RTK, insulin receptor. Overall, our study uncovered a mechanism of tumor-associated monocyte survival and demonstrated that cancer cell-derived exosomes can stimulate the MAPK pathway in monocytes through transport of functional RTKs, leading to inactivation of apoptosis-related caspases. This work provides insights into the long sought question on monocyte survival prior to formation of plentiful TAMs in the tumor microenvironment. PMID:26895960

NK cell-released exosomes: Natural nanobullets against tumors.

PubMed

Fais, Stefano

2013-01-01

We have recently reported that human natural killer (NK) cells release exosomes that express both NK-cell markers and cytotoxic molecules. Similar results were obtained with circulating exosomes from human healthy donors. Both NK-cell derived and circulating exosomes exerted a full functional activity and killed both tumor and activated immune cells. These findings indicate that NK-cell derived exosomes might constitute a new promising therapeutic tool.

Tumor-derived exosomes regulate expression of immune function-related genes in human T cell subsets.

PubMed

Muller, Laurent; Mitsuhashi, Masato; Simms, Patricia; Gooding, William E; Whiteside, Theresa L

2016-02-04

Tumor cell-derived exosomes (TEX) suppress functions of immune cells. Here, changes in the gene profiles of primary human T lymphocytes exposed in vitro to exosomes were evaluated. CD4(+) Tconv, CD8(+) T or CD4(+) CD39(+) Treg were isolated from normal donors' peripheral blood and co-incubated with TEX or exosomes isolated from supernatants of cultured dendritic cells (DEX). Expression levels of 24-27 immune response-related genes in these T cells were quantified by qRT-PCR. In activated T cells, TEX and DEX up-regulated mRNA expression levels of multiple genes. Multifactorial data analysis of ΔCt values identified T cell activation and the immune cell type, but not exosome source, as factors regulating gene expression by exosomes. Treg were more sensitive to TEX-mediated effects than other T cell subsets. In Treg, TEX-mediated down-regulation of genes regulating the adenosine pathway translated into high expression of CD39 and increased adenosine production. TEX also induced up-regulation of inhibitory genes in CD4(+) Tconv, which translated into a loss of CD69 on their surface and a functional decline. Exosomes are not internalized by T cells, but signals they carry and deliver to cell surface receptors modulate gene expression and functions of human T lymphocytes.

Lung cancer exosomes as drivers of epithelial mesenchymal transition

PubMed Central

Rahman, Mohammad A.; Barger, Jennifer F.; Lovat, Francesca; Gao, Min; Otterson, Gregory A.; Nana-Sinkam, Patrick

2016-01-01

Exosomes, a subgroup of extracellular vesicles (EVs), have been shown to serve as a conduit for the exchange of genetic information between cells. Exosomes are released from all types of cells but in abundance from cancer cells. The contents of exosomes consist of proteins and genetic material (mRNA, DNA and miRNA) from the cell of origin. In this study, we examined the effects of exosomes derived from human lung cancer serum and both highly metastatic and non-metastatic cells on recipient human bronchial epithelial cells (HBECs). We found that exosomes derived from highly metastatic lung cancer cells and human late stage lung cancer serum induced vimentin expression, and epithelial to mesenchymal transition (EMT) in HBECs. Exosomes derived from highly metastatic cancer cells as well as late stage lung cancer serum induce migration, invasion and proliferation in non-cancerous recipient cells. Our results suggest that cancer derived exosomes could be a potential mediator of EMT in the recipient cells. PMID:27363026

Lung cancer exosomes as drivers of epithelial mesenchymal transition.

PubMed

Rahman, Mohammad A; Barger, Jennifer F; Lovat, Francesca; Gao, Min; Otterson, Gregory A; Nana-Sinkam, Patrick

2016-08-23

Exosomes, a subgroup of extracellular vesicles (EVs), have been shown to serve as a conduit for the exchange of genetic information between cells. Exosomes are released from all types of cells but in abundance from cancer cells. The contents of exosomes consist of proteins and genetic material (mRNA, DNA and miRNA) from the cell of origin. In this study, we examined the effects of exosomes derived from human lung cancer serum and both highly metastatic and non-metastatic cells on recipient human bronchial epithelial cells (HBECs). We found that exosomes derived from highly metastatic lung cancer cells and human late stage lung cancer serum induced vimentin expression, and epithelial to mesenchymal transition (EMT) in HBECs. Exosomes derived from highly metastatic cancer cells as well as late stage lung cancer serum induce migration, invasion and proliferation in non-cancerous recipient cells. Our results suggest that cancer derived exosomes could be a potential mediator of EMT in the recipient cells.

Interspecies communication between plant and mouse gut host cells through edible plant derived exosome-like nanoparticles.

PubMed

Mu, Jingyao; Zhuang, Xiaoying; Wang, Qilong; Jiang, Hong; Deng, Zhong-Bin; Wang, Baomei; Zhang, Lifeng; Kakar, Sham; Jun, Yan; Miller, Donald; Zhang, Huang-Ge

2014-07-01

Exosomes, small vesicles participating in intercellular communication, have been extensively studied recently; however, the role of edible plant derived exosomes in interspecies communication has not been investigated. Here, we investigate the biological effects of edible plant derived exosome-like nanoparticles (EPDENs) on mammalian cells. In this study, exosome-like nanoparticles from four edible plants were isolated and characterized. We show that these EPDENs contain proteins, lipids, and microRNA. EPDENs are taken up by intestinal macrophages and stem cells. The results generated from EPDEN-transfected macrophages indicate that ginger EPDENs preferentially induce the expression of the antioxidation gene, heme oxygenase-1 and the anti-inflammatory cytokine, IL-10; whereas grapefruit, ginger, and carrot EPDENs promote activation of nuclear factor like (erythroid-derived 2). Furthermore, analysis of the intestines of canonical Wnt-reporter mice, i.e. B6.Cg-Tg(BAT-lacZ)3Picc/J mice, revealed that the numbers of β-galactosidase(+) (β-Gal) intestinal crypts are increased, suggesting that EPDEN treatment of mice leads to Wnt-mediated activation of the TCF4 transcription machinery in the crypts. The data suggest a role for EPDEN-mediated interspecies communication by inducing expression of genes for anti-inflammation cytokines, antioxidation, and activation of Wnt signaling, which are crucial for maintaining intestinal homeostasis. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Interspecies communication between plant and mouse gut host cells through edible plant derived exosome-like nanoparticles

PubMed Central

Mu, Jingyao; Zhuang, Xiaoying; Wang, Qilong; Jiang, Hong; Deng, Zhong-Bin; Wang, Baomei; Zhang, Lifeng; Kakar, Sham; Jun, Yan; Miller, Donald; Zhang, Huang-Ge

2015-01-01

Scope Exosomes, small vesicles participating in intercellular communication have been extensively studied recently; however, the role of edible plant derived exosomes in interspecies communication has not been investigated. Here, we investigate the biological effects of edible plant derived exosome-like nanoparticles (EPDEN) on mammalian cells. Methods and results In this study, exosome-like nanoparticles from four edible plants were isolated and characterized. We show that these EPDENs contain proteins, lipids and microRNA. EPDENs are taken up by intestinal macrophages and stem cells. The results generated from EPDEN transfected macrophages indicate that ginger EPDENs preferentially induce the expression of the anti-oxidation gene, heme oxygenase-1 (HO-1) and the anti-inflammatory cytokine, IL-10; whereas grapefruit, ginger, and carrot EPDENs promote activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Furthermore, analysis of the intestines of canonical Wnt reporter mice, i.e., B6.Cg-Tg(BAT-lacZ)3Picc/J mice, revealed that the numbers of β-galactosidase+ (β-Gal) intestinal crypts are increased, suggesting that EPDEN treatment of mice leads to Wnt mediated activation of the Tcf4 transcription machinery in the crypts. Conclusion The data suggest a role for EPDEN mediated interspecies communication by inducing expression of genes for anti-inflammation cytokines, anti-oxidation and activation of Wnt signaling, which are crucial for maintaining intestinal homeostasis. PMID:24842810

Cancer Cell-derived Exosomes Induce Mitogen-activated Protein Kinase-dependent Monocyte Survival by Transport of Functional Receptor Tyrosine Kinases.

PubMed

Song, Xiao; Ding, Yanping; Liu, Gang; Yang, Xiao; Zhao, Ruifang; Zhang, Yinlong; Zhao, Xiao; Anderson, Gregory J; Nie, Guangjun

2016-04-15

Tumor-associated macrophages (TAM) play pivotal roles in cancer initiation and progression. Monocytes, the precursors of TAMs, normally undergo spontaneous apoptosis within 2 days, but can subsist in the inflammatory tumor microenvironment for continuous survival and generation of sufficient TAMs. The mechanisms underlying tumor-driving monocyte survival remain obscure. Here we report that cancer cell-derived exosomes were crucial mediators for monocyte survival in the inflammatory niche. Analysis of the survival-promoting molecules in monocytes revealed that cancer cell-derived exosomes activated Ras and extracellular signal-regulated kinases in the mitogen-activated protein kinase (MAPK) pathway, resulting in the prevention of caspase cleavage. Phosphorylated receptor tyrosine kinases (RTKs), such as phosphorylated epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2), were abundantly expressed in cancer cell-derived exosomes. Knock-out of EGFR or/and HER-2, or alternatively, inhibitors against their phosphorylation significantly disturbed the exosome-mediated activation of the MAPK pathway, inhibition of caspase cleavage, and increase in survival rate in monocytes. Moreover, the deprived survival-stimulating activity of exosomes due to null expression of EGFR and HER-2 could be restored by activation of another RTK, insulin receptor. Overall, our study uncovered a mechanism of tumor-associated monocyte survival and demonstrated that cancer cell-derived exosomes can stimulate the MAPK pathway in monocytes through transport of functional RTKs, leading to inactivation of apoptosis-related caspases. This work provides insights into the long sought question on monocyte survival prior to formation of plentiful TAMs in the tumor microenvironment. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The role of pancreatic cancer-derived exosomes in cancer progress and their potential application as biomarkers.

PubMed

Jin, H; Wu, Y; Tan, X

2017-08-01

Pancreatic cancer is one of the most deadly cancers, with dismal prognosis due to its poor early detection rate and high metastatic rate. Thus, elucidation of the molecular mechanisms accounting for its metastasis and discovery of competent biomarkers is required. Exosomes are multivesicular body-derived small extracellular vesicles released by various cell types that serve as important message carriers during intercellular communication. They are also known to play critical roles during cancer-genesis, cancer-related immune reactions, and metastasis. They also possess promising potential as novel biomarkers for cancer early detection. Therefore, extensive studies on pancreatic cancer-derived exosomes are currently being performed because they hold the promising potential of elevating the overall survival rate of patients with pancreatic cancer. In the present review, we focus on the role of exosomes in pancreatic cancer-related immune reactions, metastasis, and complications, and on their potential application as pancreatic cancer biomarkers.

Diagnostic profiling of salivary exosomal microRNAs in oral lichen planus patients.

PubMed

Byun, J-S; Hong, S-H; Choi, J-K; Jung, J-K; Lee, H-J

2015-11-01

Oral lichen planus is a chronic inflammatory oral mucosal disease whose exact cause is unclear and which requires efficient diagnostic and therapeutic strategies. Identification of disease-specific biomarkers in saliva is an easy, quick, and non-invasive approach for molecular diagnosis. This study was designed to examine salivary exosomal microRNAs (miRNAs) that could be candidates for diagnosing and elucidating the pathogenesis of oral lichen planus. We compared miRNA profiles of salivary exosomes of patients with oral lichen planus with those of healthy controls. Saliva samples from 16 patients with oral lichen planus and eight healthy controls were divided into two sets and examined using miRNA microarray analysis and TaqMan quantitative PCR. The three miRNAs identified (miR-4484, miR-1246, and miR-1290) were further validated. Of these, miR-4484 was significantly upregulated in the salivary exosomes of patients with oral lichen planus. This study thus identifies a potential miRNA biomarker for oral lichen planus and provides insight into the functions of miRNAs in the pathogenesis of oral inflammatory diseases. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Maximizing Exosome Colloidal Stability Following Electroporation

PubMed Central

Hood, Joshua L.; Scott, Michael J.; Wickline, Samuel A.

2014-01-01

Development of exosome based semi-synthetic nanovesicles for diagnostic and therapeutic purposes requires novel approaches to load exosomes with cargo. Electroporation has previously been used to load exosomes with RNA. However, investigations into exosome colloidal stability following electroporation have not been considered. Herein, we report the development of a unique trehalose pulse media (TPM) that minimizes exosome aggregation following electroporation. Dynamic light scattering (DLS) and RNA absorbance were employed to determine the extent of exosome aggregation and electroextraction post electroporation in TPM compared to common PBS pulse media or sucrose pulse media (SPM). Use of TPM to disaggregate melanoma exosomes post electroporation was dependent on both exosome concentration and electric field strength. TPM maximized exosome dispersal post electroporation for both homogenous B16 melanoma and heterogeneous human serum derived populations of exosomes. Moreover, TPM enabled heavy cargo loading of melanoma exosomes with 5 nm superparamagnetic iron oxide nanoparticles (SPION5) while maintaining original exosome size and minimizing exosome aggregation as evidenced by transmission electron microscopy. Loading exosomes with SPION5 increased exosome density on sucrose gradients. This provides a simple, label free means to enrich exogenously modified exosomes and introduces the potential for MRI driven theranostic exosome investigations in vivo. PMID:24333249

Maximizing exosome colloidal stability following electroporation.

PubMed

Hood, Joshua L; Scott, Michael J; Wickline, Samuel A

2014-03-01

Development of exosome-based semisynthetic nanovesicles for diagnostic and therapeutic purposes requires novel approaches to load exosomes with cargo. Electroporation has previously been used to load exosomes with RNA. However, investigations into exosome colloidal stability following electroporation have not been considered. Herein, we report the development of a unique trehalose pulse media (TPM) that minimizes exosome aggregation following electroporation. Dynamic light scattering (DLS) and RNA absorbance were employed to determine the extent of exosome aggregation and electroextraction post electroporation in TPM compared to common PBS pulse media or sucrose pulse media (SPM). Use of TPM to disaggregate melanoma exosomes post electroporation was dependent on both exosome concentration and electric field strength. TPM maximized exosome dispersal post electroporation for both homogenous B16 melanoma and heterogeneous human serum-derived populations of exosomes. Moreover, TPM enabled heavy cargo loading of melanoma exosomes with 5nm superparamagnetic iron oxide nanoparticles (SPION5) while maintaining original exosome size and minimizing exosome aggregation as evidenced by transmission electron microscopy. Loading exosomes with SPION5 increased exosome density on sucrose gradients. This provides a simple, label-free means of enriching exogenously modified exosomes and introduces the potential for MRI-driven theranostic exosome investigations in vivo. Copyright © 2013 Elsevier Inc. All rights reserved.

Exosome Theranostics: Biology and Translational Medicine

PubMed Central

He, Chuanjiang; Zheng, Shu; Luo, Yan; Wang, Ben

2018-01-01

Exosomes are common membrane-bound nanovesicles that contain diverse biomolecules, such as lipids, proteins, and nucleic acids. Exosomes are derived from cells through exocytosis, are ingested by target cells, and can transfer biological signals between local or distant cells. Exosome secretion is a constitutive phenomenon that is involved in both physiological and pathological processes and determines both the exosomal surface molecules and the contents. Hence, we can exploit exosomes as biomarkers, vaccines and drug carriers and modify them rationally for therapeutic interventions. However, it is still a challenge to identify, isolate and quantify exosomes accurately, efficiently and selectively. Further studies on exosomes will explore their potential in translational medicine and provide new avenues for the creation of effective clinical diagnostics and therapeutic strategies; the use of exosomes in these applications can be called exosome theranostics. This review describes the fundamental processes of exosome formation and uptake. In addition, the physiological and pathological roles of exosomes in biology are also illustrated with a focus on how exosomes can be exploited or engineered as powerful tools in translational medicine. PMID:29290805

Mesenchymal stem cell and derived exosome as small RNA carrier and Immunomodulator to improve islet transplantation.

PubMed

Wen, Di; Peng, Yang; Liu, Di; Weizmann, Yossi; Mahato, Ram I

2016-09-28

Human bone marrow mesenchymal stem cells (hBMSCs) and their exosomes can suppress immune reaction and deliver small RNAs. Thus, they may improve islet transplantation by delivering small RNAs for promoting islet function and inhibiting immune rejection. Here, we proposed an hBMSC and its exosome-based therapy to overcome immune rejection and poor islet function, both of which hinder the success of islet transplantation. We found overexpressed siFas and anti-miR-375 in plasmid encoding shFas and anti-miR-375 transfected hBMSC-derived exosomes, which silenced Fas and miR-375 of human islets and improved their viability and function against inflammatory cytokines. This plasmid transfected hBMSCs downregulated Fas and miR-375 of human islets in a humanized NOD scid gamma (NSG) mouse model, whose immune reaction was inhibited by injecting hBMSC and peripheral blood mononuclear cell (PBMC) co-cultured exosomes. These exosomes suppressed immune reaction by inhibiting PBMC proliferation and enhancing regulatory T cell (Treg) function. Collectively, our studies elucidated the mechanisms of RNA delivery from hBMSCs to human islets and the immunosuppressive effect of hBMSC and peripheral blood mononuclear cell co-cultured exosomes for improving islet transplantation. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular profiling of prostate cancer derived exosomes may reveal a predictive signature for response to docetaxel

PubMed Central

Kharaziha, Pedram; Chioureas, Dimitris; Rutishauser, Dorothea; Baltatzis, George; Lennartsson, Lena; Fonseca, Pedro; Azimi, Alireza; Hultenby, Kjell; Zubarev, Roman; Ullén, Anders; Yachnin, Jeffrey; Nilsson, Sten; Panaretakis, Theocharis

2015-01-01

Docetaxel is a cornerstone treatment for metastatic, castration resistant prostate cancer (CRPC) which remains a leading cause of cancer-related deaths, worldwide. The clinical usage of docetaxel has resulted in modest gains in survival, primarily due to the development of resistance. There are currently no clinical biomarkers available that predict whether a CRPC patient will respond or acquire resistance to this therapy. Comparative proteomics analysis of exosomes secreted from DU145 prostate cancer cells that are sensitive (DU145 Tax-Sen) or have acquired resistance (DU145 Tax-Res) to docetaxel, demonstrated significant differences in the amount of exosomes secreted and in their molecular composition. A panel of proteins was identified by proteomics to be differentially enriched in DU145 Tax-Res compared to DU145 Tax-Sen exosomes and was validated by western blotting. Importantly, we identified MDR-1, MDR-3, Endophilin-A2 and PABP4 that were enriched only in DU145 Tax-Res exosomes. We validated the presence of these proteins in the serum of a small cohort of patients. DU145 cells that have uptaken DU145 Tax-Res exosomes show properties of increased matrix degradation. In summary, exosomes derived from DU145 Tax-Res cells may be a valuable source of biomarkers for response to therapy. PMID:25844599

Molecular profiling of prostate cancer derived exosomes may reveal a predictive signature for response to docetaxel.

PubMed

Kharaziha, Pedram; Chioureas, Dimitris; Rutishauser, Dorothea; Baltatzis, George; Lennartsson, Lena; Fonseca, Pedro; Azimi, Alireza; Hultenby, Kjell; Zubarev, Roman; Ullén, Anders; Yachnin, Jeffrey; Nilsson, Sten; Panaretakis, Theocharis

2015-08-28

Docetaxel is a cornerstone treatment for metastatic, castration resistant prostate cancer (CRPC) which remains a leading cause of cancer-related deaths, worldwide. The clinical usage of docetaxel has resulted in modest gains in survival, primarily due to the development of resistance. There are currently no clinical biomarkers available that predict whether a CRPC patient will respond or acquire resistance to this therapy. Comparative proteomics analysis of exosomes secreted from DU145 prostate cancer cells that are sensitive (DU145 Tax-Sen) or have acquired resistance (DU145 Tax-Res) to docetaxel, demonstrated significant differences in the amount of exosomes secreted and in their molecular composition. A panel of proteins was identified by proteomics to be differentially enriched in DU145 Tax-Res compared to DU145 Tax-Sen exosomes and was validated by western blotting. Importantly, we identified MDR-1, MDR-3, Endophilin-A2 and PABP4 that were enriched only in DU145 Tax-Res exosomes. We validated the presence of these proteins in the serum of a small cohort of patients. DU145 cells that have uptaken DU145 Tax-Res exosomes show properties of increased matrix degradation. In summary, exosomes derived from DU145 Tax-Res cells may be a valuable source of biomarkers for response to therapy.

Proteomic profiling of human plasma exosomes identifies PPAR{gamma} as an exosome-associated protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Looze, Christopher; Yui, David; Leung, Lester

Exosomes are nanovesicles that are released from cells as a mechanism of cell-free intercellular communication. Only a limited number of proteins have been identified from the plasma exosome proteome. Here, we developed a multi-step fractionation scheme incorporating gel exclusion chromatography, rate zonal centrifugation through continuous sucrose gradients, and high-speed centrifugation to purify exosomes from human plasma. Exosome-associated proteins were separated by SDS-PAGE and 66 proteins were identified by LC-MS/MS, which included both cellular and extracellular proteins. Furthermore, we identified and characterized peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}), a nuclear receptor that regulates adipocyte differentiation and proliferation, as well as immune and inflammatorymore » cell functions, as a novel component of plasma-derived exosomes. Given the important role of exosomes as intercellular messengers, the discovery of PPAR{gamma} as a component of human plasma exosomes identifies a potential new pathway for the paracrine transfer of nuclear receptors.« less

Bone Marrow Mesenchymal Stem Cell-Derived CD63+ Exosomes Transport Wnt3a Exteriorly and Enhance Dermal Fibroblast Proliferation, Migration, and Angiogenesis In Vitro.

PubMed

McBride, Jeffrey D; Rodriguez-Menocal, Luis; Guzman, Wellington; Candanedo, Ambar; Garcia-Contreras, Marta; Badiavas, Evangelos V

2017-10-01

Wnts are secreted glycoproteins that regulate stem cell self-renewal, differentiation, and cell-to-cell communication during embryonic development and in adult tissues. Bone marrow mesenchymal stem cells (BM-MSCs) have been shown to stimulate dermis repair and regeneration; however, it is unclear how BM-MSCs may modulate downstream Wnt signaling. While recent reports implicate that Wnt ligands and Wnt messenger RNAs (such as Wnt4) exist within the interior compartment of exosomes, it has been debated whether or not Wnts exist on the exterior surface of exosomes to travel in the extracellular space. To help answer this question, we utilized flow cytometry of magnetic beads coated with anti-CD63 antibodies and found, for the first time, that Wnt3a protein is detectable exteriorly on CD63 + exosomes derived from BM-MSCs over-secreting Wnt3a into serum-free conditioned media (Wnt3a CM). Our data suggest that CD63 + exosomes significantly help transport exterior Wnt3a signal to recipient cells to promote fibroblast and endothelial functions. During purification of exosomes, we unexpectedly found that use of ultracentrifugation alone significantly decreased the ability to detect exteriorly bound Wnt3a on CD63 + exosomes, however, polyethylene glycol (PEG)-mediated exosome-enrichment before exosome-purification (with ultracentrifugation into a sucrose cushion) resulted in exosomes more likely to retain exterior Wnt3a detectability and downstream Wnt/beta-catenin activity. Our findings indicate the important role that purification methods may have on stem cell-derived Wnt-exosome activity in downstream assays. The ability for BM-MSC Wnt3a CM and exosomes to stimulate dermal fibroblast proliferation and migration, and endothelial angiogenesis in vitro, was significantly decreased after CD63 + -exosome depletion or knockdown of Wnt coreceptor LRP6 in recipient cells, suggesting both are required for optimal Wnt-exosome activity in our system. Thus, BM-MSC-derived CD63

Role of TIM-4 in exosome-dependent entry of HIV-1 into human immune cells

PubMed Central

Sims, Brian; Farrow, Anitra L; Williams, Sparkle D; Bansal, Anju; Krendelchtchikov, Alexandre; Gu, Linlin; Matthews, Qiana L

2017-01-01

Exosomes, 30–200 nm nanostructures secreted from donor cells and internalized by recipient cells, can play an important role in the cellular entry of some viruses. These microvesicles are actively secreted into various body fluids, including blood, urine, saliva, cerebrospinal fluid, and breast milk. We successfully isolated exosomes from human breast milk and plasma. The size and concentration of purified exosomes were measured by nanoparticle tracking, while Western blotting confirmed the presence of the exosomal-associated proteins CD9 and CD63, clathrin, and T cell immunoglobulin and mucin proteins (TIMs). Through viral infection assays, we determined that HIV-1 utilizes an exosome-dependent mechanism for entry into human immune cells. The virus contains high amounts of phosphatidylserine (PtdSer) and may bind PtdSer receptors, such as TIMs. This mechanism is supported by our findings that exosomes from multiple sources increased HIV-1 entry into T cells and macrophages, and viral entry was potently blocked with anti-TIM-4 antibodies. PMID:28740388

Post isolation modification of exosomes for nanomedicine applications.

PubMed

Hood, Joshua L

2016-07-01

Exosomes are extracellular nanovesicles. They innately possess ideal structural and biocompatible nanocarrier properties. Exosome components can be engineered at the cellular level. Alternatively, when exosome source cells are unavailable for customized exosome production, exosomes derived from a variety of biological origins can be modified post isolation which is the focus of this article. Modification of exosome surface structures allows for exosome imaging and tracking in vivo. Exosome membranes can be loaded with hydrophobic therapeutics to increase drug stability and efficacy. Hydrophilic therapeutics such as RNA can be encapsulated in exosomes to improve cellular delivery. Despite advances in post isolation exosome modification strategies, many challenges to effectively harnessing their therapeutic potential remain. Future topics of exploration include: matching exosome subtypes with nanomedicine applications, optimizing exosomal nanocarrier formulation and investigating how modified exosomes interface with the immune system. Research into these areas will greatly facilitate personalized exosome-based nanomedicine endeavors.

Post isolation modification of exosomes for nanomedicine applications

PubMed Central

Hood, Joshua L

2016-01-01

Exosomes are extracellular nanovesicles. They innately possess ideal structural and biocompatible nanocarrier properties. Exosome components can be engineered at the cellular level. Alternatively, when exosome source cells are unavailable for customized exosome production, exosomes derived from a variety of biological origins can be modified post isolation which is the focus of this article. Modification of exosome surface structures allows for exosome imaging and tracking in vivo. Exosome membranes can be loaded with hydrophobic therapeutics to increase drug stability and efficacy. Hydrophilic therapeutics such as RNA can be encapsulated in exosomes to improve cellular delivery. Despite advances in post isolation exosome modification strategies, many challenges to effectively harnessing their therapeutic potential remain. Future topics of exploration include: matching exosome subtypes with nanomedicine applications, optimizing exosomal nanocarrier formulation and investigating how modified exosomes interface with the immune system. Research into these areas will greatly facilitate personalized exosome-based nanomedicine endeavors. PMID:27348448

Plasma exosomal α-synuclein is likely CNS-derived and increased in Parkinson’s disease

PubMed Central

Cook, Travis J.; Bullock, Kristin M.; Zhao, Yanchun; Ginghina, Carmen; Li, Yanfei; Aro, Patrick; Dator, Romel; He, Chunmei; Hipp, Michael J.; Zabetian, Cyrus P.; Peskind, Elaine R.; Hu, Shu-Ching; Quinn, Joseph F.; Galasko, Douglas R.; Banks, William A.; Zhang, Jing

2014-01-01

Extracellular α-synuclein is important in the pathogenesis of Parkinson disease (PD) and also as a potential biomarker when tested in the cerebrospinal fluid (CSF). The performance of blood plasma or serum α-synuclein as a biomarker has been found to be inconsistent and generally ineffective, largely due to the contribution of peripherally derived α-synuclein. In this study, we discovered, via an intracerebroventricular injection of radiolabeled α-synuclein into mouse brain, that CSF α-synuclein was readily transported to blood, with a small portion being contained in exosomes that are relatively specific to the central nervous system (CNS). Consequently, we developed a technique to evaluate the levels of α-synuclein in these exosomes in individual plasma samples. When applied to a large cohort of clinical samples (267 PD, 215 controls), we found that in contrast to CSF α-synuclein concentrations, which are consistently reported to be lower in PD patients compared to controls, the levels of plasma exosomal α-synuclein were substantially higher in PD patients, suggesting an increased efflux of the protein to the peripheral blood of these patients. Furthermore, although no association was observed between plasma exosomal and CSF α-synuclein, a significant correlation between plasma exosomal α-synuclein and disease severity (r=0.176, p=0.004) was observed, and the diagnostic sensitivity and specificity achieved by plasma exosomal α-synuclein were comparable to those determined by CSF α-synuclein. Further studies are clearly needed to elucidate the mechanism involved in the transport of CNS α-synuclein to the periphery, which may lead to a more convenient and robust assessment of PD clinically. PMID:24997849

Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: results of thefirst phase I clinical trial

PubMed Central

Escudier, Bernard; Dorval, Thierry; Chaput, Nathalie; André, Fabrice; Caby, Marie-Pierre; Novault, Sophie; Flament, Caroline; Leboulaire, Christophe; Borg, Christophe; Amigorena, Sebastian; Boccaccio, Catherine; Bonnerot, Christian; Dhellin, Olivier; Movassagh, Mojgan; Piperno, Sophie; Robert, Caroline; Serra, Vincent; Valente, Nancy; Le Pecq, Jean-Bernard; Spatz, Alain; Lantz, Olivier; Tursz, Thomas; Angevin, Eric; Zitvogel, Laurence

2005-01-01

Background DC derived-exosomes are nanomeric vesicles harboring functional MHC/peptide complexes capable of promoting T cell immune responses and tumor rejection. Here we report the feasability and safety of the first Phase I clinical trial using autologous exosomes pulsed with MAGE 3 peptides for the immunization of stage III/IV melanoma patients. Secondary endpoints were the monitoring of T cell responses and the clinical outcome. Patients and methods Exosomes were purified from day 7 autologous monocyte derived-DC cultures. Fifteen patients fullfilling the inclusion criteria (stage IIIB and IV, HLA-A1+, or -B35+ and HLA-DPO4+ leukocyte phenotype, tumor expressing MAGE3 antigen) were enrolled from 2000 to 2002 and received four exosome vaccinations. Two dose levels of either MHC class II molecules (0.13 versus 0.40 × 1014 molecules) or peptides (10 versus 100 μg/ml) were tested. Evaluations were performed before and 2 weeks after immunization. A continuation treatment was performed in 4 cases of non progression. Results The GMP process allowed to harvest about 5 × 1014 exosomal MHC class II molecules allowing inclusion of all 15 patients. There was no grade II toxicity and the maximal tolerated dose was not achieved. One patient exhibited a partial response according to the RECIST criteria. This HLA-B35+/A2+ patient vaccinated with A1/B35 defined CTL epitopes developed halo of depigmentation around naevi, a MART1-specific HLA-A2 restricted T cell response in the tumor bed associated with progressive loss of HLA-A2 and HLA-BC molecules on tumor cells during therapy with exosomes. In addition, one minor, two stable and one mixed responses were observed in skin and lymph node sites. MAGE3 specific CD4+ and CD8+ T cell responses could not be detected in peripheral blood. Conclusion The first exosome Phase I trial highlighted the feasibility of large scale exosome production and the safety of exosome administration. PMID:15740633

Prostate tumor-induced angiogenesis is blocked by exosomes derived from menstrual stem cells through the inhibition of reactive oxygen species

PubMed Central

Alcayaga-Miranda, Francisca; González, Paz L.; Lopez-Verrilli, Alejandra; Varas-Godoy, Manuel; Aguila-Díaz, Carolina; Contreras, Luis; Khoury, Maroun

2016-01-01

Mesenchymal stem cells (MSCs) secrete exosomes that are capable of modifying the tumor environment through different mechanisms including changes in the cancer-cell secretome. This activity depends on their cargo content that is largely defined by their cellular origin. Endometrial cells are fine regulators of the angiogenic process during the menstrual cycle that includes an angiostatic condition that is associated with the end of the cycle. Hence, we studied the angiogenic activity of menstrual stem cells (MenSCs)-secreted exosomes on prostate PC3 tumor cells. Our results showed that exosomes induce a reduction in VEGF secretion and NF-κB activity. Lower reactive oxygen species (ROS) production in exosomes-treated cells was detected by the DCF method, suggesting that the inhibition of the intracellular ROS impacts both NF-κB and VEGF pathways. We confirmed using tubule formation and plug transplantation assays that MenSCs-exosomes suppress the secretion of pro-angiogenic factors by the PC3 cells in a ROS-dependent manner. The inhibition of the tumor angiogenesis and, consequently, the tumor growth was also confirmed using a xenograft mouse model. Additionally, the anti-tumoral effect was associated with a reduction of tumor hemoglobin content, vascular density and inhibition of VEGF and HIF-1α expression. Importantly, we demonstrate that the exosomes anti-angiogenic effect is specific to the menstrual cell source, as bone marrow MSCs-derived exosomes showed an opposite effect on the VEGF and bFGF expression in tumor cells. Altogether, our results indicate that MenSCs-derived exosomes acts as blockers of the tumor-induced angiogenesis and therefore could be suitable for anti-cancer therapies. PMID:27286448

Myostatin inhibits osteoblastic differentiation by suppressing osteocyte-derived exosomal microRNA-218: A novel mechanism in muscle-bone communication.

PubMed

Qin, Yiwen; Peng, Yuanzhen; Zhao, Wei; Pan, Jianping; Ksiezak-Reding, Hanna; Cardozo, Christopher; Wu, Yingjie; Divieti Pajevic, Paola; Bonewald, Lynda F; Bauman, William A; Qin, Weiping

2017-06-30

Muscle and bone are closely associated in both anatomy and function, but the mechanisms that coordinate their synergistic action remain poorly defined. Myostatin, a myokine secreted by muscles, has been shown to inhibit muscle growth, and the disruption of the myostatin gene has been reported to cause muscle hypertrophy and increase bone mass. Extracellular vesicle-exosomes that carry microRNA (miRNA), mRNA, and proteins are known to perform an important role in cell-cell communication. We hypothesized that myostatin may play a crucial role in muscle-bone interactions and may promote direct effects on osteocytes and on osteocyte-derived exosomal miRNAs, thereby indirectly influencing the function of other bone cells. We report herein that myostatin promotes expression of several bone regulators such as sclerostin (SOST), DKK1, and RANKL in cultured osteocytic (Ocy454) cells, concomitant with the suppression of miR-218 in both parent Ocy454 cells and derived exosomes. Exosomes produced by Ocy454 cells that had been pretreated with myostatin could be taken up by osteoblastic MC3T3 cells, resulting in a marked reduction of Runx2, a key regulator of osteoblastic differentiation, and in decreased osteoblastic differentiation via the down-regulation of the Wnt signaling pathway. Importantly, the inhibitory effect of myostatin-modified osteocytic exosomes on osteoblast differentiation is completely reversed by expression of exogenous miR-218, through a mechanism involving miR-218-mediated inhibition of SOST. Together, our findings indicate that myostatin directly influences osteocyte function and thereby inhibits osteoblastic differentiation, at least in part, through the suppression of osteocyte-derived exosomal miR-218, suggesting a novel mechanism in muscle-bone communication. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Altered MicroRNA Expression Profile in Exosomes during Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

PubMed Central

Zhang, Shui-Jun; Zhao, Chen; Qiu, Bin-Song; Gu, Hai-Feng; Hong, Jian-Fei; Cao, Li; Chen, Yu; Xia, Bing; Bi, Qin; Wang, Ya-Ping

2014-01-01

The physiological role of microRNAs (miRNAs) in osteoblast differentiation remains elusive. Exosomal miRNAs isolated from human bone marrow-derived mesenchymal stem cells (BMSCs) culture were profiled using miRNA arrays containing probes for 894 human matured miRNAs. Seventy-nine miRNAs (∼8.84%) could be detected in exosomes isolated from BMSC culture supernatants when normalized to endogenous control genes RNU44. Among them, nine exosomal miRNAs were up regulated and 4 miRNAs were under regulated significantly (Relative fold>2, p<0.05) when compared with the values at 0 day with maximum changes at 1 to 7 days. Five miRNAs (miR-199b, miR-218, miR-148a, miR-135b, and miR-221) were further validated and differentially expressed in the individual exosomal samples from hBMSCs cultured at different time points. Bioinformatic analysis by DIANA-mirPath demonstrated that RNA degradation, mRNA surveillance pathway, Wnt signaling pathway, RNA transport were the most prominent pathways enriched in quantiles with differential exosomal miRNA patterns related to osteogenic differentiation. These data demonstrated exosomal miRNA is a regulator of osteoblast differentiation. PMID:25503309

Tumour exosome integrins determine organotropic metastasis.

PubMed

Hoshino, Ayuko; Costa-Silva, Bruno; Shen, Tang-Long; Rodrigues, Goncalo; Hashimoto, Ayako; Tesic Mark, Milica; Molina, Henrik; Kohsaka, Shinji; Di Giannatale, Angela; Ceder, Sophia; Singh, Swarnima; Williams, Caitlin; Soplop, Nadine; Uryu, Kunihiro; Pharmer, Lindsay; King, Tari; Bojmar, Linda; Davies, Alexander E; Ararso, Yonathan; Zhang, Tuo; Zhang, Haiying; Hernandez, Jonathan; Weiss, Joshua M; Dumont-Cole, Vanessa D; Kramer, Kimberly; Wexler, Leonard H; Narendran, Aru; Schwartz, Gary K; Healey, John H; Sandstrom, Per; Labori, Knut Jørgen; Kure, Elin H; Grandgenett, Paul M; Hollingsworth, Michael A; de Sousa, Maria; Kaur, Sukhwinder; Jain, Maneesh; Mallya, Kavita; Batra, Surinder K; Jarnagin, William R; Brady, Mary S; Fodstad, Oystein; Muller, Volkmar; Pantel, Klaus; Minn, Andy J; Bissell, Mina J; Garcia, Benjamin A; Kang, Yibin; Rajasekhar, Vinagolu K; Ghajar, Cyrus M; Matei, Irina; Peinado, Hector; Bromberg, Jacqueline; Lyden, David

2015-11-19

Ever since Stephen Paget's 1889 hypothesis, metastatic organotropism has remained one of cancer's greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6β4 and α6β1 were associated with lung metastasis, while exosomal integrin αvβ5 was linked to liver metastasis. Targeting the integrins α6β4 and αvβ5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.

The association of exosomes with lymph nodes.

PubMed

Hood, Joshua L

2017-07-01

Cells produce extracellular nanovesicles known as exosomes that transport information between tissue microenvironments. Exosomes can engage and regulate the function of various immune cell types facilitating both normal and pathological processes. It follows that exosomes should also associate with lymph nodes containing immune cells. Herein, data derived from investigations that incorporate experiments pertaining to the trafficking of exosomes to lymph nodes is reviewed. Within lymph nodes, direct evidence demonstrates that exosomes associate with dendritic cells, subcapsular sinus macrophages, B lymphocytes and stromal cells. Interactions with endothelial cells are also likely. The functional significance of these associations depends on exosome type. Continued investigations into the relationship between exosomes and lymph nodes will further our understanding of how exosomes regulate immune cells subsets and may serve to inspire new exosome based therapeutics to treat a variety of diseases. Copyright © 2016 Elsevier Ltd. All rights reserved.

Investigation of the roles of exosomes in colorectal cancer liver metastasis.

PubMed

Wang, Xia; Ding, Xiaoling; Nan, Lijuan; Wang, Yiting; Wang, Jing; Yan, Zhiqiang; Zhang, Wei; Sun, Jihong; Zhu, Wei; Ni, Bing; Dong, Suzhen; Yu, Lei

2015-05-01

The leading cause of death among cancer patients is tumor metastasis. Tumor-derived exosomes are emerging as mediators of metastasis. In the present study, we demonstrated that exosomes play a pivotal role in the metastatic progression of colorectal cancer. First, a nude mouse model of colorectal cancer liver metastasis was established and characterized. Then, we demonstrated that exosomes from a highly liver metastatic colorectal cancer cell line (HT-29) could significantly increase the metastatic tumor burden and distribution in the mouse liver of Caco-2 colorectal cancer cells, which ordinarily exhibit poor liver metastatic potential. We further investigated the mechanisms by which HT-29-derived-exosomes influence the liver metastasis of colorectal cancer and found that mice treated with HT-29-derived exosomes had a relatively higher level of CXCR4 in the metastatic microenvironment, indicating that exosomes may promote colorectal cancer metastasis by recruiting CXCR4-expressing stromal cells to develop a permissive metastatic microenvironment. Finally, the migration of Caco-2 cells was significantly increased following treatment with HT-29-derived exosomes in vitro, further supporting a role for exosomes in modulating colorectal tumor-derived liver metastasis. The data from the present study may facilitate further translational medicine research into the prevention and treatment of colorectal cancer liver metastasis.

Designer exosomes as next-generation cancer immunotherapy.

PubMed

Bell, Brandon M; Kirk, Isabel D; Hiltbrunner, Stefanie; Gabrielsson, Susanne; Bultema, Jarred J

2016-01-01

Exosomes are small 40-120 nm vesicles secreted by nearly all cells and are an important form of intercellular communication. Exosomes are abundant, stable, and highly bioavailable to tissues in vivo. Increasingly, exosomes are being recognized as potential therapeutics as they have the ability to elicit potent cellular responses in vitro and in vivo. Patient-derived exosomes have been employed as a novel cancer immunotherapy in several clinical trials, but at this point lack sufficient efficacy. Still other researchers have focused on modifying the content and function of exosomes in various ways, toward the end-goal of specialized therapeutic exosomes. Here we highlight major advances in the use of exosomes for cancer immunotherapy and exosome bioengineering followed by a discussion of focus areas for future research to generate potent therapeutic exosomes. From the Clinical Editor: Exosomes are small vesicles used by cells for intercellular communication. In this short article, the authors described the current status and the potential use of exosomes in the clinical setting. Copyright © 2015 Elsevier Inc. All rights reserved.

Exosomes Function in Tumor Immune Microenvironment.

PubMed

Huang, Yin; Liu, Keli; Li, Qing; Yao, Yikun; Wang, Ying

2018-01-01

Immune cells and mesenchymal stem/stromal cells are the major cellular components in tumor microenvironment that actively migrate to tumor sites by sensing "signals" released from tumor cells. Together with other stromal cells, they form the soil for malignant cell progression. In the crosstalk between tumor cells and its surrounded microenvironment, exosomes exert multiple functions in shaping tumor immune responses. In tumor cells, their exosomes can lead to pro-tumor immune responses, whereas in immune cells, their derived exosomes can operate on tumor cells and regulate their ability to growth, metastasis, even reaction to chemotherapy. Employing exosomes as vehicles for the delivery products to initiate anti-tumor immune responses has striking therapeutic effects on tumor progression. Thus, exosomes are potential therapeutic targets in tumor-related clinical conditions. Here we discuss the role of exosomes in regulating tumor immune microenvironment and future indications for the clinical application of exosomes.

BAT Exosomes: Metabolic Crosstalk with Other Organs and Biomarkers for BAT Activity.

PubMed

Goody, Deborah; Pfeifer, Alexander

2018-04-10

In the last decade, exosomes have gained interest as a new type of intercellular communication between cells and tissues. Exosomes are circulating, cell-derived lipid vesicles smaller than 200 nm that contain proteins and nucleic acids, including microRNAs (miRNAs), and are able to modify cellular targets. Exosomal miRNAs function as signalling molecules that regulate the transcription of their target genes and can cause phenotypic transformation of recipient cells. Recent studies have shown that brown fat secretes exosomes as a form of communication with other metabolic organs such as the liver. Moreover, it has been shown that levels of miRNAs in BAT-derived exosomes change after BAT activation in vitro and in vivo. Thus, BAT-derived exosomes can be used as potential biomarkers of BAT activity. Here, we review the present knowledge about BAT-derived exosomes and their role in metabolism.

Isolation of biologically-active exosomes from human plasma.

PubMed

Muller, Laurent; Hong, Chang-Sook; Stolz, Donna B; Watkins, Simon C; Whiteside, Theresa L

2014-09-01

Effects of exosomes present in human plasma on immune cells have not been examined in detail. Immunological studies with plasma-derived exosomes require their isolation by procedures involving ultracentrifugation. These procedures were largely developed using supernatants of cultured cells. To test biologic activities of plasma-derived exosomes, methods are necessary that ensure adequate recovery of exosome fractions free of contaminating larger vesicles, cell fragments and protein/nucleic acid aggregates. Here, an optimized method for exosome isolation from human plasma/serum specimens of normal controls (NC) or cancer patients and its advantages and pitfalls are described. To remove undesirable plasma-contaminating components, ultrafiltration of differentially-centrifuged plasma/serum followed by size-exclusion chromatography prior to ultracentrifugation facilitated the removal of contaminants. Plasma or serum was equally acceptable as a source of exosomes based on the recovered protein levels (in μg protein/mL plasma) and TEM image quality. Centrifugation on sucrose density gradients led to large exosome losses. Fresh plasma was the best source of morphologically-intact exosomes, while the use of frozen/thawed plasma decreased exosome purity but not their biologic activity. Treatments of frozen plasma with DNAse, RNAse or hyaluronidase did not improve exosome purity and are not recommended. Cancer patients' plasma consistently yielded more isolated exosomes than did NCs' plasma. Cancer patients' exosomes also mediated higher immune suppression as evidenced by decreased CD69 expression on responder CD4+ T effector cells. Thus, the described procedure yields biologically-active, morphologically-intact exosomes that have reasonably good purity without large protein losses and can be used for immunological, biomarker and other studies. Copyright © 2014 Elsevier B.V. All rights reserved.

Smartphone-enabled optofluidic exosome diagnostic for concussion recovery

NASA Astrophysics Data System (ADS)

Ko, Jina; Hemphill, Matthew A.; Gabrieli, David; Wu, Leon; Yelleswarapu, Venkata; Lawrence, Gladys; Pennycooke, Wesley; Singh, Anup; Meaney, Dave F.; Issadore, David

2016-08-01

A major impediment to improving the treatment of concussion is our current inability to identify patients that will experience persistent problems after the injury. Recently, brain-derived exosomes, which cross the blood-brain barrier and circulate following injury, have shown great potential as a noninvasive biomarker of brain recovery. However, clinical use of exosomes has been constrained by their small size (30-100 nm) and the extensive sample preparation (>24 hr) needed for traditional exosome measurements. To address these challenges, we developed a smartphone-enabled optofluidic platform to measure brain-derived exosomes. Sample-to-answer on our chip is 1 hour, 10x faster than conventional techniques. The key innovation is an optofluidic device that can detect enzyme amplified exosome biomarkers, and is read out using a smartphone camera. Using this approach, we detected and profiled GluR2+ exosomes in the post-injury state using both in vitro and murine models of concussion.

Smartphone-enabled optofluidic exosome diagnostic for concussion recovery

PubMed Central

Ko, Jina; Hemphill, Matthew A.; Gabrieli, David; Wu, Leon; Yelleswarapu, Venkata; Lawrence, Gladys; Pennycooke, Wesley; Singh, Anup; Meaney, Dave F.; Issadore, David

2016-01-01

A major impediment to improving the treatment of concussion is our current inability to identify patients that will experience persistent problems after the injury. Recently, brain-derived exosomes, which cross the blood-brain barrier and circulate following injury, have shown great potential as a noninvasive biomarker of brain recovery. However, clinical use of exosomes has been constrained by their small size (30–100 nm) and the extensive sample preparation (>24 hr) needed for traditional exosome measurements. To address these challenges, we developed a smartphone-enabled optofluidic platform to measure brain-derived exosomes. Sample-to-answer on our chip is 1 hour, 10x faster than conventional techniques. The key innovation is an optofluidic device that can detect enzyme amplified exosome biomarkers, and is read out using a smartphone camera. Using this approach, we detected and profiled GluR2+ exosomes in the post-injury state using both in vitro and murine models of concussion. PMID:27498963

Smartphone-enabled optofluidic exosome diagnostic for concussion recovery.

PubMed

Ko, Jina; Hemphill, Matthew A; Gabrieli, David; Wu, Leon; Yelleswarapu, Venkata; Lawrence, Gladys; Pennycooke, Wesley; Singh, Anup; Meaney, Dave F; Issadore, David

2016-08-08

A major impediment to improving the treatment of concussion is our current inability to identify patients that will experience persistent problems after the injury. Recently, brain-derived exosomes, which cross the blood-brain barrier and circulate following injury, have shown great potential as a noninvasive biomarker of brain recovery. However, clinical use of exosomes has been constrained by their small size (30-100 nm) and the extensive sample preparation (>24 hr) needed for traditional exosome measurements. To address these challenges, we developed a smartphone-enabled optofluidic platform to measure brain-derived exosomes. Sample-to-answer on our chip is 1 hour, 10x faster than conventional techniques. The key innovation is an optofluidic device that can detect enzyme amplified exosome biomarkers, and is read out using a smartphone camera. Using this approach, we detected and profiled GluR2+ exosomes in the post-injury state using both in vitro and murine models of concussion.

Neurotransmitter-Triggered Transfer of Exosomes Mediates Oligodendrocyte–Neuron Communication

PubMed Central

Kuo, Wen Ping; Amphornrat, Jesa; Thilemann, Sebastian; Saab, Aiman S.; Kirchhoff, Frank; Möbius, Wiebke; Goebbels, Sandra; Nave, Klaus-Armin; Schneider, Anja; Simons, Mikael; Klugmann, Matthias; Trotter, Jacqueline; Krämer-Albers, Eva-Maria

2013-01-01

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. PMID:23874151

Neurotransmitter-triggered transfer of exosomes mediates oligodendrocyte-neuron communication.

PubMed

Frühbeis, Carsten; Fröhlich, Dominik; Kuo, Wen Ping; Amphornrat, Jesa; Thilemann, Sebastian; Saab, Aiman S; Kirchhoff, Frank; Möbius, Wiebke; Goebbels, Sandra; Nave, Klaus-Armin; Schneider, Anja; Simons, Mikael; Klugmann, Matthias; Trotter, Jacqueline; Krämer-Albers, Eva-Maria

2013-07-01

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.

Tumour exosome integrins determine organotropic metastasis

PubMed Central

Hoshino, Ayuko; Costa-Silva, Bruno; Shen, Tang-Long; Rodrigues, Goncalo; Hashimoto, Ayako; Mark, Milica Tesic; Molina, Henrik; Kohsaka, Shinji; Di Giannatale, Angela; Ceder, Sophia; Singh, Swarnima; Williams, Caitlin; Soplop, Nadine; Uryu, Kunihiro; Pharmer, Lindsay; King, Tari; Bojmar, Linda; Davies, Alexander E.; Ararso, Yonathan; Zhang, Tuo; Zhang, Haiying; Hernandez, Jonathan; Weiss, Joshua M.; Dumont-Cole, Vanessa D.; Kramer, Kimberly; Wexler, Leonard H.; Narendran, Aru; Schwartz, Gary K.; Healey, John H.; Sandstrom, Per; Labori, Knut Jørgen; Kure, Elin H.; Grandgenett, Paul M.; Hollingsworth, Michael A.; de Sousa, Maria; Kaur, Sukhwinder; Jain, Maneesh; Mallya, Kavita; Batra, Surinder K.; Jarnagin, William R.; Brady, Mary S.; Fodstad, Oystein; Muller, Volkmar; Pantel, Klaus; Minn, Andy J.; Bissell, Mina J.; Garcia, Benjamin A.; Kang, Yibin; Rajasekhar, Vinagolu K.; Ghajar, Cyrus M.; Matei, Irina; Peinado, Hector; Bromberg, Jacqueline; Lyden, David

2015-01-01

Ever since Stephen Paget’s 1889 hypothesis, metastatic organotropism has remained one of cancer’s greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6β4 and α6β1 were associated with lung metastasis, while exosomal integrin αvβ5 was linked to liver metastasis. Targeting the integrins α6β4 and αvβ5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis. PMID:26524530

Tumour exosome integrins determine organotropic metastasis

DOE PAGES

Hoshino, Ayuko; Costa-Silva, Bruno; Shen, Tang-Long; ...

2015-10-28

Ever since Stephen Paget’s 1889 hypothesis, metastatic organotropism has remained one of cancer’s greatest mysteries. In this paper, we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α 6β 4 and α 6β 1 weremore » associated with lung metastasis, while exosomal integrin α vβ 5 was linked to liver metastasis. Targeting the integrins α 6β 4 and α vβ 5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. In conclusion, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.« less

Tumour exosome integrins determine organotropic metastasis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hoshino, Ayuko; Costa-Silva, Bruno; Shen, Tang-Long

Ever since Stephen Paget’s 1889 hypothesis, metastatic organotropism has remained one of cancer’s greatest mysteries. In this paper, we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α 6β 4 and α 6β 1 weremore » associated with lung metastasis, while exosomal integrin α vβ 5 was linked to liver metastasis. Targeting the integrins α 6β 4 and α vβ 5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. In conclusion, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.« less

Induction of Protective Immunity against Eimeria tenella, Eimeria maxima, and Eimeria acervulina Infections Using Dendritic Cell-Derived Exosomes

PubMed Central

Gallego, Margarita; Lee, Sung Hyen; Lillehoj, Hyun Soon; Quilez, Joaquin; Lillehoj, Erik P.; Sánchez-Acedo, Caridad

2012-01-01

This study describes a novel immunization strategy against avian coccidiosis using exosomes derived from Eimeria parasite antigen (Ag)-loaded dendritic cells (DCs). Chicken intestinal DCs were isolated and pulsed in vitro with a mixture of sporozoite-extracted Ags from Eimeria tenella, E. maxima, and E. acervulina, and the cell-derived exosomes were isolated. Chickens were nonimmunized or immunized intramuscularly with exosomes and subsequently noninfected or coinfected with E. tenella, E. maxima, and E. acervulina oocysts. Immune parameters compared among the nonimmunized/noninfected, nonimmunized/infected, and immunized/infected groups were the numbers of cells secreting Th1 cytokines, Th2 cytokines, interleukin-16 (IL-16), and Ag-reactive antibodies in vitro and in vivo readouts of protective immunity against Eimeria infection. Cecal tonsils, Peyer's patches, and spleens of immunized and infected chickens had increased numbers of cells secreting the IL-16 and the Th1 cytokines IL-2 and gamma interferon, greater Ag-stimulated proliferative responses, and higher numbers of Ag-reactive IgG- and IgA-producing cells following in vitro stimulation with the sporozoite Ags compared with the nonimmunized/noninfected and nonimmunized/infected controls. In contrast, the numbers of cells secreting the Th2 cytokines IL-4 and IL-10 were diminished in immunized and infected chickens compared with the nonimmunized/noninfected and the nonimmunized/infected controls. Chickens immunized with Ag-loaded exosomes and infected in vivo with Eimeria oocysts had increased body weight gains, reduced feed conversion ratios, diminished fecal oocyst shedding, lessened intestinal lesion scores, and reduced mortality compared with the nonimmunized/infected controls. These results suggest that successful field vaccination against avian coccidiosis using exosomes derived from DCs incubated with Ags isolated from Eimeria species may be possible. PMID:22354026

HFE genotype affects exosome phenotype in cancer.

PubMed

Mrowczynski, Oliver D; Madhankumar, A B; Slagle-Webb, Becky; Lee, Sang Y; Zacharia, Brad E; Connor, James R

2017-08-01

Neuroblastoma is the third most common childhood cancer, and timely diagnosis and sensitive therapeutic monitoring remain major challenges. Tumor progression and recurrence is common with little understanding of mechanisms. A major recent focus in cancer biology is the impact of exosomes on metastatic behavior and the tumor microenvironment. Exosomes have been demonstrated to contribute to the oncogenic effect on the surrounding tumor environment and also mediate resistance to therapy. The effect of genotype on exosomal phenotype has not yet been explored. We interrogated exosomes from human neuroblastoma cells that express wild-type or mutant forms of the HFE gene. HFE, one of the most common autosomal recessive polymorphisms in the Caucasian population, originally associated with hemochromatosis, has also been associated with increased tumor burden, therapeutic resistance boost, and negative impact on patient survival. Herein, we demonstrate that changes in genotype cause major differences in the molecular and functional properties of exosomes; specifically, HFE mutant derived exosomes have increased expression of proteins relating to invasion, angiogenesis, and cancer therapeutic resistance. HFE mutant derived exosomes were also shown to transfer this cargo to recipient cells and cause an increased oncogenic functionality in those recipient cells. Copyright © 2017. Published by Elsevier B.V.

Exosomes Derived from Squamous Head and Neck Cancer Promote Cell Survival after Ionizing Radiation

PubMed Central

Mutschelknaus, Lisa; Peters, Carsten; Winkler, Klaudia; Yentrapalli, Ramesh; Heider, Theresa; Atkinson, Michael John; Moertl, Simone

2016-01-01

Exosomes are nanometer-sized extracellular vesicles that are believed to function as intercellular communicators. Here, we report that exosomes are able to modify the radiation response of the head and neck cancer cell lines BHY and FaDu. Exosomes were isolated from the conditioned medium of irradiated as well as non-irradiated head and neck cancer cells by serial centrifugation. Quantification using NanoSight technology indicated an increased exosome release from irradiated compared to non-irradiated cells 24 hours after treatment. To test whether the released exosomes influence the radiation response of other cells the exosomes were transferred to non-irradiated and irradiated recipient cells. We found an enhanced uptake of exosomes isolated from both irradiated and non-irradiated cells by irradiated recipient cells compared to non-irradiated recipient cells. Functional analyses by exosome transfer indicated that all exosomes (from non-irradiated and irradiated donor cells) increase the proliferation of non-irradiated recipient cells and the survival of irradiated recipient cells. The survival-promoting effects are more pronounced when exosomes isolated from irradiated compared to non-irradiated donor cells are transferred. A possible mechanism for the increased survival after irradiation could be the increase in DNA double-strand break repair monitored at 6, 8 and 10 h after the transfer of exosomes isolated from irradiated cells. This is abrogated by the destabilization of the exosomes. Our results demonstrate that radiation influences both the abundance and action of exosomes on recipient cells. Exosomes transmit prosurvival effects by promoting the proliferation and radioresistance of head and neck cancer cells. Taken together, this study indicates a functional role of exosomes in the response of tumor cells to radiation exposure within a therapeutic dose range and encourages that exosomes are useful objects of study for a better understanding of tumor

Exosomes promote cetuximab resistance via the PTEN/Akt pathway in colon cancer cells.

PubMed

Zhang, S; Zhang, Y; Qu, J; Che, X; Fan, Y; Hou, K; Guo, T; Deng, G; Song, N; Li, C; Wan, X; Qu, X; Liu, Y

2017-11-13

Cetuximab is widely used in patients with metastatic colon cancer expressing wildtype KRAS. However, acquired drug resistance limits its clinical efficacy. Exosomes are nanosized vesicles secreted by various cell types. Tumor cell-derived exosomes participate in many biological processes, including tumor invasion, metastasis, and drug resistance. In this study, exosomes derived from cetuximab-resistant RKO colon cancer cells induced cetuximab resistance in cetuximab-sensitive Caco-2 cells. Meanwhile, exosomes from RKO and Caco-2 cells showed different levels of phosphatase and tensin homolog (PTEN) and phosphor-Akt. Furthermore, reduced PTEN and increased phosphorylated Akt levels were found in Caco-2 cells after exposure to RKO cell-derived exosomes. Moreover, an Akt inhibitor prevented RKO cell-derived exosome-induced drug resistance in Caco-2 cells. These findings provide novel evidence that exosomes derived from cetuximab-resistant cells could induce cetuximab resistance in cetuximab-sensitive cells, by downregulating PTEN and increasing phosphorylated Akt levels.

Exosomes and the emerging field of exosome-based gene therapy.

PubMed

O'Loughlin, Aisling J; Woffindale, Caroline A; Wood, Matthew J A

2012-08-01

Exosomes are a subtype of membrane vesicle released from the endocytic compartment of live cells. They play an important role in endogenous cell-to-cell communication. Previously shown to be capable of traversing biological barriers and to naturally transport functional nucleic acids between cells, they potentially represent a novel and exciting drug delivery vehicle for the field of gene therapy. Existing delivery vehicles are limited by concerns regarding their safety, toxicity and efficacy. In contrast, exosomes, as a natural cell-derived nanocarrier, are immunologically inert if purified from a compatible cell source and possess an intrinsic ability to cross biological barriers. Already utilised in a number of clinical trials, exosomes appear to be well-tolerated, even following repeat administration. Recent studies have shown that exosomes may be used to encapsulate and protect exogenous oligonucleotides for delivery to target cells. They therefore may be valuable for the delivery of RNA interference and microRNA regulatory molecules in addition to other single-stranded oligonucleotides. Prior to clinical translation, this nanotechnology requires further development by refinement of isolation, purification, loading, delivery and targeting protocols. Thus, exosome-mediated nanodelivery is highly promising and may fill the void left by current delivery methods for systemic gene therapy.

Exosomes in cancer theranostic: Diamonds in the rough

PubMed Central

Cordonnier, Marine; Chanteloup, Gaëtan; Isambert, Nicolas; Seigneuric, Renaud; Fumoleau, Pierre; Garrido, Carmen; Gobbo, Jessica

2017-01-01

ABSTRACT During the last 10 years, exosomes, which are small vesicles of 50–200 nm diameter of endosomal origin, have aroused a great interest in the scientific and clinical community for their roles in intercellular communication in almost all physiological and pathological processes. Most cells can potentially release these nanovesicles that share with the parent cell a similar lipid bilayer with transmembrane proteins and a panel of enclosed soluble proteins such as heat shock proteins and genetic material, thus acting as potential nanoshuttles of biomarkers. Exosomes surface proteins allow their targeting and capture by recipient cells, while the exosomes' content can modify the physiological state of recipient cells. Tumor derived exosomes by interacting with other cells of the tumor microenvironment modulate tumor progression, angiogenic switch, metastasis, and immune escape. Targeting tumor-derived exosomes might be an interesting approach in cancer therapy. Furthermore, because a key issue to improve cancer patients' outcome relies on earlier cancer diagnosis (metastases, as opposed to the primary tumor, are responsible for most cancer deaths) exosomes have been put forward as promising biomarker candidates for cancer diagnosis and prognosis. This review summarizes the roles of exosomes in cancer and clinical interest, focusing on the importance of exosomal heat shock proteins (HSP). The challenges of clinical translation of HSP-exosomes as therapeutic targets and biomarkers for early cancer detection are also discussed. PMID:28166442

Exosomes in cancer theranostic: Diamonds in the rough.

PubMed

Cordonnier, Marine; Chanteloup, Gaëtan; Isambert, Nicolas; Seigneuric, Renaud; Fumoleau, Pierre; Garrido, Carmen; Gobbo, Jessica

2017-03-04

During the last 10 years, exosomes, which are small vesicles of 50-200 nm diameter of endosomal origin, have aroused a great interest in the scientific and clinical community for their roles in intercellular communication in almost all physiological and pathological processes. Most cells can potentially release these nanovesicles that share with the parent cell a similar lipid bilayer with transmembrane proteins and a panel of enclosed soluble proteins such as heat shock proteins and genetic material, thus acting as potential nanoshuttles of biomarkers. Exosomes surface proteins allow their targeting and capture by recipient cells, while the exosomes' content can modify the physiological state of recipient cells. Tumor derived exosomes by interacting with other cells of the tumor microenvironment modulate tumor progression, angiogenic switch, metastasis, and immune escape. Targeting tumor-derived exosomes might be an interesting approach in cancer therapy. Furthermore, because a key issue to improve cancer patients' outcome relies on earlier cancer diagnosis (metastases, as opposed to the primary tumor, are responsible for most cancer deaths) exosomes have been put forward as promising biomarker candidates for cancer diagnosis and prognosis. This review summarizes the roles of exosomes in cancer and clinical interest, focusing on the importance of exosomal heat shock proteins (HSP). The challenges of clinical translation of HSP-exosomes as therapeutic targets and biomarkers for early cancer detection are also discussed.

Pancreatic cancer-derived exosomes suppress the production of GIP and GLP-1 from STC-1 cells in vitro by down-regulating the PCSK1/3.

PubMed

Zhang, Yuefeng; Huang, Shifei; Li, Pengping; Chen, Qing; Li, Yongzhou; Zhou, Yizhao; Wang, Lantian; Kang, Muxing; Zhang, Bo; Yang, Bin; Wu, Yulian

2018-05-22

One hallmark of pancreatic cancer (PC) is the high prevalence of pancreatic cancer-associated diabetes mellitus (PC-DM), but the mechanisms remain to be elucidated. Patients with PC who are diagnosed with new-onset diabetes/prediabetes have recently been shown to display significantly lower levels of glucose-dependent insulinotropic peptide (GIP) secreted mainly by enteroendocrine cells. We hypothesized that PC-derived exosomes are responsible for the decreased levels of incretins in patients with PC-DM. In this study, exosomes were successfully isolated from PANC-1, MIA PaCa-2 and SW620 cells and characterized. Only the exosomes from MIA PaCa-2 cells (Exo-Mia) reduce the production of GIP and glucagon-like peptide-1 (GLP-1) from STC-1 cells in vitro in a concentration- and time-dependent manner. Moreover, Exo-Mia increased the levels of the Gip and proglucagon mRNAs and decreased the expression of proprotein convertase subtilisin/kexin type 1/3 (PCSK1/3), which is responsible for the post-translational processing of Gip and proglucagon. Furthermore, differentially expressed exosomal miRNAs (miR-6796-3p, miR-6763-5p, miR-4750-3p and miR-197-3p) were identified and considered to be responsible for the inhibitory effects on GIP and GLP-1 production. To further determine the approach of cancer-derived exosomes reaching enteroendocrine cells, we analyzed the uptake and distribution of exosomes in animal model. It was observed that exosomes infused into the intestinal cavity were more easily internalized by the intestinal epithelium than exosomes injected into blood. In conclusion, pancreatic cancer-derived exosomes (Exo-Mia) suppress the synthesis of GIP and GLP-1 from STC-1 cells in vitro by down-regulating the PCSK1/3. Moreover, it may be the pancreatic juice that transport cancer-derived exosomes to target cells (K and L cells) in the gut. Copyright © 2018 Elsevier B.V. All rights reserved.

SELEX-Based Screening of Exosome-Tropic RNA.

PubMed

Yamashita, Takuma; Shinotsuka, Haruka; Takahashi, Yuki; Kato, Kana; Nishikawa, Makiya; Takakura, Yoshinobu

2017-01-01

Cell-derived nanosized vesicles or exosomes are expected to become delivery carriers for functional RNAs, such as small interfering RNA (siRNA). A method to efficiently load functional RNAs into exosomes is required for the development of exosome-based delivery carriers of functional RNAs. However, there is no method to find exosome-tropic exogenous RNA sequences. In this study, we used a systematic evolution of ligands by exponential enrichment (SELEX) method to screen exosome-tropic RNAs that can be used to load functional RNAs into exosomes by conjugation. Pooled single stranded 80-base RNAs, each of which contains a randomized 40-base sequence, were transfected into B16-BL6 murine melanoma cells and exosomes were collected from the cells. RNAs extracted from the exosomes were subjected to next round of SELEX. Cloning and sequencing of RNAs in SELEX-screened RNA pools showed that 29 of 56 clones had a typical RNA sequence. The sequence found by SELEX was enriched in exosomes after transfection to B16-BL6 cells. The results show that the SELEX-based method can be used for screening of exosome-tropic RNAs.

[Hydrogel microchip as a tool for studying exosomes in human serum].

PubMed

Butvilovskaya, V I; Tikhonov, A A; Savvateeva, E N; Ragimov, A A; Salimov, E L; Voloshin, S A; Sidorov, D V; Chernichenko, M A; Polyakov, A P; Filushin, M M; Tsybulskaya, M V; Rubina, A Yu

2017-01-01

Exosomes are cell-derived vesicles that are secreted by both normal and cancer cells. Over the last decade, a few studies have revealed that exosomes cross talk and/or influence major tumor-related pathways such as angiogenesis and metastasis involving many cell types within the tumor microenvironment. The protein composition of the membrane of an exosome reflects that of the membrane of the cell of origin. Because of this, tumor-derived exosomes differ from exosomes that are derived from normal cells. The detection of tumor exosomes and analysis of their molecular composition hold promise for diagnosis and prognosis of cancer. Here, we present hydrogel microarrays (biochips), which contain a panel of immobilized antibodies that recognize tetraspanins (CD9, CD63, CD81) and prognostic markers for colorectal cancer (A33, CD147). These biochips make it possible to analyze the surface proteins of either isolated exosomes or exosomes that are present in the serum samples without isolation. These biochips were successfully used to analyze the surface proteins of exosomes from serum that was collected from a colorectal cancer patient and healthy donor. Biochip-guided immunofluorescent analysis of the exosomes has made it possible for us to detect the A33 antigen and CD147 in the serum sample of the colorectal cancer patient with normal levels of CEA and CA19-9.

3D plasmonic nanobowl platform for the study of exosomes in solution

NASA Astrophysics Data System (ADS)

Lee, Changwon; Carney, Randy P.; Hazari, Sidhartha; Smith, Zachary J.; Knudson, Alisha; Robertson, Christopher S.; Lam, Kit S.; Wachsmann-Hogiu, Sebastian

2015-05-01

Thin silver film coated nanobowl Surface Enhanced Raman Spectroscopy (SERS) substrates are used to capture exosomes in solution for SERS measurements that can provide biochemical analysis of intact and ruptured exosomes. Exosomes derived via Total Exosome Isolation Reagent (TEIR) as well as ultracentrifugation (UC) from the SKOV3 cell line were analyzed. Spectra of exosomes derived via TEIR are dominated by a signal characteristic for the TEIR kit that needs to be subtracted for all measurements. Differences in SERS spectra recorded at different times during the drying of the exosome solution are statistically analyzed with Principal Component Analysis (PCA). At the beginning of the drying process, SERS spectra of exosomes exhibit peaks characteristic for both lipids and proteins. Later on during the drying process, new SERS peaks develop, suggesting that the initially intact exosome ruptures over time. This time-dependent evolution of SERS peaks enables analysis of exosomal membrane contents and the contents inside the exosomes.

Macrophages play an essential role in antigen-specific immune suppression mediated by T CD8⁺ cell-derived exosomes.

PubMed

Nazimek, Katarzyna; Ptak, Wlodzimierz; Nowak, Bernadeta; Ptak, Maria; Askenase, Philip W; Bryniarski, Krzysztof

2015-09-01

Murine contact sensitivity (CS) reaction could be antigen-specifically regulated by T CD8(+) suppressor (Ts) lymphocytes releasing microRNA-150 in antibody light-chain-coated exosomes that were formerly suggested to suppress CS through action on macrophages (Mφ). The present studies investigated the role of Mφ in Ts cell-exosome-mediated antigen-specific suppression as well as modulation of Mφ antigen-presenting function in humoral and cellular immunity by suppressive exosomes. Mice depleted of Mφ by clodronate liposomes could not be tolerized and did not produce suppressive exosomes. Moreover, isolated T effector lymphocytes transferring CS were suppressed by exosomes only in the presence of Mφ, demonstrating the substantial role of Mφ in the generation and action of Ts cell regulatory exosomes. Further, significant decrease of number of splenic B cells producing trinitrophenyl (TNP) -specific antibodies with the alteration of the ratio of serum titres of IgM to IgG was observed in recipients of exosome-treated, antigen-pulsed Mφ and the significant suppression of CS was demonstrated in recipients of exosome-treated, TNP-conjugated Mφ. Additionally, exosome-pulsed, TNP-conjugated Mφ mediated suppression of CS in mice pre-treated with a low-dose of cyclophosphamide, suggesting de novo induction of T regulatory (Treg) lymphocytes. Treg cell involvement in the effector phase of the studied suppression mechanism was proved by unsuccessful tolerization of DEREG mice depleted of Treg lymphocytes. Furthermore, the inhibition of proliferation of CS effector cells cultured with exosome-treated Mφ in a transmembrane manner was observed. Our results demonstrated the essential role of Mφ in antigen-specific immune suppression mediated by Ts cell-derived exosomes and realized by induction of Treg lymphocytes and inhibition of T effector cell proliferation. © 2015 John Wiley & Sons Ltd.

Colorectal cancer cell-derived exosomes containing miR-10b regulate fibroblast cells via the PI3K/Akt pathway.

PubMed

Dai, Guangyao; Yao, Xiaoguang; Zhang, Yubin; Gu, Jianbin; Geng, Yunfeng; Xue, Fei; Zhang, Jingcheng

2018-04-01

Cancer-associated fibroblasts (CAFs) contribute to the proliferation of colorectal cancer(CRC) cells. However, the mechanism by which CAFs develop in the tumor microenvironment remains unknown. Exosomes may be involved in activating CAFs. Using a miRNA expression profiling array, we determined the miRNA expression profile of secretory exosomes in CRC cells and then identified potential miRNAs with significant differential expression compared to normal cells via enrichment analysis. Predicted targets of candidate miRNAs were then assessed via bioinformatics analysis. Realtime qPCR, western blot, and cell cycle analyses were performed to evaluate the role of candidate exosomal miRNAs. Luciferase reporter assays were applied to confirm whether candidate exosomal miRNAs control target pathway expression. A CRC xenograft mouse model was constructed to evaluate tumor growth in vivo. Exosomes from CRC cells contained significantly higher levels of miR-10b than did exosomes from normal colorectal epithelial cells. Moreover, exosomes containing miR-10b were transferred to fibroblasts. Bioinformatics analysis identified PIK3CA, as a potential target of miR-10b. Luciferase reporter assays confirmed that miR-10b directly inhibited PIK3CA expression. Co-culturing fibroblasts with exosomes containing miR-10b significantly suppressed PIK3CA expression and decreased PI3K/Akt/mTOR pathway activity. Finally, exosomes containing miR-10b reduced fibroblast proliferation but promoted expression of TGF-β and SM α-actin, suggesting that exosomal miR-10b may activate fibroblasts to become CAFs that express myofibroblast markers. These activated fibroblasts were able to promote CRC growth in vitro and in vivo. CRC-derived exosomes actively promote disease progression by modulating surrounding stromal cells, which subsequently acquire features of CAFs. Copyright © 2018 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.

Identifying Exosome-Derived MicroRNAs as Candidate Biomarkers of Frailty.

PubMed

Ipson, B R; Fletcher, M B; Espinoza, S E; Fisher, A L

2018-01-01

Frailty is a geriatric syndrome associated with progressive physical decline and significantly increases risk for falls, disability, hospitalizations, and death. However, much remains unknown regarding the biological mechanisms that contribute to aging and frailty, and to date, there are no clinically used prognostic or diagnostic molecular biomarkers. The present study profiled exosome-derived microRNAs isolated from the plasma of young, robust older, and frail older individuals and identified eight miRNAs that are uniquely enriched in frailty: miR-10a-3p, miR-92a-3p, miR-185-3p, miR-194-5p, miR-326, miR-532-5p, miR-576-5p, and miR-760. Furthermore, since exosomes can deliver miRNAs to alter cellular activity and behavior, these miRNAs may also provide insights into the biological mechanisms underlying frailty; KEGG analysis of their target genes revealed multiple pathways implicated in aging and age-related processes. Although further validation and research studies are warranted, our study identified eight novel candidate biomarkers of frailty that may help to elucidate the multifactorial pathogenesis of frailty.

Exosomes derived from hypoxia-preconditioned mesenchymal stromal cells ameliorate cognitive decline by rescuing synaptic dysfunction and regulating inflammatory responses in APP/PS1 mice.

PubMed

Cui, Guo-Hong; Wu, Jing; Mou, Fang-Fang; Xie, Wei-Hua; Wang, Fu-Bo; Wang, Qiang-Li; Fang, Jie; Xu, Yan-Wu; Dong, You-Rong; Liu, Jian-Ren; Guo, Hai-Dong

2018-02-01

Administration of exosomes derived from mesenchymal stromal cells (MSCs) could improve some neurologic conditions by transferring functional biomolecules to recipient cells. Furthermore, exosomes from hypoxic progenitor cells exerted better therapeutic effects in organ injury through specific cargoes. However, there are no related reports about whether exosomes derived from MSCs or hypoxia-preconditioned MSCs (PC-MSCs) could prevent memory deficits in Alzheimer disease (AD). In this study, the exosomes derived from MSCs or PC-MSCs were systemically administered to transgenic APP/PS1 mice. The expression of miR-21 in MSCs was significantly increased after hypoxic treatment. Injection of exosomes from normoxic MSCs could rescue cognition and memory impairment according to results of the Morris water maze test, reduced plaque deposition, and Aβ levels in the brain; could decrease the activation of astrocytes and microglia; could down-regulate proinflammatory cytokines (TNF-α and IL-1β); and could up-regulate anti-inflammatory cytokines (IL-4 and -10) in AD mice, as well as reduce the activation of signal transducer and activator of transcription 3 (STAT3) and NF-κB. Compared to the group administered exosomes from normoxic MSCs, in the group administered exosomes from PC-MSCs, learning and memory capabilities were significantly improved; the plaque deposition and Aβ levels were lower, and expression of growth-associated protein 43, synapsin 1, and IL-10 was increased; and the levels of glial fibrillary acidic protein, ionized calcium-binding adaptor molecule 1, TNF-α, IL-1β, and activation of STAT3 and NF-κB were sharply decreased. More importantly, exosomes from PC-MSCs effectively increased the level of miR-21 in the brain of AD mice. Additionally, replenishment of miR-21 restored the cognitive deficits in APP/PS1 mice and prevented pathologic features. Taken together, these findings suggest that exosomes from PC-MSCs could improve the learning and memory

Cardiac myocyte exosomes: stability, HSP60, and proteomics.

PubMed

Malik, Z A; Kott, K S; Poe, A J; Kuo, T; Chen, L; Ferrara, K W; Knowlton, A A

2013-04-01

Exosomes, which are 50- to 100-nm-diameter lipid vesicles, have been implicated in intercellular communication, including transmitting malignancy, and as a way for viral particles to evade detection while spreading to new cells. Previously, we demonstrated that adult cardiac myocytes release heat shock protein (HSP)60 in exosomes. Extracellular HSP60, when not in exosomes, causes cardiac myocyte apoptosis via the activation of Toll-like receptor 4. Thus, release of HSP60 from exosomes would be damaging to the surrounding cardiac myocytes. We hypothesized that 1) pathological changes in the environment, such as fever, change in pH, or ethanol consumption, would increase exosome permeability; 2) different exosome inducers would result in different exosomal protein content; 3) ethanol at "physiological" concentrations would cause exosome release; and 4) ROS production is an underlying mechanism of increased exosome production. We found the following: first, exosomes retained their protein cargo under different physiological/pathological conditions, based on Western blot analyses. Second, mass spectrometry demonstrated that the protein content of cardiac exosomes differed significantly from other types of exosomes in the literature and contained cytosolic, sarcomeric, and mitochondrial proteins. Third, ethanol did not affect exosome stability but greatly increased the production of exosomes by cardiac myocytes. Fourth, ethanol- and hypoxia/reoxygenation-derived exosomes had different protein content. Finally, ROS inhibition reduced exosome production but did not completely inhibit it. In conclusion, exosomal protein content is influenced by the cell source and stimulus for exosome formation. ROS stimulate exosome production. The functions of exosomes remain to be fully elucidated.

Exosomes in Cancer Disease.

PubMed

Zöller, Margot

2016-01-01

Cancer diagnosis and therapy is steadily improving. Still, diagnosis is frequently late and diagnosis and follow-up procedures mostly are time-consuming and expensive. Searching for tumor-derived exosomes (TEX) in body fluids may provide an alternative, minimally invasive, yet highly reliable diagnostic tool. Beyond this, there is strong evidence that TEX could become a potent therapeutics. Exosomes, small vesicles delivered by many cells of the organism, are found in all body fluids. Exosomes are characterized by lipid composition, common and donor cell specific proteins, mRNA, small non-coding RNA including miRNA and DNA. Particularly the protein and miRNA markers received much attention as they may allow for highly specific diagnosis and can provide hints toward tumor aggressiveness and progression, where exosome-based diagnosis and follow-up is greatly facilitated by the recovery of exosomes in body fluids, particularly the peripheral blood. Beyond this, exosomes are the most important intercellular communicators that modulate, instruct, and reprogram their surrounding as well as distant organs. In concern about TEX this includes message transfer from tumor cells toward the tumor stroma, the premetastatic niche, the hematopoietic system and, last but not least, the instruction of non-cancer stem cells by cancer-initiating cells (CIC). Taking this into account, it becomes obvious that "tailored" exosomes offer themselves as potent therapeutic delivery system. In brief, during the last 4-5 years there is an ever-increasing, overwhelming interest in exosome research. This boom appears fully justified provided the content of the exosomes becomes most thoroughly analyzed and their mode of intercellular interaction can be unraveled in detail as this knowledge will open new doors toward cancer diagnosis and therapy including immunotherapy and CIC reprogramming.

Increasing the immune activity of exosomes: the effect of miRNA-depleted exosome proteins on activating dendritic cell/cytokine-induced killer cells against pancreatic cancer.

PubMed

Que, Ri-Sheng; Lin, Cheng; Ding, Guo-Ping; Wu, Zheng-Rong; Cao, Li-Ping

2016-05-01

Tumor-derived exosomes were considered to be potential candidates for tumor vaccines because they are abundant in immune-regulating proteins, whereas tumor exosomal miRNAs may induce immune tolerance, thereby having an opposite immune function. This study was designed to separate exosomal protein and depleted exosomal microRNAs (miRNAs), increasing the immune activity of exosomes for activating dendritic cell/cytokine-induced killer cells (DC/CIKs) against pancreatic cancer (PC). PC-derived exosomes (PEs) were extracted from cultured PANC-1 cell supernatants and then ruptured; this was followed by ultrafiltered exosome lysates (UELs). DCs were stimulated with lipopolysaccharide (LPS), PE, and UEL, followed by co-culture with CIKs. The anti-tumor effects of DC/CIKs against PC were evaluated by proliferation and killing rates, tumor necrosis factor-α (TNF-α) and perforin secretion. Exosomal miRNAs were depleted after lysis and ultrafiltration, while 128 proteins were retained, including several immune-activating proteins. UEL-stimulated DC/CIKs showed a higher killing rate than LPS- and PE-stimulated DC/CIKs. miRNA-depleted exosome proteins may be promising agonists for specifically activating DC/CIKs against PC.

Vascular smooth muscle cell calcification is mediated by regulated exosome secretion.

PubMed

Kapustin, Alexander N; Chatrou, Martijn L L; Drozdov, Ignat; Zheng, Ying; Davidson, Sean M; Soong, Daniel; Furmanik, Malgorzata; Sanchis, Pilar; De Rosales, Rafael Torres Martin; Alvarez-Hernandez, Daniel; Shroff, Rukshana; Yin, Xiaoke; Muller, Karin; Skepper, Jeremy N; Mayr, Manuel; Reutelingsperger, Chris P; Chester, Adrian; Bertazzo, Sergio; Schurgers, Leon J; Shanahan, Catherine M

2015-04-10

Matrix vesicles (MVs), secreted by vascular smooth muscle cells (VSMCs), form the first nidus for mineralization and fetuin-A, a potent circulating inhibitor of calcification, is specifically loaded into MVs. However, the processes of fetuin-A intracellular trafficking and MV biogenesis are poorly understood. The objective of this study is to investigate the regulation, and role, of MV biogenesis in VSMC calcification. Alexa488-labeled fetuin-A was internalized by human VSMCs, trafficked via the endosomal system, and exocytosed from multivesicular bodies via exosome release. VSMC-derived exosomes were enriched with the tetraspanins CD9, CD63, and CD81, and their release was regulated by sphingomyelin phosphodiesterase 3. Comparative proteomics showed that VSMC-derived exosomes were compositionally similar to exosomes from other cell sources but also shared components with osteoblast-derived MVs including calcium-binding and extracellular matrix proteins. Elevated extracellular calcium was found to induce sphingomyelin phosphodiesterase 3 expression and the secretion of calcifying exosomes from VSMCs in vitro, and chemical inhibition of sphingomyelin phosphodiesterase 3 prevented VSMC calcification. In vivo, multivesicular bodies containing exosomes were observed in vessels from chronic kidney disease patients on dialysis, and CD63 was found to colocalize with calcification. Importantly, factors such as tumor necrosis factor-α and platelet derived growth factor-BB were also found to increase exosome production, leading to increased calcification of VSMCs in response to calcifying conditions. This study identifies MVs as exosomes and shows that factors that can increase exosome release can promote vascular calcification in response to environmental calcium stress. Modulation of the exosome release pathway may be as a novel therapeutic target for prevention. © 2015 American Heart Association, Inc.

Exosome function: from tumor immunology to pathogen biology.

PubMed

Schorey, Jeffrey S; Bhatnagar, Sanchita

2008-06-01

Exosomes are the newest family member of 'bioactive vesicles' that function to promote intercellular communication. Exosomes are derived from the fusion of multivesicular bodies with the plasma membrane and extracellular release of the intraluminal vesicles. Recent studies have focused on the biogenesis and composition of exosomes as well as regulation of exosome release. Exosomes have been shown to be released by cells of hematopoietic and non-hematopoietic origin, yet their function remains enigmatic. Much of the prior work has focused on exosomes as a source of tumor antigens and in presentation of tumor antigens to T cells. However, new studies have shown that exosomes might also promote cell-to-cell spread of infectious agents. Moreover, exosomes isolated from cells infected with various intracellular pathogens, including Mycobacterium tuberculosis and Toxoplasma gondii, have been shown to contain microbial components and can promote antigen presentation and macrophage activation, suggesting that exosomes may function in immune surveillance. In this review, we summarize our understanding of exosome biogenesis but focus primarily on new insights into exosome function. We also discuss their possible use as disease biomarkers and vaccine candidates.

[Effects of human amniotic epithelial stem cells-derived exosomes on healing of wound with full-thickness skin defect in rats].

PubMed

Zhao, B; Wu, G F; Zhang, Y J; Zhang, W; Yang, F F; Xiao, D; Zeng, K X; Shi, J H; Su, L L; Hu, D H

2017-01-20

Objective: To investigate the effects of human amniotic epithelial stem cells-derived exosomes on healing of wound with full-thickness skin defect in rats. Methods: (1) Human amniotic epithelial stem cells were isolated from the amnion tissue of 5 full-term pregnant women in Department of Obstetrics of our hospital by the method of trypsin digestion, and their morphology was observed. The third passage of cells were stained with rhodamine-phalloidin for cytoskeleton observation. The third passage of cells were identified with flow cytometry through the detection of expressions of cell surface markers CD29, CD31, CD34, CD90, CD105, SSEA3, SSEA4 and immunity-related marker human leukocyte antigen-D related site (HLA-DR). The third passage of cells were also assessed the ability of adipogenic and osteogenic differentiation. (2) The third passage of human amniotic epithelial stem cells were cultured in DMEM medium supplemented with 10% exosome-free fetal bovine serum. Exosomes were isolated from culture supernatant by the method of ultracentrifugation and represented with scanning electron microscope for morphologic observation. (3) Six adult SD rats were anesthetized, and four 1 cm×1 cm sized wounds with full-thickness skin defect were made on the back of each rat. The wounds on the back of each rat were divided into control group, 25 μg/mL exosomes group, 50 μg/mL exosomes group, and 100 μg/mL exosomes group according to the random number table (with 6 wounds in each group), and a total volume of 100 μL phosphate buffered saline, 25 μg/mL exosomes, 50 μg/mL exosomes, and 100 μg/mL exosomes were evenly injected around the wound through multiple subcutaneous sites, respectively. The wound healing rate was calculated based on measurement on post injury day (PID) 7, 14, and 21. On PID 21, the healed wound tissue of each group was collected and stained with HE to observe and count skin accessories, and the arrangement of collagen fibers was observed with Masson

Hypoxic exosomes facilitate bladder tumor growth and development through transferring long non-coding RNA-UCA1.

PubMed

Xue, Mei; Chen, Wei; Xiang, An; Wang, Ruiqi; Chen, He; Pan, Jingjing; Pang, Huan; An, Hongli; Wang, Xiang; Hou, Huilian; Li, Xu

2017-08-25

To overcome the hostile hypoxic microenvironment of solid tumors, tumor cells secrete a large number of non-coding RNA-containing exosomes that facilitate tumor development and metastasis. However, the precise mechanisms of tumor cell-derived exosomes during hypoxia are unknown. Here, we aim to clarify whether hypoxia affects tumor growth and progression by transferring long non-coding RNA-urothelial cancer-associated 1 (lncRNA-UCA1) enriched exosomes secreted from bladder cancer cells. We used bladder cancer 5637 cells with high expression of lncRNA-UCA1 as exosome-generating cells and bladder cancer UMUC2 cells with low expression of lncRNA-UCA1 as recipient cells. Exosomes derived from 5637 cells cultured under normoxic or hypoxic conditions were isolated and identified by transmission electron microscopy, nanoparticle tracking analysis and western blotting analysis. These exosomes were co-cultured with UMUC2 cells to evaluate cell proliferation, migration and invasion. We further investigated the roles of exosomal lncRNA-UCA1 derived from hypoxic 5637 cells by xenograft models. The availability of lncRNA-UCA1 in serum-derived exosomes as a biomarker for bladder cancer was also assessed. We found that hypoxic exosomes derived from 5637 cells promoted cell proliferation, migration and invasion, and hypoxic exosomal RNAs could be internalized by three bladder cancer cell lines. Importantly, lncRNA-UCA1 was secreted in hypoxic 5637 cell-derived exosomes. Compared with normoxic exosomes, hypoxic exosomes derived from 5637 cells showed the higher expression levels of lncRNA-UCA1. Moreover, Hypoxic exosomal lncRNA-UCA1 could promote tumor growth and progression though epithelial-mesenchymal transition, in vitro and in vivo. In addition, the expression levels of lncRNA-UCA1 in the human serum-derived exosomes of bladder cancer patients were higher than that in the healthy controls. Together, our results demonstrate that hypoxic bladder cancer cells remodel tumor

Altered expression of CD63 and exosomes in scleroderma dermal fibroblasts.

PubMed

Nakamura, Kayo; Jinnin, Masatoshi; Harada, Miho; Kudo, Hideo; Nakayama, Wakana; Inoue, Kuniko; Ogata, Aki; Kajihara, Ikko; Fukushima, Satoshi; Ihn, Hironobu

2016-10-01

Exosomes are small vesicles shed from various cells. They contain proteins, lipids, and nucleic acids, and are regarded as a tool of cell-cell communication. To reveal the putative role of exosomes in systemic sclerosis (SSc), and to elucidate the effect of exosomes on wound healing. The expression of common markers for exosomes (CD63, CD9, and CD81) and type I collagen were examined with real-time PCR, immunohistochemical analysis, ELISA, immunoblotting, and flow cytometry. The effect of serum-derived exosomes on wound healing was tested on full-thickness wounds in the mid-dorsal skin of BALB/c mice. The expression levels of CD63 as well as CD9 and CD81 tended to be increased in SSc dermal fibroblasts compared to normal fibroblasts. Increased exosomes in a cultured media of SSc fibroblasts stimulated the expression levels of type I collagen in normal fibroblasts. As the mechanism, collagen-related microRNA levels in SSc fibroblast-derived exosomes were dysregulated, indicating that both the amount and the content of exosomes were altered in SSc. On the other hand, SSc sera showed significantly decreased exosome levels compared to normal sera. The frequencies of vascular involvements, including skin ulcers or pitting scars, were significantly increased in patients with decreased serum exosome levels. The healing of mice wounds was accelerated by treatment with serum-derived exosomes. Vascular abnormalities in SSc may account for the decreased serum exosome levels by the disturbed transfer of exosomes from the skin tissue to the blood stream. Our study suggests the possibility that SSc patients with vascular involvements have decreased serum exosome levels, which causes the delay of wound healing due to down-regulation of collagen, resulting in higher susceptibility to pitting scars and/or ulcers. Exosome research will lead to a detailed understanding of SSc pathogenesis and new therapeutic approaches. Copyright © 2016 Japanese Society for Investigative Dermatology

Exosome Adherence and Internalization by Hepatic Stellate Cells Triggers Sphingosine 1-Phosphate-dependent Migration*

PubMed Central

Wang, Ruisi; Ding, Qian; Yaqoob, Usman; de Assuncao, Thiago M.; Verma, Vikas K.; Hirsova, Petra; Cao, Sheng; Mukhopadhyay, Debabrata; Huebert, Robert C.; Shah, Vijay H.

2015-01-01

Exosomes are cell-derived extracellular vesicles thought to promote intercellular communication by delivering specific content to target cells. The aim of this study was to determine whether endothelial cell (EC)-derived exosomes could regulate the phenotype of hepatic stellate cells (HSCs). Initial microarray studies showed that fibroblast growth factor 2 induced a 2.4-fold increase in mRNA levels of sphingosine kinase 1 (SK1). Exosomes derived from an SK1-overexpressing EC line increased HSC migration 3.2-fold. Migration was not conferred by the dominant negative SK1 exosome. Incubation of HSCs with exosomes was also associated with an 8.3-fold increase in phosphorylation of AKT and 2.5-fold increase in migration. Exosomes were found to express the matrix protein and integrin ligand fibronectin (FN) by Western blot analysis and transmission electron microscopy. Blockade of the FN-integrin interaction with a CD29 neutralizing antibody or the RGD peptide attenuated exosome-induced HSC AKT phosphorylation and migration. Inhibition of endocytosis with transfection of dynamin siRNA, the dominant negative dynamin GTPase construct Dyn2K44A, or the pharmacological inhibitor Dynasore significantly attenuated exosome-induced AKT phosphorylation. SK1 levels were increased in serum exosomes derived from mice with experimental liver fibrosis, and SK1 mRNA levels were up-regulated 2.5-fold in human liver cirrhosis patient samples. Finally, S1PR2 inhibition protected mice from CCl4-induced liver fibrosis. Therefore, EC-derived SK1-containing exosomes regulate HSC signaling and migration through FN-integrin-dependent exosome adherence and dynamin-dependent exosome internalization. These findings advance our understanding of EC/HSC cross-talk and identify exosomes as a potential target to attenuate pathobiology signals. PMID:26534962

Exosome Adherence and Internalization by Hepatic Stellate Cells Triggers Sphingosine 1-Phosphate-dependent Migration.

PubMed

Wang, Ruisi; Ding, Qian; Yaqoob, Usman; de Assuncao, Thiago M; Verma, Vikas K; Hirsova, Petra; Cao, Sheng; Mukhopadhyay, Debabrata; Huebert, Robert C; Shah, Vijay H

2015-12-25

Exosomes are cell-derived extracellular vesicles thought to promote intercellular communication by delivering specific content to target cells. The aim of this study was to determine whether endothelial cell (EC)-derived exosomes could regulate the phenotype of hepatic stellate cells (HSCs). Initial microarray studies showed that fibroblast growth factor 2 induced a 2.4-fold increase in mRNA levels of sphingosine kinase 1 (SK1). Exosomes derived from an SK1-overexpressing EC line increased HSC migration 3.2-fold. Migration was not conferred by the dominant negative SK1 exosome. Incubation of HSCs with exosomes was also associated with an 8.3-fold increase in phosphorylation of AKT and 2.5-fold increase in migration. Exosomes were found to express the matrix protein and integrin ligand fibronectin (FN) by Western blot analysis and transmission electron microscopy. Blockade of the FN-integrin interaction with a CD29 neutralizing antibody or the RGD peptide attenuated exosome-induced HSC AKT phosphorylation and migration. Inhibition of endocytosis with transfection of dynamin siRNA, the dominant negative dynamin GTPase construct Dyn2K44A, or the pharmacological inhibitor Dynasore significantly attenuated exosome-induced AKT phosphorylation. SK1 levels were increased in serum exosomes derived from mice with experimental liver fibrosis, and SK1 mRNA levels were up-regulated 2.5-fold in human liver cirrhosis patient samples. Finally, S1PR2 inhibition protected mice from CCl4-induced liver fibrosis. Therefore, EC-derived SK1-containing exosomes regulate HSC signaling and migration through FN-integrin-dependent exosome adherence and dynamin-dependent exosome internalization. These findings advance our understanding of EC/HSC cross-talk and identify exosomes as a potential target to attenuate pathobiology signals. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of pre-clinical Alzheimer’s disease by a profile of pathogenic proteins in neurally-derived blood exosomes: a case-control study

PubMed Central

Fiandaca, Massimo S.; Kapogiannis, Dimitrios; Mapstone, Mark; Boxer, Adam; Eitan, Erez; Schwartz, Janice B.; Abner, Erin L.; Petersen, Ronald C.; Federoff, Howard J.; Miller, Bruce L.; Goetzl, Edward J.

2014-01-01

Background Proteins pathogenic in Alzheimer’s disease (AD) were extracted from neurally-derived blood exosomes and quantified to develop biomarkers for staging of sporadic AD. Methods Blood exosomes obtained at one time-point from patients with AD (n=57) or frontotemporal dementia (FTD) (n=16), and at two time-points from others (n=24) when cognitively normal and one-ten years later when diagnosed with AD were enriched for neural sources by immunoabsorption. AD-pathogenic exosomal proteins were extracted and quantified by ELISAs. Results Mean exosomal levels of total Tau, P-T181-tau, P-S396-tau and Aβ1-42 for AD and levels of P-T181-tau and Aβ1-42 for FTD were significantly higher than for case-controls. Stepwise discriminant modeling incorporated P-T181-tau, P-S396-tau and Aβ1-42 in AD, but only P-T181-tau in FTD. Classification of 96.4% of AD patients and 87.5% of FTD patients was correct. In 24 AD patients, exosomal levels of P-S396-tau, P-T181-tau and Aβ1-42 were significantly higher than for controls both one to ten years before and when diagnosed with AD. Conclusions Levels of P-S396-tau, P-T181-tau and Aβ1-42 in extracts of neurally-derived blood exosomes predict development of AD up to 10 years prior to clinical onset. PMID:25130657

Exosome secretion affects social motility in Trypanosoma brucei

PubMed Central

Shaked, Hadassa; Arvatz, Gil; Tkacz, Itai Dov; Binder, Lior; Waldman Ben-Asher, Hiba; Okalang, Uthman; Chikne, Vaibhav; Cohen-Chalamish, Smadar; Michaeli, Shulamit

2017-01-01

Extracellular vesicles (EV) secreted by pathogens function in a variety of biological processes. Here, we demonstrate that in the protozoan parasite Trypanosoma brucei, exosome secretion is induced by stress that affects trans-splicing. Following perturbations in biogenesis of spliced leader RNA, which donates its spliced leader (SL) exon to all mRNAs, or after heat-shock, the SL RNA is exported to the cytoplasm and forms distinct granules, which are then secreted by exosomes. The exosomes are formed in multivesicular bodies (MVB) utilizing the endosomal sorting complexes required for transport (ESCRT), through a mechanism similar to microRNA secretion in mammalian cells. Silencing of the ESCRT factor, Vps36, compromised exosome secretion but not the secretion of vesicles derived from nanotubes. The exosomes enter recipient trypanosome cells. Time-lapse microscopy demonstrated that cells secreting exosomes or purified intact exosomes affect social motility (SoMo). This study demonstrates that exosomes are delivered to trypanosome cells and can change their migration. Exosomes are used to transmit stress signals for communication between parasites. PMID:28257521

Neuroblastoma SH-SY5Y cell-derived exosomes stimulate dendrite-like outgrowths and modify the differentiation of A375 melanoma cells.

PubMed

Park, Seyeon; Ahn, Eun Sook; Kim, Yunjoo

2015-04-01

The identification of small vesicles released by many cell types as tools of intercellular communication is proposed. Here, we identify SH-SY5Y neuroblastoma-derived exosomes comprised of major histocompatibility complex II (MHC II), Hsp90 and flotillin-1. Our data also suggest that, when applied extracellularly, exosomes released from neuronal cells stimulate dendrite-like outgrowth and melanogenesis of A375 melanoma cells through the mitogen-activated protein kinase (MAP kinase), extracellular signal-regulated kinase 1 (ERK1) activation. These results suggest a modification of differentiation of melanocyte by the treatment of neuronal cell exosomes. Since exosomes from neuronal cells have the capacity to affect melanoma cells, they could be generally implicated in intercellular communication between different types of cells. © 2014 International Federation for Cell Biology.

Low pH increases the yield of exosome isolation.

PubMed

Ban, Jae-Jun; Lee, Mijung; Im, Wooseok; Kim, Manho

2015-05-22

Exosomes are the extracellular vesicles secreted by various cells. Exosomes mediate intercellular communication by delivering a variety of molecules between cells. Cancer cell derived exosomes seem to be related with tumor progression and metastasis. Tumor microenvironment is thought to be acidic and this low pH controls exosome physiology, leading to tumor progression. Despite the importance of microenvironmental pH on exosome, most of exosome studies have been performed without regard to pH. Therefore, the difference of exosome stability and yield of isolation by different pH need to be studied. In this research, we investigated the yield of total exosomal protein and RNA after incubation in acidic, neutral and alkaline conditioned medium. Representative exosome markers were investigated by western blot after incubation of exosomes in different pH. As a result, the concentrations of exosomal protein and nucleic acid were significantly increased after incubation in the acidic medium compared with neutral medium. The higher levels of exosome markers including CD9, CD63 and HSP70 were observed after incubation in an acidic environment. On the other hand, no exosomal protein, exosomal RNA and exosome markers have been detected after incubation in an alkaline condition. In summary, our results indicate that the acidic condition is the favorable environment for existence and isolation of exosomes. Copyright © 2015 Elsevier Inc. All rights reserved.

Ascent of atomic force microscopy as a nanoanalytical tool for exosomes and other extracellular vesicles

NASA Astrophysics Data System (ADS)

Sharma, S.; LeClaire, M.; Gimzewski, J. K.

2018-04-01

Over the last 30 years, atomic force microscopy (AFM) has made several significant contributions to the field of biology and medicine. In this review, we draw our attention to the recent applications and promise of AFM as a high-resolution imaging and force sensing technology for probing subcellular vesicles: exosomes and other extracellular vesicles. Exosomes are naturally occurring nanoparticles found in several body fluids such as blood, saliva, cerebrospinal fluid, amniotic fluid and urine. Exosomes mediate cell-cell communication, transport proteins and genetic content between distant cells, and are now known to play important roles in progression of diseases such as cancers, neurodegenerative disorders and infectious diseases. Because exosomes are smaller than 100 nm (about 30-120 nm), the structural and molecular characterization of these vesicles at the individual level has been challenging. AFM has revealed a new degree of complexity in these nanosized vesicles and generated growing interest as a nanoscale tool for characterizing the abundance, morphology, biomechanics, and biomolecular make-up of exosomes. With the recent interest in exosomes for diagnostic and therapeutic applications, AFM-based characterization promises to contribute towards improved understanding of these particles at the single vesicle and sub-vesicular levels. When coupled with complementary methods like optical super resolution STED and Raman, AFM could further unlock the potential of exosomes as disease biomarkers and as therapeutic agents.

Exosomes released by keratinocytes modulate melanocyte pigmentation

PubMed Central

Cicero, Alessandra Lo; Delevoye, Cédric; Gilles-Marsens, Floriane; Loew, Damarys; Dingli, Florent; Guéré, Christelle; André, Nathalie; Vié, Katell; van Niel, Guillaume; Raposo, Graça

2015-01-01

Cells secrete extracellular vesicles (EVs), exosomes and microvesicles, which transfer proteins, lipids and RNAs to regulate recipient cell functions. Skin pigmentation relies on a tight dialogue between keratinocytes and melanocytes in the epidermis. Here we report that exosomes secreted by keratinocytes enhance melanin synthesis by increasing both the expression and activity of melanosomal proteins. Furthermore, we show that the function of keratinocyte-derived exosomes is phototype-dependent and is modulated by ultraviolet B. In sum, this study uncovers an important physiological function for exosomes in human pigmentation and opens new avenues in our understanding of how pigmentation is regulated by intercellular communication in both healthy and diseased states. PMID:26103923

High-grade ovarian cancer secreting effective exosomes in tumor angiogenesis.

PubMed

Yi, Huan; Ye, Jun; Yang, Xiao-Mei; Zhang, Li-Wen; Zhang, Zhi-Gang; Chen, Ya-Ping

2015-01-01

Ovarian cancer, the most lethal gynecological cancer, related closely to tumor stage. High-grade ovarian cancer always results in a late diagnose and high recurrence, which reduce survival within five years. Until recently, curable therapy is still under research and anti-angiogenesis proves a promising way. Tumor-derived exosomes are essential in tumor migration and metastases such as angiogenesis is enhanced by exosomes. In our study, we have made comparison between high-grade and unlikely high-grade serous ovarian cancer cells on exosomal function of endothelial cells proliferation, migration and tube formation. Exosomes derived from high-grade ovarian cancer have a profound impact on angiogenesis with comparison to unlikely high-grade ovarian cancer. Proteomic profiles revealed some potential proteins involved in exosomal function of angiogenesis such as ATF2, MTA1, ROCK1/2 and so on. Therefore, exosomes plays an influential role in angiogenesis in ovarian serous cancer and also function more effectively in high-grade ovarian cancer cells.

"Exosomics"-A Review of Biophysics, Biology and Biochemistry of Exosomes With a Focus on Human Breast Milk.

PubMed

de la Torre Gomez, Carolina; Goreham, Renee V; Bech Serra, Joan J; Nann, Thomas; Kussmann, Martin

2018-01-01

Exosomes are biomolecular nanostructures released from cells. They carry specific biomolecular information and are mainly researched for their exquisite properties as a biomarker source and delivery system. We introduce exosomes in the context of other extracellular vesicles, describe their biophysical isolation and characterisation and discuss their biochemical profiling. Motivated by our interest in early-life nutrition and health, and corresponding studies enrolling lactating mothers and their infants, we zoom into exosomes derived from human breast milk. We argue that these should be more extensively studied at proteomic and micronutrient profiling level, because breast milk exosomes provide a more specific window into breast milk quality from an immunological (proteomics) and nutritional (micronutrient) perspective. Such enhanced breast milk exosome profiling would thereby complement and enrich the more classical whole breast milk analysis and is expected to deliver more functional insights than the rather descriptive analysis of human milk, or larger fractions thereof, such as milk fat globule membrane. We substantiate our arguments by a bioinformatic analysis of two published proteomic data sets of human breast milk exosomes.

Comparison of exosomes secreted by induced pluripotent stem cell-derived mesenchymal stem cells and synovial membrane-derived mesenchymal stem cells for the treatment of osteoarthritis.

PubMed

Zhu, Yu; Wang, Yuchen; Zhao, Bizeng; Niu, Xin; Hu, Bin; Li, Qing; Zhang, Juntao; Ding, Jian; Chen, Yunfeng; Wang, Yang

2017-03-09

Osteoarthritis (OA) is the most common joint disease worldwide. In the past decade, mesenchymal stem cells (MSCs) have been used widely for the treatment of OA. A potential mechanism of MSC-based therapies has been attributed to the paracrine secretion of trophic factors, in which exosomes may play a major role. In this study, we aimed to compare the effectiveness of exosomes secreted by synovial membrane MSCs (SMMSC-Exos) and exosomes secreted by induced pluripotent stem cell-derived MSCs (iMSC-Exos) on the treatment of OA. Induced pluripotent stem cell-derived MSCs and synovial membrane MSCs were characterized by flow cytometry. iMSC-Exos and SMMSC-Exos were isolated using an ultrafiltration method. Tunable resistive pulse-sensing analysis, transmission electron microscopy, and western blots were used to identify exosomes. iMSC-Exos and SMMSC-Exos were injected intra-articularly in a mouse model of collagenase-induced OA and the efficacy of exosome injections was assessed by macroscopic, histological, and immunohistochemistry analysis. We also evaluated the effects of iMSC-Exos and SMMSC-Exos on proliferation and migration of human chondrocytes by cell-counting and scratch assays, respectively. The majority of iMSC-Exos and SMMSC-Exos were approximately 50-150 nm in diameter and expressed CD9, CD63, and TSG101. The injection of iMSC-Exos and SMMSC-Exos both attenuated OA in the mouse OA model, but iMSC-Exos had a superior therapeutic effect compared with SMMSC-Exos. Similarly, chondrocyte migration and proliferation were stimulated by both iMSC-Exos and SMMSC-Exos, with iMSC-Exos exerting a stronger effect. The present study demonstrated that iMSC-Exos have a greater therapeutic effect on OA than SMMSC-Exos. Because autologous iMSCs are theoretically inexhaustible, iMSC-Exos may represent a novel therapeutic approach for the treatment of OA.

Real time and label free profiling of clinically relevant exosomes.

PubMed

Sina, Abu Ali Ibn; Vaidyanathan, Ramanathan; Dey, Shuvashis; Carrascosa, Laura G; Shiddiky, Muhammad J A; Trau, Matt

2016-07-28

Tumor-derived exosomes possess significant clinical relevance due to their unique composition of genetic and protein material that is representative of the parent tumor. Specific isolation as well as identification of proportions of these clinically relevant exosomes (CREs) from biological samples could help to better understand their clinical significance as cancer biomarkers. Herein, we present a simple approach for quantification of the proportion of CREs within the bulk exosome population isolated from patient serum. This proportion of CREs can potentially inform on the disease stage and enable non-invasive monitoring of inter-individual variations in tumor-receptor expression levels. Our approach utilises a Surface Plasmon Resonance (SPR) platform to quantify the proportion of CREs in a two-step strategy that involves (i) initial isolation of bulk exosome population using tetraspanin biomarkers (i.e., CD9, CD63), and (ii) subsequent detection of CREs within the captured bulk exosomes using tumor-specific markers (e.g., human epidermal growth factor receptor 2 (HER2)). We demonstrate the isolation of bulk exosome population and detection of as low as 10% HER2(+) exosomes from samples containing designated proportions of HER2(+) BT474 and HER2(-) MDA-MB-231 cell derived exosomes. We also demonstrate the successful isolation of exosomes from a small cohort of breast cancer patient samples and identified that approximately 14-35% of their bulk population express HER2.

Exosomes as therapeutics: The implications of molecular composition and exosomal heterogeneity.

PubMed

Ferguson, Scott W; Nguyen, Juliane

2016-04-28

Harnessing exosomes as therapeutic drug delivery vehicles requires a better understanding of exosomal composition and their mode of action. A full appreciation of all the exosomal components (proteins, lipids, and RNA content) will be important for the design of effective exosome-based or exosome-mimicking drug carriers. In this review we describe the presence of rarely studied, non-coding RNAs that exist in high numbers in exosomes. We discuss the implications of the molecular composition and heterogeneity of exosomes on their biological and therapeutic effects. Finally, we highlight outstanding questions with regard to RNA loading into exosomes, analytical methods to sort exosomes and their sub-populations, and the effects of exosomal proteins and lipids on recipient cells. Investigations into these facets of exosome biology will further advance the field, could lead to the clinical translation of exosome-based therapeutics, and aid in the reverse-engineering of synthetic exosomes. Although synthetic exosomes are still an underexplored area, they could offer researchers a way to manufacture exosomes with highly defined structure, composition, and function. Copyright © 2016 Elsevier B.V. All rights reserved.

The contribution of tumour-derived exosomes to the hallmarks of cancer.

PubMed

Meehan, Katie; Vella, Laura J

2016-01-01

Exosomes are small, biologically active extracellular vesicles and over the last decade, both stromal and tumour-derived exosomes (TDE) have been implicated in cancer onset, progression and metastases. Cancer is a complex disease that is underpinned by several "cancer hallmarks", originally described by Hanahan and Weinberg in 2000 and then revised in 2011. The hallmarks of cancer comprise six biological capabilities, along with two emerging hallmarks and two enabling characteristics that facilitate tumour growth and metastatic dissemination. Ample evidence supports a clear role for TDE in four of the original biological hallmarks (sustaining proliferative signalling, resisting cell death, inducing angiogenesis and activating invasion and metastases). A less-defined role exists for TDE in evading growth suppressors, and currently, there is no evidence to suggest a role for TDE in enabling replicative immortality. TDE are intimately involved in the newly defined hallmarks of cancer and enabling characteristics, most evidently in immune inhibition and tumour-promoting inflammation, which ultimately enable escape from immune destruction and tumour progression. Herein, we discuss the role of TDE in the context of the hallmarks and enabling characteristics of cancer as defined by Hanahan and Weinberg.

Exosomes as potential alternatives to stem cell therapy for intervertebral disc degeneration: in-vitro study on exosomes in interaction of nucleus pulposus cells and bone marrow mesenchymal stem cells.

PubMed

Lu, Kang; Li, Hai-Yin; Yang, Kuang; Wu, Jun-Long; Cai, Xiao-Wei; Zhou, Yue; Li, Chang-Qing

2017-05-10

The stem cell-based therapies for intervertebral disc degeneration have been widely studied. However, the mechanisms of mesenchymal stem cells interacting with intervertebral disc cells, such as nucleus pulposus cells (NPCs), remain unknown. Exosomes as a vital paracrine mechanism in cell-cell communication have been highly focused on. The purpose of this study was to detect the role of exosomes derived from bone marrow mesenchymal stem cells (BM-MSCs) and NPCs in their interaction with corresponding cells. The exosomes secreted by BM-MSCs and NPCs were purified by differential centrifugation and identified by transmission electron microscope and immunoblot analysis of exosomal marker proteins. Fluorescence confocal microscopy was used to examine the uptake of exosomes by recipient cells. The effects of NPC exosomes on the migration and differentiation of BM-MSCs were determined by transwell migration assays and quantitative RT-PCR analysis of NPC phenotypic genes. Western blot analysis was performed to examine proteins such as aggrecan, sox-9, collagen II and hif-1α in the induced BM-MSCs. Proliferation and the gene expression profile of NPCs induced by BM-MSC exosomes were measured by Cell Counting Kit-8 and qRT-PCR analysis, respectively. Both the NPCs and BM-MSCs secreted exosomes, and these exosomes underwent uptake by the corresponding cells. NPC-derived exosomes promoted BM-MSC migration and induced BM-MSC differentiation to a nucleus pulposus-like phenotype. BM-MSC-derived exosomes promoted NPC proliferation and healthier extracellular matrix production in the degenerate NPCs. Our study indicates that the exosomes act as an important vehicle in information exchange between BM-MSCs and NPCs. Given a variety of functions and multiple advantages, exosomes alone or loaded with specific genes and drugs would be an appropriate option in a cell-free therapy strategy for intervertebral disc degeneration.

Placenta-derived mesenchymal stromal cells and their exosomes exert therapeutic effects in Duchenne muscular dystrophy.

PubMed

Bier, Ariel; Berenstein, Peter; Kronfeld, Noam; Morgoulis, Daria; Ziv-Av, Amotz; Goldstein, Hodaya; Kazimirsky, Gila; Cazacu, Simona; Meir, Rinat; Popovtzer, Rachela; Dori, Amir; Brodie, Chaya

2018-05-03

Duchenne muscular dystrophy (DMD) is a degenerative lethal, X-linked disease of skeletal and cardiac muscles caused by mutations in the dystrophin gene. Cell therapy using different cell types, including mesenchymal stromal cells (MSCs), has been considered as a potential approach for the treatment of DMD. MSCs can be obtained from autologous sources such as bone marrow and adipose tissues or from allogeneic placenta and umbilical cord. The safety and therapeutic impact of these cells has been demonstrated in pre-clinical and clinical studies and their functions are attributed to paracrine effects that are mediated by secreted cytokines and extracellular vesicles. Here, we studied the therapeutic effects of placenta-derived MSCs (PL-MSCs) and their secreted exosomes using mouse and human myoblasts from healthy controls, Duchenne patients and mdx mice. Treatment of myoblasts with conditioned medium or exosomes secreted by PL-MSCs increased the differentiation of these cells and decreased the expression of fibrogenic genes in DMD patient myoblasts. In addition, these treatments also increased the expression of utrophin in these cells. Using a quantitative miR-29c reporter, we demonstrated that the PL-MSC effects were partly mediated by the transfer of exosomal miR-29c. Intramuscular transplantation of PL-MSCs in mdx mice resulted in decreased creatine kinase levels. PL-MSCs significantly decreased the expression of TGF-β and the level of fibrosis in the diaphragm and cardiac muscles, inhibited inflammation and increased utrophin expression. In vivo imaging analyses using MSCs labeled with gold nanoparticles or fluorescent dyes demonstrated localization of the cells in the muscle tissues up to 3 weeks post treatment. Altogether, these results demonstrate that PL-MSCs and their secreted exosomes have important clinical applications in cell therapy of DMD partly via the targeted delivery of exosomal miR-29c. Copyright © 2018. Published by Elsevier Ltd.

Stabilization of exosome-targeting peptides via engineered glycosylation.

PubMed

Hung, Michelle E; Leonard, Joshua N

2015-03-27

Exosomes are secreted extracellular vesicles that mediate intercellular transfer of cellular contents and are attractive vehicles for therapeutic delivery of bimolecular cargo such as nucleic acids, proteins, and even drugs. Efficient exosome-mediated delivery in vivo requires targeting vesicles for uptake by specific recipient cells. Although exosomes have been successfully targeted to several cellular receptors by displaying peptides on the surface of the exosomes, identifying effective exosome-targeting peptides for other receptors has proven challenging. Furthermore, the biophysical rules governing targeting peptide success remain poorly understood. To evaluate one factor potentially limiting exosome delivery, we investigated whether peptides displayed on the exosome surface are degraded during exosome biogenesis, for example by endosomal proteases. Indeed, peptides fused to the N terminus of exosome-associated transmembrane protein Lamp2b were cleaved in samples derived from both cells and exosomes. To suppress peptide loss, we engineered targeting peptide-Lamp2b fusion proteins to include a glycosylation motif at various positions. Introduction of this glycosylation motif both protected the peptide from degradation and led to an increase in overall Lamp2b fusion protein expression in both cells and exosomes. Moreover, glycosylation-stabilized peptides enhanced targeted delivery of exosomes to neuroblastoma cells, demonstrating that such glycosylation does not ablate peptide-target interactions. Thus, we have identified a strategy for achieving robust display of targeting peptides on the surface of exosomes, which should facilitate the evaluation and development of new exosome-based therapeutics. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Multimodal transfer of MDR by exosomes in human osteosarcoma.

PubMed

Torreggiani, Elena; Roncuzzi, Laura; Perut, Francesca; Zini, Nicoletta; Baldini, Nicola

2016-07-01

Exosomes are extracellular vesicles released by both normal and tumour cells which are involved in a new intercellular communication pathway by delivering cargo (e.g., proteins, microRNAs, mRNAs) to recipient cells. Tumour-derived exosomes have been shown to play critical roles in different stages of tumour growth and progression. In this study, we investigated the potential role of exosomes to transfer the multidrug resistance (MDR) phenotype in human osteosarcoma cells. Exosomes were isolated by differential centrifugation of culture media from multidrug resistant human osteosarcoma MG-63DXR30 (Exo/DXR) and MG-63 parental cells (Exo/S). Exosome purity was examined by transmission electron microscopy and confirmed by immunoblot analysis for the expression of specific exosomal markers. Our data showed that exosomes derived from doxorubicin-resistant osteosarcoma cells could be taken up into secondary cells and induce a doxorubicin-resistant phenotype. The incubation of osteosarcoma cells with Exo/DXR decreased the sensitivity of parental cells to doxorubicin, while exposure with Exo/S was ineffective. In addition, we demonstrated that Exo/DXR expressed higher levels of MDR-1 mRNA and P-glycoprotein compared to Exo/S (p=0.03). Interestingly, both MDR-1 mRNA and P-gp increased in MG-63 cells after incubation with Exo/DXR, suggesting this as the main mechanism of exosome-mediated transfer of drug resistance. Our findings suggest that multidrug resistant osteosarcoma cells are able to spread their ability to resist the effects of doxorubicin treatment on sensitive cells by transferring exosomes carrying MDR-1 mRNA and its product P-glycoprotein.

Exosomes as a liquid biopsy for lung cancer.

PubMed

Cui, Shaohua; Cheng, Zhuoan; Qin, Wenxin; Jiang, Liyan

2018-02-01

In lung cancer and other malignancies, the so-called "liquid biopsy" is quickly moving into clinical practice. Its full potential has not yet been fully identified, but the "liquid biopsy" is no longer a promise but has become a reality that allows for better treatment selection and monitoring of lung cancer. This emerging field has significant potential to make up for the limitations of the traditional tissue-derived biomaterials. Exosomes are spherical nano-sized vesicles with a diameter of 40-100 nm and a density of 1.13-1.19 g/ml. In both physiological and pathological conditions, exosomes can be released by different cell types, including immune cells, stem cells and tumor cells. These small molecules may serve as promising biomarkers in lung cancer "liquid biopsy" as they can be easily obtained from most body fluids. In addition, the lipid bilayer of exosomes allows for stable cargoes which are relatively hard to degrade. Furthermore, the composition of exosomes reflects that of their parental cells, suggesting that exosomes are potential surrogates of the original cells and, therefore, are useful for understanding cell biology. Previous studies have demonstrated that exosomes play important roles in cell-to-cell communication. Moreover, tumor-derived exosomes are evolved in tumor-specific biological process, including tumor proliferation and progression. Recently, a growing number of studies has focused on exosomal cargo and their use in lung cancer genesis and progression. In addition, their utility as lung cancer diagnostic, prognostic and predictive biomarkers have also been studied. The current review primarily summaries lung cancer-related exosomal biomarkers that have recently been identified and discusses their potential in clinical practice. Copyright © 2017 Elsevier B.V. All rights reserved.

Lipidomic and proteomic analysis of exosomes from mouse cortical collecting duct cells.

PubMed

Dang, Viet D; Jella, Kishore Kumar; Ragheb, Ragy R T; Denslow, Nancy D; Alli, Abdel A

2017-12-01

Exosomes are endosome-derived nanovesicles that are involved in cellular communication and signaling. Exosomes are produced by epithelial cells and are found in biologic fluids including blood and urine. The packaged material within exosomes includes proteins and lipids, but the molecular comparison within exosome subtypes is largely unknown. The purpose of this study was to investigate differences between exosomes derived from the apical plasma membrane and basolateral plasma membrane of polarized murine cortical collecting duct principal cells. Nanoparticle tracking analysis showed that the size and concentration of apical and basolateral exosomes remained relatively stable across 3 different temperatures (23, 37, and 42°C). Liquid chromatography-tandem mass spectrometry analysis revealed marked differences between the proteins packaged within the two types of exosomes from the same cells. Several proteins expressed at the inner leaflet of the plasma membrane, including α-actinin-1, moesin, 14-3-3 protein ζ/δ, annexin A1/A3/A4/A5/A6, clathrin heavy chain 1, glyceraldehyde-3-phosphate dehydrogenase, α-enolase, filamin-A, and heat shock protein 90, were identified in samples of apical plasma membrane-derived exosomes, but not in basolateral plasma membrane exosomes from mouse cortical collecting duct cells. In addition to differences at the protein level, mass spectrometry-based shotgun lipidomics analysis showed significant differences in the lipid classes and fatty acid composition of the two types of exosomes. We found higher levels of sphingomyelin and lower levels of cardiolipin, among other phospholipids in the apical plasma membrane compared to the basolateral plasma membrane exosomes. The molecular analyses of exosome subtypes presented herein will contribute to our understanding of exosome biogenesis, and the results may have potential implications for biomarker discovery.-Dang, V. D., Jella, K. K., Ragheb, R. R. T., Denslow, N. D., Alli, A. A

Hepatocyte-derived exosomes promote T follicular regulatory cell expansion during hepatitis C virus infection.

PubMed

Cobb, Dustin A; Kim, Ok-Kyung; Golden-Mason, Lucy; Rosen, Hugo R; Hahn, Young S

2018-01-01

Hepatitis C virus (HCV) is a global health concern that can cause severe liver disease, such as cirrhosis and hepatocellular carcinoma. Control of HCV requires vigorous T-cell responses, yet CD4 + T cells in chronic HCV patients are dysfunctional. T follicular regulatory (Tfr) cells are a subset of regulatory T cells that suppress T follicular helper (Tfh) cells and the generation of high affinity antibody-producing B cells. In this study, we examined the accumulation of Tfr cells in the liver compartment during chronic HCV infection and defined the cellular and molecular mechanisms underlying their expansion. Our analysis revealed a substantial population of Tfr cells in livers of chronic HCV patients that is absent in liver tissues from nonviral hepatitis or healthy subjects. Coculture of PBMCs from healthy subjects with HCV-infected hepatoma cells resulted in preferential expansion of circulating Tfr cells, leading to suppression of Tfh cells. Additionally, coculture of tonsillar cells with infected hepatoma cells lead to an expansion of germinal center Tfr. Notably, expansion was mediated by transforming growth factor beta (TGF-β)-containing exosomes released from HCV-infected hepatocytes given that blockade of exosome-associated TGF-β or inhibition of exosome release abrogated Tfr expansion. These results show that liver-derived exosomes play a pivotal role in the accumulation of Tfr cells, likely leading to suppression of Tfh responses in HCV-infected patients. Our study identifies a novel pathway in which HCV infection in hepatocytes exacerbates Tfr cell responses to subvert antiviral immunity. (Hepatology 2018;67:71-85). © 2017 by the American Association for the Study of Liver Diseases.

Proteomic content of circulating exosomes in dairy cows with or without uterine infection.

PubMed

Almughlliq, Fatema B; Koh, Yong Q; Peiris, Hassendrini N; Vaswani, Kanchan; McDougall, Scott; Graham, Elizabeth M; Burke, Chris R; Arachchige, Buddhika J; Reed, Sarah; Mitchell, Murray D

2018-07-01

In the past few decades, there has been a global decrease in dairy cow reproductive performance. An activated inflammatory system, due to uterine infection, has been associated with decreased cow fertility and as such, there is a need to detect uterine disease earlier. Early detection could be achieved by identifying biomarkers for uterine disease. Exosomes are small nanovesicles known to package and deliver protein, mRNA, and miRNAs to near and distant sites. Therefore, the content of circulating exosomes may have the potential to carry biomarkers for earlier diagnosis of disease. We hypothesized that circulating exosomes from cows with and without uterine infection may contain information representative of endometrial health or disease. We compared the proteomic content of circulating exosomes derived from plasma of dairy cows with (n = 10) or without (n = 10) induced uterine infection, using high-performance liquid chromatography tandem mass spectrometry (HPLC MS/MS). Our results demonstrate that there were a total of 103 bovine and 9 Trueperella pyogenes proteins found in plasma exosomes derived from infected cows (infected exosomes), and 90 bovine and 5 T. pyogenes proteins found in exosomes derived from plasma of non-infected cows (non-infected exosomes). 71 bovine proteins were found to be unique to the infected exosomes while only 4 bovine proteins were found to be unique to the non-infected exosomes. 8 unique T. pyogenes proteins were identified in infected exosomes and 4 were found to be unique to the non-infected exosomes. Pathway analysis showed that infected exosomes had more proteins involved in structural molecule activity and immune system processes than non-infected exosomal protein. Additionally, proteins from infected exosomes were involved in unique pathways: angiogenesis and integrin signaling pathway. Our data provide preliminary evidence of a potential role for exosomes in the early diagnosis of uterine infection in dairy cows

Characterization and Comprehensive Proteome Profiling of Exosomes Secreted by Hepatocytes

PubMed Central

Conde-Vancells, Javier; Rodriguez-Suarez, Eva; Embade, Nieves; Gil, David; Matthiesen, Rune; Valle, Mikel; Elortza, Felix; Lu, Shelly C.; Mato, Jose M.; Falcon-Perez, Juan M.

2009-01-01

Synopsis Exosomes constitute a discrete population of nanometer-sized (30-150 nm) vesicles formed in endocytic compartments and released to the extracellular environment by different cell types. In this work we demonstrated by electron microscopic, western blotting and proteomic analyses that primary hepatocytes secrete exosome-like vesicles containing proteins involved in metabolizing lipoproteins, endogenous compounds as well as xenobiotics. These new findings contribute to improve our knowledge about biology's hepatocyte and may have important diagnostic, prognosis and therapeutic implications in liver diseases Exosomes represent a discrete population of vesicles that are secreted from various cell types to the extracellular media. Their protein and lipid composition are a consequence of sorting events at the level of the multivesicular body, a central organelle which integrates endocytic and secretory pathways. Characterization of exosomes from different biological samples has shown the presence of common as well as cell-type specific proteins. Remarkably, the protein content of the exosomes is modified upon pathological or stress conditions. Hepatocytes play a central role in the body response to stress metabolizing potentially harmful endogenous substances as well as xenobiotics. In the present study we described and characterized for first time exosome secretion in non-tumoral hepatocytes, and using a systematic proteomic approach, we establish the first extensive proteome of a hepatocyte-derived exosome population which should be useful in furthering our understanding of the hepatic function and in the identification of components that may serve as biomarkers for hepatic alterations. Our analysis identifies a significant number of proteins previously described among exosomes derived from others cell types as well as proteins involved in metabolizing lipoproteins, endogenous compounds and xenobiotics, not previously described in exosomes. Furthermore, we

Real time and label free profiling of clinically relevant exosomes

PubMed Central

Sina, Abu Ali Ibn; Vaidyanathan, Ramanathan; Dey, Shuvashis; Carrascosa, Laura G.; Shiddiky, Muhammad J. A.; Trau, Matt

2016-01-01

Tumor-derived exosomes possess significant clinical relevance due to their unique composition of genetic and protein material that is representative of the parent tumor. Specific isolation as well as identification of proportions of these clinically relevant exosomes (CREs) from biological samples could help to better understand their clinical significance as cancer biomarkers. Herein, we present a simple approach for quantification of the proportion of CREs within the bulk exosome population isolated from patient serum. This proportion of CREs can potentially inform on the disease stage and enable non-invasive monitoring of inter-individual variations in tumor-receptor expression levels. Our approach utilises a Surface Plasmon Resonance (SPR) platform to quantify the proportion of CREs in a two-step strategy that involves (i) initial isolation of bulk exosome population using tetraspanin biomarkers (i.e., CD9, CD63), and (ii) subsequent detection of CREs within the captured bulk exosomes using tumor-specific markers (e.g., human epidermal growth factor receptor 2 (HER2)). We demonstrate the isolation of bulk exosome population and detection of as low as 10% HER2(+) exosomes from samples containing designated proportions of HER2(+) BT474 and HER2(−) MDA-MB-231 cell derived exosomes. We also demonstrate the successful isolation of exosomes from a small cohort of breast cancer patient samples and identified that approximately 14–35% of their bulk population express HER2. PMID:27464736

Long Distance Metabolic Regulation through Adipose-Derived Circulating Exosomal miRNAs: A Trail for RNA-Based Therapies?

PubMed Central

Fatima, Farah; Nawaz, Muhammad

2017-01-01

The contribution of non-coding RNAs, such as microRNAs (miRNAs) in regulating physiological and pathological states has been intensively elucidated during last 15 years. The discovery of circulating miRNAs (cir-miRNAs) in variety of body fluids, is, however a recent focus of interest in understanding pathophysiological states of their originating cells/organs. Yet another stimulating debate that takes miRNAs to the next level is their presence in exosomes, and this is truly interesting area of research. Exosomes are cell-derived extracellular vesicles, and are naturally equipped biological vehicles that not only enable functional transfer of miRNAs between cells (horizontal transfer) but also foster inter-organ communication, presumably guided by organ specific receptors—decorated on their surface. However, understandings on inter-organ communication elicited by tissue specific exosomal-miRNA fingerprints remain elusive. Recently, Thomou et al., has discovered that adipose tissue contributes a large fraction of adipose specific exosomal-miRNA fingerprints in blood circulation. Experimental evidence emphasize adipose tissue as major depot of cir-miRNAs that sail through blood flow and reach to distal organs—primarily in the liver, where they regulate gene expression of host tissue and elicit metabolic control. This appears to be a genetic form of adipokines (endocrine factors secreted from adipose tissue). We review such offshore metabolic insults, and make an effort to address few important missing links between miRNAs processing and their incorporation into exosomes. We provide potential perspectives on how this knowledge could be steered towards RNA-based therapeutics for monitoring complex metabolic diseases and beyond. PMID:28824444

Exosomes derived from human umbilical cord blood mesenchymal stem cells stimulates rejuvenation of human skin.

PubMed

Kim, Yoon-Jin; Yoo, Sae Mi; Park, Hwan Hee; Lim, Hye Jin; Kim, Yu-Lee; Lee, Seunghee; Seo, Kwang-Won; Kang, Kyung-Sun

2017-11-18

Human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) play an important role in cutaneous wound healing, and recent studies suggested that MSC-derived exosomes activate several signaling pathways, which are conducive in wound healing and cell growth. In this study, we investigated the roles of exosomes that are derived from USC-CM (USC-CM Exos) in cutaneous collagen synthesis and permeation. We found that USC-CM has various growth factors associated with skin rejuvenation. Our in vitro results showed that USC-CM Exos integrate in Human Dermal Fibroblasts (HDFs) and consequently promote cell migration and collagen synthesis of HDFs. Moreover, we evaluated skin permeation of USC-CM Exos by using human skin tissues. Results showed that Exo-Green labeled USC-CM Exos approached the outermost layer of the epidermis after 3 h and gradually approached the epidermis after 18 h. Moreover, increased expressions of Collagen I and Elastin were found after 3 days of treatment on human skin. The results showed that USC-CM Exos is absorbed into human skin, it promotes Collagen I and Elastin synthesis in the skin, which are essential to skin rejuvenation and shows the potential of USC-CM integration with the cosmetics or therapeutics. Copyright © 2017 Elsevier Inc. All rights reserved.

Exosomes carrying immunoinhibitory proteins and their role in cancer.

PubMed

Whiteside, T L

2017-09-01

Recent emergence of exosomes as information carriers between cells has introduced us to a new previously unknown biological communication system. Multi-directional cross-talk mediated by exosomes carrying proteins, lipids and nucleic acids between normal cells, cells harbouring a pathogen or cancer and immune cells has been instrumental in determining outcomes of physiological as well as pathological conditions. Exosomes play a key role in the broad spectrum of human diseases. In cancer, tumour-derived exosomes carry multiple immunoinhibitory signals, disable anti-tumour immune effector cells and promote tumour escape from immune control. Exosomes delivering negative signals to immune cells in cancer, viral infections, autoimmune or other diseases may interfere with therapy and influence outcome. Exosomes can activate tissue cells to produce inhibitory factors and thus can suppress the host immune responses indirectly. Exosomes also promise to be non-invasive disease biomarkers with a dual capability to provide insights into immune dysfunction as well as disease progression and outcome. © 2017 British Society for Immunology.

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

PubMed Central

Peinado, Héctor; Alečković, Maša; Lavotshkin, Simon; Matei, Irina; Costa-Silva, Bruno; Moreno-Bueno, Gema; Hergueta-Redondo, Marta; Williams, Caitlin; García-Santos, Guillermo; Nitadori-Hoshino, Ayuko; Hoffman, Caitlin; Badal, Karen; Garcia, Benjamin A.; Callahan, Margaret K.; Yuan, Jianda; Martins, Vilma R.; Skog, Johan; Kaplan, Rosandra N.; Brady, Mary S.; Wolchok, Jedd D.; Chapman, Paul B.; Kang, Yibin; Bromberg, Jacqueline; Lyden, David

2013-01-01

Tumor-derived exosomes are emerging mediators of tumorigenesis with tissue-specific addresses and messages. We explored the function of melanoma-derived exosomes in the formation of primary tumor and metastases in mouse and human subjects. Exosomes from highly metastatic melanoma increased the metastatic behavior of primary tumors by permanently “educating” bone marrow (BM) progenitors via the MET receptor. Melanoma-derived exosomes also induced vascular leakiness at pre-metastatic sites, and reprogrammed BM progenitors towards a c-Kit+Tie2+Met+ pro-vasculogenic phenotype. Reducing Met expression in exosomes diminished the pro-metastatic behavior of BM cells. Importantly, MET expression was elevated in circulating CD45−C-KITlow/+TIE2+ BM progenitors from metastatic melanoma subjects. RAB1a, RAB5b, RAB7, and RAB27a were highly expressed in melanoma cells and Rab27a RNA interference decreased exosome production, preventing BM education, tumor growth and metastasis. Finally, 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. PMID:22635005

Effect of exosome isolation methods on physicochemical properties of exosomes and clearance of exosomes from the blood circulation.

PubMed

Yamashita, Takuma; Takahashi, Yuki; Nishikawa, Makiya; Takakura, Yoshinobu

2016-01-01

Exosomes, which are expected to be delivery systems for biomolecules such as nucleic acids, are collected by several methods. However, the effect of exosome isolation methods on the characteristics of exosomes as drug carriers, such as recovery efficiency after sterile filtration and pharmacokinetics, has not been investigated despite the importance of these characteristics for the development of exosome-based delivery systems. In the present study, exosomes collected from murine melanoma B16-BL6 cells by several methods were compared with respect to dispersibility, recovery rate after filtering, and clearance from the blood circulation in mice. The exosomes were collected by three ultracentrifugation-based methods: simple ultracentrifugation/pelleting (pelleting method), ultracentrifugation with an iodixanol cushion (cushion method), and ultracentrifugation on an iodixanol density gradient (gradient method). The isolation methods had little effect on the particle number of exosomes. In contrast, transmission electron microscopy observation and size distribution measurement using tunable resistive pulse sensing indicated that the exosomes of the gradient method were more dispersed than the others. The exosomes were labeled with Gaussia luciferase and intravenously injected into mice. Clearance of injected exosomes from the blood circulation did not significantly change with isolation methods. When the exosomes were filtered using a 0.2-μm filter, the recovery rate was 82% for the exosomes of the gradient method, whereas it was less than 50% for the others. These results indicate that the exosome isolation method markedly affects the dispersibility and filtration efficiency of the exosomes. Copyright © 2015 Elsevier B.V. All rights reserved.

Tumour-derived exosomes as a signature of pancreatic cancer - liquid biopsies as indicators of tumour progression.

PubMed

Nuzhat, Zarin; Kinhal, Vyjayanthi; Sharma, Shayna; Rice, Gregory E; Joshi, Virendra; Salomon, Carlos

2017-03-07

Pancreatic cancer is the fourth most common cause of death due to cancer in the world. It is known to have a poor prognosis, mostly because early stages of the disease are generally asymptomatic. Progress in pancreatic cancer research has been slow, leaving several fundamental questions pertaining to diagnosis and treatment unanswered. Recent studies highlight the putative utility of tissue-specific vesicles (i.e. extracellular vesicles) in the diagnosis of disease onset and treatment monitoring in pancreatic cancer. Extracellular vesicles are membrane-limited structures derived from the cell membrane. They contain specific molecules including proteins, mRNA, microRNAs and non-coding RNAs that are secreted in the extracellular space. Extracellular vesicles can be classified according to their size and/or origin into microvesicles (~150-1000 nm) and exosomes (~40-120 nm). Microvesicles are released by budding from the plasmatic membrane, whereas exosomes are released via the endocytic pathway by fusion of multivesicular bodies with the plasmatic membrane. This endosomal origin means that exosomes contain an abundance of cell-specific biomolecules which may act as a 'fingerprint' of the cell of origin. In this review, we discuss our current knowledge in the diagnosis and treatment of pancreatic cancer, particularly the potential role of EVs in these facets of disease management. In particular, we suggest that as exosomes contain cellular protein and RNA molecules in a cell type-specific manner, they may provide extensive information about the signature of the tumour and pancreatic cancer progression.

Exosomes released by chronic lymphocytic leukemia cells induce the transition of stromal cells into cancer-associated fibroblasts

PubMed Central

Paggetti, Jerome; Haderk, Franziska; Seiffert, Martina; Janji, Bassam; Distler, Ute; Ammerlaan, Wim; Kim, Yeoun Jin; Adam, Julien; Lichter, Peter; Solary, Eric; Berchem, Guy

2015-01-01

Exosomes derived from solid tumor cells are involved in immune suppression, angiogenesis, and metastasis, but the role of leukemia-derived exosomes has been less investigated. The pathogenesis of chronic lymphocytic leukemia (CLL) is stringently associated with a tumor-supportive microenvironment and a dysfunctional immune system. Here, we explore the role of CLL-derived exosomes in the cellular and molecular mechanisms by which malignant cells create this favorable surrounding. We show that CLL-derived exosomes are actively incorporated by endothelial and mesenchymal stem cells ex vivo and in vivo and that the transfer of exosomal protein and microRNA induces an inflammatory phenotype in the target cells, which resembles the phenotype of cancer-associated fibroblasts (CAFs). As a result, stromal cells show enhanced proliferation, migration, and secretion of inflammatory cytokines, contributing to a tumor-supportive microenvironment. Exosome uptake by endothelial cells increased angiogenesis ex vivo and in vivo, and coinjection of CLL-derived exosomes and CLL cells promoted tumor growth in immunodeficient mice. Finally, we detected α-smooth actin–positive stromal cells in lymph nodes of CLL patients. These findings demonstrate that CLL-derived exosomes actively promote disease progression by modulating several functions of surrounding stromal cells that acquire features of cancer-associated fibroblasts. PMID:26100252

Tumor exosomes: cellular postmen of cancer diagnosis and personalized therapy.

PubMed

Sharma, Aman; Khatun, Zamila; Shiras, Anjali

2016-02-01

Nanosized (30-150 nm) extracellular vesicles 'exosomes' are secreted by cells for intercellular communication during normal and pathological conditions. Exosomes carry biomacromolecules from cell-of-origin and, therefore, represent molecular bioprint of the cell. Tumor-derived exosomes or TDEx modulate tumor microenvironment by transfer of macromolecules locally as well as at distant metastatic sites. Due to their biological stability, TDEx are rich source of biomarkers in cancer patients. TDEx focused cancer diagnosis allows liquid biopsy-based tumor typing and may facilitate therapy response monitoring by developing novel exosomes diagnostics. Therefore, efficient and specific capturing of exosomes for subsequent amplification of the biomessages; for example, DNA, RNA, miRNA can reinvent cancer diagnosis. Here, in this review, we discuss advancements in exosomes isolation strategies, presence of exosomes biomarkers and importance of TDEx in gauging tumor heterogeneity for their potential use in cancer diagnosis, therapy.

Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer.

PubMed

Kamerkar, Sushrut; LeBleu, Valerie S; Sugimoto, Hikaru; Yang, Sujuan; Ruivo, Carolina F; Melo, Sonia A; Lee, J Jack; Kalluri, Raghu

2017-06-22

The mutant form of the GTPase KRAS is a key driver of pancreatic cancer but remains a challenging therapeutic target. Exosomes are extracellular vesicles generated by all cells, and are naturally present in the blood. Here we show that enhanced retention of exosomes, compared to liposomes, in the circulation of mice is likely due to CD47-mediated protection of exosomes from phagocytosis by monocytes and macrophages. Exosomes derived from normal fibroblast-like mesenchymal cells were engineered to carry short interfering RNA or short hairpin RNA specific to oncogenic Kras G12D , a common mutation in pancreatic cancer. Compared to liposomes, the engineered exosomes (known as iExosomes) target oncogenic KRAS with an enhanced efficacy that is dependent on CD47, and is facilitated by macropinocytosis. Treatment with iExosomes suppressed cancer in multiple mouse models of pancreatic cancer and significantly increased overall survival. Our results demonstrate an approach for direct and specific targeting of oncogenic KRAS in tumours using iExosomes.

Exosomes Facilitate Therapeutic Targeting of Oncogenic Kras in Pancreatic Cancer

PubMed Central

Kamerkar, Sushrut; LeBleu, Valerie S.; Sugimoto, Hikaru; Yang, Sujuan; Ruivo, Carolina F.; Melo, Sonia A.; Lee, J. Jack; Kalluri, Raghu

2017-01-01

Summary The mutant form of the GTPase KRAS is a key driver of pancreatic cancer but remains a challenging therapeutic target. Exosomes, extracellular vesicles generated by all cells, are naturally present in the blood. Here we demonstrate that enhanced retention of exosomes in circulation, compared to liposomes, is due to CD47 mediated protection of exosomes from phagocytosis by monocytes and macrophages. Exosomes derived from normal fibroblast-like mesenchymal cells were engineered to carry siRNA or shRNA specific to oncogenic KRASG12D (iExosomes), a common mutation in pancreatic cancer. Compared to liposomes, iExosomes target oncogenic Kras with an enhanced efficacy that is dependent on CD47, and is facilitated by macropinocytosis. iExosomes treatment suppressed cancer in multiple mouse models of pancreatic cancer and significantly increased their overall survival. Our results inform on a novel approach for direct and specific targeting of oncogenic Kras in tumors using iExosomes. PMID:28607485

Organ tropic metastatic secretomes and exosomes in breast cancer

DTIC Science & Technology

2017-10-01

integrins, functionally relevant in organ- 11 tropic, specifically lung-tropic breast cancer metastasis, and tested these samples by ELISA (protein...guided by the results of our exosomal proteomics analysis, we had previously performed ELISA assays for plasma-derived exosomal integrins in breast

Induction of protective immunity against Eimeria tenella, Eimeria maxima, and Eimeria acervulina infections using DC-derived exosomes

USDA-ARS?s Scientific Manuscript database

This study describes a novel immunization strategy against avian coccidiosis using exosomes derived from Eimeria parasite antigen (Ag)-loaded dendritic cells (DCs) in the absence of soluble Ag. Chicken intestinal DCs were isolated and pulsed in vitro with a mixture of sporozoite-extracted Ags from E...

Pharmacokinetics of Exosomes-An Important Factor for Elucidating the Biological Roles of Exosomes and for the Development of Exosome-Based Therapeutics.

PubMed

Morishita, Masaki; Takahashi, Yuki; Nishikawa, Makiya; Takakura, Yoshinobu

2017-09-01

Exosomes are small membrane vesicles containing lipids, proteins, and nucleic acids. Recently, researchers have uncovered that exosomes are involved in various biological events, such as tumor growth, metastasis, and the immune response, by delivering their cargos to exosome-receiving cells. Moreover, exosomes are expected to be used in therapeutic treatments, such as tissue regeneration therapy and antitumor immunotherapy, because exosomes are effective delivery vehicles for proteins, nucleic acids, and other bioactive compounds. To elucidate the biological functions of exosomes, and for the development of exosome-based therapeutics, the pharmacokinetics of exosomes is important. In this review, we aim to summarize current knowledge about the pharmacokinetics and biodistribution of exosomes. The pharmacokinetics of exogenously administered exosomes is discussed based on the tissue distribution, types of cells taking up exosomes, and key molecules in the pharmacokinetics of exosomes. In addition, recent progress in the methods to control the pharmacokinetics of exosomes is reviewed. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Exosomes derived from human adipose mensenchymal stem cells accelerates cutaneous wound healing via optimizing the characteristics of fibroblasts.

PubMed

Hu, Li; Wang, Juan; Zhou, Xin; Xiong, Zehuan; Zhao, Jiajia; Yu, Ran; Huang, Fang; Zhang, Handong; Chen, Lili

2016-09-12

Prolonged healing and scar formation are two major challenges in the treatment of soft tissue trauma. Adipose mesenchymal stem cells (ASCs) play an important role in tissue regeneration, and recent studies have suggested that exosomes secreted by stem cells may contribute to paracrine signaling. In this study, we investigated the roles of ASCs-derived exosomes (ASCs-Exos) in cutaneous wound healing. We found that ASCs-Exos could be taken up and internalized by fibroblasts to stimulate cell migration, proliferation and collagen synthesis in a dose-dependent manner, with increased genes expression of N-cadherin, cyclin-1, PCNA and collagen I, III. In vivo tracing experiments demonstrated that ASCs-Exos can be recruited to soft tissue wound area in a mouse skin incision model and significantly accelerated cutaneous wound healing. Histological analysis showed increased collagen I and III production by systemic administration of exosomes in the early stage of wound healing, while in the late stage, exosomes might inhibit collagen expression to reduce scar formation. Collectively, our findings indicate that ASCs-Exos can facilitate cutaneous wound healing via optimizing the characteristics of fibroblasts. Our results provide a new perspective and therapeutic strategy for the use of ASCs-Exos in soft tissue repair.

Exosomes from eosinophils autoregulate and promote eosinophil functions.

PubMed

Cañas, José Antonio; Sastre, Beatriz; Mazzeo, Carla; Fernández-Nieto, Mar; Rodrigo-Muñoz, José Manuel; González-Guerra, Andrés; Izquierdo, Manuel; Barranco, Pilar; Quirce, Santiago; Sastre, Joaquín; Del Pozo, Victoria

2017-05-01

Eosinophils are able to secrete exosomes that have an undefined role in asthma pathogenesis. We hypothesized that exosomes released by eosinophils autoregulate and promote eosinophil function. Eosinophils of patients with asthma ( n = 58) and healthy volunteers ( n = 16) were purified from peripheral blood, and exosomes were isolated and quantified from eosinophils of the asthmatic and healthy populations. Apoptosis, adhesion, adhesion molecules expression, and migration assays were performed with eosinophils in the presence or absence of exosomes from healthy and asthmatic individuals. Reactive oxygen species (ROS) were evaluated by flow cytometry with an intracellular fluorescent probe and nitric oxide (NO) and a colorimetric kit. In addition, exosomal proteins were analyzed by mass spectrometry. Eosinophil-derived exosomes induced an increase in NO and ROS production on eosinophils. Moreover, exosomes could act as a chemotactic factor on eosinophils, and they produced an increase in cell adhesion, giving rise to a specific augmentation of adhesion molecules, such as ICAM-1 and integrin α2. Protein content between exosomes from healthy and asthmatic individuals seems to be similar in both groups. In conclusion, we found that exosomes from the eosinophils of patients with asthma could modify several specific eosinophil functions related to asthma pathogenesis and that they could contribute fundamentally to the development and maintenance of asthma. © Society for Leukocyte Biology.

Adiponectin is partially associated with exosomes in mouse serum.

PubMed

Phoonsawat, Worrawalan; Aoki-Yoshida, Ayako; Tsuruta, Takeshi; Sonoyama, Kei

2014-06-06

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. Copyright © 2014 Elsevier Inc. All rights reserved.

Exosomal and Non-Exosomal Transport of Extra-Cellular microRNAs in Follicular Fluid: Implications for Bovine Oocyte Developmental Competence

PubMed Central

Sohel, Md. Mahmodul Hasan; Hoelker, Michael; Noferesti, Sina Seifi; Salilew-Wondim, Dessie; Tholen, Ernst; Looft, Christian; Rings, Franca; Uddin, Muhammad Jasim; Spencer, Thomas E.; Schellander, Karl; Tesfaye, Dawit

2013-01-01

Cell-cell communication within the follicle involves many signaling molecules, and this process may be mediated by secretion and uptake of exosomes that contain several bioactive molecules including extra-cellular miRNAs. Follicular fluid and cells from individual follicles of cattle were grouped based on Brilliant Cresyl Blue (BCB) staining of the corresponding oocytes. Both Exoquick precipitation and differential ultracentrifugation were used to separate the exosome and non-exosomal fraction of follicular fluid. Following miRNA isolation from both fractions, the human miRCURY LNA™ Universal RT miRNA PCR array system was used to profile miRNA expression. This analysis found that miRNAs were present in both exosomal and non-exosomal fraction of bovine follicular fluid. We found 25 miRNAs differentially expressed (16 up and 9 down) in exosomes and 30 miRNAs differentially expressed (21 up and 9 down) in non-exosomal fraction of follicular fluid in comparison of BCB- versus BCB+ oocyte groups. Expression of selected miRNAs was detected in theca, granulosa and cumulus oocyte complex. To further explore the potential roles of these follicular fluid derived extra-cellular miRNAs, the potential target genes were predicted, and functional annotation and pathway analysis revealed most of these pathways are known regulators of follicular development and oocyte growth. In order to validate exosome mediated cell-cell communication within follicular microenvironment, we demonstrated uptake of exosomes and resulting increase of endogenous miRNA level and subsequent alteration of mRNA levels in follicular cells in vitro. This study demonstrates for the first time, the presence of exosome or non-exosome mediated transfer of miRNA in the bovine follicular fluid, and oocyte growth dependent variation in extra-cellular miRNA signatures in the follicular environment. PMID:24223816

[Regulation of immune responses by exosomes derived from antigen presenting cells].

PubMed

Maravillas-Montero, José Luis; Martínez-Cortés, Ismael

2017-01-01

Cells release several biomolecules to the extracellular environment using them as a communication alternative with neighbor cells. Besides these molecules, cells also release more complex elements, like vesicles; structures composed of a lipidic bilayer with transmembrane proteins that protect a hydrophilic content. Exosomes are a small subtype of vesicles (30-150 nm), produced by many cell types, such as tumor cells, neurons, epithelial cells and immune cells. Included in this last group, antigen presenting cells produce exosomes that contain different types of molecules depending on their activation and/or maturation state. In recent years there has been an exponential interest in exosomes due to the recent evidences that show the immunomodulatory properties of these vesicles and therefore, their great potential in diagnostic approaches and development of therapies for different inflammation-associated pathologies.

Exosomes Mediate Intercellular Transmission of Porcine Reproductive and Respiratory Syndrome Virus.

PubMed

Wang, Ting; Fang, Liurong; Zhao, Fuwei; Wang, Dang; Xiao, Shaobo

2018-02-15

Exosomes are small membrane-enclosed vesicles produced by various cells and actively released into the extracellular space. They participate in intercellular communication and transfer of biologically active proteins, lipids, and nucleic acids. Accumulating evidence suggests that exosomes derived from cells infected by some viruses selectively encapsulate viral proteins, genetic materials, or even virions to mediate cell-to-cell communication and/or virus transmission. Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has been devastating the global swine industry since the late 1980s. Recent studies have shown that major proteins secreted from PRRSV-infected cells are exosomal proteins and that the serum-derived exosomes from PRRSV-infected pigs contain viral proteins. However, the role of exosomes in PRRSV infection remains unclear. In this study, purified exosomes isolated from PRRSV-infected cells were shown with reverse transcription-PCR and mass spectrometry to contain viral genomic RNA and partial viral proteins. Furthermore, exosomes from PRRSV-infected cells established productive infection in both PRRSV-susceptible and -nonsusceptible cells. More importantly, exosome-mediated infection was not completely blocked by PRRSV-specific neutralizing antibodies. In summary, this study demonstrated that exosomes can mediate PRRSV transmission and are even resistant to antibody neutralization, identifying a potential immune evasion mechanism utilized by PRRSV. IMPORTANCE Exosomes have recently been characterized as bioactive vesicles that function to promote intercellular communication. The exosomes from virally infected cells containing altered compositions confer numerous novel functionalities. A study of the secretome of cells infected with PRRSV indicated that the exosomal pathway is strongly activated by PRRSV infection. Here, we demonstrate that PRRSV can utilize host exosomes to infect naive healthy cells. Furthermore

Cancer Exosomes Perform Cell-Independent MicroRNA Biogenesis and Promote Tumorigenesis

PubMed Central

Melo, Sonia A.; Sugimoto, Hikaru; O’Connell, Joyce T.; Kato, Noritoshi; Villanueva, Alberto; Vidal, August; Qiu, Le; Vitkin, Edward; Perelman, Lev T.; Melo, Carlos A.; Lucci, Anthony; Ivan, Cristina; Calin, George A.; Kalluri, Raghu

2014-01-01

SUMMARY Exosomes are secreted by all cell types and contain proteins and nucleic acids. Here, we report that breast cancer associated exosomes contain microRNAs (miRNAs) associated with the RISC Loading Complex (RLC) and display cell-independent capacity to process precursor microRNAs (pre-miRNAs) into mature miRNAs. Pre-miRNAs, along with Dicer, AGO2, and TRBP, are present in exosomes of cancer cells. CD43 mediates the accumulation of Dicer specifically in cancer exosomes. Cancer exosomes mediate an efficient and rapid silencing of mRNAs to reprogram the target cell transcriptome. Exosomes derived from cells and sera of patients with breast cancer instigate non-tumorigenic epithelial cells to form tumors in a Dicer-dependent manner. These findings offer opportunities for the development of exosomes based biomarkers and therapies. PMID:25446899

A Comprehensive Proteomics Analysis Reveals a Secretory Path- and Status-Dependent Signature of Exosomes Released from Tumor-Associated Macrophages.

PubMed

Zhu, Yinghui; Chen, Xianwei; Pan, Qingfei; Wang, Yang; Su, Siyuan; Jiang, Cuicui; Li, Yang; Xu, Ningzhi; Wu, Lin; Lou, Xiaomin; Liu, Siqi

2015-10-02

Exosomes are 30-120 nm-sized membrane vesicles of endocytic origin that are released into the extracellular environment and play roles in cell-cell communication. Tumor-associated macrophages (TAMs) are important constituents of the tumor microenvironment; thus, it is critical to study the features and complex biological functions of TAM-derived exosomes. Here, we constructed a TAM cell model from a mouse macrophage cell line, Ana-1, and performed comparative proteomics on exosomes, exosome-free media, and cells between TAMs and Ana-1. Proteomic analysis between exosome and exosome-free fractions indicated that the functions of exosome dominant proteins were mainly enriched in RNA processing and proteolysis. TAM status dramatically affected the abundances of 20S proteasome subunits and ribosomal proteins in their exosomes. The 20S proteasome activity assay strongly indicated that TAM exosomes possessed higher proteolytic activity. In addition, Ana-1- and TAM-derived exosomes have different RNA profiles, which may result from differential RNA processing proteins. Taken together, our comprehensive proteomics study provides novel views for understanding the complicated roles of macrophage-derived exosomes in the tumor microenvironment.

Tumour-derived exosomes as a signature of pancreatic cancer - liquid biopsies as indicators of tumour progression

PubMed Central

Nuzhat, Zarin; Kinhal, Vyjayanthi; Sharma, Shayna; Rice, Gregory E.; Joshi, Virendra; Salomon, Carlos

2017-01-01

Pancreatic cancer is the fourth most common cause of death due to cancer in the world. It is known to have a poor prognosis, mostly because early stages of the disease are generally asymptomatic. Progress in pancreatic cancer research has been slow, leaving several fundamental questions pertaining to diagnosis and treatment unanswered. Recent studies highlight the putative utility of tissue-specific vesicles (i.e. extracellular vesicles) in the diagnosis of disease onset and treatment monitoring in pancreatic cancer. Extracellular vesicles are membrane-limited structures derived from the cell membrane. They contain specific molecules including proteins, mRNA, microRNAs and non-coding RNAs that are secreted in the extracellular space. Extracellular vesicles can be classified according to their size and/or origin into microvesicles (∼150-1000 nm) and exosomes (∼40-120 nm). Microvesicles are released by budding from the plasmatic membrane, whereas exosomes are released via the endocytic pathway by fusion of multivesicular bodies with the plasmatic membrane. This endosomal origin means that exosomes contain an abundance of cell-specific biomolecules which may act as a fingerprint of the cell of origin. In this review, we discuss our current knowledge in the diagnosis and treatment of pancreatic cancer, particularly the potential role of EVs in these facets of disease management. In particular, we suggest that as exosomes contain cellular protein and RNA molecules in a cell type-specific manner, they may provide extensive information about the signature of the tumour and pancreatic cancer progression. PMID:27999198

ET-33PLACENTA-DERIVED MESENCHYMAL STEM CELLS AND THEIR SECRETED EXOSOMES INHIBIT THE SELF-RENEWAL AND STEMNESS OF GLIOMA STEM CELLS IN VITRO AND IN VIVO

PubMed Central

Lee, Hae Kyung; Buchris, Efrat; Finniss, Susan; Cazacu, Simona; Xiang, Cunli; Poisson, Laila; Brodie, Chaya

2014-01-01

Mesenchymal stromal cells (MSCs) are multipotent stem cells that can be obtained from bone marrow and adipose tissues or from other sources such as placenta and umbilical cord. The latter allow the potential use of universal, allogeneic cell therapy because to reduced antigenicity due to low expression of MHC class II molecules. MSCs can be easily expanded in vitro for therapeutic applications and their safety and therapeutic impact have been demonstrated in various pre-clinical and clinical studies. MSCs have been shown to cross the blood brain barrier and migrate to sites of experimental GBM and can deliver cytotoxic compounds that exert anti-tumor effects. In this study we examined the effects of placenta-derived MSCs and their secreted exosomes on GSCs in vitro and in vivo. Conditioned medium of placenta MSCs or their derived exosomes decreased the self-renewal, stemness markers, Sox2 and Oct4 and the migration of these cells. Similarly, intracranial administration of the MSCs decreased the tumor volume of GSC-derived xenografts and prolonged animal survival. miRNA sequencing analysis of placenta MSC-derived exosomes revealed a set of specific miRNAs that were downregulated in GSCs and that acted as tumor suppressor in these cells. We demonstrated delivery of some of these miRNAs to GSCs following treatments with MSC-derived exosomes. We further demonstrated that MSCs or exosomes that were loaded with exogenous miR-124 delivered high levels of this miRNA into glioma cells as detected by a novel quantitative miRNA reporter. Moreover, administration of placenta MSCs loaded with exogenous miR-124 exerted a strong inhibitory effect on GSC-derived xenograft growth. These results demonstrate that placenta-derived MSCs may have important clinical applications in stem cell-based glioma therapeutics. Moreover, these studies provide a novel approach for the targeted delivery of endogenous and exogenous anti-tumor miRNAs to glioma cells as a miRNA replacement therapy for

Exosomes enriched in stemness/metastatic-related mRNAS promote oncogenic potential in breast cancer.

PubMed

Rodríguez, Marta; Silva, Javier; Herrera, Alberto; Herrera, Mercedes; Peña, Cristina; Martín, Paloma; Gil-Calderón, Beatriz; Larriba, María Jesús; Coronado, M Josés; Soldevilla, Beatriz; Turrión, Víctor S; Provencio, Mariano; Sánchez, Antonio; Bonilla, Félix; García-Barberán, Vanesa

2015-12-01

Cancer cells efficiently transfer exosome contents (essentially mRNAs and microRNAs) to other cell types, modifying immune responses, cell growth, angiogenesis and metastasis. Here we analyzed the exosomes release by breast tumor cells with different capacities of stemness/metastasis based on CXCR4 expression, and evaluated their capacity to generate oncogenic features in recipient cells. Breast cancer cells overexpressing CXCR4 showed an increase in stemness-related markers, and in proliferation, migration and invasion capacities. Furthermore, recipient cells treated with exosomes from CXCR4-cells showed increased in the same abilities. Moreover, inoculation of CXCR4-cell-derived exosomes in immunocompromised mice stimulated primary tumor growth and metastatic potential. Comparison of nucleic acids contained into exosomes isolated from patients revealed a "stemness and metastatic" signature in exosomes of patients with worse prognosis. Finally, our data supported the view that cancer cells with stem-like properties show concomitant metastatic behavior, and their exosomes stimulate tumor progression and metastasis. Exosomes-derived nucleic acids from plasma of breast cancer patients are suitable markers in the prognosis of such patients.

High-resolution proteomic and lipidomic analysis of exosomes and microvesicles from different cell sources

PubMed Central

Haraszti, Reka A.; Didiot, Marie-Cecile; Sapp, Ellen; Leszyk, John; Shaffer, Scott A.; Rockwell, Hannah E.; Gao, Fei; Narain, Niven R.; DiFiglia, Marian; Kiebish, Michael A.; Aronin, Neil; Khvorova, Anastasia

2016-01-01

Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are explored for use in diagnostics, therapeutics and drug delivery. However, little is known about the relationship of protein and lipid composition of EVs and their source cells. Here, we report high-resolution lipidomic and proteomic analyses of exosomes and MVs derived by differential ultracentrifugation from 3 different cell types: U87 glioblastoma cells, Huh7 hepatocellular carcinoma cells and human bone marrow-derived mesenchymal stem cells (MSCs). We identified 3,532 proteins and 1,961 lipid species in the screen. Exosomes differed from MVs in several different areas: (a) The protein patterns of exosomes were more likely different from their cells of origin than were the protein patterns of MVs; (b) The proteomes of U87 and Huh7 exosomes were similar to each other but different from the proteomes of MSC exosomes, whereas the lipidomes of Huh7 and MSC exosomes were similar to each other but different from the lipidomes of U87 exosomes; (c) exosomes exhibited proteins of extracellular matrix, heparin-binding, receptors, immune response and cell adhesion functions, whereas MVs were enriched in endoplasmic reticulum, proteasome and mitochondrial proteins. Exosomes and MVs also differed in their types of lipid contents. Enrichment in glycolipids and free fatty acids characterized exosomes, whereas enrichment in ceramides and sphingomyelins characterized MVs. Furthermore, Huh7 and MSC exosomes were specifically enriched in cardiolipins; U87 exosomes were enriched in sphingomyelins. This study comprehensively analyses the protein and lipid composition of exosomes, MVs and source cells in 3 different cell types. PMID:27863537

Regulation of exosome release from mammary epithelial and breast cancer cells - a new regulatory pathway.

PubMed

Riches, Andrew; Campbell, Elaine; Borger, Eva; Powis, Simon

2014-03-01

Exosomes are small 50-100nm sized extracellular vesicles released from normal and tumour cells and are a source of a new intercellular communication pathway. Tumour exosomes promote tumour growth and progression. What regulates the release and homoeostatic levels of exosomes, in cancer, in body fluids remains undefined. We utilised a human mammary epithelial cell line (HMEC B42) and a breast cancer cell line derived from it (B42 clone 16) to investigate exosome production and regulation. Exosome numbers were quantified using a Nanosight LM10 and measured in culture supernatants in the absence and presence of exosomes in the medium. Concentrated suspensions of exosomes from the normal mammary epithelial cells, the breast cancer cells and bladder cancer cells were used. The interaction of exosomes with tumour cells was also investigated using fluorescently labelled exosomes. Exosome release from normal human mammary epithelial cells and breast cancer cells is regulated by the presence of exosomes, derived from their own cells, in the extracellular environment of the cells. Exosomes from normal mammary epithelial cells also inhibit exosome secretion by breast cancer cells, which occurs in a tissue specific manner. Labelled exosomes from mammary epithelial cells are internalised into the tumour cells implicating a dynamic equilibrium and suggesting a mechanism for feedback control. These data suggest a previously unknown novel feedback regulatory mechanism for controlling exosome release, which may highlight a new therapeutic approach to controlling the deleterious effects of tumour exosomes. This regulatory mechanism is likely to be generic to other tumours. Copyright © 2014 Elsevier Ltd. All rights reserved.

Exosome delivered anticancer drugs across the blood-brain barrier for brain cancer therapy in Danio rerio.

PubMed

Yang, Tianzhi; Martin, Paige; Fogarty, Brittany; Brown, Alison; Schurman, Kayla; Phipps, Roger; Yin, Viravuth P; Lockman, Paul; Bai, Shuhua

2015-06-01

The blood-brain barrier (BBB) essentially restricts therapeutic drugs from entering into the brain. This study tests the hypothesis that brain endothelial cell derived exosomes can deliver anticancer drug across the BBB for the treatment of brain cancer in a zebrafish (Danio rerio) model. Four types of exosomes were isolated from brain cell culture media and characterized by particle size, morphology, total protein, and transmembrane protein markers. Transport mechanism, cell uptake, and cytotoxicity of optimized exosome delivery system were tested. Brain distribution of exosome delivered anticancer drugs was evaluated using transgenic zebrafish TG (fli1: GFP) embryos and efficacies of optimized formations were examined in a xenotransplanted zebrafish model of brain cancer model. Four exosomes in 30-100 diameters showed different morphologies and exosomes derived from brain endothelial cells expressed more CD63 tetraspanins transmembrane proteins. Optimized exosomes increased the uptake of fluorescent marker via receptor mediated endocytosis and cytotoxicity of anticancer drugs in cancer cells. Images of the zebrafish showed exosome delivered anticancer drugs crossed the BBB and entered into the brain. In the brain cancer model, exosome delivered anticancer drugs significantly decreased fluorescent intensity of xenotransplanted cancer cells and tumor growth marker. Brain endothelial cell derived exosomes could be potentially used as a carrier for brain delivery of anticancer drug for the treatment of brain cancer.

Optimizing exosomal RNA isolation for RNA-Seq analyses of archival sera specimens.

PubMed

Prendergast, Emily N; de Souza Fonseca, Marcos Abraão; Dezem, Felipe Segato; Lester, Jenny; Karlan, Beth Y; Noushmehr, Houtan; Lin, Xianzhi; Lawrenson, Kate

2018-01-01

Exosomes are endosome-derived membrane vesicles that contain proteins, lipids, and nucleic acids. The exosomal transcriptome mediates intercellular communication, and represents an understudied reservoir of novel biomarkers for human diseases. Next-generation sequencing enables complex quantitative characterization of exosomal RNAs from diverse sources. However, detailed protocols describing exosome purification for preparation of exosomal RNA-sequence (RNA-Seq) libraries are lacking. Here we compared methods for isolation of exosomes and extraction of exosomal RNA from human cell-free serum, as well as strategies for attaining equal representation of samples within pooled RNA-Seq libraries. We compared commercial precipitation with ultracentrifugation for exosome purification and confirmed the presence of exosomes via both transmission electron microscopy and immunoblotting. Exosomal RNA extraction was compared using four different RNA purification methods. We determined the minimal starting volume of serum required for exosome preparation and showed that high quality exosomal RNA can be isolated from sera stored for over a decade. Finally, RNA-Seq libraries were successfully prepared with exosomal RNAs extracted from human cell-free serum, cataloguing both coding and non-coding exosomal transcripts. This method provides researchers with strategic options to prepare RNA-Seq libraries and compare RNA-Seq data quantitatively from minimal volumes of fresh and archival human cell-free serum for disease biomarker discovery.

A Gestational Profile of Placental Exosomes in Maternal Plasma and Their Effects on Endothelial Cell Migration

PubMed Central

Salomon, Carlos; Torres, Maria Jose; Kobayashi, Miharu; Scholz-Romero, Katherin; Sobrevia, Luis; Dobierzewska, Aneta; Illanes, Sebastian E.; Mitchell, Murray D.; Rice, Gregory E.

2014-01-01

Studies completed to date provide persuasive evidence that placental cell-derived exosomes play a significant role in intercellular communication pathways that potentially contribute to placentation and development of materno-fetal vascular circulation. The aim of this study was to establish the gestational-age release profile and bioactivity of placental cell-derived exosome in maternal plasma. Plasma samples (n = 20 per pregnant group) were obtained from non-pregnant and pregnant women in the first (FT, 6–12 weeks), second (ST, 22–24 weeks) and third (TT, 32–38 weeks) trimester. The number of exosomes and placental exosome contribution were determined by quantifying immunoreactive exosomal CD63 and placenta-specific marker (PLAP), respectively. The effect of exosomes isolated from FT, ST and TT on endothelial cell migration were established using a real-time, live-cell imaging system (Incucyte). Exosome plasma concentration was more than 50-fold greater in pregnant women than in non-pregnant women (p<0.001). During normal healthy pregnancy, the number of exosomes present in maternal plasma increased significantly with gestational age by more that two-fold (p<0.001). Exosomes isolated from FT, ST and TT increased endothelial cell migration by 1.9±0.1, 1.6±0.2 and 1.3±0.1-fold, respectively compared to the control. Pregnancy is associated with a dramatic increase in the number of exosomes present in plasma and maternal plasma exosomes are bioactive. While the role of placental cell-derived exosome in regulating maternal and/or fetal vascular responses remains to be elucidated, changes in exosome profile may be of clinical utility in the diagnosis of placental dysfunction. PMID:24905832

Comparison of Plasma Exosomes by Differential Ultracentrifugation and Solvent Precipitation Methods.

PubMed

Peng, Qiao; Zhang, Jing; Zhou, Gang

2018-06-01

Emerging evidence has identified that exosomes play a pivotal role in intercellular signal transmission. However, the standardized purification techniques to isolate high quality exosomes are still deficient at present. This study was to evaluate reproducibility and efficiency of differential ultracentrifugation and solvent precipitation-based kits by isolating plasma-derived exosomes from oral lichen planus patients. Morphology, exosomal biomarkers, particle size distribution, proteomic components, and protein yield of isolated exosomes were evaluated by transmission electron microscope, western blot, laser diffraction instrument, Coomassie staining, and BCA protein assay kit, respectively. TEM displayed representative cup-shaped morphology of exosomes and western blot identified exosomal biomarkers CD9 and CD63. The size distribution showed that particles by differential ultracentrifugation were mainly from 26.15 nm to 166.5 nm, while some of the particles obtained by solvent precipitation kits were larger than 1,000 nm. In addition, exosomes isolated by solvent precipitation kits showed a significantly higher amount of protein yield due to plasma albumin contamination. Both differential ultracentrifugation and precipitation based kits could successfully isolate plasma exosomes, and exosomes by differential ultracentrifugation were purer and more appropriate for further proteomic analysis.

Exosomal Signaling during Hypoxia Mediates Microvascular Endothelial Cell Migration and Vasculogenesis

PubMed Central

Salomon, Carlos; Ryan, Jennifer; Sobrevia, Luis; Kobayashi, Miharu; Ashman, Keith; Mitchell, Murray; Rice, Gregory E.

2013-01-01

Vasculogenesis and angiogenesis are critical processes in fetal circulation and placental vasculature development. Placental mesenchymal stem cells (pMSC) are known to release paracrine factors (some of which are contained within exosomes) that promote angiogenesis and cell migration. The aims of this study were: to determine the effects of oxygen tension on the release of exosomes from pMSC; and to establish the effects of pMSC-derived exosomes on the migration and angiogenic tube formation of placental microvascular endothelial cells (hPMEC). pMSC were isolated from placental villi (8–12 weeks of gestation, n = 6) and cultured under an atmosphere of 1%, 3% or 8% O2. Cell-conditioned media were collected and exosomes (exo-pMSC) isolated by differential and buoyant density centrifugation. The dose effect (5–20 µg exosomal protein/ml) of pMSC-derived exosomes on hPMEC migration and tube formation were established using a real-time, live-cell imaging system (Incucyte™). The exosome pellet was resuspended in PBS and protein content was established by mass spectrometry (MS). Protein function and canonical pathways were identified using the PANTHER program and Ingenuity Pathway Analysis, respectively. Exo-pMSC were identified, by electron microscopy, as spherical vesicles, with a typical cup-shape and diameters around of 100 nm and positive for exosome markers: CD63, CD9 and CD81. Under hypoxic conditions (1% and 3% O2) exo-pMSC released increased by 3.3 and 6.7 folds, respectively, when compared to the controls (8% O2; p<0.01). Exo-pMSC increased hPMEC migration by 1.6 fold compared to the control (p<0.05) and increased hPMEC tube formation by 7.2 fold (p<0.05). MS analysis identified 390 different proteins involved in cytoskeleton organization, development, immunomodulatory, and cell-to-cell communication. The data obtained support the hypothesis that pMSC-derived exosomes may contribute to placental vascular adaptation to low oxygen tension under both

Exosomes and their roles in immune regulation and cancer.

PubMed

Greening, David W; Gopal, Shashi K; Xu, Rong; Simpson, Richard J; Chen, Weisan

2015-04-01

Exosomes, a subset of extracellular vesicles (EVs), function as a mode of intercellular communication and molecular transfer. Exosomes facilitate the direct extracellular transfer of proteins, lipids, and miRNA/mRNA/DNAs between cells in vitro and in vivo. The immunological activities of exosomes affect immunoregulation mechanisms including modulating antigen presentation, immune activation, immune suppression, immune surveillance, and intercellular communication. Besides immune cells, cancer cells secrete immunologically active exosomes that influence both physiological and pathological processes. The observation that exosomes isolated from immune cells such as dendritic cells (DCs) modulate the immune response has enforced the way these membranous vesicles are being considered as potential immunotherapeutic reagents. Indeed, tumour- and immune cell-derived exosomes have been shown to carry 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 malignant tumour development. Further understanding of these areas of exosome biology, and especially of molecular mechanisms involved in immune cell targeting, interaction and manipulation, is likely to provide significant insights into immunorecognition and therapeutic intervention. Here, we review the emerging roles of exosomes in immune regulation and the therapeutic potential in cancer. Copyright © 2015 Elsevier Ltd. All rights reserved.

Comparison of commercial exosome isolation kits for circulating exosomal microRNA profiling.

PubMed

Ding, Meng; Wang, Cheng; Lu, Xiaolan; Zhang, Cuiping; Zhou, Zhen; Chen, Xi; Zhang, Chen-Yu; Zen, Ke; Zhang, Chunni

2018-06-01

Circulating exosomal microRNAs (miRNAs) are valuable biomarker candidates; however, information on the characterization and mutual agreement of commercial kits for circulating exosomal miRNA profiling is scarce. Here, we analyzed the advantages and weaknesses of four commonly used commercial kits for exosomal miRNA profiling and their application to the sample of serum and/or plasma, respectively. NanoSight and Western blotting were conducted to evaluate the efficiency and purity of the isolated exosomes. In our conditions, the size distribution of the isolated particles was appropriate (40-150 nm), and ExoQuick™ Exosome Precipitation Solution (EXQ) generated a relatively high yield of exosomes. Nevertheless, albumin impurity was ubiquitous for all the four kits, and Total Exosome Isolation for serum or plasma (TEI) yielded a relatively pure isolation. We further performed Illumina sequencing combined with RT-qPCR to determine the ability of these kits for miRNA profiling. There was significant correlation of the exosomal miRNA profile and specific miRNAs between kits, but with differences depending on methods. exoRNeasy Serum/Plasma Midi Kit (EXR) and EXQ performed better in the specific exosomal miRNAs recovery. Intraassay CVs for specific miRNA measurement were 0.88-3.82, 1.19-3.77, 0-2.70, and 1.23-9.11% for EXR, TEI, EXQ, and RIBO™ Exosome Isolation Reagent (REI), respectively. In each kit, serum yielded a higher abundance of exosomes and exosomal miRNAs than plasma, yet with more albumin impurity. In conclusion, our data provide some valuable guidance for the methodology of disease biomarker identification of circulation exosomal miRNAs. Graphical abstract Circulating exosomal microRNAs (miRNAs) are valuable biomarker candidates; however, information on the characterization and mutual agreement of commercial kits for circulating exosomal miRNA profiling is scarce. In this study, we compared four commonly used commercially available kits for exosomal mi

Isolation of Biologically Active Exosomes from Plasma of Patients with Cancer.

PubMed

Hong, Chang-Sook; Funk, Sonja; Whiteside, Theresa L

2017-01-01

A method for exosome isolation from human plasma was developed for rapid, high-throughput processing of plasma specimens obtained from patients with cancer. This method removes the bulk of plasma proteins associated with exosomes and can be used for comparative examinations of exosomes and their content in serial specimens of patients' plasma, allowing for monitoring changes in exosome numbers, profiles, and functions in the course of cancer progression or during therapy. The plasma-derived exosomes can be recovered in quantities sufficient for the characterization of their morphology by transmission electron microscopy (TEM), size and concentration by qNano, protein/lipid ratios, nucleic acid extraction, molecular profiling by Western blots or immune arrays, and functional assays.

Exosomes From Adipose-derived Mesenchymal Stem Cells Protect the Myocardium Against Ischemia/Reperfusion Injury Through Wnt/β-Catenin Signaling Pathway.

PubMed

Cui, Xiaojun; He, Zhangyou; Liang, Zihao; Chen, Zhenyi; Wang, Haifeng; Zhang, Jiankai

2017-10-01

Mesenchymal stem cells (MSCs) and their secreted exosomes exert a cardioprotective role in jeopardized myocardium. However, the specific effects and underlying mechanisms of exosomes derived from adipose-derived MSCs (ADMSCs) on myocardial ischemia/reperfusion (I/R) injury remain largely unclear. In this study, ADMSC-derived exosomes (ADMSCs-ex) were administrated into the rats subjected to I/R injury and H9c2 cells exposed to hypoxia/reoxygenation (H/R). Consequently, administration of ADMSCs-ex significantly reduced I/R-induced myocardial infarction, accompanied with a decrease in serum levels of creatine kinase-myocardial band, lactate dehydrogenase, and cardiac troponin I (cTnI). Simultaneously, ADMSCs-ex dramatically antagonized I/R-induced myocardial apoptosis, along with the upregulation of Bcl-2 and downregulation of Bax, and inhibition of Caspase 3 activity in rat myocardium. Similarly, ADMSCs-ex significantly reduced cell apoptosis and the expression of Bax, but markedly increased cell viability and the expression of Bcl-2 and Cyclin D1 under H/R. Furthermore, ADMSCs-ex observably induced the activation of Wnt/β-catenin signaling by attenuating I/R- and H/R-induced inhibition of Wnt3a, p-GSK-3β (Ser9), and β-catenin expression. Importantly, treatment with Wnt/β-catenin inhibitor XAV939 partly neutralized ADMSC-ex-induced antiapoptotic and prosurvival effects in H9c2 cells. In conclusion, we confirmed that ADMSCs-ex protect ischemic myocardium from I/R injury through the activation of Wnt/β-catenin signaling pathway.

Proteome profiling of exosomes derived from plasma of heifers with divergent genetic merit for fertility.

PubMed

Koh, Yong Qin; Peiris, Hassendrini N; Vaswani, Kanchan; Almughlliq, Fatema B; Meier, Susanne; Burke, Chris R; Roche, John R; Reed, Charlotte B; Arachchige, Buddhika J; Reed, Sarah; Mitchell, Murray D

2018-04-25

The current study evaluated exosomes isolated from plasma of heifers bred to have high or low fertility through developing extreme diversity in fertility breeding values, however, key animal traits (e.g., body weight, milk production, and percentage of North American genetics) remained similar between the 2 groups. The exosomes were isolated by a combined ultracentrifugation and size exclusion chromatography approach and characterized by their size distribution (nanoparticle tracking analysis), morphology (transmission electron microscopy), and presence of exosomal markers (immunoblotting). In addition, a targeted mass spectrometry approach was used to confirm the presence of 2 exosomal markers, tumor susceptibility gene 101 and flotillin 1. The number of exosomes from plasma of high fertility heifers was greater compared with low fertility heifers. Interestingly, the exosomal proteomic profile, evaluated using mass spectrometry, identified 89 and 116 proteins in the high and low fertility heifers respectively, of which 4 and 31 were unique, respectively. These include proteins associated with specific biological processes and molecular functions of fertility. Most notably, the tetratricopeptide repeat protein 41-related, glycodelin, and kelch-like protein 8 were identified in plasma exosomes unique to the low fertility heifers. These proteins are suggested to play a role in reproduction; however, the role of these proteins in dairy cow reproduction remains to be elucidated. Their identification underscores the potential for proteins within exosomes to provide information on the fertility status and physiological condition of the cow. This may potentially lead to the development of prognostic tools and interventions to improving dairy cow fertility. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

The biological significance and clinical applications of exosomes in ovarian cancer

PubMed Central

Dorayappan, Kalpana Deepa Priya; Wallbillich, John J.; Cohn, David E.; Selvendiran, Karuppaiyah

2016-01-01

Exosomes are nano-sized (20–100 nm) vesicles released by a variety of cells and are generated within the endosomal system or at the plasma membrane. There is emerging evidence that exosomes play a key role in intercellular communication in ovarian and other cancers. The protein and microRNA content of exosomes has been implicated in various intracellular processes that mediate oncogenesis, tumor spread, and drug resistance. Exosomes may prime distant tissue sites for reception of future metastases and their release can be mediated by the tumor microenvironment (e.g., hypoxia). Ovarian cancer-derived exosomes have unique features that could be leveraged for use as biomarkers to facilitate improved detection and treatment of the disease. Further, exosomes have the potential to serve as targets and/or drug delivery vehicles in the treatment of ovarian cancer. In this review we discuss the biological and clinical significance of exosomes relevant to the progression, detection, and treatment of ovarian cancer. PMID:27058839

The biological significance and clinical applications of exosomes in ovarian cancer.

PubMed

Dorayappan, Kalpana Deepa Priya; Wallbillich, John J; Cohn, David E; Selvendiran, Karuppaiyah

2016-07-01

Exosomes are nano-sized (20-100nm) vesicles released by a variety of cells and are generated within the endosomal system or at the plasma membrane. There is emerging evidence that exosomes play a key role in intercellular communication in ovarian and other cancers. The protein and microRNA content of exosomes has been implicated in various intracellular processes that mediate oncogenesis, tumor spread, and drug resistance. Exosomes may prime distant tissue sites for reception of future metastases and their release can be mediated by the tumor microenvironment (e.g., hypoxia). Ovarian cancer-derived exosomes have unique features that could be leveraged for use as biomarkers to facilitate improved detection and treatment of the disease. Further, exosomes have the potential to serve as targets and/or drug delivery vehicles in the treatment of ovarian cancer. In this review we discuss the biological and clinical significance of exosomes relevant to the progression, detection, and treatment of ovarian cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

Exosomes derived from human amniotic epithelial cells accelerate wound healing and inhibit scar formation.

PubMed

Zhao, Bin; Zhang, Yijie; Han, Shichao; Zhang, Wei; Zhou, Qin; Guan, Hao; Liu, Jiaqi; Shi, Jihong; Su, Linlin; Hu, Dahai

2017-04-01

Wound healing is a highly orchestrated physiological process consisting of a complex events, and scarless wound healing is highly desired for the development and application in clinical medicine. Recently, we have demonstrated that human amniotic epithelial cells (hAECs) promoted wound healing and inhibited scar formation through a paracrine mechanism. However, exosomes (Exo) are one of the most important paracrine factors. Whether exosomes derived from human amniotic epithelial cells (hAECs-Exo) have positive effects on scarless wound healing have not been reported yet. In this study, we examined the role of hAECs-Exo on wound healing in a rat model. We found that hAECs, which exhibit characteristics of both embryonic and mesenchymal stem cells, have the potential to differentiate into all three germ layers. hAECs-Exo ranged from 50 to 150 nm in diameter, and positive for exosomal markers CD9, CD63, CD81, Alix, TSG101 and HLA-G. Internalization of hAECs-Exo promoted the migration and proliferation of fibroblasts. Moreover, the deposition of extracellular matrix (ECM) were partly abolished by the treatment of high concentration of hAECs-Exo (100 μg/mL), which may be through stimulating the expression of matrix metalloproteinase-1 (MMP-1). In vivo animal experiments showed that hAECs-Exo improved the skin wound healing with well-organized collagen fibers. Taken together, These findings represent that hAECs-Exo can be used as a novel hope in cell-free therapy for scarless wound healing.

Exosomes as nanocarriers for immunotherapy of cancer and inflammatory diseases.

PubMed

Tran, Thanh-Huyen; Mattheolabakis, George; Aldawsari, Hibah; Amiji, Mansoor

2015-09-01

Cell secreted exosomes (30-100nm vesicles) play a major role in intercellular communication due to their ability to transfer proteins and nucleic acids from one cell to another. Depending on the originating cell type and the cargo, exosomes can have immunosuppressive or immunostimulatory effects, which have potential application as immunotherapies for cancer and autoimmune diseases. Cellular components shed from tumor cells or antigen presenting cells (APCs), such as dendritic cells, macrophages and B cells, have been shown to be efficiently packaged in exosomes. In this review, we focus on the application of exosomes as nanocarriers and immunological agents for cancer and autoimmune immunotherapy. APC-derived exosomes demonstrate effective therapeutic efficacy for the treatment of cancer and experimental autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease, and multiple sclerosis. In addition to their intrinsic immunomodulating activity, exosomes have many advantages over conventional nanocarriers for drug and gene delivery. Copyright © 2015 Elsevier Inc. All rights reserved.

Exosomal DNMT1 mediates cisplatin resistance in ovarian cancer.

PubMed

Cao, Ya-Lei; Zhuang, Ting; Xing, Bao-Heng; Li, Na; Li, Qin

2017-08-01

Ovarian cancer is the most common malignancy in women. Owing to late syndromic presentation and lack of efficient early detection, most cases are diagnosed at advanced stages. Surgery and platinum-based chemotherapy are still the standard care currently. However, resistance invoked often compromises the clinical value of the latter. Expression of DNA methyltransferase 1 (DNMT1) was analysed by gene array. Protein was determined by immunoblotting. Exosome was isolated with commercial kit. Cell proliferation was measured by CCK8 method. Annexin V-PI double staining was performed for apoptosis evaluation. Xenograft model was established and administrated with exosome. Tumour growth and overall survival were monitored. We demonstrated the upregulation of DNMT1 in both tumour and derived cell line. DNMT1 transcripts were highly enriched in exosomes from conditioned medium of ovarian cells. Co-incubation with exosomes stimulated endogenous expression and rendered host cell the resistance to cytotoxicity of cisplatin. In vivo administration of DNMT1-containing exosomes exacerbated xenograft progression and reduced overall survival significantly. Moreover, treatment with exosome inhibitor GW4869 almost completely restored sensitivity in resistant cells. Our data elucidated an unappreciated mechanism of exosomal DNMT1 in cisplatin resistance in ovarian cancer, also indicating the potential of the combination of exosome inhibitor with cisplatin in resistant patients. Copyright © 2017 John Wiley & Sons, Ltd.

Interactions between resin monomers and commercial composite resins with human saliva derived esterases.

PubMed

Jaffer, F; Finer, Y; Santerre, J P

2002-04-01

Cholesterol esterase (CE) and pseudocholinesterase (PCE) have been reported to degrade commercial and model composite resins containing bisphenylglycidyl dimethacrylate (BisGMA), triethylene glycol dimethacrylate (TEGDMA) or the latter in combination with urethane modified BisGMA monomer systems. In addition, human saliva has been shown to contain esterase like activities similar to CE and PCE. Hence, it was the aim of the current study to determine to what extent human saliva could degrade two common commercial composite resins (Z250 from 3M Inc. and Spectrum TPH from L.D. Caulk) which contain the above monomer systems. Saliva samples from different volunteers were collected, processed, pooled, and freeze-dried. TEGDMA and BisGMA monomers were incubated with human saliva derived esterase activity (HSDEA) and their respective hydrolysis was monitored using high performance liquid chromatography (HPLC). Both monomers were completely hydrolyzed within 25 h by HSDEA. Photopolymerized composites were incubated with buffer or human saliva (pH 7.0 and 37 C) for 2, 8 and 16 days. The incubation solutions were analyzed using HPLC and mass spectrometry. Surface morphology characterization was carried out using scanning electron microscopy. Upon biodegradation, the Z250 composite yielded higher amounts of BisGMA and TEGDMA related products relative to the TPH composite. However, there were higher amounts of ethoxylated bis-phenol A released from the TPH material. In terms of total mass of products released, human saliva demonstrated a greater ability to degrade Z250. In summary, HSDEA has been shown to contain esterase activities that can readily catalyze the biodegradation of current commercial composite resins.

Tumor-derived exosomes promote tumor self-seeding in hepatocellular carcinoma by transferring miRNA-25-5p to enhance cell motility.

PubMed

Liu, Hao; Chen, Wei; Zhi, Xiao; Chen, En-Jiang; Wei, Tao; Zhang, Jian; Shen, Jian; Hu, Li-Qiang; Zhao, Bin; Feng, Xin-Hua; Bai, Xue-Li; Liang, Ting-Bo

2018-05-22

Tumor self-seeding occurs when circulating malignant cells reinfiltrate the original tumor. The process may breed more aggressive tumor cells, which may contribute to cancer progression. In this study, we observed tumor self-seeding in mouse xenograft models of hepatocellular carcinoma (HCC) for the first time. We confirmed that circulating tumor cell uptake of tumor-derived exosomes, which are increasingly recognized as key instigators of cancer progression by facilitating cell-cell communication, promoted tumor self-seeding by enhancing the invasive and migration capability of recipient HCC cells. Horizontal transfer of exosomal microRNA-25-5p to anoikis-resistant HCC cells significantly enhanced their migratory and invasive abilities, whereas inhibiting microRNA-25-5p alleviated these effects. Our experiments delineate an exosome-based novel pathway employed by functional microRNA from the original tumor cells that can influence the biological fate of circulating tumor cells.

Exosomes confer pro-survival signals to alter the phenotype of prostate cells in their surrounding environment

PubMed Central

Hosseini-Beheshti, Elham; Choi, Wendy; Weiswald, Louis-Bastien; Kharmate, Geetanjali; Ghaffari, Mazyar; Roshan-Moniri, Mani; Hassona, Mohamed D.; Chan, Leslie; Chin, Mei Yieng; Tai, Isabella T.; Rennie, Paul S.; Fazli, Ladan; Guns, Emma S. Tomlinson

2016-01-01

Prostate cancer (PCa) is the most frequently diagnosed cancer in men. Current research on tumour-related extracellular vesicles (EVs) suggests that exosomes play a significant role in paracrine signaling pathways, thus potentially influencing cancer progression via multiple mechanisms. In fact, during the last decade numerous studies have revealed the role of EVs in the progression of various pathological conditions including cancer. Moreover, differences in the proteomic, lipidomic, and cholesterol content of exosomes derived from PCa cell lines versus benign prostate cell lines confirm that exosomes could be excellent biomarker candidates. As such, as part of an extensive proteomic analysis using LCMS we previously described a potential role of exosomes as biomarkers for PCa. Current evidence suggests that uptake of EV's into the local tumour microenvironment encouraging us to further examine the role of these vesicles in distinct mechanisms involved in the progression of PCa and castration resistant PCa. For the purpose of this study, we hypothesized that exosomes play a pivotal role in cell-cell communication in the local tumour microenvironment, conferring activation of numerous survival mechanisms during PCa progression and development of therapeutic resistance. Our in vitro results demonstrate that PCa derived exosomes significantly reduce apoptosis, increase cancer cell proliferation and induce cell migration in LNCaP and RWPE-1 cells. In conjunction with our in vitro findings, we have also demonstrated that exosomes increased tumor volume and serum PSA levels in vivo when xenograft bearing mice were administered DU145 cell derived exosomes intravenously. This research suggests that, regardless of androgen receptor phenotype, exosomes derived from PCa cells significantly enhance multiple mechanisms that contribute to PCa progression. PMID:26840259

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

PubMed Central

Konadu, Kateena Addae; Huang, Ming Bo; Roth, William; Armstrong, Wendy; Powell, Michael; Villinger, Francois; Bond, Vincent

2016-01-01

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated. Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge. To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions. PMID:26780239

Targeting regenerative exosomes to myocardial infarction using cardiac homing peptide

PubMed Central

Vandergriff, Adam; Huang, Ke; Shen, Deliang; Hu, Shiqi; Hensley, Michael Taylor; Caranasos, Thomas G.; Qian, Li; Cheng, Ke

2018-01-01

Rationale: Cardiac stem cell-derived exosomes have been demonstrated to promote cardiac regeneration following myocardial infarction in preclinical studies. Recent studies have used intramyocardial injection in order to concentrate exosomes in the infarct. Though effective in a research setting, this method is not clinically appealing due to its invasive nature. We propose the use of a targeting peptide, cardiac homing peptide (CHP), to target intravenously-infused exosomes to the infarcted heart. Methods: Exosomes were conjugated with CHP through a DOPE-NHS linker. Ex vivo targeting was analyzed by incubating organ sections with the CHP exosomes and analyzing with fluorescence microscopy. In vitro assays were performed on neonatal rat cardiomyocytes and H9C2 cells. For the animal study, we utilized an ischemia/reperfusion rat model. Animals were treated with either saline, scramble peptide exosomes, or CHP exosomes 24 h after surgery. Echocardiography was performed 4 h after surgery and 21 d after surgery. At 21 d, animals were sacrificed, and organs were collected for analysis. Results: By conjugating the exosomes with CHP, we demonstrate increased retention of the exosomes within heart sections ex vivo and in vitro with neonatal rat cardiomyocytes. In vitro studies showed improved viability, reduced apoptosis and increased exosome uptake when using CHP-XOs. Using an animal model of ischemia/reperfusion injury, we measured the heart function, infarct size, cellular proliferation, and angiogenesis, with improved outcomes with the CHP exosomes. Conclusions: Our results demonstrate a novel method for increasing delivery of for treatment of myocardial infarction. By targeting exosomes to the infarcted heart, there was a significant improvement in outcomes with reduced fibrosis and scar size, and increased cellular proliferation and angiogenesis. PMID:29556361

Exosomes as mediators of platinum resistance in ovarian cancer

PubMed Central

Crow, Jennifer; Atay, Safinur; Banskota, Samagya; Artale, Brittany; Schmitt, Sarah; Godwin, Andrew K

2017-01-01

Exosomes have been implicated in the cell-cell transfer of oncogenic proteins and genetic material. We speculated this may be one mechanism by which an intrinsically platinum-resistant population of epithelial ovarian cancer (EOC) cells imparts its influence on surrounding tumor cells. To explore this possibility we utilized a platinum-sensitive cell line, A2780 and exosomes derived from its resistant subclones, and an unselected, platinum-resistant EOC line, OVCAR10. A2780 cells demonstrate a ~2-fold increase in viability upon treatment with carboplatin when pre-exposed to exosomes from platinum-resistant cells as compared to controls. This coincided with increased epithelial to mesenchymal transition (EMT). DNA sequencing of EOC cell lines revealed previously unreported somatic mutations in the Mothers Against Decapentaplegic Homolog 4 (SMAD4) within platinum-resistant cells. A2780 cells engineered to exogenously express these SMAD4 mutations demonstrate up-regulation of EMT markers following carboplatin treatment, are more resistant to carboplatin, and release exosomes which impart a ~1.7-fold increase in resistance in naive A2780 recipient cells as compared to controls. These studies provide the first evidence that acquired SMAD4 mutations enhance the chemo-resistance profile of EOC and present a novel mechanism in which exchange of tumor-derived exosomes perpetuates an EMT phenotype, leading to the development of subpopulations of platinum-refractory cells. PMID:28060758

Exosomes as mediators of platinum resistance in ovarian cancer.

PubMed

Crow, Jennifer; Atay, Safinur; Banskota, Samagya; Artale, Brittany; Schmitt, Sarah; Godwin, Andrew K

2017-02-14

Exosomes have been implicated in the cell-cell transfer of oncogenic proteins and genetic material. We speculated this may be one mechanism by which an intrinsically platinum-resistant population of epithelial ovarian cancer (EOC) cells imparts its influence on surrounding tumor cells. To explore this possibility we utilized a platinum-sensitive cell line, A2780 and exosomes derived from its resistant subclones, and an unselected, platinum-resistant EOC line, OVCAR10. A2780 cells demonstrate a ~2-fold increase in viability upon treatment with carboplatin when pre-exposed to exosomes from platinum-resistant cells as compared to controls. This coincided with increased epithelial to mesenchymal transition (EMT). DNA sequencing of EOC cell lines revealed previously unreported somatic mutations in the Mothers Against Decapentaplegic Homolog 4 (SMAD4) within platinum-resistant cells. A2780 cells engineered to exogenously express these SMAD4 mutations demonstrate up-regulation of EMT markers following carboplatin treatment, are more resistant to carboplatin, and release exosomes which impart a ~1.7-fold increase in resistance in naive A2780 recipient cells as compared to controls. These studies provide the first evidence that acquired SMAD4 mutations enhance the chemo-resistance profile of EOC and present a novel mechanism in which exchange of tumor-derived exosomes perpetuates an EMT phenotype, leading to the development of subpopulations of platinum-refractory cells.

Tumor exosomes induce tunneling nanotubes in lipid raft-enriched regions of human mesothelioma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thayanithy, Venugopal; Babatunde, Victor; Dickson, Elizabeth L.

Tunneling nanotubes (TnTs) are long, non-adherent, actin-based cellular extensions that act as conduits for transport of cellular cargo between connected cells. The mechanisms of nanotube formation and the effects of the tumor microenvironment and cellular signals on TnT formation are unknown. In the present study, we explored exosomes as potential mediators of TnT formation in mesothelioma and the potential relationship of lipid rafts to TnT formation. Mesothelioma cells co-cultured with exogenous mesothelioma-derived exosomes formed more TnTs than cells cultured without exosomes within 24–48 h; and this effect was most prominent in media conditions (low-serum, hyperglycemic medium) that support TnT formationmore » (1.3–1.9-fold difference). Fluorescence and electron microscopy confirmed the purity of isolated exosomes and revealed that they localized predominantly at the base of and within TnTs, in addition to the extracellular environment. Time-lapse microscopic imaging demonstrated uptake of tumor exosomes by TnTs, which facilitated intercellular transfer of these exosomes between connected cells. Mesothelioma cells connected via TnTs were also significantly enriched for lipid rafts at nearly a 2-fold higher number compared with cells not connected by TnTs. Our findings provide supportive evidence of exosomes as potential chemotactic stimuli for TnT formation, and also lipid raft formation as a potential biomarker for TnT-forming cells. - Highlights: • Exosomes derived from malignant cells can stimulate an increased rate in the formation of tunneling nanotubes. • Tunneling nanotubes can serve as conduits for intercellular transfer of these exosomes. • Most notably, exosomes derived from benign mesothelial cells had no effect on nanotube formation. • Cells forming nanotubes were enriched in lipid rafts at a greater number compared with cells not forming nanotubes. • Our findings suggest causal and potentially synergistic association of exosomes

Labeling and tracking exosomes within the brain using gold nanoparticles

NASA Astrophysics Data System (ADS)

Betzer, Oshra; Perets, Nisim; Barnoy, Eran; Offen, Daniel; Popovtzer, Rachela

2018-02-01

Cell-to-cell communication system involves Exosomes, small, membrane-enveloped nanovesicles. Exosomes are evolving as effective therapeutic tools for different pathologies. These extracellular vesicles can bypass biological barriers such as the blood-brain barrier, and can function as powerful nanocarriers for drugs, proteins and gene therapeutics. However, to promote exosomes' therapy development, especially for brain pathologies, a better understanding of their mechanism of action, trafficking, pharmacokinetics and bio-distribution is needed. In this research, we established a new method for non-invasive in-vivo neuroimaging of mesenchymal stem cell (MSC)-derived exosomes, based on computed tomography (CT) imaging with glucose-coated gold nanoparticle (GNP) labeling. We demonstrated that the exosomes were efficiently and directly labeled with GNPs, via an energy-dependent mechanism. Additionally, we found the optimal parameters for exosome labeling and neuroimaging, wherein 5 nm GNPs enhanced labeling, and intranasal administration produced superior brain accumulation. We applied our technique in a mouse model of focal ischemia. Imaging and tracking of intranasally-administered GNP-labeled exosomes revealed specific accumulation and prolonged presence at the lesion area, up to 24 hrs. We propose that this novel exosome labeling and in-vivo neuroimaging technique can serve as a general platform for brain theranostics.

Exosome-derived microRNAs in cancer metabolism: possible implications in cancer diagnostics and therapy.

PubMed

Tomasetti, Marco; Lee, Wan; Santarelli, Lory; Neuzil, Jiri

2017-01-20

Malignant progression is greatly affected by dynamic cross-talk between stromal and cancer cells. Exosomes are secreted nanovesicles that have key roles in cell-cell communication by transferring nucleic acids and proteins to target cells and tissues. Recently, MicroRNAs (miRs) and their delivery in exosomes have been implicated in physiological and pathological processes. Tumor-delivered miRs, interacting with stromal cells in the tumor microenvironment, modulate tumor progression, angiogenesis, metastasis and immune escape. Altered cell metabolism is one of the hallmarks of cancer. A number of different types of tumor rely on mitochondrial metabolism by triggering adaptive mechanisms to optimize their oxidative phosphorylation in relation to their substrate supply and energy demands. Exogenous exosomes can induce metabolic reprogramming by restoring the respiration of cancer cells and supress tumor growth. The exosomal miRs involved in the modulation of cancer metabolism may be potentially utilized for better diagnostics and therapy.

The Origin and Functions of Exosomes in Cancer

PubMed Central

Rajagopal, Chitra; Harikumar, K. B.

2018-01-01

Exosomes are nanovesicles having a maximum size of 150 nm and is a newly emerging focus in various fields of research. Its role in cargo trafficking along with its differential expression is associated with the disrupted homeostasis and provides an opportunity to defend against different diseases like cancer. Furthermore, exosomes are rich in cargos, which contain proteins and nucleic acids that directly reflect the metabolic state of the cells from which it originates. This review summarizes recent studies on tumor-derived exosomes with an overview about biogenesis, their functions and potential of using as diagnostic and prognostic markers. We also discussed the current challenges and microfluidic-based detection approaches that might improve the detection of exosomes in different settings. More intricate studies of the molecular mechanisms in angiogenesis, pre-metastatic niche formation, and metastasis can give more promising insights and novel strategies in oncotherapeutics. PMID:29616188

[Analysis and Control of in Vivo Kinetics of Exosomes for the Development of Exosome-based DDS].

PubMed

Takahashi, Yuki; Nishikawa, Makiya; Takakura, Yoshinobu

2016-01-01

  Exosomes are secretory membrane vesicles containing lipids, proteins, and nucleic acids. They act as intercellular transporters by delivering their components to exosome recipient cells. Based on their endogenous delivery system properties, exosomes are expected to become drug delivery systems (DDS) for various molecules such as nucleic acid-based drugs. Important factors such as drug loading to exosomes, production, and pharmacokinetics of exosomes need to be considered for the development of exosome-based DDS. Of these, the pharmacokinetics of exosomes have rarely been studied, probably because of the lack of quantitative evaluation methods of in vivo exosomal pharmacokinetics. We selected lactadherin as an exosome tropic protein and developed it as a fusion protein with Gaussia luciferase to label exosomes for in vivo imaging. In addition, a fusion protein of lactadherin and streptavidin was developed, and the tissue distribution of exosomes was quantitatively evaluated by radiolabeling the exosomes using (125)I-labeled biotin. Using labeled exosomes, we found that intravenously injected exosomes were rapidly cleared from the systemic circulation by macrophages. In addition, the exosomes were mainly distributed to the liver, lung, and spleen. We also examined the effect of exosome isolation methods on their physicochemical and pharmacokinetic properties. We found that exosomes collected by the ultracentrifugation-based density-gradient method were more dispersed than exosomes collected by other methods, including the ultracentrifugation-based pelleting method. The gradient method is more time-consuming than others; therefore the development of a more efficient method for exosome isolation will advance the development of exosome-based DDS.

Differential expression of ligands for NKG2D and DNAM-1 receptors by epithelial ovarian cancer-derived exosomes and its influence on NK cell cytotoxicity.

PubMed

Labani-Motlagh, Alireza; Israelsson, Pernilla; Ottander, Ulrika; Lundin, Eva; Nagaev, Ivan; Nagaeva, Olga; Dehlin, Eva; Baranov, Vladimir; Mincheva-Nilsson, Lucia

2016-04-01

Cancers constitutively produce and secrete into the blood and other biofluids 30-150 nm-sized endosomal vehicles called exosomes. Cancer-derived exosomes exhibit powerful influence on a variety of biological mechanisms to the benefit of the tumors that produce them. We studied the immunosuppressive ability of epithelial ovarian cancer (EOC) exosomes on two cytotoxic pathways of importance for anticancer immunity-the NKG2D receptor-ligand pathway and the DNAM-1-PVR/nectin-2 pathway. Using exosomes, isolated from EOC tumor explant and EOC cell-line culture supernatants, and ascitic fluid from EOC patients, we studied the expression of NKG2D and DNAM-1 ligands on EOC exosomes and their ability to downregulate the cognate receptors. Our results show that EOC exosomes differentially and constitutively express NKG2D ligands from both MICA/B and ULBP families on their surface, while DNAM-1 ligands are more seldom expressed and not associated with the exosomal membrane surface. Consequently, the NKG2D ligand-bearing EOC exosomes significantly downregulated the NKG2D receptor expression on peripheral blood mononuclear cells (PBMC) while the DNAM-1 receptor was unaffected. The downregulation of NKG2D receptor expression was coupled to inhibition of NKG2D receptor-ligand-mediated degranulation and cytotoxicity measured in vitro with OVCAR-3 and K562 cells as targets. The EOC exosomes acted as a decoy impairing the NKG2D mediated cytotoxicity while the DNAM-1 receptor-ligand system remained unchanged. Taken together, our results support and explain the mechanism behind the recently reported finding that in EOC, NK-cell recognition and killing of tumor cells was mainly dependent on DNAM-1 signaling while the contribution of the NKG2D receptor-ligand pathway was complementary and uncertain.

Exosomes Promote Ovarian Cancer Cell Invasion through Transfer of CD44 to Peritoneal Mesothelial Cells.

PubMed

Nakamura, Koji; Sawada, Kenjiro; Kinose, Yasuto; Yoshimura, Akihiko; Toda, Aska; Nakatsuka, Erika; Hashimoto, Kae; Mabuchi, Seiji; Morishige, Ken-Ichirou; Kurachi, Hirohisa; Lengyel, Ernst; Kimura, Tadashi

2017-01-01

Epithelial ovarian cancer (EOC) cells metastasize within the peritoneal cavity and directly encounter human peritoneal mesothelial cells (HPMC) as the initial step of metastasis. The contact between ovarian cancer cells and the single layer of mesothelial cells involves direct communications that modulate cancer progression but the mechanisms are unclear. One candidate mediating cell-cell communications is exosomes, 30-100 nm membrane vesicles of endocytic origin, through the cell-cell transfer of proteins, mRNAs, or microRNAs. Therefore, the goal was to mechanistically characterize how EOC-derived exosomes modulate metastasis. Exosomes from ovarian cancer cells were fluorescently labeled and cocultured with HPMCs which internalized the exosomes. Upon exosome uptake, HPMCs underwent a change in cellular morphology to a mesenchymal, spindle phenotype. CD44, a cell surface glycoprotein, was found to be enriched in the cancer cell-derived exosomes, transferred, and internalized to HPMCs, leading to high levels of CD44 in HPMCs. This increased CD44 expression in HPMCs promoted cancer invasion by inducing the HPMCs to secrete MMP9 and by cleaning the mesothelial barrier for improved cancer cell invasion. When CD44 expression was knocked down in cancer cells, exosomes had fewer effects on HPMCs. The inhibition of exosome release from cancer cells blocked CD44 internalization in HPMCs and suppressed ovarian cancer invasion. In ovarian cancer omental metastasis, positive CD44 expression was observed in those mesothelial cells that directly interacted with cancer cells, whereas CD44 expression was negative in the mesothelial cells remote from the invading edge. This study indicates that ovarian cancer-derived exosomes transfer CD44 to HPMCs, facilitating cancer invasion. Mechanistic insight from the current study suggests that therapeutic targeting of exosomes may be beneficial in treating ovarian cancer. Mol Cancer Res; 15(1); 78-92. ©2016 AACR. ©2016 American

Plasma exosome microRNAs are indicative of breast cancer.

PubMed

Hannafon, Bethany N; Trigoso, Yvonne D; Calloway, Cameron L; Zhao, Y Daniel; Lum, David H; Welm, Alana L; Zhao, Zhizhuang J; Blick, Kenneth E; Dooley, William C; Ding, W Q

2016-09-08

microRNAs are promising candidate breast cancer biomarkers due to their cancer-specific expression profiles. However, efforts to develop circulating breast cancer biomarkers are challenged by the heterogeneity of microRNAs in the blood. To overcome this challenge, we aimed to develop a molecular profile of microRNAs specifically secreted from breast cancer cells. Our first step towards this direction relates to capturing and analyzing the contents of exosomes, which are small secretory vesicles that selectively encapsulate microRNAs indicative of their cell of origin. To our knowledge, circulating exosome microRNAs have not been well-evaluated as biomarkers for breast cancer diagnosis or monitoring. Exosomes were collected from the conditioned media of human breast cancer cell lines, mouse plasma of patient-derived orthotopic xenograft models (PDX), and human plasma samples. Exosomes were verified by electron microscopy, nanoparticle tracking analysis, and western blot. Cellular and exosome microRNAs from breast cancer cell lines were profiled by next-generation small RNA sequencing. Plasma exosome microRNA expression was analyzed by qRT-PCR analysis. Small RNA sequencing and qRT-PCR analysis showed that several microRNAs are selectively encapsulated or highly enriched in breast cancer exosomes. Importantly, the selectively enriched exosome microRNA, human miR-1246, was detected at significantly higher levels in exosomes isolated from PDX mouse plasma, indicating that tumor exosome microRNAs are released into the circulation and can serve as plasma biomarkers for breast cancer. This observation was extended to human plasma samples where miR-1246 and miR-21 were detected at significantly higher levels in the plasma exosomes of 16 patients with breast cancer as compared to the plasma exosomes of healthy control subjects. Receiver operating characteristic curve analysis indicated that the combination of plasma exosome miR-1246 and miR-21 is a better indicator of

The eukaryotic RNA exosome: same scaffold but variable catalytic subunits.

PubMed

Lykke-Andersen, Søren; Tomecki, Rafal; Jensen, Torben Heick; Dziembowski, Andrzej

2011-01-01

The RNA exosome is a versatile ribonucleolytic protein complex that participates in a multitude of cellular RNA processing and degradation events. It consists of an invariable nine-subunit core that associates with a variety of enzymatically active subunits and co-factors. These contribute to or even provide the catalytic activity and substrate specificity of the complex. The S. cerevisiae exosome has been intensively studied since its discovery in 1997 and thus serves as the archetype of eukaryotic exosomes. Notably, its catalytic potential, derived exclusively from associated subunits, differs between the nuclear and cytoplasmic versions of the complex. The same holds true for other eukaryotes, however, recent discoveries from various laboratories including our own have revealed that there are variations on this theme. Here, we review the latest findings concerning catalytic subunits of eukaryotic exosomes, and we discuss the apparent need for differential composition and subcellular distribution of exosome variants.

Secretion modification region-derived peptide blocks exosome release and mediates cell cycle arrest in breast cancer cells.

PubMed

Huang, Ming-Bo; Gonzalez, Ruben R; Lillard, James; Bond, Vincent C

2017-02-14

Discovery and development of a novel anticancer PEG-SMR-Clu peptide to prevent breast cancer metastasis. How breast cancer cells and primary mammary epithelial cells interact and communicate with each other to promote tumorigenesis and how to prevent tumor metastasis has long been a concern of researchers. Cancer cells secrete exosomes containing proteins and RNA. These factors can influence tumor development by directly targeting cancer cells and tumor stroma. In this study, we determined the effects of a peptide as an inhibitor of exosome secretion on breast tumors. We developed a peptide derived from the Secretion Modification Region (SMR) of HIV-1 Nef protein that was modified with PEG on the N-terminus and with a Clusterin (Clu)-binding peptide on the C-terminus. Attachment of PEG to the SMR peptide, termed PEGylation, offers improved water solubility and stability as well as reduced clearance through the kidneys, leading to a longer circulation time. The 12-mer Clu-binding peptide plays multiple roles in tumor development and metastasis. The Clu peptide can be detected by antibody in vivo, thus it has the potential to be used to monitor tumor status and treatment efficacy in animal studies and eventually in cancer patients. PEG-SMRwt-Clu and PEG-SMRwt peptides inhibited the growth of both of MCF-7 (estrogen responsive, ER+) and MDA-MD-231 (estrogen non-responsive, ER-) human breast cancer cells in a dose and time-dependent manner, without inducing cytotoxic effects. The SMRwt peptide, combined with paclitaxel, induced G2/M phase cell cycle arrest on MCF-7 and MDA-MB-231 cells but did not promote apoptosis. PEG-SMRwt-Clu peptide treatment blocked exosome release from both MCF-7 and MDA-MB-231 cells. This effect was blocked by knockdown of the chaperone protein mortalin by either antibody or siRNA. MCF-7 and MDA-MB-231 breast tumor cells were treated with PEG-SMR-Clu peptide alone and in combination with paclitaxel and cisplatin. Cell proliferation and viabilty

The secretion and biological function of tumor suppressor maspin as an exosome cargo protein.

PubMed

Dean, Ivory; Dzinic, Sijana H; Bernardo, M Margarida; Zou, Yi; Kimler, Vickie; Li, Xiaohua; Kaplun, Alexander; Granneman, James; Mao, Guangzhao; Sheng, Shijie

2017-01-31

Maspin is an epithelial-specific tumor suppressor shown to exert its biological effects as an intracellular, cell membrane-associated, and secreted free molecule. A recent study suggests that upon DNA-damaging g-irradiation, tumor cells can secrete maspin as an exosome-associated protein. To date, the biological significance of exosomal secretion of maspin is unknown. The current study aims at addressing whether maspin is spontaneously secreted as an exosomal protein to regulate tumor/stromal interactions. We prepared exosomes along with cell extracts and vesicle-depleted conditioned media (VDCM) from normal epithelial (CRL2221, MCF-10A and BEAS-2B) and cancer (LNCaP, PC3 and SUM149) cell lines. Atomic force microscopy and dynamic light scattering analysis revealed similar size distribution patterns and surface zeta potentials between the normal cells-derived and tumor cells-derived exosomes. Electron microscopy revealed that maspin was encapsulated by the exosomal membrane as a cargo protein. While western blotting revealed that the level of exosomal maspin from tumor cell lines was disproportionally lower relative to the levels of corresponding intracellular and VDCM maspin, as compared to that from normal cell lines, maspin knockdown in MCF-10A cells led to maspin-devoid exosomes, which exhibited significantly reduced suppressive effects on the chemotaxis activity of recipient NIH3T3 fibroblast cells. These data are the first to demonstrate the potential of maspin delivered by exosomes to block tumor-induced stromal response, and support the clinical application of exosomal maspin in cancer diagnosis and treatment.

[Biological characteristics of exosomes secreted by human bone marrow mesenchymal stem cells].

PubMed

Feng, Ying; Lu, Shi-Hong; Wang, Xin; Cui, Jun-Jie; Li, Xue; DU, Wen-Jing; Wang, Ying; Li, Juan-Juan; Song, Bao-Quan; Chen, Fang; Ma, Feng-Xia; Chi, Ying; Yang, Shao-Guang; Han, Zhong-Chao

2014-06-01

This study was aimed to explore the immunoregulatory function and capability supporting the angiogenesis of exosomes secreted by bone marrow mesenchymal stem cells (BMMSC) from healthy persons. Supernatant of BMMSC (P4-P6) was collected for exosome purification. Transmission electron microscopy (TEM) and Western blot were used to identify the quality of isolated exosomes. The amount of exosomes was quantified through bicinchoninic acid (BCA) protein assay. Human peripheral blood mononuclear cells (PBMNC) were isolated from healthy donor and added with isolating exosomes. After co-cultured for 72 h, IFN-γ from the co-culture system was detected by ELISA. The expression of miRNA-associated with immunity were detected by real-time reverse transcription polymerase chain reaction (Real-time RT-PCR). The interactions between exosomes and human umbilical vein endothelial cells (HUVEC) were observed with confocal microscopy. Subconfluent HUVEC were harvested and treated with the indicated concentration of exosomes. Nude mice were injected subcutaneously with exosomes or PBS as control to verify the ability of angiogenesis. The results showed that diameter range of exosomes was range from 40 to 160 nm. The isolated exosomes expressed the CD9. There was approximately linear relation between the secretion of exosomes and cell density. The exosomes suppressed the production of IFN-γ from PBMNC, and contained miRNA associated with immune regulation such as miR301, miR22 and miR-let-7a. Exosomes induced vascular tube formation in vitro and vascularization of Matrigel plugs in vivo. It is concluded that the BMMSC-derived exosomes can regulate immunity and support vascularization.

Exosomes derived from human umbilical cord mesenchymal stem cells protect against cisplatin-induced ovarian granulosa cell stress and apoptosis in vitro.

PubMed

Sun, Liping; Li, Dong; Song, Kun; Wei, Jianlu; Yao, Shu; Li, Zhao; Su, Xuantao; Ju, Xiuli; Chao, Lan; Deng, Xiaohui; Kong, Beihua; Li, Li

2017-05-31

Human umbilical cord mesenchymal stem cells (huMSCs) can treat primary ovarian insufficiency (POI) related to ovarian granulosa cell (OGC) apoptosis caused by cisplatin chemotherapy. Exosomes are a class of membranous vesicles with diameters of 30-200 nm that are constitutively released by eukaryotic cells. Exosomes mediate local cell-to-cell communication by transferring microRNAs and proteins. In the present study, we demonstrated the effects of exosomes derived from huMSCs (huMSC-EXOs) on a cisplatin-induced OGC model in vitro and discussed the preliminary mechanisms involved in these effects. We successfully extracted huMSC-EXOs from huMSC culture supernatant and observed the effective uptake of exosomes by cells with fluorescent staining. Using flow cytometry (with annexin-V/PI labelling), we found that huMSC-EXOs increased the number of living cells. Western blotting showed that the expression of Bcl-2 and caspase-3 were upregulated, whilst the expression of Bax, cleaved caspase-3 and cleaved PARP were downregulated to protect OGCs. These results suggest that huMSC-EXOs can be used to prevent and treat chemotherapy-induced OGC apoptosis in vitro. Therefore, this work provides insight and further evidence of stem cell function and indicates that huMSC-EXOs protect OGCs from cisplatin-induced injury in vitro.

Exosomes in cancer: small vesicular transporters for cancer progression and metastasis, biomarkers in cancer therapeutics

PubMed Central

Abhari, Alireza; Rahimzadeh, Sevda

2018-01-01

Cancer progression is a polygenic procedure in which the exosomes can function as substantial roles. Exosomes are tiny, phospholipid bilayer membrane nanovesicles of endocytic derivation with a diameter of 40–100 nm. These nanovesicles can transport bioactive molecules containing mRNAs, proteins, DNA fragments, and non-coding RNAs from a donor cell to recipient cells, and cause the alteration in genetic and epigenetic factors and reprogramming of the target cells. Many diverse cell types such as mesenchymal cells, immune cells, and cancer cells can induce the release of exosomes. Increasing evidence illustrated that the exosomes derived from tumor cells might trigger the tumor initiation, tumor cell growth and progression, metastasis, and drug resistance. The secreted nanovesicles of exosomes can play significant roles in cells communicate via shuttling the nucleic acid molecules and proteins to target cells and tissues. In this review, we discussed multiple mechanisms related to biogenesis, load, and shuttle of the exosomes. Also, we illustrated the diverse roles of exosomes in several types of human cancer development, tumor immunology, angiogenesis, and metastasis. The exosomes may act as the promising biomarkers for the prognosis of various types of cancers which suggested a new pathway for anti-tumor therapeutic of these nanovesicles and promoted exosome-based cancer for clinical diagnostic and remedial procedures. PMID:29868251

Integration of stem cell-derived exosomes with in situ hydrogel glue as a promising tissue patch for articular cartilage regeneration.

PubMed

Liu, Xiaolin; Yang, Yunlong; Li, Yan; Niu, Xin; Zhao, Bizeng; Wang, Yang; Bao, Chunyan; Xie, Zongping; Lin, Qiuning; Zhu, Linyong

2017-03-30

The regeneration of articular cartilage, which scarcely shows innate self-healing ability, is a great challenge in clinical treatment. Stem cell-derived exosomes (SC-Exos), an important type of extracellular nanovesicle, exhibit great potential for cartilage regeneration to replace stem cell-based therapy. Cartilage regeneration often takes a relatively long time and there is currently no effective administration method to durably retain exosomes at cartilage defect sites to effectively exert their reparative effect. Therefore, in this study, we exploited a photoinduced imine crosslinking hydrogel glue, which presents excellent operation ability, biocompatibility and most importantly, cartilage-integration, as an exosome scaffold to prepare an acellular tissue patch (EHG) for cartilage regeneration. It was found that EHG can retain SC-Exos and positively regulate both chondrocytes and hBMSCs in vitro. Furthermore, EHG can integrate with native cartilage matrix and promote cell deposition at cartilage defect sites, finally resulting in the promotion of cartilage defect repair. The EHG tissue patch therefore provides a novel, cell-free scaffold material for wound repair.

Quantitative Proteomic Analysis of Serum Exosomes from Patients with Locally Advanced Pancreatic Cancer Undergoing Chemoradiotherapy.

PubMed

An, Mingrui; Lohse, Ines; Tan, Zhijing; Zhu, Jianhui; Wu, Jing; Kurapati, Himabindu; Morgan, Meredith A; Lawrence, Theodore S; Cuneo, Kyle C; Lubman, David M

2017-04-07

Pancreatic cancer is the third leading cause of cancer-related death in the USA. Despite extensive research, minimal improvements in patient outcomes have been achieved. Early identification of treatment response and metastasis would be valuable to determine the appropriate therapeutic course for patients. In this work, we isolated exosomes from the serum of 10 patients with locally advanced pancreatic cancer at serial time points over a course of therapy, and quantitative analysis was performed using the iTRAQ method. We detected approximately 700-800 exosomal proteins per sample, several of which have been implicated in metastasis and treatment resistance. We compared the exosomal proteome of patients at different time points during treatment to healthy controls and identified eight proteins that show global treatment-specific changes. We then tested the effect of patient-derived exosomes on the migration of tumor cells and found that patient-derived exosomes, but not healthy controls, induce cell migration, supporting their role in metastasis. Our data show that exosomes can be reliably extracted from patient serum and analyzed for protein content. The differential loading of exosomes during a course of therapy suggests that exosomes may provide novel insights into the development of treatment resistance and metastasis.

Exosomes in Cancer Nanomedicine and Immunotherapy: Prospects and Challenges.

PubMed

Syn, Nicholas L; Wang, Lingzhi; Chow, Edward Kai-Hua; Lim, Chwee Teck; Goh, Boon-Cher

2017-07-01

Exosomes (versatile, cell-derived nanovesicles naturally endowed with exquisite target-homing specificity and the ability to surmount in vivo biological barriers) hold substantial promise for developing exciting approaches in drug delivery and cancer immunotherapy. Specifically, bioengineered exosomes are being successfully deployed to deliver potent tumoricidal drugs (siRNAs and chemotherapeutic compounds) preferentially to cancer cells, while a new generation of exosome-based therapeutic cancer vaccines has produced enticing results in early-phase clinical trials. Here, we review the state-of-the-art technologies and protocols, and discuss the prospects and challenges for the clinical development of this emerging class of therapeutics. Copyright © 2017 Elsevier Ltd. All rights reserved.

Bladder cancer exosomes contain EDIL-3/Del1 and facilitate cancer progression.

PubMed

Beckham, Carla J; Olsen, Jayme; Yin, Peng-Nien; Wu, Chia-Hao; Ting, Huei-Ju; Hagen, Fred K; Scosyrev, Emelian; Messing, Edward M; Lee, Yi-Fen

2014-08-01

High grade bladder cancer is an extremely aggressive malignancy associated with high rates of morbidity and mortality. Understanding how exosomes may affect bladder cancer progression could reveal novel therapeutic targets. Exosomes derived from human bladder cancer cell lines and the urine of patients with high grade bladder cancer were assessed for the ability to promote cancer progression in standard assays. Exosomes purified from the high grade bladder cancer cell line TCC-SUP and the nonmalignant urothelial cell line SV-HUC were submitted for mass spectrometry analysis. EDIL-3 was identified and selected for further analysis. Western blot was done to determine EDIL-3 levels in urinary exosomes from patients with high grade bladder cancer. shRNA gene knockdown and recombinant EDIL-3 were applied to study EDIL-3 function. Exosomes isolated from high grade bladder cancer cells and the urine of patients with high grade bladder cancer promoted angiogenesis and migration of bladder cancer cells and endothelial cells. We silenced EDIL-3 expression and found that shEDIL-3 exosomes did not facilitate angiogenesis, and urothelial and endothelial cell migration. Moreover, exosomes purified from the urine of patients with high grade bladder cancer contained significantly higher EDIL-3 levels than exosomes from the urine of healthy controls. EDIL-3 activated epidermal growth factor receptor signaling while blockade of epidermal growth factor receptor signaling abrogated this EDIL-3 induced bladder cell migration. Exosomes derived from the urine of patients with bladder cancer contains bioactive molecules such as EDIL-3. Identifying these components and their associated oncogenic pathways could lead to novel therapeutic targets and treatment strategies. Copyright © 2014 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Heat shock protein 70 surface-positive tumor exosomes stimulate migratory and cytolytic activity of natural killer cells.

PubMed

Gastpar, Robert; Gehrmann, Mathias; Bausero, Maria A; Asea, Alexzander; Gross, Catharina; Schroeder, Josef A; Multhoff, Gabriele

2005-06-15

Detergent-soluble membrane vesicles are actively released by human pancreas (Colo-/Colo+) and colon (CX-/CX+) carcinoma sublines, differing in their capacity to present heat shock protein 70 (Hsp70)/Bag-4 on their plasma membranes. Floating properties, acetylcholine esterase activity, and protein composition characterized them as exosomes. An enrichment of Rab-4 documented their intracellular transport route from early endosomes to the plasma membrane. After solubilization, comparable amounts of cytosolic proteins, including tubulin, Hsp70, Hsc70, and Bag-4, but not ER-residing Grp94 and calnexin, were detectable in tumor-derived exosomes. However, with respect to the exosomal surface, only Colo+/CX+ but not Colo-/CX- derived exosomes were Hsp70 membrane positive. Therefore, concomitant with an up-regulated cell surface density of activation markers, migration and Hsp70 reactivity of natural killer (NK) cells was stimulated selectively by Hsp70/Bag-4 surface-positive exosomes, but not by their negative counterparts and tumor cell lysates. Moreover, the exosome-mediated lytic activity of NK cells was blockable by Hsp70-specific antibody. As already shown for TKD stimulation, NK cells preincubated with Hsp70 surface-positive exosomes initiated apoptosis in tumors through granzyme B release. In summary, our data provide an explanation how Hsp70 reactivity in NK cells is induced by tumor-derived exosomes.

Heat Shock Protein 70 Surface-Positive Tumor Exosomes Stimulate Migratory and Cytolytic Activity of Natural Killer Cells

PubMed Central

Gastpar, Robert; Gehrmann, Mathias; Bausero, Maria A.; Asea, Alexzander; Gross, Catharina; Schroeder, Josef A.

2006-01-01

Detergent-soluble membrane vesicles are actively released by human pancreas (Colo−/Colo+) and colon (CX−/CX+) carcinoma sublines, differing in their capacity to present heat shock protein 70 (Hsp70)/Bag-4 on their plasma membranes. Floating properties, acetylcholine esterase activity, and protein composition characterized them as exosomes. An enrichment of Rab-4 documented their intracellular transport route from early endosomes to the plasma membrane. After solubilization, comparable amounts of cytosolic proteins, including tubulin, Hsp70, Hsc70, and Bag-4, but not ER-residing Grp94 and calnexin, were detectable in tumor-derived exosomes. However, with respect to the exosomal surface, only Colo+/CX+ but not Colo−/CX exosomes were Hsp70 membrane derived positive. Therefore, concomitant with an up-regulated cell surface density of activation markers, migration and Hsp70 reactivity of natural killer (NK) cells was stimulated selectively by Hsp70/Bag-4 surface-positive exosomes, but not by their negative counterparts and tumor cell lysates. Moreover, the exosome-mediated lytic activity of NK cells was blockable by Hsp70-specific antibody. As already shown for TKD stimulation, NK cells preincubated with Hsp70 surface-positive exosomes initiated apoptosis in tumors through granzyme B release. In summary, our data provide an explanation how Hsp70 reactivity in NK cells is induced by tumor-derived exosomes. PMID:15958569

Dendritic cells loaded with HeLa-derived exosomes simulate an antitumor immune response.

PubMed

Ren, Guoping; Wang, Yanhong; Yuan, Shexia; Wang, Baolian

2018-05-01

The aim of the present study was to investigate the effect of loading dendritic cells (DCs) with HeLa-derived exosomes on cytotoxic T-lymphocyte (CTL) responses, and the cytotoxic effects of CTL responses on the HeLa cell line. Ultrafiltration centrifugation combined with sucrose density gradient ultracentrifugation was applied to isolate exosomes (HeLa-exo) from the supernatant of HeLa cells. Morphological features of HeLa-exo were identified by transmission electron microscopy (TEM), and the expression of cluster of differentiation (CD)63 was detected by western blotting. Next, monocytes were isolated from peripheral blood and cultured with the removal of adherent cells to induce DC proliferation. DCs were then phenotypically characterized by flow cytometry. Finally, MTT assays were performed to analyze the effects of DCs loaded with HeLa-exo on T cell proliferation and cytotoxicity assays to evaluate the effect of CTL responses on HeLa cells. TEM revealed that HeLa-exo exhibit typical cup-shaped morphology with a diameter range of 30-100 nm. It was also identified that the CD63 surface antigen is expressed on HeLa-exo. Furthermore, monocyte-derived DCs were able to express CD1a, suggesting that DC induction was a success. DCs exhibited hair-like protrusions and other typical dendritic cell morphology. Furthermore, DCs loaded with HeLa-exo could enhance CTL proliferation and the cytotoxic activity of CTLs compared with DCs without HeLa-exo (P<0.05). In conclusion, DCs loaded with HeLa-exo may promote T cell proliferation and induce CTL responses to inhibit the growth of cervical cancer cells in vitro .

Immunomodulatory molecules are released from the first trimester and term placenta via exosomes

PubMed Central

Kshirsagar, S.K.; Alam, S.M.; Jasti, S.; Hodes, H.; Nauser, T.; Gilliam, M.; Billstrand, C.; Hunt, J.S.; Petroff, M.G.

2012-01-01

The semiallogenic fetus is tolerated by the maternal immune system through control of innate and adaptive immune responses. Trophoblast cells secrete nanometer scale membranous particles called exosomes, which have been implicated in modulation of the local and systemic maternal immune system. Here we investigate the possibility that exosomes secreted from the first trimester and term placenta carry HLA-G and B7 family immunomodulators. Confocal microscopy of placental sections revealed intracellular colocalization of B7-H1 with CD63, suggesting that B7-H1 associates with subcellular vesicles that give rise to exosomes. First trimester and term placental explants were then cultured for 24 hours. B7H-1 (CD274), B7-H3 (CD276) and HLA-G5 were abundant in pelleted supernatants of these cultures that contained microparticles and exosomes; the latter, however, was observed only in first trimester pellets and was nearly undetectable in term explant-derived pellets. Further purification of exosomes by sucrose density fractionation confirmed the association of these proteins specifically with exosomes. Finally, culture of purified trophoblast cells in the presence or absence of EGF suggested that despite the absence of HLA-G5 association with term explant-derived exosomes, it is present in exosomes secreted from mononuclear cytotrophoblast cells. Further, differentiation of cytotrophoblast cells reduced the presence of HLA-G5 in secreted exosomes. Together, the results suggest that the immunomodulatory proteins HLA-G5, B7-H1 and B7-H3, are secreted from early and term placenta, and have important implications in the mechanisms by which trophoblast immunomodulators modify the maternal immunological environment. PMID:23107341

Circulating exosomes potentiate tumor malignant properties in a mouse model of chronic sleep fragmentation

PubMed Central

Khalyfa, Abdelnaby; Almendros, Isaac; Gileles-Hillel, Alex; Akbarpour, Mahzad; Trzepizur, Wojciech; Mokhlesi, Babak; Huang, Lei; Andrade, Jorge; Farré, Ramon; Gozal, David

2016-01-01

Background Chronic sleep fragmentation (SF) increases cancer aggressiveness in mice. Exosomes exhibit pleiotropic biological functions, including immune regulatory functions, antigen presentation, intracellular communication and inter-cellular transfer of RNA and proteins. We hypothesized that SF-induced alterations in biosynthesis and cargo of plasma exosomes may affect tumor cell properties. Results SF-derived exosomes increased tumor cell proliferation (~13%), migration (~2.3-fold) and extravasation (~10%) when compared to exosomes from SC-exposed mice. Similarly, Pre exosomes from OSA patients significantly enhanced proliferation and migration of human adenocarcinoma cells compared to Post. SF-exosomal cargo revealed 3 discrete differentially expressed miRNAs, and exploration of potential mRNA targets in TC1 tumor cells uncovered 132 differentially expressed genes that encode for multiple cancer-related pathways. Methods Plasma-derived exosomes from C57/B6 mice exposed to 6 wks of SF or sleep control (SC), and from adult human patients with obstructive sleep apnea (OSA) before (Pre) and after adherent treatment for 6 wks (Post) were co-cultured with mouse lung TC1 or human adenocarcinoma tumor cell lines, respectively. Proliferation, migration, invasion, endothelial barrier integrity and extravasation assays of tumor cells were performed. Plasma mouse exosomal miRNAs were profiled with arrays, and transcriptomic assessments of TC1 cells exposed to SF or SC exosomes were conducted to identify gene targets. Conclusions Chronic SF induces alterations in exosomal miRNA cargo that alter the biological properties of TC1 lung tumor cells to enhance their proliferative, migratory and extravasation properties, and similar findings occur in OSA patients, in whom SF is a constitutive component of their sleep disorder. Thus, exosomes could participate, at least in part, in the adverse cancer outcomes observed in OSA. PMID:27419627

Nano-plasmonic exosome diagnostics

PubMed Central

Im, Hyungsoon; Shao, Huilin; Weissleder, Ralph; Castro, Cesar M.; Lee, Hakho

2015-01-01

Exosomes have emerged as a promising biomarker. These vesicles abound in biofluids and harbor molecular constituents from their parent cells, thereby offering a minimally-invasive avenue for molecular analyses. Despite such clinical potential, routine exosomal analysis, particularly the protein assay, remains challenging, due to requirements for large sample volumes and extensive processing. We have been developing miniaturized systems to facilitate clinical exosome studies. These systems can be categorized into two components: microfluidics for sample preparation and analytical tools for protein analyses. In this report, we review a new assay platform, nano-plasmonic exosome (nPLEX), in which sensing is based on surface plasmon resonance to achieve label-free exosome detection. Looking forward, we also discuss some potential challenges and improvements in exosome studies. PMID:25936957

Purified horse milk exosomes contain an unpredictable small number of major proteins.

PubMed

Sedykh, Sergey E; Purvinish, Lada V; Monogarov, Artem S; Burkova, Evgeniya E; Grigor'eva, Alina E; Bulgakov, Dmitrii V; Dmitrenok, Pavel S; Vlassov, Valentin V; Ryabchikova, Elena I; Nevinsky, Georgy A

2017-06-01

Exosomes are 40-100 nm nanovesicles containing RNA and different proteins. Exosomes containing proteins, lipids, mRNAs, and microRNAs are important in intracellular communication and immune function. Exosomes from different sources are usually obtained by combination of centrifugation and ultracentrifugation and according to published data can contain from a few dozens to thousands of different proteins. Crude exosome preparations from milk of eighteen horses were obtained for the first time using several standard centrifugations. Exosome preparations were additionally purified by FPLC gel filtration. Individual preparations demonstrated different profiles of gel filtration showing well or bad separation of exosome peaks and one or two peaks of co-isolating proteins and their complexes. According to the electron microscopy, well purified exosomes displayed a typical exosome-like size (30-100 nm) and morphology. It was shown that exosomes may have several different biological functions, but detection of their biological functions may vary significantly depending on the presence of exosome contaminating proteins and proteins directly into exosomes. Exosome proteins were identified before and after gel filtration by MALDI MS and MS/MS spectrometry of protein tryptic hydrolyzates derived by SDS PAGE and 2D electrophoresis. The results of protein identification were unexpected: one or two peaks co-isolating proteins after gel-filtration mainly contained kappa-, beta-, alpha-S1-caseins and its precursors, but these proteins were not found in well-purified exosomes. Well-purified exosomes contained from five to eight different major proteins: CD81, CD63 receptors, beta-lactoglobulin and lactadherin were common to all preparations, while actin, butyrophilin, lactoferrin, and xanthine dehydrogenase were found only in some of them. The article describes the morphology and the protein content of major horse milk exosomes for the first time. Our results on the decrease of

Mass spectrometric detection of 27-hydroxycholesterol in breast cancer exosomes.

PubMed

Roberg-Larsen, Hanne; Lund, Kaja; Seterdal, Kristina Erikstad; Solheim, Stian; Vehus, Tore; Solberg, Nina; Krauss, Stefan; Lundanes, Elsa; Wilson, Steven Ray

2017-05-01

Exosomes from cancer cells are rich sources of biomarkers and may contain elevated levels of lipids of diagnostic value. 27-Hydroxycholesterol (27-OHC) is associated with proliferation and metastasis in estrogen receptor positive (ER+) breast cancer. In this study, we investigated the levels of 27-OHC, and other sidechain-hydroxylated oxysterols in exosomes. To study both cytoplasmic and exosomal oxysterol samples of limited size, we have developed a capillary liquid chromatography-mass spectrometry platform that outperforms our previously published systems regarding chromatographic resolution, analysis time and sensitivity. In the analyzed samples, the quantified level of cytoplasmic 27-OHC using this platform fitted with mRNA levels of 27-OHC's corresponding enzyme, CYP27A1. We find clearly increased levels of 27-OHC in exosomes (i.e., enrichment) from an ER+ breast cancer cell line (MCF-7) compared to exosomes derived from an estrogen receptor (ER-) breast cancer cell line (MDA-MB-231) and other control exosomes (non-cancerous cell line (HEK293) and human pooled serum). The exosomal oxysterol profile did not reflect cytoplasmic oxysterol profiles in the cells of origin; cytoplasmic 27-OHC was low in ER+ MCF-7 cells while high in MDA-MB-231 cells. Other control cancer cells showed varied cytoplasmic oxysterol levels. Hence, exosome profiling in cancer cells might provide complementary information with the possibility of diagnostic value. Copyright © 2016 Elsevier Ltd. All rights reserved.

Simultaneous and multiplexed detection of exosome microRNAs using molecular beacons.

PubMed

Lee, Ji Hye; Kim, Jeong Ah; Jeong, Seunga; Rhee, Won Jong

2016-12-15

Simultaneous and multiplexed detection of microRNAs (miRNAs) in a whole exosome is developed, which can be utilized as a PCR-free efficient diagnosis method for various diseases. Exosomes are small extracellular vesicles that contain biomarker miRNAs from parental cells. Because they circulate throughout bodily fluids, exosomal biomarkers offer great advantages for diagnosis in many aspects. In general, PCR-based methods can be used for exosomal miRNA detection but they are laborious, expensive, and time-consuming, which make them unsuitable for high-throughput diagnosis of diseases. Previously, we reported that single miRNA in the exosomes can be detected specifically using an oligonucleotide probe or molecular beacon. Herein, we demonstrate for the first time that multiple miRNAs can be detected simultaneously in exosomes using miRNA-targeting molecular beacons. Exosomes from a breast cancer cell line, MCF-7, were used for the production of exosomes because MCF-7 has a high level of miR-21, miR-375, and miR-27a as target miRNAs. Molecular beacons successfully hybridized with multiple miRNAs in the cancer cell-derived exosomes even in the presence of high human serum concentration. In addition, it is noteworthy that the choice of fluorophores for multiplexing biomarkers in an exosome is crucial because of its small size. The proposed method described in this article is beneficial to high-throughput analysis for disease diagnosis, prognosis, and response to treatment because it is a time-, labor-, and cost-saving technique. Copyright © 2016 Elsevier B.V. All rights reserved.

Exosome-Like Vesicles Derived from Adipose Tissue Provide Biochemical Cues for Adipose Tissue Regeneration.

PubMed

Dai, Minjia; Yu, Mei; Zhang, Yan; Tian, Weidong

2017-11-01

There is an emerging need for soft tissue replacements in the field of reconstructive surgery for the treatment of congenital deformities, posttraumatic repair, and cancer rehabilitation. Previous studies have shown that the bioactive adipose tissue extract can induce adipogenesis without additional stem cells or growth factors. In this study, we innovatively investigated whether exosome-like vesicles derived from adipose tissue (ELV-AT) could direct stem cell differentiation and trigger adipose tissue regeneration. In vitro, ELV-AT can induce adipogenesis of adipose-derived stem cells and promote proliferation, migration, and angiogenic potential of the aorta endothelial cells. In vivo, ELV-AT were transplanted to a chamber on the back of nude mice and neoadipose tissues were formed. Our findings indicated that ELV-AT could be used as a cell-free therapeutic approach for adipose tissue regeneration.

Isolation of Exosome-Like Nanoparticles and Analysis of MicroRNAs Derived from Coconut Water Based on Small RNA High-Throughput Sequencing.

PubMed

Zhao, Zhehao; Yu, Siran; Li, Min; Gui, Xin; Li, Ping

2018-03-21

In this study, the presence of microRNAs in coconut water was identified by real-time polymerase chain reaction (PCR) based on the results of high-throughput small RNA sequencing. In addition, the differences in microRNA content between immature and mature coconut water were compared. A total of 47 known microRNAs belonging to 25 families and 14 new microRNAs were identified in coconut endosperm. Through analysis using a target gene prediction software, potential microRNA target genes were identified in the human genome. Real-time PCR showed that the level of most microRNAs was higher in mature coconut water than in immature coconut water. Then, exosome-like nanoparticles were isolated from coconut water. After ultracentrifugation, some particle structures were seen in coconut water samples using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate fluorescence staining. Subsequent scanning electron microscopy observation and dynamic light scattering analysis also revealed some exosome-like nanoparticles in coconut water, and the mean diameters of the particles detected by the two methods were 13.16 and 59.72 nm, respectively. In conclusion, there are extracellular microRNAs in coconut water, and their levels are higher in mature coconut water than in immature coconut water. Some exosome-like nanoparticles were isolated from coconut water, and the diameter of these particles was smaller than that of animal-derived exosomes.

Quantification of plasma exosome is a potential prognostic marker for esophageal squamous cell carcinoma.

PubMed

Matsumoto, Yasunori; Kano, Masayuki; Akutsu, Yasunori; Hanari, Naoyuki; Hoshino, Isamu; Murakami, Kentaro; Usui, Akihiro; Suito, Hiroshi; Takahashi, Masahiko; Otsuka, Ryota; Xin, Hu; Komatsu, Aki; Iida, Keiko; Matsubara, Hisahiro

2016-11-01

Exosomes play important roles in cancer progression. Although its contents (e.g., proteins and microRNAs) have been focused on in cancer research, particularly as potential diagnostic markers, the exosome behavior and methods for exosome quantification remain unclear. In the present study, we analyzed the tumor-derived exosome behavior and assessed the quantification of exosomes in patient plasma as a biomarker for esophageal squamous cell carcinoma (ESCC). A CD63-GFP expressing human ESCC cell line (TE2-CD63-GFP) was made by transfection, and mouse subcutaneous tumor models were established. Fluorescence imaging was performed on tumors and plasma exosomes harvested from mice. GFP-positive small vesicles were confirmed in the plasma obtained from TE2-CD63-GFP tumor-bearing mice. Patient plasma was collected in Chiba University Hospital (n=86). Exosomes were extracted from 100 µl of the plasma and quantified by acetylcholinesterase (AChE) activity. The relationship between exosome quantification and the patient clinical characteristics was assessed. The quantification of exosomes isolated from the patient plasma revealed that esophageal cancer patients (n=66) expressed higher exosome levels than non-malignant patients (n=20) (P=0.0002). Although there was no correlation between the tumor progression and the exosome levels, exosome number was the independent prognostic marker and low levels of exosome predicted a poor prognosis (P=0.03). In conclusion, exosome levels may be useful as an independent prognostic factor for ESCC patients.

Exosome-associated release, uptake, and neurotoxicity of HIV-1 Tat protein.

PubMed

Rahimian, Pejman; He, Johnny J

2016-12-01

HIV-1 Tat is an indispensible transactivator for HIV gene transcription and replication. It has been shown to exit cells as a free protein and enter neighboring cells or interact with surface receptors of neighboring cells to regulate gene expression and cell function. In this study, we report, for the first time, exosome-associated Tat release and uptake. Using a HIV-1 LTR-driven luciferase reporter-based cell assay and Western blotting or in combination with exosome inhibitor, OptiPrep gradient fractionation, and exosome depletion, we demonstrated significant presence of HIV-1 Tat in exosomes derived from Tat-expressing primary astrocytes, Tat-transfected U373.MG and 293T, and HIV-infected MT4. We further showed that exosome-associated Tat from Tat-expressing astrocytes was capable of causing neurite shortening and neuron death, further supporting that this new form of extracellular Tat is biologically active. Lastly, we constructed a Tat mutant deleted of its basic domain and determined the role of the basic domain in Tat trafficking into exosomes. Basic domain-deleted Tat exhibited no apparent effects on Tat trafficking into exosomes, while maintained its dominant-negative function in Tat-mediated LTR transactivation. Taken together, these results show a significant fraction of Tat is secreted and present in the form of exosomes and may contribute to the stability of extracellular Tat and broaden the spectrum of its target cells.

Exosome-like vesicles in Gloydius blomhoffii blomhoffii venom.

PubMed

Ogawa, Yuko; Kanai-Azuma, Masami; Akimoto, Yoshihiro; Kawakami, Hayato; Yanoshita, Ryohei

2008-05-01

Exosomes are small membrane vesicles (30-100 nm) with an endosome-derived limiting membrane that are secreted by a diverse range of cell types. We provide here the first evidence for the presence of exosome-like vesicles in snake venom. We isolated vesicles from fresh venom from Gloydius blomhoffii blomhoffii by gel-filtration. We found that the vesicles showed a typical exosome-like size and morphology as analyzed by electron microscopy. We observed that the vesicles contained dipeptidyl peptidase IV, aminopeptidase A, ecto-5'-nucleotidase and actin. Vesicle preparations truncated bioactive peptides such as angiotensin II, substance P, cholecystokinin-octapeptide, glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1. The role of these vesicles is still unknown, but they may affect blood pressure and glucose homeostasis following envenomation.

Exosomes: Improved methods to characterize their morphology, RNA content, and surface protein biomarkers

PubMed Central

Wu, Yueting; Deng, Wentao; Klinke, David J.

2016-01-01

As a type of secreted membrane vesicle, exosomes are an emerging mode of cell-to-cell communication. Yet as exosome samples are commonly contaminated with other extracellular vesicles, the biological roles of exosomes in regulating immunity and promoting oncogenesis remain controversial. Wondering whether existing methods could distort our view of exosome biology, we compared two direct methods for imaging extracellular vesicles and quantified the impact of different production and storage conditions on the quality of exosome samples. Scanning electron microscope (SEM) was compared to transmission electron microscope (TEM) as alternatives to examine the morphology of exosomes. Using SEM, we were able to distinguish exosomes from other contaminating extracellular vesicles based on the size distribution. More importantly, freezing of samples prior to SEM imaging made it more difficult to distinguish exosomes from extracellular vesicles secreted during cell death. In addition to morphology, the quality of RNA contained within the exosomes was characterized under different storage conditions, where freezing of samples also degraded RNA. Finally, we developed a new flow cytometry approach to assay transmembrane proteins on exosomes. While high-copy-number proteins could be readily detected, detecting low-copy-number proteins was improved using a lipophilic tracer that clustered exosomes. To illustrate this, we observed that exosomes derived from SKBR3 cells, a cell model for human HER2+ breast cancer, contained both HER1 and HER2 but at different levels of abundance. Collectively, these new methods will help to ensure a consistent framework to identify specific roles that exosomes play in regulating cell-to-cell communication. PMID:26332016

Secreted primary human malignant mesothelioma exosome signature reflects oncogenic cargo

PubMed Central

Greening, David W.; Ji, Hong; Chen, Maoshan; Robinson, Bruce W. S.; Dick, Ian M.; Creaney, Jenette; Simpson, Richard J.

2016-01-01

Malignant mesothelioma (MM) is a highly-aggressive heterogeneous malignancy, typically diagnosed at advanced stage. An important area of mesothelioma biology and progression is understanding intercellular communication and the contribution of the secretome. Exosomes are secreted extracellular vesicles shown to shuttle cellular cargo and direct intercellular communication in the tumour microenvironment, facilitate immunoregulation and metastasis. In this study, quantitative proteomics was used to investigate MM-derived exosomes from distinct human models and identify select cargo protein networks associated with angiogenesis, metastasis, and immunoregulation. Utilising bioinformatics pathway/network analyses, and correlation with previous studies on tumour exosomes, we defined a select mesothelioma exosomal signature (mEXOS, 570 proteins) enriched in tumour antigens and various cancer-specific signalling (HPGD/ENO1/OSMR) and secreted modulators (FN1/ITLN1/MAMDC2/PDGFD/GBP1). Notably, such circulating cargo offers unique insights into mesothelioma progression and tumour microenvironment reprogramming. Functionally, we demonstrate that oncogenic exosomes facilitate the migratory capacity of fibroblast/endothelial cells, supporting the systematic model of MM progression associated with vascular remodelling and angiogenesis. We provide biophysical and proteomic characterisation of exosomes, define a unique oncogenic signature (mEXOS), and demonstrate the regulatory capacity of exosomes in cell migration/tube formation assays. These findings contribute to understanding tumour-stromal crosstalk in the context of MM, and potential new diagnostic and therapeutic extracellular targets. PMID:27605433

Proteomic Analysis of Saliva in HIV-positive Heroin Addicts Reveals Proteins Correlated with Cognition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dominy, Stephen; Brown, Joseph N.; Ryder, Mark I.

The prevalence of HIV-associated neurocognitive disorders (HAND) remains high despite effective antiretroviral therapies. Multiple etiologies have been proposed over the last few years to account for this phenomenon, including the neurotoxic effects of antiretrovirals and co-morbid substance abuse. However, no underlying molecular mechanism has been identified. Emerging evidence in several fields has linked the gut to brain diseases, but the effect of the gut on the brain during HIV infection has not been explored. Saliva is the most accessible gut biofluid, and is therefore of great scientific interest for diagnostic and prognostic purposes. This study presents a longitudinal, liquid chromatography-massmore » spectrometry-based quantitative proteomics study investigating saliva samples taken from 8 HIV-positive (HIV+) and 11 -negative (HIV-) heroin addicts. In the HIV+ group, 58 proteins were identified that show significant correlations with cognitive scores and that implicate disruption of protein quality control pathways by HIV. Notably, no proteins from the HIV- heroin addict cohort showed significant correlations with cognitive scores. In addition, the majority of correlated proteins have been shown to be associated with exosomes, allowing us to propose that the salivary glands and/or oral epithelium may modulate brain function during HIV infection through the release of discrete packets of proteins in the form of exosomes.« less

Exosomes: novel implications in diagnosis and treatment of gastrointestinal cancer.

PubMed

Rahbari, Mohammad; Rahbari, Nuh; Reissfelder, Christoph; Weitz, Juergen; Kahlert, Christoph

2016-12-01

Amongst all cancer subtypes, gastrointestinal tumours are responsible for most cancer-related deaths. In most of the cases, the limitation of the prognosis of patients with malignant gastrointestinal tumours can be attributed to delayed diagnosis of the disease. In the last decade, secondary prevention strategies, in particular tumour screenings, have been identified to significantly improve the identification of patients with early-stage disease, leading to more effective therapeutic interventions. Therefore, new screening methods and further innovative treatment approaches may lead to an increase in progression-free and overall survival rates. Exosomes are small microvesicles with a size of 50-150 nm. They are formed in the endosomal system of many different cell types, where they are packed with nucleotides and proteins from the parental cell. After their release into the extracellular space, exosomes can deliver their cargo into recipient cells. By this mechanism, tumour cells can recruit and manipulate the adjacent and systemic microenvironment in order to support invasion and dissemination. Cancer-derived exosomes in the blood may provide detailed information about the tumour biology of each individual patient. Moreover, tumour-derived exosomes can be used as targetable factors and drug delivery agents in clinical practice. In this review, we summarise new aspects about novel implications in the diagnosis and treatment of gastrointestinal cancer and show how circulating exosomes have come into the spotlight of research as a high potential source of 'liquid biopsies'.

Progress in Exosome Isolation Techniques.

PubMed

Li, Pin; Kaslan, Melisa; Lee, Sze Han; Yao, Justin; Gao, Zhiqiang

2017-01-01

Exosomes are one type of membrane vesicles secreted into extracellular space by most types of cells. In addition to performing many biological functions particularly in cell-cell communication, cumulative evidence has suggested that several biological entities in exosomes like proteins and microRNAs are closely associated with the pathogenesis of most human malignancies and they may serve as invaluable biomarkers for disease diagnosis, prognosis, and therapy. This provides a commanding impetus and growing demands for simple, efficient, and affordable techniques to isolate exosomes. Capitalizing on the physicochemical and biochemical properties of exosomes, a number of techniques have been developed for the isolation of exosomes. This article summarizes the advances in exosome isolation techniques with an emphasis on their isolation mechanism, performance, challenges, and prospects. We hope that this article will provide an overview of exosome isolation techniques, opening up new perspectives towards the development more innovative strategies and devices for more time saving, cost effective, and efficient isolations of exosomes from a wide range of biological matrices.

Inhibition of apoptosis using exosomes in Chinese hamster ovary cell culture.

PubMed

Han, Seora; Rhee, Won Jong

2018-05-01

Animal cell culture technology for therapeutic protein production has shown significant improvement over the last few decades. Chinese hamster ovary (CHO) cells have been widely adapted for the production of biopharmaceutical drugs. In the biopharmaceutical industry, it is crucial to develop cell culture media and culturing conditions to achieve the highest productivity and quality. However, CHO cells are significantly affected by apoptosis in the bioreactors, resulting in a substantial decrease in product quantity and quality. Thus, to overcome the obstacle of apoptosis in CHO cell culture, it is critical to develop a novel method that does not have minimal concern of safety or cost. Herein, we showed for the first time that exosomes, which are nano-sized extracellular vesicles, derived from CHO cells inhibited apoptosis in CHO cell culture when supplemented to the culture medium. Flow cytometric and microscopic analyses revealed that substantial amounts of exosomes were delivered to CHO cells. Higher cell viability after staurosporine treatment was observed by exosome supplementation (67.3%) as compared to control (41.1%). Furthermore, exosomes prevented the mitochondrial membrane potential loss and caspase-3 activation, meaning that the exosomes enhanced cellular activities under pro-apoptotic condition. As the exosomes supplements are derived from CHO cells themselves, it is not only beneficial for the biopharmaceutical productivity of CHO cell culture to inhibit apoptosis, but also from a regulatory standpoint to diminish any safety concerns. Thus, we conclude that the method developed in this research may contribute to the biopharmaceutical industry where minimizing apoptosis in CHO cell culture is beneficial. © 2018 Wiley Periodicals, Inc.

Exosomes as divine messengers: are they the Hermes of modern molecular oncology?

PubMed Central

Braicu, C; Tomuleasa, C; Monroig, P; Cucuianu, A; Berindan-Neagoe, I; Calin, G A

2015-01-01

Exosomes are cell-derived vesicles that convey key elements with the potential to modulate intercellular communication. They are known to be secreted from all types of cells, and are crucial messengers that can regulate cellular processes by ‘trafficking' molecules from cells of one tissue to another. The exosomal content has been shown to be broad, composed of different types of cytokines, growth factors, proteins, or nucleic acids. Besides messenger RNA (mRNA) they can also contain noncoding transcripts such as microRNAs (miRNAs), which are small endogenous cellular regulators of protein expression. In diseases such as cancer, exosomes can facilitate tumor progression by altering their vesicular content and supplying the tumor niche with molecules that favor the progression of oncogenic processes such as proliferation, invasion and metastasis, or even drug resistance. The packaging of their molecular content is known to be tissue specific, a fact that makes them interesting tools in clinical diagnostics and ideal candidates for biomarkers. In the current report, we describe the main properties of exosomes and explain their involvement in processes such as cell differentiation and cell death. Furthermore, we emphasize the need of developing patient-targeted treatments by applying the conceptualization of exosomal-derived miRNA-based therapeutics. PMID:25236394

Human T-lymphotropic Virus Type 1-infected Cells Secrete Exosomes That Contain Tax Protein*

PubMed Central

Jaworski, Elizabeth; Narayanan, Aarthi; Van Duyne, Rachel; Shabbeer-Meyering, Shabana; Iordanskiy, Sergey; Saifuddin, Mohammed; Das, Ravi; Afonso, Philippe V.; Sampey, Gavin C.; Chung, Myung; Popratiloff, Anastas; Shrestha, Bindesh; Sehgal, Mohit; Jain, Pooja; Vertes, Akos; Mahieux, Renaud; Kashanchi, Fatah

2014-01-01

Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. The HTLV-1 transactivator protein Tax controls many critical cellular pathways, including host cell DNA damage response mechanisms, cell cycle progression, and apoptosis. Extracellular vesicles called exosomes play critical roles during pathogenic viral infections as delivery vehicles for host and viral components, including proteins, mRNA, and microRNA. We hypothesized that exosomes derived from HTLV-1-infected cells contain unique host and viral proteins that may contribute to HTLV-1-induced pathogenesis. We found exosomes derived from infected cells to contain Tax protein and proinflammatory mediators as well as viral mRNA transcripts, including Tax, HBZ, and Env. Furthermore, we observed that exosomes released from HTLV-1-infected Tax-expressing cells contributed to enhanced survival of exosome-recipient cells when treated with Fas antibody. This survival was cFLIP-dependent, with Tax showing induction of NF-κB in exosome-recipient cells. Finally, IL-2-dependent CTLL-2 cells that received Tax-containing exosomes were protected from apoptosis through activation of AKT. Similar experiments with primary cultures showed protection and survival of peripheral blood mononuclear cells even in the absence of phytohemagglutinin/IL-2. Surviving cells contained more phosphorylated Rb, consistent with the role of Tax in regulation of the cell cycle. Collectively, these results suggest that exosomes may play an important role in extracellular delivery of functional HTLV-1 proteins and mRNA to recipient cells. PMID:24939845

Exosome production and its regulation of EGFR during wound healing in renal tubular cells.

PubMed

Zhou, Xiangjun; Zhang, Wei; Yao, Qisheng; Zhang, Hao; Dong, Guie; Zhang, Ming; Liu, Yutao; Chen, Jian-Kang; Dong, Zheng

2017-06-01

Kidney repair following injury involves the reconstitution of a structurally and functionally intact tubular epithelium. Growth factors and their receptors, such as EGFR, are important in the repair of renal tubules. Exosomes are cell-produced small (~100 nm in diameter) vesicles that contain and transfer proteins, lipids, RNAs, and DNAs between cells. In this study, we examined the relationship between exosome production and EGFR activation and the potential role of exosome in wound healing. EGFR activation occurred shortly after scratch wounding in renal tubular cells. Wound repair after scratching was significantly promoted by EGF and suppressed by EGFR inhibitor gefitinib. Interestingly, scratch wounding induced a significant increase of exosome production. The exosome production was decreased by EGF and increased by gefitinib, suggesting a suppressive role of EGFR signaling in exosome production. Conversely, inhibition of exosome release by GW4869 and manumycin A markedly increased EGFR activation and promoted wound healing. Moreover, exosomes derived from scratch-wounding cells could inhibit wound healing. Collectively, the results indicate that wound healing in renal tubular cells is associated with EGFR activation and exosome production. Although EGFR activation promotes wound healing, released exosomes may antagonize EGFR activation and wound healing. Copyright © 2017 the American Physiological Society.

High-purity capture and release of circulating exosomes using an exosome-specific dual-patterned immunofiltration (ExoDIF) device.

PubMed

Kang, Yoon-Tae; Kim, Young Jun; Bu, Jiyoon; Cho, Young-Ho; Han, Sae-Won; Moon, Byung-In

2017-09-21

We present a microfluidic device for the capture and release of circulating exosomes from human blood. The exosome-specific dual-patterned immunofiltration (ExoDIF) device is composed of two distinct immuno-patterned layers, and is capable of enhancing the chance of binding between the antibody and exosomes by generating mechanical whirling, thus achieving high-throughput exosome isolation with high specificity. Moreover, follow-up recovery after the immuno-affinity based isolation, via cleavage of a linker, enables further downstream analysis. We verified the performance of the present device using MCF-7 secreted exosomes and found that both the concentration and proportion of exosome-sized vesicles were higher than in the samples obtained from the conventional exosome isolation kit. We then isolated exosomes from the human blood samples with our device to compare the exosome level between cancer patients and healthy donors. Cancer patients show a significantly higher exosome level with higher selectivity when validating the exosome-sized vesicles using both electron microscopy and nanoparticle tracking analysis. The captured exosomes from cancer patients also express abundant cancer-associated antigens, the epithelial cell adhesion molecule (EpCAM) on their surface. Our simple and rapid exosome recovery technique has huge potential to elucidate the function of exosomes in cancer patients and can thus be applied for various exosome-based cancer research studies.

Melanoma exosomes deliver a complex biological payload that upregulates PTPN11 to suppress T lymphocyte function

PubMed Central

Wu, Yueting; Deng, Wentao; McGinley, Emily Chambers; Klinke, David J.

2017-01-01

Summary As exosomes are emerging as a new mode of intercellular communication, we hypothesized that the payload contained within exosomes is shaped by somatic evolution. To test this, we assayed the impact on primary CD8+ T cell function, a key mechanism for anti-tumor immunity, of exosomes derived from three melanoma-related cell lines. While morphologically similar, exosomes from each cell line were functionally different, as B16F0 exosomes dose-dependently suppressed T cell proliferation. In contrast, Cloudman S91 exosomes promoted T cell proliferation and Melan-A exosomes had a negligible effect on primary CD8+ T cells. Mechanistically, transcript profiling suggested that exosomal mRNA is enriched for full-length mRNAs that target immune-related pathways. Interestingly, B16F0 exosomes were unique in that they contained both protein and mRNA for Ptpn11, which inhibited T cell proliferation. Collectively, the results suggest that upregulation of PTPN11 by B16F0 exosomes to tumor infiltrating lymphocytes would bypass the extracellular control of the immune checkpoints. PMID:27930879

Microenvironmental pH is a key factor for exosome traffic in tumor cells.

PubMed

Parolini, Isabella; Federici, Cristina; Raggi, Carla; Lugini, Luana; Palleschi, Simonetta; De Milito, Angelo; Coscia, Carolina; Iessi, Elisabetta; Logozzi, Mariantonia; Molinari, Agnese; Colone, Marisa; Tatti, Massimo; Sargiacomo, Massimo; Fais, Stefano

2009-12-04

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.

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

PubMed Central

Parolini, Isabella; Federici, Cristina; Raggi, Carla; Lugini, Luana; Palleschi, Simonetta; De Milito, Angelo; Coscia, Carolina; Iessi, Elisabetta; Logozzi, Mariantonia; Molinari, Agnese; Colone, Marisa; Tatti, Massimo; Sargiacomo, Massimo; Fais, Stefano

2009-01-01

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. PMID:19801663

Effective internalization of U251-MG-secreted exosomes into cancer cells and characterization of their lipid components.

PubMed

Toda, Yuki; Takata, Kazuyuki; Nakagawa, Yuko; Kawakami, Hikaru; Fujioka, Shusuke; Kobayashi, Kazuya; Hattori, Yasunao; Kitamura, Yoshihisa; Akaji, Kenichi; Ashihara, Eishi

2015-01-16

Exosomes, the natural vehicles of various biological molecules, have been examined in several research fields including drug delivery. Although understanding of the biological functions of exosomes has increased, how exosomes are transported between cells remains unclear. We hypothesized that cell tropism is important for effective exosomal intercellular communication and that parental cells regulate exosome movement by modulating constituent exosomal molecules. Herein, we demonstrated the strong translocation of glioblastoma-derived exosomes (U251exo) into their parental (U251) cells, breast cancer (MDA-MB-231) cells, and fibrosarcoma (HT-1080). Furthermore, disruption of proteins of U251exo by enzymatic treatment did not affect their uptake. Therefore, we focused on lipid molecules of U251exo with the expectation that they are crucial for effective incorporation of U251exo by cancer cells. Phosphatidylethanolamine was identified as a unique lipid component of U251-MG cell-derived extracellular vesicles. From these results, valuable insight is provided into the targeting of U251exo to cancer cells, which will help to develop a cancer-targeted drug delivery system. Copyright © 2014 Elsevier Inc. All rights reserved.

Combining Machine Learning and Nanofluidic Technology To Diagnose Pancreatic Cancer Using Exosomes.

PubMed

Ko, Jina; Bhagwat, Neha; Yee, Stephanie S; Ortiz, Natalia; Sahmoud, Amine; Black, Taylor; Aiello, Nicole M; McKenzie, Lydie; O'Hara, Mark; Redlinger, Colleen; Romeo, Janae; Carpenter, Erica L; Stanger, Ben Z; Issadore, David

2017-11-28

Circulating exosomes contain a wealth of proteomic and genetic information, presenting an enormous opportunity in cancer diagnostics. While microfluidic approaches have been used to successfully isolate cells from complex samples, scaling these approaches for exosome isolation has been limited by the low throughput and susceptibility to clogging of nanofluidics. Moreover, the analysis of exosomal biomarkers is confounded by substantial heterogeneity between patients and within a tumor itself. To address these challenges, we developed a multichannel nanofluidic system to analyze crude clinical samples. Using this platform, we isolated exosomes from healthy and diseased murine and clinical cohorts, profiled the RNA cargo inside of these exosomes, and applied a machine learning algorithm to generate predictive panels that could identify samples derived from heterogeneous cancer-bearing individuals. Using this approach, we classified cancer and precancer mice from healthy controls, as well as pancreatic cancer patients from healthy controls, in blinded studies.

Progress in Exosome Isolation Techniques

PubMed Central

Li, Pin; Kaslan, Melisa; Lee, Sze Han; Yao, Justin; Gao, Zhiqiang

2017-01-01

Exosomes are one type of membrane vesicles secreted into extracellular space by most types of cells. In addition to performing many biological functions particularly in cell-cell communication, cumulative evidence has suggested that several biological entities in exosomes like proteins and microRNAs are closely associated with the pathogenesis of most human malignancies and they may serve as invaluable biomarkers for disease diagnosis, prognosis, and therapy. This provides a commanding impetus and growing demands for simple, efficient, and affordable techniques to isolate exosomes. Capitalizing on the physicochemical and biochemical properties of exosomes, a number of techniques have been developed for the isolation of exosomes. This article summarizes the advances in exosome isolation techniques with an emphasis on their isolation mechanism, performance, challenges, and prospects. We hope that this article will provide an overview of exosome isolation techniques, opening up new perspectives towards the development more innovative strategies and devices for more time saving, cost effective, and efficient isolations of exosomes from a wide range of biological matrices. PMID:28255367

Detection of phosphatidylserine-positive exosomes for the diagnosis of early-stage malignancies.

PubMed

Sharma, Raghava; Huang, Xianming; Brekken, Rolf A; Schroit, Alan J

2017-08-08

There has been increasing interest in the detection of tumour exosomes in blood for cancer diagnostics. Most studies have focussed on miRNA and protein signatures that are surrogate markers for specific tumour types. Because tumour cells and tumour-derived exosomes display phosphatidylserine (PS) in their outer membrane leaflet, we developed a highly sensitive ELISA-based system that detects picogram amounts of exosomal phospholipid in plasma as a cancer biomarker. This report describes the development of a highly specific and sensitive ELISA for the capture of PS-expressing tumour exosomes in the blood of tumour-bearing mice. To monitor the relationship between tumour burden and tumour exosome plasma concentrations, plasma from one transplantable breast cancer model (MDA-MB-231) and three genetic mouse models (MMTV-PyMT; breast and KIC and KPC; pancreatic) were screened for captured exosomal phospholipid. We show that quantitative assessment of PS-expressing tumour exosomes detected very early-stage malignancies before clinical evidence of disease in all four model systems. Tumour exosome levels showed significant increases by day 7 after tumour implantation in the MDA-MB-231 model while palpable tumours appeared only after day 27. For the MMTV-PyMT and KIC models, tumour exosome levels increased significantly by day 49 (P⩽0.0002) and day 21 (P⩽0.001) while tumours developed only after days 60 and 40, respectively. For the KPC model, a significant increase in blood exosome levels was detected by day 70 (P=0.023) when only preinvasive lesions are microscopically detectable. These data indicate that blood PS exosome levels is a specific indicator of cancer and suggest that blood PS is a biomarker for early-stage malignancies.

STAT3-regulated exosomal miR-21 promotes angiogenesis and is involved in neoplastic processes of transformed human bronchial epithelial cells.

PubMed

Liu, Yi; Luo, Fei; Wang, Bairu; Li, Huiqiao; Xu, Yuan; Liu, Xinlu; Shi, Le; Lu, Xiaolin; Xu, Wenchao; Lu, Lu; Qin, Yu; Xiang, Quanyong; Liu, Qizhan

2016-01-01

Although microRNA (miRNA) enclosed in exosomes can mediate intercellular communication, the roles of exosomal miRNA and angiogenesis in lung cancer remain unclear. We investigated functions of STAT3-regulated exosomal miR-21 derived from cigarette smoke extract (CSE)-transformed human bronchial epithelial (HBE) cells in the angiogenesis of CSE-induced carcinogenesis. miR-21 levels in serum were higher in smokers than those in non-smokers. The medium from transformed HBE cells promoted miR-21 levels in normal HBE cells and angiogenesis of human umbilical vein endothelial cells (HUVEC). Transformed cells transferred miR-21 into normal HBE cells via exosomes. Knockdown of STAT3 reduced miR-21 levels in exosomes derived from transformed HBE cells, which blocked the angiogenesis. Exosomes derived from transformed HBE cells elevated levels of vascular endothelial growth factor (VEGF) in HBE cells and thereby promoted angiogenesis in HUVEC cells. Inhibition of exosomal miR-21, however, decreased VEGF levels in recipient cells, which blocked exosome-induced angiogenesis. Thus, miR-21 in exosomes leads to STAT3 activation, which increases VEGF levels in recipient cells, a process involved in angiogenesis and malignant transformation of HBE cells. These results, demonstrating the function of exosomal miR-21 from transformed HBE cells, provide a new perspective for intervention strategies to prevent carcinogenesis of lung cancer. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Exosomes isolated from cancer patients' sera transfer malignant traits and confer the same phenotype of primary tumors to oncosuppressor-mutated cells.

PubMed

Abdouh, Mohamed; Hamam, Dana; Gao, Zu-Hua; Arena, Vincenzo; Arena, Manuel; Arena, Goffredo Orazio

2017-08-30

Horizontal transfer of malignant traits from the primary tumor to distant organs, through blood circulating factors, has recently become a thoroughly studied metastatic pathway to explain cancer dissemination. Recently, we reported that oncosuppressor gene-mutated human cells undergo malignant transformation when exposed to cancer patients' sera. We also observed that oncosuppressor mutated cells would show an increased uptake of cancer-derived exosomes and we suggested that oncosuppressor genes might protect the integrity of the cell genome by blocking integration of cancer-derived exosomes. In the present study, we tested the hypothesis that cancer patients' sera-derived exosomes might be responsible for the malignant transformation of target cells and that oncosuppressor mutation would promote their increased uptake. We also sought to unveil the mechanisms behind the hypothesized phenomena. We used human BRCA1 knockout (BRCA1-KO) fibroblasts as target cells. Cells were treated in vitro with cancer patients' sera or cancer patients' sera-derived exosomes. Treated cells were injected into NOD-SCID mice. Immunohistochemical analyses were performed to determine the differentiation state of the xenotransplants. Mass spectrometry analyses of proteins from cancer exosomes and the BRCA1-KO fibroblasts' membrane were performed to investigate possible de novo expression of molecules involved in vesicles uptake. Blocking of the identified molecules in vitro was performed and in vivo experiments were conducted to confirm the role of these molecules in the malignant transformation carried out by cancer-derived exosomes. Cells treated with exosomes isolated from cancer patients' sera underwent malignant transformation and formed tumors when transplanted into immunodeficient mice. Histological analyses showed that the tumors were carcinomas that differentiated into the same lineage of the primary tumors of blood donors. Oncosuppressor mutation promoted the de novo expression

Tumor-derived exosomes in ovarian cancer – liquid biopsies for early detection and real-time monitoring of cancer progression

PubMed Central

Sharma, Shayna; Zuñiga, Felipe; Rice, Gregory E.; Perrin, Lewis C.; Hooper, John D.; Salomon, Carlos

2017-01-01

Ovarian cancer usually has a poor prognosis because it predominantly presents as high stage disease. New approaches are required to develop more effective early detection strategies and real-time treatment response monitoring. Nano-sized extracellular vesicles (EVs, including exosomes) may provide an approach to enrich tumor biomarker detection and address this clinical need. Exosomes are membranous extracellular vesicles of approximately 100 nm in diameter that have potential to be used as biomarkers and therapeutic delivery tools for ovarian cancer. Exosomal content (proteins and miRNA) is often parent cell specific thus providing an insight or “fingerprint” of the intracellular environment. Furthermore, exosomes can aid cell-cell communication and have the ability to modify target cells by transferring their content. Additionally, via the capacity to evade the immune system and remain stable over long periods in circulation, exosomes have potential as natural drug agents. This review examines the potential role of exosomes in diagnosis, drug delivery and real-time monitoring in ovarian cancer. PMID:29262670

Label-free Proteomic Analysis of Exosomes Derived from Inducible Hepatitis B Virus-Replicating HepAD38 Cell Line*

PubMed Central

Jia, Xiaofang; Chen, Jieliang; Megger, Dominik A.; Zhang, Xiaonan; Kozlowski, Maya; Zhang, Lijun; Fang, Zhong; Li, Jin; Chu, Qiaofang; Wu, Min; Li, Yaming; Sitek, Barbara; Yuan, Zhenghong

2017-01-01

Hepatitis B virus (HBV) infection is a major health problem worldwide. Recent evidence suggests that some viruses can manipulate the infection process by packing specific viral and cellular components into exosomes, small nanometer-sized (30–150 nm) vesicles secreted from various cells. However, the impact of HBV replication on the content of exosomes produced by hepatocytes has not been fully delineated. In this work, an HBV-inducible cell line HepAD38 was used to directly compare changes in the protein content of exosomes secreted from HepAD38 cells with or without HBV replication. Exosomes were isolated from supernantants of HepAD38 cells cultured with or without doxycycline (dox) and their purity was confirmed by transmission electron microscopy (TEM) and Western immunoblotting assays. Ion-intensity based label-free LC-MS/MS quantitation technologies were applied to analyze protein content of exosomes from HBV replicating cells [referred as HepAD38 (dox−)-exo] and from HBV nonreplicating cells [referred as HepAD38 (dox+)-exo]. A total of 1412 exosomal protein groups were identified, among which the abundance of 35 proteins was significantly changed following HBV replication. Strikingly, 5 subunit proteins from the 26S proteasome complex, including PSMC1, PSMC2, PSMD1, PSMD7 and PSMD14 were consistently enhanced in HepAD38 (dox−)-exo. Bioinformatic analysis of differential exosomal proteins confirmed the significant enrichment of components involved in the proteasomal catabolic process. Proteasome activity assays further suggested that HepAD38 (dox−)-exo had enhanced proteolytic activity compared with HepAD38 (dox+)-exo. Furthermore, human peripheral monocytes incubated with HepAD38 (dox−)-exo induced a significantly lower level of IL-6 secretion compared with IL-6 levels from HepAD38 (dox+)-exo. Irreversible inhibition of proteasomal activity within exosomes restored higher production of IL-6 by monocytes, suggesting that transmission of proteasome

Label-free Proteomic Analysis of Exosomes Derived from Inducible Hepatitis B Virus-Replicating HepAD38 Cell Line.

PubMed

Jia, Xiaofang; Chen, Jieliang; Megger, Dominik A; Zhang, Xiaonan; Kozlowski, Maya; Zhang, Lijun; Fang, Zhong; Li, Jin; Chu, Qiaofang; Wu, Min; Li, Yaming; Sitek, Barbara; Yuan, Zhenghong

2017-04-01

Hepatitis B virus (HBV) infection is a major health problem worldwide. Recent evidence suggests that some viruses can manipulate the infection process by packing specific viral and cellular components into exosomes, small nanometer-sized (30-150 nm) vesicles secreted from various cells. However, the impact of HBV replication on the content of exosomes produced by hepatocytes has not been fully delineated. In this work, an HBV-inducible cell line HepAD38 was used to directly compare changes in the protein content of exosomes secreted from HepAD38 cells with or without HBV replication. Exosomes were isolated from supernantants of HepAD38 cells cultured with or without doxycycline (dox) and their purity was confirmed by transmission electron microscopy (TEM) and Western immunoblotting assays. Ion-intensity based label-free LC-MS/MS quantitation technologies were applied to analyze protein content of exosomes from HBV replicating cells [referred as HepAD38 (dox - )-exo] and from HBV nonreplicating cells [referred as HepAD38 (dox + )-exo]. A total of 1412 exosomal protein groups were identified, among which the abundance of 35 proteins was significantly changed following HBV replication. Strikingly, 5 subunit proteins from the 26S proteasome complex, including PSMC1, PSMC2, PSMD1, PSMD7 and PSMD14 were consistently enhanced in HepAD38 (dox - )-exo. Bioinformatic analysis of differential exosomal proteins confirmed the significant enrichment of components involved in the proteasomal catabolic process. Proteasome activity assays further suggested that HepAD38 (dox - )-exo had enhanced proteolytic activity compared with HepAD38 (dox + )-exo. Furthermore, human peripheral monocytes incubated with HepAD38 (dox - )-exo induced a significantly lower level of IL-6 secretion compared with IL-6 levels from HepAD38 (dox + )-exo. Irreversible inhibition of proteasomal activity within exosomes restored higher production of IL-6 by monocytes, suggesting that transmission of

Morphologic and proteomic characterization of exosomes released by cultured extravillous trophoblast cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Atay, Safinur; Gercel-Taylor, Cicek; Kesimer, Mehmet

Exosomes represent an important intercellular communication vehicle, mediating events essential for the decidual microenvironment. While we have demonstrated exosome induction of pro-inflammatory cytokines, to date, no extensive characterization of trophoblast-derived exosomes has been provided. Our objective was to provide a morphologic and proteomic characterization of these exosomes. Exosomes were isolated from the conditioned media of Swan71 human trophoblast cells by ultrafiltration and ultracentrifugation. These were analyzed for density (sucrose density gradient centrifugation), morphology (electron microscopy), size (dynamic light scattering) and protein composition (Ion Trap mass spectrometry and western immunoblotting). Based on density gradient centrifugation, microvesicles from Sw71 cells exhibit amore » density between 1.134 and 1.173 g/ml. Electron microscopy demonstrated that microvesicles from Sw71 cells exhibit the characteristic cup-shaped morphology of exosomes. Dynamic light scattering showed a bell-shaped curve, indicating a homogeneous population with a mean size of 165 nm {+-} 0.5 nm. Ion Trap mass spectrometry demonstrated the presence of exosome marker proteins (including CD81, Alix, cytoskeleton related proteins, and Rab family). The MS results were confirmed by western immunoblotting. Based on morphology, density, size and protein composition, we defined the release of exosomes from extravillous trophoblast cells and provide their first extensive characterization. This characterization is essential in furthering our understanding of 'normal' early pregnancy.« less

Human T-lymphotropic virus type 1-infected cells secrete exosomes that contain Tax protein.

PubMed

Jaworski, Elizabeth; Narayanan, Aarthi; Van Duyne, Rachel; Shabbeer-Meyering, Shabana; Iordanskiy, Sergey; Saifuddin, Mohammed; Das, Ravi; Afonso, Philippe V; Sampey, Gavin C; Chung, Myung; Popratiloff, Anastas; Shrestha, Bindesh; Sehgal, Mohit; Jain, Pooja; Vertes, Akos; Mahieux, Renaud; Kashanchi, Fatah

2014-08-08

Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. The HTLV-1 transactivator protein Tax controls many critical cellular pathways, including host cell DNA damage response mechanisms, cell cycle progression, and apoptosis. Extracellular vesicles called exosomes play critical roles during pathogenic viral infections as delivery vehicles for host and viral components, including proteins, mRNA, and microRNA. We hypothesized that exosomes derived from HTLV-1-infected cells contain unique host and viral proteins that may contribute to HTLV-1-induced pathogenesis. We found exosomes derived from infected cells to contain Tax protein and proinflammatory mediators as well as viral mRNA transcripts, including Tax, HBZ, and Env. Furthermore, we observed that exosomes released from HTLV-1-infected Tax-expressing cells contributed to enhanced survival of exosome-recipient cells when treated with Fas antibody. This survival was cFLIP-dependent, with Tax showing induction of NF-κB in exosome-recipient cells. Finally, IL-2-dependent CTLL-2 cells that received Tax-containing exosomes were protected from apoptosis through activation of AKT. Similar experiments with primary cultures showed protection and survival of peripheral blood mononuclear cells even in the absence of phytohemagglutinin/IL-2. Surviving cells contained more phosphorylated Rb, consistent with the role of Tax in regulation of the cell cycle. Collectively, these results suggest that exosomes may play an important role in extracellular delivery of functional HTLV-1 proteins and mRNA to recipient cells. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Suppression of Lymphocyte Functions by Plasma Exosomes Correlates with Disease Activity in Patients with Head and Neck Cancer.

PubMed

Ludwig, Sonja; Floros, Theofanis; Theodoraki, Marie-Nicole; Hong, Chang-Sook; Jackson, Edwin K; Lang, Stephan; Whiteside, Theresa L

2017-08-15

Purpose: Head and neck cancers (HNCs) often induce profound immunosuppression, which contributes to disease progression and interferes with immune-based therapies. Body fluids of patients with HNC are enriched in exosomes potentially engaged in negative regulation of antitumor immune responses. The presence and content of exosomes derived from plasma of patients with HNC are evaluated for the ability to induce immune dysfunction and influence disease activity. Experimental Design: Exosomes were isolated by size-exclusion chromatography from plasma of 38 patients with HNC and 14 healthy donors. Morphology, size, numbers, and protein and molecular contents of the recovered exosomes were determined. Coculture assays were performed to measure exosome-mediated effects on functions of normal human lymphocyte subsets and natural killer (NK) cells. The results were correlated with disease stage and activity. Results: The presence, quantity, and molecular content of isolated, plasma-derived exosomes discriminated patients with HNC with active disease (AD) from those with no evident disease (NED) after oncologic therapies. Exosomes of patients with AD were significantly more effective than exosomes of patients with NED in inducing apoptosis of CD8 + T cells, suppression of CD4 + T-cell proliferation, and upregulation of regulatory T-cell (Treg) suppressor functions (all at P < 0.05). Exosomes of patients with AD also downregulated NKG2D expression levels in NK cells. Conclusions: Exosomes in plasma of patients with HNC carry immunosuppressive molecules and interfere with functions of immune cells. Exosome-induced immune suppression correlates with disease activity in HNC, suggesting that plasma exosomes could be useful as biomarkers of HNC progression. Clin Cancer Res; 23(16); 4843-54. ©2017 AACR . ©2017 American Association for Cancer Research.

Tetraspanin-3 regulates protective immunity against Eimera tenella infection following immunization with dendritic cell-derived exosomes

USDA-ARS?s Scientific Manuscript database

The effects of immunization with dendritic cell (DC) exosomes, which had been incubated or non-incubated with an anti-tetraspanin-3 (Tspan-3) blocking antibody (Ab), were studied using an experimental model of Eimeria tenella avian coccidiosis. Purified exosomes from cecal tonsil and splenic DCs exp...

Exosomes/tricalcium phosphate combination scaffolds can enhance bone regeneration by activating the PI3K/Akt signaling pathway.

PubMed

Zhang, Jieyuan; Liu, Xiaolin; Li, Haiyan; Chen, Chunyuan; Hu, Bin; Niu, Xin; Li, Qing; Zhao, Bizeng; Xie, Zongping; Wang, Yang

2016-09-20

Recently, accumulating evidence has shown that exosomes, the naturally secreted nanocarriers of cells, can exert therapeutic effects in various disease models in the absence of parent cells. However, application of exosomes in bone defect repair and regeneration has been rarely reported, and little is known regarding their underlying mechanisms. Exosomes derived from human-induced pluripotent stem cell-derived mesenchymal stem cells (hiPS-MSC-Exos) were combined with tricalcium phosphate (β-TCP) to repair critical-sized calvarial bone defects, and the efficacy was assessed by histological examination. We evaluated the in vitro effects of hiPSC-MSC-Exos on the proliferation, migration, and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) by cell-counting, scratch assays, and qRT-PCR, respectively. Gene expression profiling and bioinformatics analyses were also used to identify the underlying mechanisms in the repair. We found that the exosome/β-TCP combination scaffolds could enhance osteogenesis as compared to pure β-TCP scaffolds. In vitro assays showed that the exosomes could release from β-TCP and could be internalized by hBMSCs. In addition, the internalization of exosomes into hBMSCs could profoundly enhance the proliferation, migration, and osteogenic differentiation of hBMSCs. Furthermore, gene expression profiling and bioinformatics analyses demonstrated that exosome/β-TCP combination scaffolds significantly altered the expression of a network of genes involved in the PI3K/Akt signaling pathway. Functional studies further confirmed that the PI3K/Akt signaling pathway was the critical mediator during the exosome-induced osteogenic responses of hBMSCs. We propose that the exosomes can enhance the osteoinductivity of β-TCP through activating the PI3K/Akt signaling pathway of hBMSCs, which means that the exosome/β-TCP combination scaffolds possess better osteogenesis activity than pure β-TCP scaffolds. These

Molecular lipid species in urinary exosomes as potential prostate cancer biomarkers.

PubMed

Skotland, Tore; Ekroos, Kim; Kauhanen, Dimple; Simolin, Helena; Seierstad, Therese; Berge, Viktor; Sandvig, Kirsten; Llorente, Alicia

2017-01-01

Exosomes have recently appeared as a novel source of noninvasive cancer biomarkers, since these nanovesicles contain molecules from cancer cells and can be detected in biofluids. We have here investigated the potential use of lipids in urinary exosomes as prostate cancer biomarkers. A high-throughput mass spectrometry quantitative lipidomic analysis was performed to reveal the lipid composition of urinary exosomes in prostate cancer patients and healthy controls. Control samples were first analysed to characterise the lipidome of urinary exosomes and test the reproducibility of the method. In total, 107 lipid species were quantified in urinary exosomes. Several differences, for example, in cholesterol and phosphatidylcholine, were found between urinary exosomes and exosomes derived from cell lines, thus showing the importance of in vivo studies for biomarker analysis. The 36 most abundant lipid species in urinary exosomes were then quantified in 15 prostate cancer patients and 13 healthy controls. Interestingly, the levels of nine lipids species were found to be significantly different when the two groups were compared. The highest significance was shown for phosphatidylserine (PS) 18:1/18:1 and lactosylceramide (d18:1/16:0), the latter also showed the highest patient-to-control ratio. Furthermore, combinations of these lipid species and PS 18:0-18:2 distinguished the two groups with 93% sensitivity and 100% specificity. Finally, in agreement with the reported dysregulation of sphingolipid metabolism in cancer cells, alteration in specific sphingolipid lipid classes were observed. This study shows for the first time the potential use of exosomal lipid species in urine as prostate cancer biomarkers. Copyright © 2016 Elsevier Ltd. All rights reserved.

Melanoma exosomes deliver a complex biological payload that upregulates PTPN11 to suppress T lymphocyte function.

PubMed

Wu, Yueting; Deng, Wentao; McGinley, Emily Chambers; Klinke, David J

2017-03-01

As exosomes are emerging as a new mode of intercellular communication, we hypothesized that the payload contained within exosomes is shaped by somatic evolution. To test this, we assayed the impact on primary CD8+ T-cell function, a key mechanism for antitumor immunity, of exosomes derived from three melanoma-related cell lines. While morphologically similar, exosomes from each cell line were functionally different, as B16F0 exosomes dose-dependently suppressed T-cell proliferation. In contrast, Cloudman S91 exosomes promoted T-cell proliferation and Melan-A exosomes had a negligible effect on primary CD8+ T cells. Mechanistically, transcript profiling suggested that exosomal mRNA is enriched for full-length mRNAs that target immune-related pathways. Interestingly, B16F0 exosomes were unique in that they contained both protein and mRNA for PTPN11, which inhibited T-cell proliferation. Collectively, the results suggest that upregulation of PTPN11 by B16F0 exosomes to tumor infiltrating lymphocytes would bypass the extracellular control of the immune checkpoints. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Exosomes serve as novel modes of tick-borne flavivirus transmission from arthropod to human cells and facilitates dissemination of viral RNA and proteins to the vertebrate neuronal cells

PubMed Central

Neupane, Biswas; Bai, Fengwei; Sherman, Michael B.; Choi, Kyung H.; Neelakanta, Girish

2018-01-01

Molecular determinants and mechanisms of arthropod-borne flavivirus transmission to the vertebrate host are poorly understood. In this study, we show for the first time that a cell line from medically important arthropods, such as ticks, secretes extracellular vesicles (EVs) including exosomes that mediate transmission of flavivirus RNA and proteins to the human cells. Our study shows that tick-borne Langat virus (LGTV), a model pathogen closely related to tick-borne encephalitis virus (TBEV), profusely uses arthropod exosomes for transmission of viral RNA and proteins to the human- skin keratinocytes and blood endothelial cells. Cryo-electron microscopy showed the presence of purified arthropod/neuronal exosomes with the size range of 30 to 200 nm in diameter. Both positive and negative strands of LGTV RNA and viral envelope-protein were detected inside exosomes derived from arthropod, murine and human cells. Detection of Nonstructural 1 (NS1) protein in arthropod and neuronal exosomes further suggested that exosomes contain viral proteins. Viral RNA and proteins in exosomes derived from tick and mammalian cells were secured, highly infectious and replicative in all tested evaluations. Treatment with GW4869, a selective inhibitor that blocks exosome release affected LGTV loads in both arthropod and mammalian cell-derived exosomes. Transwell-migration assays showed that exosomes derived from infected-brain-microvascular endothelial cells (that constitute the blood-brain barrier) facilitated LGTV RNA and protein transmission, crossing of the barriers and infection of neuronal cells. Neuronal infection showed abundant loads of both tick-borne LGTV and mosquito-borne West Nile virus RNA in exosomes. Our data also suggest that exosome-mediated LGTV viral transmission is clathrin-dependent. Collectively, our results suggest that flaviviruses uses arthropod-derived exosomes as a novel means for viral RNA and protein transmission from the vector, and the vertebrate

Adipose-derived mesenchymal stem cell-derived exosomes alleviate overwhelming systemic inflammatory reaction and organ damage and improve outcome in rat sepsis syndrome

PubMed Central

Chang, Chia-Lo; Sung, Pei-Hsun; Chen, Kuan-Hung; Shao, Pei-Lin; Yang, Chih-Chao; Cheng, Ben-Chung; Lin, Kun-Chen; Chen, Chih-Hung; Chai, Han-Tan; Chang, Hsueh-Wen; Yip, Hon-Kan; Chen, Hong-Hwa

2018-01-01

This study tested the hypothesis that healthy adipose-derived mesenchymal stem cell (ADMSC)-derived exosomes (HMSCEXO) and apoptotic (A) (induced by 12 h hypoxia/12 h starvation)-ADMSC-derived exosomes (AMSCEXO) were comparably effective at alleviating sepsis syndrome [SS; induced by cecal-ligation and puncture (CLP)]-induced systemic inflammation and reduced organ damage and unfavorable outcomes in rats. SD rats were divided into sham control (SC), SS only, SS + HMSCEXO (100 µg intravenous administration 3 h after CLP), and AMSCEXO. By day 5 after CLP procedure, the mortality rate was significantly higher in SS than in SC and HMSCEXO (all P < 0.01), but it showed no significant different between SC and HMSCEXO, between AMSCEXO and HMSCEXO or between SS and AMSCEXO (P > 0.05). The levels of inflammatory mediators in circulation (CD11b/c/Ly6G/MIF), bronchioalveolar lavage (CD11b/c/Ly6G) and abdominal ascites (CD11b/c/CD14/Ly6G/MIF) were highest in SS, lowest in SC and significantly higher in AMSCEXO than in HMSCEXO (all P < 0.001). The circulating/splenic levels of immune cells (CD34+/CD4+/CD3+/CD8+) were expressed in an identical pattern whereas the T-reg+ cells exhibited an opposite pattern of inflammation among the groups (all P < 0.001). The protein expressions of inflammation (MMP-9/MIF/TNF-α/NF-κB/IL-1β) and oxidative stress (NOX-1/NOX-2/oxidized protein), and cellular expressions (CD14+/CD68+) in lung/kidney parenchyma exhibited an identical pattern of inflammatory mediators (all P < 0.001). The kidney/lung injury scores displayed an identical pattern of inflammatory mediators among the groups (all P < 0.001). In conclusion, HMSCEXO might be superior to AMSCEXO for improving survival and suppressing the inflammatory reactions in rats after SS. PMID:29736200

Engineering Exosomes for Cancer Therapy.

PubMed

Gilligan, Katie E; Dwyer, Róisín M

2017-05-24

There remains an urgent need for novel therapeutic strategies to treat metastatic cancer, which results in over 8 million deaths annually worldwide. Following secretion, exosomes are naturally taken up by cells, and capable of the stable transfer of drugs, therapeutic microRNAs and proteins. As knowledge of the biogenesis, release and uptake of exosomes continues to evolve, and thus also has interest in these extracellular vesicles as potential tumor-targeted vehicles for cancer therapy. The ability to engineer exosome content and migratory itinerary holds tremendous promise. Studies to date have employed viral and non-viral methods to engineer the parent cells to secrete modified exosomes, or alternatively, to directly manipulate exosome content following secretion. The majority of studies have demonstrated promising results, with decreased tumor cell invasion, migration and proliferation, along with enhanced immune response, cell death, and sensitivity to chemotherapy observed. The studies outlined in this review highlight the exciting potential for exosomes as therapeutic vehicles for cancer treatment. Successful implementation in the clinical setting will be dependent upon establishment of rigorous standards for exosome manipulation, isolation, and characterisation.

Exosomes from embryonic mesenchymal stem cells alleviate osteoarthritis through balancing synthesis and degradation of cartilage extracellular matrix.

PubMed

Wang, Yafei; Yu, Dongsheng; Liu, Zhiming; Zhou, Fang; Dai, Jun; Wu, Bingbing; Zhou, Jing; Heng, Boon Chin; Zou, Xiao Hui; Ouyang, Hongwei; Liu, Hua

2017-08-14

Mesenchymal stem cell therapy for osteoarthritis (OA) has been widely investigated, but the mechanisms are still unclear. Exosomes that serve as carriers of genetic information have been implicated in many diseases and are known to participate in many physiological processes. Here, we investigate the therapeutic potential of exosomes from human embryonic stem cell-induced mesenchymal stem cells (ESC-MSCs) in alleviating osteoarthritis (OA). Exosomes were harvested from conditioned culture media of ESC-MSCs by a sequential centrifugation process. Primary mouse chondrocytes treated with interleukin 1 beta (IL-1β) were used as an in vitro model to evaluate the effects of the conditioned medium with or without exosomes and titrated doses of isolated exosomes for 48 hours, prior to immunocytochemistry or western blot analysis. Destabilization of the medial meniscus (DMM) surgery was performed on the knee joints of C57BL/6 J mice as an OA model. This was followed by intra-articular injection of either ESC-MSCs or their exosomes. Cartilage destruction and matrix degradation were evaluated with histological staining and OARSI scores at the post-surgery 8 weeks. We found that intra-articular injection of ESC-MSCs alleviated cartilage destruction and matrix degradation in the DMM model. Further in vitro studies illustrated that this effect was exerted through ESC-MSC-derived exosomes. These exosomes maintained the chondrocyte phenotype by increasing collagen type II synthesis and decreasing ADAMTS5 expression in the presence of IL-1β. Immunocytochemistry revealed colocalization of the exosomes and collagen type II-positive chondrocytes. Subsequent intra-articular injection of exosomes derived from ESC-MSCs successfully impeded cartilage destruction in the DMM model. The exosomes from ESC-MSCs exert a beneficial therapeutic effect on OA by balancing the synthesis and degradation of chondrocyte extracellular matrix (ECM), which in turn provides a new target for OA drug

The emerging roles and therapeutic potential of exosomes in epithelial ovarian cancer.

PubMed

Li, Xiaoduan; Wang, Xipeng

2017-05-15

Ovarian cancer (OC) is one of the three types of malignant tumors in the female reproductive system, and epithelial ovarian cancer (EOC) is its most typical form. Due to the asymptomatic nature of the early stages and resistance to chemotherapy, EOC has both a poor prognosis and a high fatality rate. Current treatments for OC are very limited, and the 5-years survival rate is approximately 30%. Exosomes, which are microvesicles ranging from approximately 30-100 nm in size that are secreted by living cells, can be produced from different cell types and detected in various body fluids. Cancer cells can secrete more exosomes than healthy cells, and more importantly, the content of cancer cell-derived exosomes is distinct. The exosomes shedding from tumor cells are considered to be involved in tumor progression and metastasis. As such, exosomes are expected to be potential tools for tumor diagnosis and treatment. In this review, we briefly present the emerging roles of exosomes in OC and summarize related articles about their roles as diagnostic or prognostic biomarkers and in the treatment and drug resistance of OC.

Extracellular Vesicle (EV) Array: microarray capturing of exosomes and other extracellular vesicles for multiplexed phenotyping.

PubMed

Jørgensen, Malene; Bæk, Rikke; Pedersen, Shona; Søndergaard, Evo K L; Kristensen, Søren R; Varming, Kim

2013-01-01

Exosomes are one of the several types of cell-derived vesicles with a diameter of 30-100 nm. These extracellular vesicles are recognized as potential markers of human diseases such as cancer. However, their use in diagnostic tests requires an objective and high-throughput method to define their phenotype and determine their concentration in biological fluids. To identify circulating as well as cell culture-derived vesicles, the current standard is immunoblotting or a flow cytometrical analysis for specific proteins, both of which requires large amounts of purified vesicles. Based on the technology of protein microarray, we hereby present a highly sensitive Extracellular Vesicle (EV) Array capable of detecting and phenotyping exosomes and other extracellular vesicles from unpurified starting material in a high-throughput manner. To only detect the exosomes captured on the EV Array, a cocktail of antibodies against the tetraspanins CD9, CD63 and CD81 was used. These antibodies were selected to ensure that all exosomes captured are detected, and concomitantly excluding the detection of other types of microvesicles. The limit of detection (LOD) was determined on exosomes derived from the colon cancer cell line LS180. It clarified that supernatant from only approximately 10(4) cells was needed to obtain signals or that only 2.5×10(4) exosomes were required for each microarray spot (~1 nL). Phenotyping was performed on plasma (1-10 µL) from 7 healthy donors, which were applied to the EV Array with a panel of antibodies against 21 different cellular surface antigens and cancer antigens. For each donor, there was considerable heterogeneity in the expression levels of individual markers. The protein profiles of the exosomes (defined as positive for CD9, CD63 and CD81) revealed that only the expression level of CD9 and CD81 was approximately equal in the 7 donors. This implies questioning the use of CD63 as a standard exosomal marker since the expression level of this

Proteomics Analysis of Bladder Cancer Exosomes*

PubMed Central

Welton, Joanne L.; Khanna, Sanjay; Giles, Peter J.; Brennan, Paul; Brewis, Ian A.; Staffurth, John; Mason, Malcolm D.; Clayton, Aled

2010-01-01

Exosomes are nanometer-sized vesicles, secreted by various cell types, present in biological fluids that are particularly rich in membrane proteins. Ex vivo analysis of exosomes may provide biomarker discovery platforms and form non-invasive tools for disease diagnosis and monitoring. These vesicles have never before been studied in the context of bladder cancer, a major malignancy of the urological tract. We present the first proteomics analysis of bladder cancer cell exosomes. Using ultracentrifugation on a sucrose cushion, exosomes were highly purified from cultured HT1376 bladder cancer cells and verified as low in contaminants by Western blotting and flow cytometry of exosome-coated beads. Solubilization in a buffer containing SDS and DTT was essential for achieving proteomics analysis using an LC-MALDI-TOF/TOF MS approach. We report 353 high quality identifications with 72 proteins not previously identified by other human exosome proteomics studies. Overrepresentation analysis to compare this data set with previous exosome proteomics studies (using the ExoCarta database) revealed that the proteome was consistent with that of various exosomes with particular overlap with exosomes of carcinoma origin. Interrogating the Gene Ontology database highlighted a strong association of this proteome with carcinoma of bladder and other sites. The data also highlighted how homology among human leukocyte antigen haplotypes may confound MASCOT designation of major histocompatability complex Class I nomenclature, requiring data from PCR-based human leukocyte antigen haplotyping to clarify anomalous identifications. Validation of 18 MS protein identifications (including basigin, galectin-3, trophoblast glycoprotein (5T4), and others) was performed by a combination of Western blotting, flotation on linear sucrose gradients, and flow cytometry, confirming their exosomal expression. Some were confirmed positive on urinary exosomes from a bladder cancer patient. In summary, the

Redefining the Breast Cancer Exosome Proteome by Tandem Mass Tag Quantitative Proteomics and Multivariate Cluster Analysis.

PubMed

Clark, David J; Fondrie, William E; Liao, Zhongping; Hanson, Phyllis I; Fulton, Amy; Mao, Li; Yang, Austin J

2015-10-20

Exosomes are microvesicles of endocytic origin constitutively released by multiple cell types into the extracellular environment. With evidence that exosomes can be detected in the blood of patients with various malignancies, the development of a platform that uses exosomes as a diagnostic tool has been proposed. However, it has been difficult to truly define the exosome proteome due to the challenge of discerning contaminant proteins that may be identified via mass spectrometry using various exosome enrichment strategies. To better define the exosome proteome in breast cancer, we incorporated a combination of Tandem-Mass-Tag (TMT) quantitative proteomics approach and Support Vector Machine (SVM) cluster analysis of three conditioned media derived fractions corresponding to a 10 000g cellular debris pellet, a 100 000g crude exosome pellet, and an Optiprep enriched exosome pellet. The quantitative analysis identified 2 179 proteins in all three fractions, with known exosomal cargo proteins displaying at least a 2-fold enrichment in the exosome fraction based on the TMT protein ratios. Employing SVM cluster analysis allowed for the classification 251 proteins as "true" exosomal cargo proteins. This study provides a robust and vigorous framework for the future development of using exosomes as a potential multiprotein marker phenotyping tool that could be useful in breast cancer diagnosis and monitoring disease progression.

Exosomes from dental pulp stem cells rescue human dopaminergic neurons from 6-hydroxy-dopamine-induced apoptosis.

PubMed

Jarmalavičiūtė, Akvilė; Tunaitis, Virginijus; Pivoraitė, Ugnė; Venalis, Algirdas; Pivoriūnas, Augustas

2015-07-01

Stem cells derived from the dental pulp of human exfoliated deciduous teeth (SHEDs) have unique neurogenic properties that could be potentially exploited for therapeutic use. The importance of paracrine SHED signaling for neuro-regeneration has been recognized, but the exact mechanisms behind these effects are presently unknown. In the present study, we investigated the neuro-protective potential of exosomes and micro-vesicles derived from SHEDs on human dopaminergic neurons during oxidative stress-induced by 6-hydroxy-dopamine (6-OHDA). ReNcell VM human neural stem cells were differentiated into dopaminergic neurons and treated with 100 μmol/L of 6-OHDA alone or in combination with exosomes or micro-vesicles purified by ultracentrifugation from SHEDs cultivated in serum-free medium under two conditions: in standard two-dimensional culture flasks or on laminin-coated micro-carriers in a bioreactor. Real-time monitoring of apoptosis was performed with the use of time-lapse confocal microscopy and the CellEvent Caspase-3/7 green detection reagent. Exosomes but not micro-vesicles derived from SHEDs grown on the laminin-coated three-dimensional alginate micro-carriers suppressed 6-OHDA-induced apoptosis in dopaminergic neurons by approximately 80% throughout the culture period. Strikingly, no such effects were observed for the exosomes derived from SHEDs grown under standard culture conditions. Our results suggest that exosomes derived from SHEDs are considered as new potential therapeutic tool in the treatment of Parkinson's disease. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Exosomes from Cardiomyocyte Progenitor Cells and Mesenchymal Stem Cells Stimulate Angiogenesis Via EMMPRIN.

PubMed

Vrijsen, Krijn R; Maring, Janita A; Chamuleau, Steven A J; Verhage, Vera; Mol, Emma A; Deddens, Janine C; Metz, Corina H G; Lodder, Kirsten; van Eeuwijk, Esther C M; van Dommelen, Susan M; Doevendans, Pieter A; Smits, Anke M; Goumans, Marie-José; Sluijter, Joost P G

2016-10-01

To date, cellular transplantation therapy has not yet fulfilled its high expectations for cardiac repair. A major limiting factor is lack of long-term engraftment of the transplanted cells. Interestingly, transplanted cells can positively affect their environment via secreted paracrine factors, among which are extracellular vesicles, including exosomes: small bi-lipid-layered vesicles containing proteins, mRNAs, and miRNAs. An exosome-based therapy will therefore relay a plethora of effects, without some of the limiting factors of cell therapy. Since cardiomyocyte progenitor cells (CMPC) and mesenchymal stem cells (MSC) induce vessel formation and are frequently investigated for cardiac-related therapies, the pro-angiogenic properties of CMPC and MSC-derived exosome-like vesicles are investigated. Both cell types secrete exosome-like vesicles, which are efficiently taken up by endothelial cells. Endothelial cell migration and vessel formation are stimulated by these exosomes in in vitro models, mediated via ERK/Akt-signaling. Additionally, these exosomes stimulated blood vessel formation into matrigel plugs. Analysis of pro-angiogenic factors revealed high levels of extracellular matrix metalloproteinase inducer (EMMPRIN). Knockdown of EMMPRIN on CMPCs leads to a diminished pro-angiogenic effect, both in vitro and in vivo. Therefore, CMPC and MSC exosomes have powerful pro-angiogenic effects, and this effect is largely mediated via the presence of EMMPRIN on exosomes. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

High Levels of Exosomes Expressing CD63 and Caveolin-1 in Plasma of Melanoma Patients

PubMed Central

Logozzi, Mariantonia; De Milito, Angelo; Lugini, Luana; Borghi, Martina; Calabrò, Luana; Spada, Massimo; Perdicchio, Maurizio; Marino, Maria Lucia; Federici, Cristina; Iessi, Elisabetta; Brambilla, Daria; Venturi, Giulietta; Lozupone, Francesco; Santinami, Mario; Huber, Veronica; Maio, Michele; Rivoltini, Licia; Fais, Stefano

2009-01-01

Background Metastatic melanoma is an untreatable cancer lacking reliable and non-invasive markers of disease progression. Exosomes are small vesicles secreted by normal as well as tumor cells. Human tumor-derived exosomes are involved in malignant progression and we evaluated the presence of exosomes in plasma of melanoma patients as a potential tool for cancer screening and follow-up. Methodology/Principal Findings We designed an in-house sandwich ELISA (Exotest) to capture and quantify exosomes in plasma based on expression of housekeeping proteins (CD63 and Rab-5b) and a tumor-associated marker (caveolin-1). Western blot and flow cytometry analysis of exosomes were used to confirm the Exotest-based findings. The Exotest allowed sensitive detection and quantification of exosomes purified from human tumor cell culture supernatants and plasma from SCID mice engrafted with human melanoma. Plasma levels of exosomes in melanoma-engrafted SCID mice correlated to tumor size. We evaluated the levels of plasma exosomes expressing CD63 and caveolin-1 in melanoma patients (n = 90) and healthy donors (n = 58). Consistently, plasma exosomes expressing CD63 (504±315) or caveolin-1 (619±310) were significantly increased in melanoma patients as compared to healthy donors (223±125 and 228±102, respectively). While the Exotest for CD63+ plasma exosomes had limited sensitivity (43%) the Exotest for detection of caveolin-1+ plasma exosomes showed a higher sensitivity (68%). Moreover, caveolin-1+ plasma exosomes were significantly increased with respect to CD63+ exosomes in the patients group. Conclusions/Significance We describe a new non-invasive assay allowing detection and quantification of human exosomes in plasma of melanoma patients. Our results suggest that the Exotest for detection of plasma exosomes carrying tumor-associated antigens may represent a novel tool for clinical management of cancer patients. PMID:19381331

High levels of exosomes expressing CD63 and caveolin-1 in plasma of melanoma patients.

PubMed

Logozzi, Mariantonia; De Milito, Angelo; Lugini, Luana; Borghi, Martina; Calabrò, Luana; Spada, Massimo; Perdicchio, Maurizio; Marino, Maria Lucia; Federici, Cristina; Iessi, Elisabetta; Brambilla, Daria; Venturi, Giulietta; Lozupone, Francesco; Santinami, Mario; Huber, Veronica; Maio, Michele; Rivoltini, Licia; Fais, Stefano

2009-01-01

Metastatic melanoma is an untreatable cancer lacking reliable and non-invasive markers of disease progression. Exosomes are small vesicles secreted by normal as well as tumor cells. Human tumor-derived exosomes are involved in malignant progression and we evaluated the presence of exosomes in plasma of melanoma patients as a potential tool for cancer screening and follow-up. We designed an in-house sandwich ELISA (Exotest) to capture and quantify exosomes in plasma based on expression of housekeeping proteins (CD63 and Rab-5b) and a tumor-associated marker (caveolin-1). Western blot and flow cytometry analysis of exosomes were used to confirm the Exotest-based findings. The Exotest allowed sensitive detection and quantification of exosomes purified from human tumor cell culture supernatants and plasma from SCID mice engrafted with human melanoma. Plasma levels of exosomes in melanoma-engrafted SCID mice correlated to tumor size. We evaluated the levels of plasma exosomes expressing CD63 and caveolin-1 in melanoma patients (n = 90) and healthy donors (n = 58). Consistently, plasma exosomes expressing CD63 (504+/-315) or caveolin-1 (619+/-310) were significantly increased in melanoma patients as compared to healthy donors (223+/-125 and 228+/-102, respectively). While the Exotest for CD63+ plasma exosomes had limited sensitivity (43%) the Exotest for detection of caveolin-1+ plasma exosomes showed a higher sensitivity (68%). Moreover, caveolin-1+ plasma exosomes were significantly increased with respect to CD63+ exosomes in the patients group. We describe a new non-invasive assay allowing detection and quantification of human exosomes in plasma of melanoma patients. Our results suggest that the Exotest for detection of plasma exosomes carrying tumor-associated antigens may represent a novel tool for clinical management of cancer patients.

Human Umbilical Cord MSC-Derived Exosomes Suppress the Development of CCl4-Induced Liver Injury through Antioxidant Effect.

PubMed

Jiang, Wenqian; Tan, Youwen; Cai, Mengjie; Zhao, Ting; Mao, Fei; Zhang, Xu; Xu, Wenrong; Yan, Zhixin; Qian, Hui; Yan, Yongmin

2018-01-01

Mesenchymal stem cells (MSCs) have been increasingly applied into clinical therapy. Exosomes are small (30-100 nm in diameter) membrane vesicles released by different cell types and possess the similar functions with their derived cells. Human umbilical cord MSC-derived exosomes (hucMSC-Ex) play important roles in liver repair. However, the effects and mechanisms of hucMSC-Ex on liver injury development remain elusive. Mouse models of acute and chronic liver injury and liver tumor were induced by carbon tetrachloride (CCl 4 ) injection, followed by administration of hucMSC-Ex via the tail vein. Alleviation of liver injury by hucMSC-Ex was determined. We further explored the production of oxidative stress and apoptosis in the development of liver injury and compared the antioxidant effects of hucMSC-Ex with frequently used hepatic protectant, bifendate (DDB) in liver injury. hucMSC-Ex alleviated CCl 4 -induced acute liver injury and liver fibrosis and restrained the growth of liver tumors. Decreased oxidative stress and apoptosis were found in hucMSC-Ex-treated mouse models and liver cells. Compared to bifendate (DDB) treatment, hucMSC-Ex presented more distinct antioxidant and hepatoprotective effects. hucMSC-Ex may suppress CCl 4 -induced liver injury development via antioxidant potentials and could be a more effective antioxidant than DDB in CCl 4 -induced liver tumor development.

Surface Functionalization of Exosomes Using Click Chemistry

PubMed Central

2015-01-01

A method for conjugation of ligands to the surface of exosomes was developed using click chemistry. Copper-catalyzed azide alkyne cycloaddition (click chemistry) is ideal for biocojugation of small molecules and macromolecules to the surface of exosomes, due to fast reaction times, high specificity, and compatibility in aqueous buffers. Exosomes cross-linked with alkyne groups using carbodiimide chemistry were conjugated to a model azide, azide-fluor 545. Conjugation had no effect on the size of exosomes, nor was there any change in the extent of exosome adherence/internalization with recipient cells, suggesting the reaction conditions were mild on exosome structure and function. We further investigated the extent of exosomal protein modification with alkyne groups. Using liposomes with surface alkyne groups of a similar size and concentration to exosomes, we estimated that approximately 1.5 alkyne groups were present for every 150 kDa of exosomal protein. PMID:25220352

Proteomic Profiling of Exosomes Leads to the Identification of Novel Biomarkers for Prostate Cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duijvesz, Diederick; Burnum-Johnson, Kristin E.; Gritsenko, Marina A.

Introduction: Current markers for prostate cancer, such as PSA lack specificity. Therefore, novel biomarkers are needed. Unfortunately, biomarker discovery from body fluids is often hampered by the high abundance of many proteins unrelated to disease. An attractive alternative biomarker discovery approach is the isolation of small vesicles (exosomes, ~100 nm). They contain proteins that are specific to the tissue from which they are derived and therefore can be considered as treasure chests for disease-specific marker discovery. Profiling prostate cancer-derived exosomes could reveal new markers for this malignancy. Materials and Methods: Exosomes were isolated from 2 immortalized primary prostate epithelial cellsmore » (PNT2C2 and RWPE-1) and 2 PCa cell lines (PC346C and VCaP) by ultracentrifugation. Proteomic analyses utilized a nanoLC coupled with an LTQ-Orbitrap operated in tandem MS (MS/MS) mode, followed by the Accurate Mass and Time (AMT) tag approach. Exosomal proteins were validated by Western blotting. A Tissue Micro Array, containing 481 different PCa samples (radical prostatectomy), was used to correlate candidate markers with several clinical-pathological parameters such as PSA, Gleason score, biochemical recurrence, and (PCa-related) death. Results: Proteomic characterization resulted in the identification of 263 proteins by at least 2 peptides. Specifically analysis of exosomes from PNT2C2, RWPE-1, PC346C, and VCaP identified 248, 233, 169, and 216 proteins, respectively. Statistical analyses revealed 52 proteins differently expressed between PCa and control cells, 9 of which were more abundant in PCa. Validation by Western blotting confirmed a higher abundance of FASN, XPO1 and PDCD6IP (ALIX) in PCa exosomes. The Tissue Micro 4 Array showed strong correlation of higher Gleason scores and local recurrence with increased cytoplasmic XPO1 (P<0.001). Conclusions: Differentially abundant proteins of cell line-derived exosomes make a clear subdivision

Urine Exosome Isolation and Characterization.

PubMed

Street, Jonathan M; Koritzinsky, Erik H; Glispie, Deonna M; Yuen, Peter S T

2017-01-01

Exosomes are nanometer-scale, membrane-enclosed vesicles that can potentially be used to detect nephrotoxicity, and reveal the subsequent response of the kidney. Epithelial cells of every nephron segment can contribute to the urinary exosome population, which is rich in potential biomarkers, including membrane proteins such as transporters and receptors, transcription factors, and microRNAs. These exosomal biomarkers may be up- or downregulated upon nephrotoxicant exposure. Exosome isolation is an area of ongoing research. Although faster and simpler methods have been developed, ultracentrifugation remains a mainstay for purification. A single ultracentrifugation step provides an enriched preparation suitable for biomarker discovery, and a second ultracentrifugation on a sucrose/D 2 O cushion provides the purest exosome preparation currently available and may be preferred for bioactivity assays. The concentration of exosomes can be determined using Nanosight Nanoparticle Tracking Analysis and their contents studied with a variety of approaches including western blots for proteins and RT-qPCR for microRNAs.

Circulating exosomes as new biomarkers for brain disease and injury

NASA Astrophysics Data System (ADS)

Graner, Michael W.; Epple, Laura M.; Dusto, Nathaniel L.; Lencioni, Alex M.; Nega, Meheret; Herring, Matthew; Winston, Ben; Madsen, Helen; Bemis, Lynne T.; Anchordoquy, Thomas J.

2013-05-01

Brain diseases such as cancers, neurodegenerative disorders, or trauma are frequently diagnosed with imaging modalities and sometimes with intracranial biopsies. Treatment response is similarly monitored, along with clinical indications. While these technologies provide important windows into the disease state, they fail to provide us a detailed molecular portrait of the disease and of the changes taking place during therapy. Exosomes are virus-sized nanovesicles derived from the endosomal system and are released extracellularly from essentially all cell types. Exosomes contain intracellular entities (proteins, nucleic acids, metabolites), membrane proteins and lipids, and even extracellular proteins bound to them. Exosomes may be considered as mini-surrogates of their cells of origin, with some content common to all cells/exosomes, but some of the content would be cell-specific. These vesicles are found in all biofluids in humans, and are thus accessible to "liquid biopsy" with harvest of vesicles from such fluids. Current challenges are to identify disease-related markers or panels of markers to distinguish the disease state. Here we will show examples of brain tumor markers found in/on exosomes from cell culture and patient sera, and we will suggest that aspects of the biology of disease may have a relevant place in the search for biomarkers.

Exosomes from M1-Polarized Macrophages Potentiate the Cancer Vaccine by Creating a Pro-inflammatory Microenvironment in the Lymph Node.

PubMed

Cheng, Lifang; Wang, Yuhua; Huang, Leaf

2017-07-05

Exosomes are small membrane-bound vesicular particles generated by most cells for intercellular communication and regulation. During biogenesis, specific lipids, RNAs, proteins, and carbohydrates are enriched and packaged into the vesicles so that the exosomal contents reflect not only the source but also the physiological conditions of the parental cells. These exosomes transport materials or signals to the target cells for diverse physiological purposes. Our study focused on the exosomes derived from M1-polarized, proinflammatory macrophages for the possibility of using M1 exosomes as an immunopotentiator for a cancer vaccine. The M1 exosomes displayed a tropism toward lymph nodes after subcutaneous injection, primarily taken up by the local macrophages and dendritic cells, and they induced the release of a pool of Th1 cytokines. We found that M1, but not M2, exosomes enhanced activity of lipid calcium phosphate (LCP) nanoparticle-encapsulated Trp2 vaccine, and they induced a stronger antigen-specific cytotoxic T cell response. The M1 exosomes proved to be a more potent immunopotentiator than CpG oligonucleotide when used with LCP nanoparticle vaccine in a melanoma growth inhibition study. Thus, our study indicated that exosomes derived from M1-polarized macrophages could be used as a vaccine adjuvant. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Single exosome detection in serum using microtoroid optical resonators (Conference Presentation)

NASA Astrophysics Data System (ADS)

Su, Judith

2016-03-01

Recently exosomes have attracted interest due to their potential as cancer biomarkers. We report the real time, label-free sensing of single exosomes in serum using microtoroid optical resonators. We use this approach to assay the progression of tumors implanted in mice by specifically detecting low concentrations of tumor-derived exosomes. Our approach measures the adsorption of individual exosomes onto a functionalized silica microtoroid by tracking changes in the optical resonant frequency of the microtoroid. When exosomes land on the microtoroid, they perturb its refractive index in the evanescent field and thus shift its resonance frequency. Through digital frequency locking, we are able to rapidly track these shifts with accuracies of better than 10 attometers (one part in 10^11). Samples taken from tumor-implanted mice from later weeks generated larger frequency shifts than those from earlier weeks. Control samples taken from a mouse with no tumor generated no such increase in signal between subsequent weeks. Analysis of shifts from tumor-implanted mouse samples show a distribution of unitary steps, with the maximum step having a height of ~1.2 fm, corresponding to an exosome size of 44 ± 4.8 nm. This size range corresponds to that found by performing nanoparticle tracking analysis on the same samples. Our results demonstrate development towards a minimally-invasive tumor "biopsy" that eliminates the need to find and access a tumor.

Prediction of conversion from mild cognitive impairment to dementia with neuronally derived blood exosome protein profile.

PubMed

Winston, Charisse N; Goetzl, Edward J; Akers, Johnny C; Carter, Bob S; Rockenstein, Edward M; Galasko, Douglas; Masliah, Eliezer; Rissman, Robert A

2016-01-01

Levels of Alzheimer's disease (AD)-related proteins in plasma neuronal derived exosomes (NDEs) were quantified to identify biomarkers for prediction and staging of mild cognitive impairment (MCI) and AD. Plasma exosomes were extracted, precipitated, and enriched for neuronal source by anti-L1CAM antibody absorption. NDEs were characterized by size (Nanosight) and shape (TEM) and extracted NDE protein biomarkers were quantified by ELISAs. Plasma NDE cargo was injected into normal mice, and results were characterized by immunohistochemistry to determine pathogenic potential. Plasma NDE levels of P-T181-tau, P-S396-tau, and Aβ1-42 were significantly higher, whereas those of neurogranin (NRGN) and the repressor element 1-silencing transcription factor (REST) were significantly lower in AD and MCI converting to AD (ADC) patients compared to cognitively normal controls (CNC) subjects and stable MCI patients. Mice injected with plasma NDEs from ADC patients displayed increased P-tau (PHF-1 antibody)-positive cells in the CA1 region of the hippocampus compared to plasma NDEs from CNC and stable MCI patients. Abnormal plasma NDE levels of P-tau, Aβ1-42, NRGN, and REST accurately predict conversion of MCI to AD dementia. Plasma NDEs from demented patients seeded tau aggregation and induced AD-like neuropathology in normal mouse CNS.

Different exosome cargo from plasma/bronchoalveolar lavage in non-small-cell lung cancer.

PubMed

Rodríguez, Marta; Silva, Javier; López-Alfonso, Ana; López-Muñiz, María Belen; Peña, Cristina; Domínguez, Gemma; García, Jose Miguel; López-Gónzalez, Ana; Méndez, Miriam; Provencio, Mariano; García, Vanesa; Bonilla, Félix

2014-09-01

Tumor-derived exosomes mediate tumorigenesis by facilitating tumor growth, metastasis, development of drug resistance, and immunosuppression. However, little is known about the exosomes isolated from bronchoalveolar lavage (BAL) in patients with lung neoplasm. Exosomes isolated in plasma and BAL from 30 and 75 patients with tumor and nontumor pathology were quantified by acetylcholinesterase activity and characterized by Western Blot, Electron Microscopy, and Nanoparticle Tracking Analysis. Differences in exosome cargo were analyzed by miRNA quantitative PCR in pooled samples and validated in a second series of patients. More exosomes were detected in plasma than in BAL in both groups (P < 0.001). The most miRNAs evaluated by PCR array were detected in tumor plasma, tumor BAL, and nontumor BAL pools, but only 56% were detected in the nontumor plasma pool. Comparing the top miRNAs with the highest levels detected in each pool, we found close homology only between the BAL samples of the two pathologies. In tumor plasma, we found a higher percentage of miRNAs with increased levels than in tumor BAL or in nontumor plasma. The data reveal differences between BAL and plasma exosome amount and miRNA content. © 2014 Wiley Periodicals, Inc.

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

PubMed

Bryniarski, Krzysztof; Ptak, Wlodzimierz; Martin, Emilia; Nazimek, Katarzyna; Szczepanik, Marian; Sanak, Marek; Askenase, Philip W

2015-01-01

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.

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

PubMed Central

Bryniarski, Krzysztof; Ptak, Wlodzimierz; Martin, Emilia; Nazimek, Katarzyna; Szczepanik, Marian; Sanak, Marek; Askenase, Philip W.

2015-01-01

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. PMID:25923429

Regulated capture by exosomes of mRNAs for cytoplasmic tRNA synthetases.

PubMed

Wang, Feng; Xu, Zhiwen; Zhou, Jie; Lo, Wing-Sze; Lau, Ching-Fun; Nangle, Leslie A; Yang, Xiang-Lei; Zhang, Mingjie; Schimmel, Paul

2013-10-11

Although tRNA synthetases are enzymes that catalyze the first step of translation in the cytoplasm, surprising functions unrelated to translation have been reported. These studies, and the demonstration of novel activities of splice variants, suggest a far broader reach of tRNA synthetases into cell biology than previously recognized. Here we show that mRNAs for most tRNA synthetases can be detected in exosomes. Also detected in exosomes was an mRNA encoding a unique splice variant that others had associated with prostate cancer. The exosomal mRNAs encoding the native synthetase and its cancer-associated splice variant could be translated in vitro and in mammalian cells into stable proteins. Other results showed that selection by exosomes of the splice variant mRNA could be regulated by an external stimulus. Thus, a broad and diverse regulated pool of tRNA synthetase-derived mRNAs is packaged for genetic exchange.

Glypican1 identifies cancer exosomes and facilitates early detection of cancer

PubMed Central

Melo, Sonia A.; Luecke, Linda B.; Kahlert, Christoph; Fernandez, Agustin F.; Gammon, Seth T.; Kaye, Judith; LeBleu, Valerie S.; Mittendorf, Elizabeth A.; Weitz, Juergen; Rahbari, Nuh; Reissfelder, Christoph; Pilarsky, Christian; Fraga, Mario F.; Piwnica-Worms, David; Kalluri, Raghu

2016-01-01

Summary Exosomes are lipid bilayer-enclosed extracellular vesicles (EVs) that contain proteins and nucleic acids. They are secreted by all cells and circulate in the blood. Specific detection and isolation of cancer cell-derived exosomes in circulation is currently lacking. Using mass spectrometry analyses, we identified a cell surface proteoglycan, glypican-1 (GPC1), specifically enriched on cancer cell-derived exosomes. GPC1+ circulating exosomes (crExos) were monitored and isolated using flow cytometry from the serum of cancer patients and mice with cancer. GPC1+ crExos were detected in the serum of patients with pancreas cancer with absolute specificity and sensitivity, distinguishing healthy subjects and patients with a benign pancreas disease from patients with early and late stage pancreas cancer. Levels of GPC1+ crExos correlate with tumor burden and survival in patients pre- and post-surgical tumor resection. GPC1+ crExos from patients and from mice with spontaneous pancreas tumors driven by oncogenic KRAS contained RNA with specific KRAS mutation, and it emerges as a reliable biomarker for the detection of PanIN lesions despite negative signal by MRI in mice. GPC1+ crExos may serve as a potential non-invasive diagnostic and screening tool to detect early stages of pancreas cancer to facilitate possible curative surgical therapy. PMID:26106858

Toll-Like Receptor 4 Engagement Mediates Prolyl Endopeptidase Release from Airway Epithelia via Exosomes.

PubMed

Szul, Tomasz; Bratcher, Preston E; Fraser, Kyle B; Kong, Michele; Tirouvanziam, Rabindra; Ingersoll, Sarah; Sztul, Elizabeth; Rangarajan, Sunil; Blalock, J Edwin; Xu, Xin; Gaggar, Amit

2016-03-01

Proteases are important regulators of pulmonary remodeling and airway inflammation. Recently, we have characterized the enzyme prolyl endopeptidase (PE), a serine peptidase, as a critical protease in the generation of the neutrophil chemoattractant tripeptide Pro-Gly-Pro (PGP) from collagen. However, PE has been characterized as a cytosolic enzyme, and the mechanism mediating PE release extracellularly remains unknown. We examined the role of exosomes derived from airway epithelia as a mechanism for PE release and the potential extracellular signals that regulate the release of these exosomes. We demonstrate a specific regulatory pathway of exosome release from airway epithelia and identify PE as novel exosome cargo. LPS stimulation of airway epithelial cells induces release of PE-containing exosomes, which is significantly attenuated by small interfering RNA depletion of Toll-like receptor 4 (TLR4). These differences were recapitulated upon intratracheal LPS administration in mice competent versus deficient for TLR4 signaling. Finally, sputum samples from subjects with cystic fibrosis colonized with Pseudomonas aeruginosa demonstrate elevated exosome content and increased PE levels. This TLR4-based mechanism highlights the first report of nonstochastic release of exosomes in the lung and couples TLR4 activation with matrikine generation. The increased quantity of these proteolytic exosomes in the airways of subjects with chronic lung disease highlights a new mechanism of injury and inflammation in the pathogenesis of pulmonary disorders.

Exosomes: Some approaches to cancer diagnosis and therapy

NASA Astrophysics Data System (ADS)

Shtam, T.; Samsonov, R.; Kamyshinsky, R.; Pantina, R.; Verlov, N.; Vasiliev, A.; Konevega, A. L.; Malek, A. V.

2017-09-01

Exosomes are membrane-bound, intercellular communication shuttle vesicles that are defined by their endocytic origin and size range of 30-120 nm. Secreted by nearly all mammalian cell types and present in bodily fluids, exosomes confer messages between cells, by transporting functionally relevant proteins, nucleic acids, and lipids. The capability of tumor exosomes to house tumorigenic information and induce cellular responses that promote disease pathogenesis make tumor exosomes an attractive tool in identifying cancer biomarkers and exploiting exosomes for therapy. In this paper, we sum up our previous findings to utilize exosomes as biomarkers for early detection, diagnosis and therapy selection of prostate and thyroid cancer and present our results on exosomes in colon cancer. Some of plasma exosomal miRNAs showed their potential as diagnostic markers for colon cancer. All together, the data suggested the potentials of circulating exosomal miRNAs as liquid biopsy markers for cancer. Here we also present the possibilities of delivering therapeutic molecules by exosomes. Previously, we had demonstrated the potential of exosome-mediated siRNA delivery. Here, we present the possibility of carrying the exogenous p53 protein by exosomes in vitro.

CNS tau efflux via exosomes is likely increased in Parkinson disease but not in Alzheimer disease

PubMed Central

Shi, Min; Kovac, Andrej; Korff, Ane; Cook, Travis J.; Ginghina, Carmen; Bullock, Kristin M.; Yang, Li; Stewart, Tessandra; Zheng, Danfeng; Aro, Patrick; Atik, Anzari; Kerr, Kathleen F.; Zabetian, Cyrus P.; Peskind, Elaine R.; Hu, Shu-Ching; Quinn, Joseph F.; Galasko, Douglas R.; Montine, Thomas J.; Banks, William A.; Zhang, Jing

2016-01-01

Background Alzheimer disease (AD) and Parkinson disease (PD) involve tau pathology. Tau is detectable in blood, but its clearance from neuronal cells and the brain is poorly understood. Methods Tau efflux from the brain to the blood was evaluated by administering radioactively labeled and unlabeled tau intracerebroventricularly in wild-type and tau knock-out mice, respectively. Central nervous system (CNS)-derived tau in L1CAM-containing exosomes was further characterized extensively in human plasma, including by Single Molecule Array technology with 303 subjects. Results The efflux of Tau, including a fraction via CNS-derived L1CAM exosomes, was observed in mice. In human plasma, tau was explicitly identified within L1CAM exosomes. In contrast to AD patients, L1CAM exosomal tau was significantly higher in PD patients than controls, and correlated with cerebrospinal fluid tau. Conclusions Tau is readily transported from the brain to the blood. The mechanisms of CNS tau efflux are likely different between AD and PD. PMID:27234211

Exosome secretion promotes chemotaxis of cancer cells.

PubMed

Sung, Bong Hwan; Weaver, Alissa M

2017-03-04

Migration of cells toward chemical cues, or chemotaxis, is important for many biologic processes such as immune defense, wound healing and cancer metastasis. Although chemotaxis is thought to occur in cancer cells, it is less well characterized than chemotaxis of professional immune cells such as neutrophils. Here, we show that cancer cell chemotaxis relies on secretion of exosome-type extracellular vesicles. Migration of fibrosarcoma cells toward a gradient of exosome-depleted serum was diminished by knockdown of the exosome secretion regulator Rab27a. Rescue experiments in which chemotaxis chambers were coated with purified extracellular vesicles demonstrate that exosomes but not microvesicles affect both speed and directionality of migrating cells. Chamber coating with purified fibronectin and fibronectin-depleted exosomes demonstrates that the exosome cargo fibronectin promotes cell speed but cannot account for the role of exosomes in promoting directionality of fibrosarcoma cell movement during chemotaxis. These experiments indicate that exosomes contain multiple motility-promoting cargoes that contribute to different aspects of cell motility.

Exosomes and cardiovascular cell-cell communication.

PubMed

Poe, Adam J; Knowlton, Anne A

2018-05-15

Exosomes have become an important player in intercellular signaling. These lipid microvesicles can stably transfer miRNA, protein, and other molecules between cells and circulate throughout the body. Exosomes are released by almost all cell types and are present in most if not all biological fluids. The biologically active cargo carried by exosomes can alter the phenotype of recipient cells. Exosomes increasingly are recognized as having an important role in the progression and treatment of cardiac disease states. Injured cardiac cells can release exosomes with important pathological effects on surrounding tissue, in addition to effecting other organs. But of equal interest is the possible benefit(s) conferred by exosomes released from stem cells for use in treatment and possible repair of cardiac damage. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Isolation and Characterization of Exosome from Human Embryonic Stem Cell-Derived C-Myc-Immortalized Mesenchymal Stem Cells.

PubMed

Lai, Ruenn Chai; Yeo, Ronne Wee Yeh; Padmanabhan, Jayanthi; Choo, Andre; de Kleijn, Dominique P V; Lim, Sai Kiang

2016-01-01

Mesenchymal stem cells (MSC) are currently the cell type of choice in many cell therapy trials. The number of therapeutic applications for MSCs registered as product IND submissions with the FDA and initiation of registered clinical trials has increased substantially in recent years, in particular between 2006 and 2012. However, defined mechanisms of action underpinning the therapeutic efficacy of MSCs are lacking, but they are increasingly attributed to MSC trophic secretion rather than their differentiation potential. A promising secreted therapeutic candidate is an extracellular vesicle (EV) known as the exosome. The use of exosomes instead of cells as a therapeutic agent provides several advantages. A critical advantage is the prospect of a conventional pharmaceutical manufacturing process that is highly scalable and amenable to the stringent manufacturing process. For example, MSCs used as producers of therapeutics, and not as therapeutics per se, could be immortalized to generate infinitely expansible clonal lines to enhance the reproducible production of therapeutic exosomes. In this chapter, we will describe the immortalization of MSCs, and the production, isolation, and characterization of exosomes from immortalized MSC.

Comparative proteomics evaluation of plasma exosome isolation techniques and assessment of the stability of exosomes in normal human blood plasma.

PubMed

Kalra, Hina; Adda, Christopher G; Liem, Michael; Ang, Ching-Seng; Mechler, Adam; Simpson, Richard J; Hulett, Mark D; Mathivanan, Suresh

2013-11-01

Exosomes are nanovesicles released by a variety of cells and are detected in body fluids including blood. Recent studies have highlighted the critical application of exosomes as personalized targeted drug delivery vehicles and as reservoirs of disease biomarkers. While these research applications have created significant interest and can be translated into practice, the stability of exosomes needs to be assessed and exosome isolation protocols from blood plasma need to be optimized. To optimize methods to isolate exosomes from blood plasma, we performed a comparative evaluation of three exosome isolation techniques (differential centrifugation coupled with ultracentrifugation, epithelial cell adhesion molecule immunoaffinity pull-down, and OptiPrep(TM) density gradient separation) using normal human plasma. Based on MS, Western blotting and microscopy results, we found that the OptiPrep(TM) density gradient method was superior in isolating pure exosomal populations, devoid of highly abundant plasma proteins. In addition, we assessed the stability of exosomes in plasma over 90 days under various storage conditions. Western blotting analysis using the exosomal marker, TSG101, revealed that exosomes are stable for 90 days. Interestingly, in the context of cellular uptake, the isolated exosomes were able to fuse with target cells revealing that they were indeed biologically active. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Erosion protection conferred by whole human saliva, dialysed saliva, and artificial saliva

NASA Astrophysics Data System (ADS)

Baumann, T.; Kozik, J.; Lussi, A.; Carvalho, T. S.

2016-10-01

During dental erosion, tooth minerals are dissolved, leading to a softening of the surface and consequently to irreversible surface loss. Components from human saliva form a pellicle on the tooth surface, providing some protection against erosion. To assess the effect of different components and compositions of saliva on the protective potential of the pellicle against enamel erosion, we prepared four different kinds of saliva: human whole stimulated saliva (HS), artificial saliva containing only ions (AS), human saliva dialysed against artificial saliva, containing salivary proteins and ions (HS/AS), and human saliva dialysed against deionised water, containing only salivary proteins but no ions (HS/DW). Enamel specimens underwent four cycles of immersion in either HS, AS, HS/AS, HS/DW, or a humid chamber (Ctrl), followed by erosion with citric acid. During the cycling process, the surface hardness and the calcium released from the surface of the specimens were measured. The different kinds of saliva provided different levels of protection, HS/DW exhibiting significantly better protection than all the other groups (p < 0.0001). Different components of saliva, therefore, have different effects on the protective properties of the pellicle and the right proportions of these components in saliva are critical for the ability to form a protective pellicle.

Deubiquitylation of Protein Cargo Is Not an Essential Step in Exosome Formation*

PubMed Central

Huebner, Alyssa R.; Cheng, Lei; Somparn, Poorichaya; Knepper, Mark A.; Fenton, Robert A.; Pisitkun, Trairak

2016-01-01

Exosomes, derived from multivesicular bodies (MVBs), contain proteins and genetic materials from their cell of origin and are secreted from various cells types, including kidney epithelial cells. In general, it is thought that protein cargo is ubiquitylated but that ubiquitin is cleaved by specific deubiquitylases during the process of cargo incorporation into MVBs. Here, we provide direct evidence that, in vivo, deubiquitylation is not essential. Ubiquitin was detected within human MVBs and urinary exosomes by electron microscopy. Of the >6000 proteins identified in human urinary exosomes was mass spectrometry, 15% were ubiquitylated with various topologies (Lys63>Lys48> Lys11>Lys6>Lys29>Lys33>Lys27). A significant preference for basic amino acids upstream of ubiquitylation sites suggests specific ubiquitylation motifs. The current studies demonstrate that, in vivo, deubiquitylation of proteins is not necessary for their incorporation into MVBs and highlight that urinary exosomes are an enriched source for studying ubiquitin modifications in physiological or disease states. PMID:26884507

Exosomes and their role in the micro-/macro-environment: a comprehensive review

PubMed Central

Javeed, Naureen; Mukhopadhyay, Debabrata

2017-01-01

The importance of extracellular vesicles (EVs) in cell-cell communication has long been recognized due to their ability to transfer important cellular cargoes such as DNA, mRNA, miRNAs, and proteins to target cells. Compelling evidence supports the role of EVs in the horizontal transfer of cellular material which has the potential to influence normal cellular physiology and promote various disease states. Of the different types of EVs, exosomes have garnered much attention in the past decade due to their abundance in various biological fluids and ability to affect multiple organ systems. The main focus of this review will be on cancer and how cancer-derived exosomes are important mediators of metastasis, angiogenesis, immune modulation, and the tumor macro-/microenvironment. We will also discuss exosomes as potential biomarkers for cancers due to their abundance in biological fluids, ease of uptake, and cellular content. Exosome use in diagnosis, prognosis, and in establishing treatment regimens has enormous potential to revolutionize patient care. PMID:28290182

Exosomes and their role in the micro-/macro-environment: a comprehensive review.

PubMed

Javeed, Naureen; Mukhopadhyay, Debabrata

2017-09-26

The importance of extracellular vesicles (EVs) in cell-cell communication has long been recognized due to their ability to transfer important cellular cargoes such as DNA, mRNA, miRNAs, and proteins to target cells. Compelling evidence supports the role of EVs in the horizontal transfer of cellular material which has the potential to influence normal cellular physiology and promote various disease states. Of the different types of EVs, exosomes have garnered much attention in the past decade due to their abundance in various biological fluids and ability to affect multiple organ systems. The main focus of this review will be on cancer and how cancer-derived exosomes are important mediators of metastasis, angiogenesis, immune modulation, and the tumor macro-/microenvironment. We will also discuss exosomes as potential biomarkers for cancers due to their abundance in biological fluids, ease of uptake, and cellular content. Exosome use in diagnosis, prognosis, and in establishing treatment regimens has enormous potential to revolutionize patient care.

Comparison of exosomes and ferritin protein nanocages for the delivery of membrane protein therapeutics.

PubMed

Cho, Eunji; Nam, Gi-Hoon; Hong, Yeonsun; Kim, Yoon Kyoung; Kim, Dong-Hwee; Yang, Yoosoo; Kim, In-San

2018-06-10

Exosomes are small membrane vesicles secreted by most cell types that play an important role in intercellular communication. Due to the characteristic of transferring their biomacromolecules, exosomes have potential as a new alternative for delivering protein therapeutics. Here, we investigate whether exosomes provide crucial advantages over other nanoparticles, in particular protein nanocage formulations, as a delivery system for membrane protein therapeutics. We characterized membrane-scaffold-based exosomes and protein-scaffold-based ferritin nanocages, both harboring SIRPα (signal regulatory protein α), an antagonist of CD47 on tumor cells. The efficacy of these two systems in delivering protein therapeutics was compared by testing their ability to enhance phagocytosis of tumor cells by bone-marrow-derived macrophages and subsequent inhibition of in vivo tumor growth. These analyses allowed us to comprehensively conclude that the therapeutic index of exosome-mediated CD47 blockade against tumor growth inhibition was higher than that of the same dose of ferritin-SIRPα. The results of this analysis reveal the importance of the unique characteristics of exosomes, in particular their membrane scaffold, in improving therapeutic protein delivery compared with protein-scaffold-based nanocages. Copyright © 2018 Elsevier B.V. All rights reserved.

Analysis of exosome purification methods using a model liposome system and tunable-resistive pulse sensing

NASA Astrophysics Data System (ADS)

Lane, Rebecca E.; Korbie, Darren; Anderson, Will; Vaidyanathan, Ramanathan; Trau, Matt

2015-01-01

Exosomes are vesicles which have garnered interest due to their diagnostic and therapeutic potential. Isolation of pure yields of exosomes from complex biological fluids whilst preserving their physical characteristics is critical for downstream applications. In this study, we use 100 nm-liposomes from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol as a model system as a model system to assess the effect of exosome isolation protocols on vesicle recovery and size distribution using a single-particle analysis method. We demonstrate that liposome size distribution and ζ-potential are comparable to extracted exosomes, making them an ideal model for comparison studies. Four different purification protocols were evaluated, with liposomes robustly isolated by three of them. Recovered yields varied and liposome size distribution was unaltered during processing, suggesting that these protocols do not induce particle aggregation. This leads us to conclude that the size distribution profile and characteristics of vesicles are stably maintained during processing and purification, suggesting that reports detailing how exosomes derived from tumour cells differ in size to those from normal cells are reporting a real phenomenon. However, we hypothesize that larger particles present in most purified exosome samples represent co-purified contaminating non-exosome debris. These isolation techniques are therefore likely nonspecific and may co-isolate non-exosome material of similar physical properties.

Exosome-mediated transfer of miR-10b promotes cell invasion in breast cancer.

PubMed

Singh, Ramesh; Pochampally, Radhika; Watabe, Kounosuke; Lu, Zhaohui; Mo, Yin-Yuan

2014-11-26

Exosomes are 30-100 nm membrane vesicles of endocytic origin, mediating diverse biological functions including tumor cell invasion, cell-cell communication and antigen presentation through transfer of proteins, mRNAs and microRNAs. Recent evidence suggests that microRNAs can be released through ceramide-dependent secretory machinery regulated by neutral sphingomyelinase 2 (nSMase2) enzyme encoded by the smpd3 gene that triggers exosome secretion. However, whether exosome-mediated microRNA transfer plays any role in cell invasion remains poorly understood. Thus, the aim of this study was to identify the exosomal microRNAs involved in breast cancer invasion. The expression level of endogenous and exosomal miRNAs were examined by real time PCR and the expression level of target proteins were detected by western blot. Scanning electron and confocal microscopy were used to characterize exosomes and to study its uptake and transfer. Luciferase reporter plasmids and its mutant were used to confirm direct targeting. Furthermore, the functional significance of exosomal miR-10b was estimated by invasion assay. In this study, we demonstrate that microRNA carrying exosomes can be transferred among different cell lines through direct uptake. miR-10b is highly expressed in metastatic breast cancer MDA-MB-231 cells as compared to non-metastatic breast cancer cells or non-malignant breast cells; it is actively secreted into medium via exosomes. In particular, nSMase2 or ceramide promotes the exosome-mediated miR-10b secretion whereas ceramide inhibitor suppresses this secretion. Moreover, upon uptake, miR-10b can suppress the protein level of its target genes such as HOXD10 and KLF4, indicating its functional significance. Finally, treatment with exosomes derived from MDA-MB-231 cells could induce the invasion ability of non-malignant HMLE cells. Together, our results suggest that a set of specific microRNAs may play an important role in modulating tumor microenvironment through

Exosomes: New players in cancer

PubMed Central

Guo, Wei; Gao, Yibo; Li, Ning; Shao, Fei; Wang, Chunni; Wang, Pan; Yang, Zhenlin; Li, Renda; He, Jie

2017-01-01

The past decade has witnessed an exponential increase in research on exosomes. For many years considered to be extracellular debris, exosomes are now considered important mediators in intercellular communication. The capability of exosomes to transfer proteins, DNA, mRNA, as well as non-coding RNAs has made them an attractive focus of research into the pathogenesis of different diseases, including cancer. Increasing evidence suggests that tumor cells release a large sum of exosomes, which may not only influence proximal tumor cells and stromal cells in local microenvironment, but also can exert systemic effects when participating in blood circulation. In this study, we review the current understanding on this topic. The literature outlines two broad facets of exosomes in cancer: 1) promotion of tumor growth, tumorigenesis, tumor angiogenesis, tumor immune escape, drug resistance, and metastasis and 2) their role as promising biomarkers for cancer diagnosis and even as potential treatment targets for cancer patients. PMID:28627679

Bovine milk exosome proteome

USDA-ARS?s Scientific Manuscript database

Exosomes are 40-100 nm membrane vesicles of endocytic origin and are found in blood, urine, amniotic fluid, bronchoalveolar lavage (BAL) fluid, as well as human and bovine milk. Exosomes are extracellular organelles important in intracellular communication/signaling, immune function, and biomarkers ...

High prevalence of mutant KRAS in circulating exosome-derived DNA from early-stage pancreatic cancer patients

PubMed Central

Allenson, K.; Castillo, J.; San Lucas, F. A.; Scelo, G.; Kim, D. U.; Bernard, V.; Davis, G.; Kumar, T.; Katz, M.; Overman, M. J.; Foretova, L.; Fabianova, E.; Holcatova, I.; Janout, V.; Meric-Bernstam, F.; Gascoyne, P.; Wistuba, I.; Varadhachary, G.; Brennan, P.; Hanash, S.; Li, D.; Maitra, A.; Alvarez, H.

2017-01-01

Background Exosomes arise from viable cancer cells and may reflect a different biology than circulating cell-free DNA (cfDNA) shed from dying tissues. We compare exosome-derived DNA (exoDNA) to cfDNA in liquid biopsies of patients with pancreatic ductal adenocarcinoma (PDAC). Patients and methods Patient samples were obtained between 2003 and 2010, with clinically annotated follow up to 2015. Droplet digital PCR was performed on exoDNA and cfDNA for sensitive detection of KRAS mutants at codons 12/13. A cumulative series of 263 individuals were studied, including a discovery cohort of 142 individuals: 68 PDAC patients of all stages; 20 PDAC patients initially staged with localized disease, with blood drawn after resection for curative intent; and 54 age-matched healthy controls. A validation cohort of 121 individuals (39 cancer patients and 82 healthy controls) was studied to validate KRAS detection rates in early-stage PDAC patients. Primary outcome was circulating KRAS status as detected by droplet digital PCR. Secondary outcomes were disease-free and overall survival. Results KRAS mutations in exoDNA, were identified in 7.4%, 66.7%, 80%, and 85% of age-matched controls, localized, locally advanced, and metastatic PDAC patients, respectively. Comparatively, mutant KRAS cfDNA was detected in 14.8%, 45.5%, 30.8%, and 57.9% of these individuals. Higher exoKRAS MAFs were associated with decreased disease-free survival in patients with localized disease. In the validation cohort, mutant KRAS exoDNA was detected in 43.6% of early-stage PDAC patients and 20% of healthy controls. Conclusions Exosomes are a distinct source of tumor DNA that may be complementary to other liquid biopsy DNA sources. A higher percentage of patients with localized PDAC exhibited detectable KRAS mutations in exoDNA than previously reported for cfDNA. A substantial minority of healthy samples demonstrated mutant KRAS in circulation, dictating careful consideration and application of liquid

Exosomes account for vesicle-mediated transcellular transport of activatable phospholipases and prostaglandins[S

PubMed Central

Subra, Caroline; Grand, David; Laulagnier, Karine; Stella, Alexandre; Lambeau, Gérard; Paillasse, Michael; De Medina, Philippe; Monsarrat, Bernard; Perret, Bertrand; Silvente-Poirot, Sandrine; Poirot, Marc; Record, Michel

2010-01-01

Exosomes are bioactive vesicles released from multivesicular bodies (MVB) by intact cells and participate in intercellular signaling. We investigated the presence of lipid-related proteins and bioactive lipids in RBL-2H3 exosomes. Besides a phospholipid scramblase and a fatty acid binding protein, the exosomes contained the whole set of phospholipases (A2, C, and D) together with interacting proteins such as aldolase A and Hsp 70. They also contained the phospholipase D (PLD) / phosphatidate phosphatase 1 (PAP1) pathway leading to the formation of diglycerides. RBL-2H3 exosomes also carried members of the three phospholipase A2 classes: the calcium-dependent cPLA2-IVA, the calcium-independent iPLA2-VIA, and the secreted sPLA2-IIA and V. Remarkably, almost all members of the Ras GTPase superfamily were present, and incubation of exosomes with GTPγS triggered activation of phospholipase A2 (PLA2)and PLD2. A large panel of free fatty acids, including arachidonic acid (AA) and derivatives such as prostaglandin E2 (PGE2) and 15-deoxy-Δ12,14-prostaglandinJ2 (15-d PGJ2), were detected. We observed that the exosomes were internalized by resting and activated RBL cells and that they accumulated in an endosomal compartment. Endosomal concentrations were in the micromolar range for prostaglandins; i.e., concentrations able to trigger prostaglandin-dependent biological responses. Therefore exosomes are carriers of GTP-activatable phospholipases and lipid mediators from cell to cell. PMID:20424270

Characterization of CLL exosomes reveals a distinct microRNA signature and enhanced secretion by activation of BCR signaling.

PubMed

Yeh, Yuh-Ying; Ozer, Hatice Gulcin; Lehman, Amy M; Maddocks, Kami; Yu, Lianbo; Johnson, Amy J; Byrd, John C

2015-05-21

Multiple studies show that chronic lymphocytic leukemia (CLL) cells are heavily dependent on their microenvironment for survival. Communication between CLL cells and the microenvironment is mediated through direct cell contact, soluble factors, and extracellular vesicles. Exosomes are small particles enclosed with lipids, proteins, and small RNAs that can convey biological materials to surrounding cells. Our data herein demonstrate that CLL cells release significant amounts of exosomes in plasma that exhibit abundant CD37, CD9, and CD63 expression. Our work also pinpoints the regulation of B-cell receptor (BCR) signaling in the release of CLL exosomes: BCR activation by α-immunoglobulin (Ig)M induces exosome secretion, whereas BCR inactivation via ibrutinib impedes α-IgM-stimulated exosome release. Moreover, analysis of serial plasma samples collected from CLL patients on an ibrutinib clinical trial revealed that exosome plasma concentration was significantly decreased following ibrutinib therapy. Furthermore, microRNA (miR) profiling of plasma-derived exosomes identified a distinct exosome microRNA signature, including miR-29 family, miR-150, miR-155, and miR-223 that have been associated with CLL disease. Interestingly, expression of exosome miR-150 and miR-155 increases with BCR activation. In all, this study successfully characterized CLL exosomes, demonstrated the control of BCR signaling in the release of CLL exosomes, and uncovered a disease-relevant exosome microRNA profile. © 2015 by The American Society of Hematology.

Reduction of saliva-promoted adhesion of Streptococcus mutans MT8148 and dental biofilm development by tragacanth gum and yeast-derived phosphomannan.

PubMed

Shimotoyodome, A; Kobayashi, H; Nakamura, J; Tokimitsu, I; Hase, T; Inoue, T; Matsukubo, T; Takaesu, Y

2006-01-01

The aim of this study was to investigate materials which reduce saliva-promoted adhesion of Streptococcus mutans onto enamel surfaces, and their potential in preventing dental biofilm development. The effects of hydroxyapatite (HA) surface pretreatment with hydrophilic polysaccharides on saliva-promoted S. mutans adhesion in vitro and de novo dental biofilm deposition in vivo were examined. Saliva-promoted adhesion of S. mutans MT8148 was significantly reduced by pretreatment of the HA surface with tragacanth gum (TG) and yeast-derived phosphoglycans. Extracellular phosphomannan (PM) from Pichia capsulata NRRL Y-1842 and TG reduced biofilm development on lower incisors in plaque-susceptible rats when administered via drinking water at concentrations of 0.5% and 0.01%, respectively. The inhibitory effect of TG on de novo dental biofilm formation was also demonstrated when administered via mouthwash in humans. It is concluded that TG and yeast-derived PM have the potential for use as anti-adherent agents and are effective in reducing de novo dental biofilm formation.

Exosomes from human colorectal cancer induce a tumor-like behavior in colonic mesenchymal stromal cells

PubMed Central

Lugini, Luana; Valtieri, Mauro; Federici, Cristina; Cecchetti, Serena; Meschini, Stefania; Condello, Maria; Signore, Michele; Fais, Stefano

2016-01-01

Background Cancer cells, including colorectal cancer ones (CRC), release high amounts of nanovesicles (exosomes), delivering biochemical messages for paracrine or systemic crosstalk. Mesenchymal stromal cells (MSCs) have been shown to play contradicting roles in tumor progression. Results CRC exosomes induce in cMSCs: i) atypical morphology, higher proliferation, migration and invasion; ii) formation of spheroids; iii) an acidic extracellular environment associated with iv) a plasma membrane redistribution of vacuolar H+-ATPase and increased expression of CEA. Colon cancer derived MSCs, which were isolated from tumor masses, produce umbilicated spheroids, a future frequently observed in the inner core of rapidly growing tumors and recapitulate the changes observed in normal colonic MSCs exposed to CRC exosomes. Materials and Methods Tissue specific colonic (c)MSCs were exposed to primary or metastatic CRC exosomes and analysed by light and electron microscopy, proliferation in 2D and 3D cultures, migration and invasion assays, Western blot and confocal microscopy for vacuolar H+-ATPase expression. Conclusions CRC exosomes are able to induce morphological and functional changes in colonic MSCs, which may favour tumor growth and its malignant progression. Our results suggest that exosomes are actively involved in cancer progression and that inhibiting tumor exosome release may represent a way to interfere with cancer. PMID:27418137

Exosomes from human colorectal cancer induce a tumor-like behavior in colonic mesenchymal stromal cells.

PubMed

Lugini, Luana; Valtieri, Mauro; Federici, Cristina; Cecchetti, Serena; Meschini, Stefania; Condello, Maria; Signore, Michele; Fais, Stefano

2016-08-02

Cancer cells, including colorectal cancer ones (CRC), release high amounts of nanovesicles (exosomes), delivering biochemical messages for paracrine or systemic crosstalk. Mesenchymal stromal cells (MSCs) have been shown to play contradicting roles in tumor progression. CRC exosomes induce in cMSCs: i) atypical morphology, higher proliferation, migration and invasion; ii) formation of spheroids; iii) an acidic extracellular environment associated with iv) a plasma membrane redistribution of vacuolar H+-ATPase and increased expression of CEA. Colon cancer derived MSCs, which were isolated from tumor masses, produce umbilicated spheroids, a future frequently observed in the inner core of rapidly growing tumors and recapitulate the changes observed in normal colonic MSCs exposed to CRC exosomes. Tissue specific colonic (c)MSCs were exposed to primary or metastatic CRC exosomes and analysed by light and electron microscopy, proliferation in 2D and 3D cultures, migration and invasion assays, Western blot and confocal microscopy for vacuolar H+-ATPase expression. CRC exosomes are able to induce morphological and functional changes in colonic MSCs, which may favour tumor growth and its malignant progression. Our results suggest that exosomes are actively involved in cancer progression and that inhibiting tumor exosome release may represent a way to interfere with cancer.

Integrated isolation and quantitative analysis of exosome shuttled proteins and nucleic acids using immunocapture approaches.

PubMed

Zarovni, Natasa; Corrado, Antonietta; Guazzi, Paolo; Zocco, Davide; Lari, Elisa; Radano, Giorgia; Muhhina, Jekatarina; Fondelli, Costanza; Gavrilova, Julia; Chiesi, Antonio

2015-10-01

Clinical implementation of exosome based diagnostic and therapeutic applications is still limited by the lack of standardized technologies that integrate efficient isolation of exosomes with comprehensive detection of relevant biomarkers. Conventional methods for exosome isolation based on their physical properties such as size and density (filtration, ultracentrifugation or density gradient), or relying on their differential solubility (chemical precipitation) are established primarily for processing of cell supernatants and later adjusted to complex biological samples such as plasma. Though still representing gold standard in the field, these methods are clearly suboptimal for processing of routine clinical samples and have intrinsic limits that impair their use in biomarker discovery and development of novel diagnostics. Immunoisolation (IA) offers unique advantages for the recovery of exosomes from complex and viscous fluids, in terms of increased efficiency and specificity of exosome capture, integrity and selective origin of isolated vesicles. We have evaluated several commercially available solutions for immunoplate- and immunobead-based affinity isolation and have further optimized protocols to decrease non-specific binding due to exosomes complexity and matrix contaminants. In order to identify best molecular targets for total exosome capture from diverse biological sources, as well as for selective enrichment in populations of interest (e.g. tumor derived exosomes) several exosome displayed proteins and respective antibodies have been evaluated for plate and bead functionalisation. Moreover, we have optimized and directly implemented downstream steps allowing on-line quantification and characterization of bound exosome markers, namely proteins and RNAs. Thus assembled assays enabled rapid overall quantification and validation of specific exosome associated targets in/on plasma exosomes, with multifold increased yield and enrichment ratio over benchmarking

Exosomal tumor microRNA modulates premetastatic organ cells.

PubMed

Rana, Sanyukta; Malinowska, Kamilla; Zöller, Margot

2013-03-01

Tumor exosomes educate selected host tissues toward a prometastatic phenotype. We demonstrated this for exosomes of the metastatic rat adenocarcinoma BSp73ASML (ASML), which modulate draining lymph nodes and lung tissue to support settlement of poorly metastatic BSp73ASML-CD44v4-v7 knockdown (ASML-CD44v(kd)) cells. Now, we profiled mRNA and microRNA (miRNA) of ASML(wt) and ASML-CD44v(kd) exosomes to define the pathway(s), whereby exosomes prepare the premetastatic niche. ASML exosomes, recovered in draining lymph nodes after subcutaneous injection, preferentially are taken up by lymph node stroma cells (LnStr) and lung fibroblasts (LuFb) that were chosen as exosome targets. ASML(wt) and ASML-CD44v(kd) exosomes contain a restricted mRNA and miRNA repertoire that differs significantly between the two lines and exosomes thereof due to CD44v6 influencing gene and miRNA transcription/posttranscriptional regulation. Exosomal mRNA and miRNA are recovered in target cells, where transferred miRNA significantly affected mRNA translation. Besides others, this was exemplified for abundant ASML(wt)-exosomal miR-494 and miR-542-3p, which target cadherin-17 (cdh17). Concomitantly, matrix metalloproteinase transcription, accompanying cdh17 down-regulation, was upregulated in LnStr transfected with miR-494 or miR-542-3p or co-cultured with tumor exosomes. Thus, tumor exosomes target non-transformed cells in premetastatic organs and modulate premetastatic organ cells predominantly through transferred miRNA, where miRNA from a metastasizing tumor prepares premetastatic organ stroma cells for tumor cell hosting. Fitting the demands of metastasizing tumor cells, transferred exosomal miRNA mostly affected proteases, adhesion molecules, chemokine ligands, cell cycle- and angiogenesis-promoting genes, and genes engaged in oxidative stress response. The demonstration of function-competent exosomal miRNA in host target cells encourages exploiting exosomes as a therapeutic gene delivery

CryoEM structure of yeast cytoplasmic exosome complex.

PubMed

Liu, Jun-Jie; Niu, Chu-Ya; Wu, Yao; Tan, Dan; Wang, Yang; Ye, Ming-Da; Liu, Yang; Zhao, Wenwei; Zhou, Ke; Liu, Quan-Sheng; Dai, Junbiao; Yang, Xuerui; Dong, Meng-Qiu; Huang, Niu; Wang, Hong-Wei

2016-07-01

The eukaryotic multi-subunit RNA exosome complex plays crucial roles in 3'-to-5' RNA processing and decay. Rrp6 and Ski7 are the major cofactors for the nuclear and cytoplasmic exosomes, respectively. In the cytoplasm, Ski7 helps the exosome to target mRNAs for degradation and turnover via a through-core pathway. However, the interaction between Ski7 and the exosome complex has remained unclear. The transaction of RNA substrates within the exosome is also elusive. In this work, we used single-particle cryo-electron microscopy to solve the structures of the Ski7-exosome complex in RNA-free and RNA-bound forms at resolutions of 4.2 Å and 5.8 Å, respectively. These structures reveal that the N-terminal domain of Ski7 adopts a structural arrangement and interacts with the exosome in a similar fashion to the C-terminal domain of nuclear Rrp6. Further structural analysis of exosomes with RNA substrates harboring 3' overhangs of different length suggests a switch mechanism of RNA-induced exosome activation in the through-core pathway of RNA processing.

Exosome cargo reflects TGF-β1-mediated epithelial-to-mesenchymal transition (EMT) status in A549 human lung adenocarcinoma cells.

PubMed

Kim, Jiyeon; Kim, Tae Yeon; Lee, Myung Shin; Mun, Ji Young; Ihm, Chunhwa; Kim, Soon Ae

2016-09-16

It has been suggested that tumor cells secrete exosomes to modify the local microenvironment, which then promotes intercellular communication and metastasis. Although exosomes derived from cancer cells may contribute to the epithelial-mesenchymal transition (EMT) in untransformed cells, few studies have defined exosome cargo upon induction of EMT. In this study, we investigated the changes in exosomal cargo from the epithelial to mesenchymal cell phenotype by inducing EMT with transforming growth factor (TGF)-β1 in A549 human lung adenocarcinoma cells. The protein content of the exosomes reflects the change in the cell phenotype. In addition, miR-23a was significantly enriched in the exosomes after mesenchymal transition. Following treatment of exosomes from mesenchymal cells via EMT induction with TGF-β1 to the epithelial cell type, phenotypic changes in protein expression level and cell morphology were observed. Autologous treatment of exosomes enhanced the transcriptional activity and abundance of β-catenin. Our results suggest that the exosomal protein and miRNA content reflects the physiological condition of its source and that exosomes induce phenotypic changes via autocrine signaling. Copyright © 2016 Elsevier Inc. All rights reserved.

Exosomes play an important role in the process of psoralen reverse multidrug resistance of breast cancer.

PubMed

Wang, Xiaohong; Xu, Chengfeng; Hua, Yitong; Sun, Leitao; Cheng, Kai; Jia, Zhongming; Han, Yong; Dong, Jianli; Cui, Yuzhen; Yang, Zhenlin

2016-12-01

Release of exosomes have been shown to play critical roles in drug resistance by delivering cargo. Targeting the transfer of exosomes from resistant cells to sensitive cells may be an approach to overcome some cases of drug resistance. In this study, we investigated the potential role of exosomes in the process of psoralen reverse multidrug resistance of MCF-7/ADR cells. Exosomes were isolated by differential centrifugation of culture media from MCF-7/ADR cells (ADR/exo) and MCF-7 parental cells (S/exo). Exosomes were characterized by morphology, exosomal markers and size distribution. The ability of ADR/exo to transfer multidrug resistance was assessed by MTT and real-time quantitative PCR. The different formation and secretion of exosomes were detected by immunofluorescence and transmission electron microscopy. Then we performed comparative transcriptomic analysis using RNA-Seq technology and real-time quantitative PCR to better understand the gene expression regulation in exosmes formation and release after psoralen treatment. Our data showed that exosomes derived from MCF-7/ADR cells were able to promote active sequestration of drugs and could induce a drug resistance phenotype by transferring drug-resistance-related gene MDR-1 and P-glycoprotein protein. Psoralen could reduce the formation and secretion of exosomes to overcome drug resistance. There were 21 differentially expressed genes. Gene ontology (GO) pathway analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the most significantly expressed genes were linked to PPAR and P53 signaling pathways which were related to exosomes formation, secretion and cargo sorting. Psoralen can affect the exosomes and induce the reduction of resistance transmission via exosomes might through PPAR and P53 signaling pathways, which might provide a novel strategy for breast cancer resistance to chemotherapy in the future.

The biology and function of exosomes in cancer.

PubMed

Kalluri, Raghu

2016-04-01

Humans circulate quadrillions of exosomes at all times. Exosomes are a class of extracellular vesicles released by all cells, with a size range of 40-150 nm and a lipid bilayer membrane. Exosomes contain DNA, RNA, and proteins. Exosomes likely remove excess and/or unnecessary constituents from the cells, functioning like garbage bags, although their precise physiological role remains unknown. Additionally, exosomes may mediate specific cell-to-cell communication and activate signaling pathways in cells they fuse or interact with. Exosomes are detected in the tumor microenvironment, and emerging evidence suggests that they play a role in facilitating tumorigenesis by regulating angiogenesis, immunity, and metastasis. Circulating exosomes can be used as liquid biopsies and noninvasive biomarkers for early detection, diagnosis, and treatment of cancer patients.

Evaluating Exosome Protein Content Changes Induced by Virus Activity Using SILAC Labeling and LC-MS/MS.

PubMed

Zhao, X; Xie, Y; Liu, J

2017-01-01

Exosomes are small membrane vesicles that are produced by cells and excreted into extracellular space. Contents of exosomes generally include lipid, membrane, and soluble proteins, and various types of coding and noncoding RNAs. Over the past decades, it has become clear that exosomes constitute an important vector for intercellular transport and communication with significant functional relevance. Evaluating exosome contents and their changes are vital for understanding its role in different physiological and pathological processes. Infection by certain pathogens, including viruses as well as intracellular bacteria, fungi, and parasites, has been shown to induce specific content changes in exosomes produced by infected cells. Evidences also indicate that exosomes produced by infected cells may actively participate in host-virus interactions, including immune responses. Studies of exosome content changes involve highly complex experimental and computational procedures, which can become even more complicated in the context of viral infections, due to the production and secretion of multiple virus-derived proteins and particles by infected cells. In this chapter, general and specific considerations relating to studies of exosome content changes induced by virus activities are discussed and illustrated with the detailed protocols previously used to identify protein content changes in Huh-7 cell exosomes induced by transfection with hepatitis B virus replicon plasmids, using SILAC labeling and LS-MS/MS. Hopefully, this would help enable more and further studies along similar lines and enhance the understanding of this new aspect of host-pathogen interactions. © 2017 Elsevier Inc. All rights reserved.

[The Functional Role of Exosomes in Cancer Biology and Their Potential as Biomarkers and Therapeutic Targets of Cancer].

PubMed

Naito, Yutaka; Yoshioka, Yusuke; Ochiya, Takahiro

2015-06-01

Intercellular communication plays an important role in the regulation of various cellular events. In particular, cancer cells and the surrounding cells communicate with each other, and this intercellular communication triggers cancer initiation and progression through the secretion of molecules, including growth factors and cytokines. Recent advances in cancer biology have indicated that small membrane vesicles, termed exosomes, also serve as regulatory agents in intercellular communications. Exosomes contain functional cellular components, including proteins and microRNAs (miRNAs), and they transfer these components to recipient cells. This exosome-mediated intercellular communication leads to increased growth, invasion, and metastasis of cancer. Thus, researchers regard exosomes as important cues to understanding the molecular mechanisms of cancer biology. Indeed, several lines of evidence have demonstrated that exosomes can explain multiple aspects of cancer biology. In addition, increasing evidence suggests that exosomes and their specific molecules are also attractive for use as biomarkers and therapeutic targets in cancer. Recent reports showed the efficacy of a novel diagnosis by detecting component molecules of cancer-derived exosomes, including miRNAs and membrane proteins. Furthermore, clinical trials that test the application of exosomes for cancer therapy have already been reported. From these points of view, we will summarize experimental data that support the role of exosomes in cancer progression and the potential of exosomes for use in novel diagnostic and therapeutic approaches for cancer.

Cell-Based and Exosome Therapy in Diabetic Stroke.

PubMed

Venkat, Poornima; Chopp, Michael; Chen, Jieli

2018-03-02

Stroke is a global health concern and it is imperative that therapeutic strategies with wide treatment time frames be developed to improve neurological outcome in patients. Patients with diabetes mellitus who suffer a stroke have worse neurological outcomes and long-term functional recovery than nondiabetic stroke patients. Diabetes induced vascular damage and enhanced inflammatory milieu likely contributes to worse post stroke outcomes. Diabetic stroke patients have an aggravated pathological cascade, and treatments that benefit nondiabetic stroke patients do not necessarily translate to diabetic stroke patients. Therefore, there is a critical need to develop therapeutics for stroke specifically in the diabetic population. Stem cell based therapy for stroke is an emerging treatment option with wide therapeutic time window. Cell-based therapies for stroke promote endogenous central nervous system repair and neurorestorative mechanisms such as angiogenesis, neurogenesis, vascular remodeling, white matter remodeling, and also modulate inflammatory and immune responses at the local and systemic level. Emerging evidence suggests that exosomes and their cargo microRNA mediate cell therapy derived neurorestorative effects. Exosomes are small vesicles containing protein and RNA characteristic of its parent cell. Exosomes are transported by biological fluids and facilitate communication between neighboring and remote cells. MicroRNAs, a class of naturally occurring, small noncoding RNA sequences, contained within exosomes can regulate recipient cell's signaling pathways and alter protein expression either acting alone or in concert with other microRNAs. In this perspective article, we summarize current knowledge and highlight the promising future of cell based and exosome therapy for stroke and specifically for diabetic stroke. Stem Cells Translational Medicine 2018. © 2018 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of

Identification of RNA-binding proteins in exosomes capable of interacting with different types of RNA: RBP-facilitated transport of RNAs into exosomes.

PubMed

Statello, Luisa; Maugeri, Marco; Garre, Elena; Nawaz, Muhammad; Wahlgren, Jessica; Papadimitriou, Alexandros; Lundqvist, Christina; Lindfors, Lennart; Collén, Anna; Sunnerhagen, Per; Ragusa, Marco; Purrello, Michele; Di Pietro, Cinzia; Tigue, Natalie; Valadi, Hadi

2018-01-01

The RNA that is packaged into exosomes is termed as exosomal-shuttle RNA (esRNA); however, the players, which take this subset of RNA (esRNA) into exosomes, remain largely unknown. We hypothesized that RNA binding proteins (RBPs) could serve as key players in this mechanism, by making complexes with RNAs and transporting them into exosomes during the biosynthesis of exosomes. Here, we demonstrate the presence of 30 RBPs in exosomes that were shown to form RNA-RBP complexes with both cellular RNA and exosomal-RNA species. To assess the involvement of these RBPs in RNA-transfer into exosomes, the gene transcripts encoding six of the proteins identified in exosomes (HSP90AB1, XPO5, hnRNPH1, hnRNPM, hnRNPA2B1, and MVP) were silenced by siRNA and subsequent effect on esRNA was assessed. A significant reduction of total esRNA was observed by post-transcriptional silencing of MVP, compared to other RBPs. Furthermore, to confirm the binding of MVP with esRNA, a biotinylated-MVP was transiently expressed in HEK293F cells. Higher levels of esRNA were recovered from MVP that was eluted from exosomes of transfected cells, as compared to those of non-transfected cells. Our data indicate that these RBPs could end up in exosomes together with RNA molecules in the form of RNA-ribonucleoprotein complexes, which could be important for the transport of RNAs into exosomes and the maintenance of RNAs inside exosomes. This type of maintenance may favor the shuttling of RNAs from exosomes to recipient cells in the form of stable complexes.