Cellular Internalization of Exosomes Occurs Through Phagocytosis

Exosomes play important roles in many physiological and pathological processes. However, the exosome–cell interaction mode and the intracellular trafficking pathway of exosomes in their recipient cells remain unclear. Here, we report that exosomes derived from K562 or MT4 cells are internalized more efficiently by phagocytes than by non‐phagocytic cells. Most exosomes were observed attached to the plasma membrane of non‐phagocytic cells, while in phagocytic cells these exosomes were found to enter via phagocytosis. Specifically, they moved to phagosomes together with phagocytic polystyrene carboxylate‐modified latex beads (biospheres) and were further sorted into phagolysosomes. Moreover, exosome internalization was dependent on the actin cytoskeleton and phosphatidylinositol 3‐kinase, and could be inhibited by the knockdown of dynamin2 or overexpression of a dominant‐negative form of dynamin2. Further, antibody pretreatment assays demonstrated that tim4 but not tim1 was involved in exosomes uptake. We also found that exosomes did not enter the internalization pathway involving caveolae, macropinocytosis and clathrin‐coated vesicles. Our observation that the cellular uptake of exosomes occurs through phagocytosis has important implications for exosome–cell interactions and the exosome intracellular trafficking pathway.     

Mesenchymal stroma/stem‐like cells of GARP knockdown inhibits cell proliferation and invasion of mouse colon cancer cells (MC38) through exosomes

Mesenchymal stroma/stem‐like cells (MSCs) have antitumour activity, and MSC‐derived exosomes play a role in the growth, metastasis and invasion of tumour cells. Additionally, glycoprotein A repetition predominant (GARP) promotes oncogenesis in breast cancer. Therefore, GARP is speculated to be a target gene for cancer therapy. We aimed to explore the therapy role of MSC‐derived exosomes targeting GARP in mouse colon cancer cell MC38. We successfully established a GARP knockdown system using three kinds of siRNA‐GARP in MSC cells. Exosomes were isolated from MSC and siGARP‐MSC cells, and verified by the exosome surface protein markers CD9, CD63 and CD81. GARP expression was significantly decreased in siGARP‐MSC exosomes compared with that of MSC exosomes. We found that siGARP‐MSC exosomes inhibited cell proliferation, migration and invasion of MC38 cells, using CCK‐8, colony formation, wound‐healing and Transwell invasion assays. Furthermore, siGARP‐MSC exosomes impeded IL‐6 secretion and partly inactivated JAK1/STAT3 pathway, measured using ELISA and RT‐qPCR. In conclusion, MSC‐derived exosomes targeting GARP are a potential strategy for cancer therapy.     

Activation-induced internalization differs for the tetraspanins CD9 and Tspan8: Impact on tumor cell motility

Exosomes are most important intercellular communicators and tetraspanins/tetraspanin-complexes have been suggested to play an important role in exosomal target cell selection. We have shown that only exosomes expressing a Tspan8-CD49d complex preferentially bind endothelial cells, which initiates angiogenesis. This finding was unexpected as in the exosome donor cell Tspan8 is associated with CD49c and the tetraspanins CD9 and CD151. In view of the discussed therapeutic power of exosomes as message/drug transporter, it became important to clarify the mechanisms accounting for the distinct Tspan8-web in the cell membrane versus exosomes. We therefore compared the route of Tspan8 and Tspan8-chimera internalization, where the N- and/or C-terminal regions were exchanged with the corresponding regions of CD9 or CD151. Activation-induced Tspan8-internalization proceeds more rapidly than CD9 internalization and is accompanied by disassembly of the Tspan8-CD9-CD151 membrane complex in resting cells. Tspan8-internalization relies on the association of the Tspan8 N-terminal region with intersectin-2, a multimodular complex involved in clathrin-coated pit internalization. Internalization and recovery of Tspan8 in early endosomes is further promoted by the recruitment of CD49d such that only in PMA-activated cells a Tspan8-INS2-CD49d-clathrin complex is recovered in cholesterol-depletion-resistant membrane microdomains. PMA-induced Tspan8-internalization promotes cell migration, but reduces matrix and cell adhesion. Thus, stimulation initiates tetraspanin-web rearrangements, which have strong functional consequences for the cell, exosome-delivery and exosome target selection. This knowledge will be essential for generating tailored therapeutic exosomes.     

Identification and characterization of hADSC-derived exosome proteins from different isolation methods

Exosomes are secreted into the extracellular space by most cell types and contain various molecular constituents, which play roles in many biological processes. Adipose-derived mesenchymal stem cells (ADSCs) can differentiate into a variety of cell types and secrete a series of paracrine factors through exosomes. ADSC-derived exosomes have shown diagnostic and therapeutic potential in many clinical diseases. The molecular components are critical for their mechanisms. Several methods have been developed for exosome purification, including ultracentrifugation, ultrafiltration, density gradient purification, size-based isolation, polymer precipitation and immuno-affinity purification. Thus, we employed four methods to isolate exosomes from the hADSC culture medium, including ultracentrifugation, size exclusion chromatography, ExoQuick-TC precipitation and ExoQuick-TC ULTRA isolation. Following exosome isolation, we performed quantitative proteomic analysis of the exosome proteins using isobaric tags for relative and absolute quantification (iTRAQ) labelling, combined with 2D-LC-MS/MS. There were 599 universal and 138 stably expressed proteins in hADSC-derived exosomes. We proved that these proteins were potential hADSC-derived exosomes markers, including CD109, CD166, HSPA4, TRAP1, RAB2A, RAB11B and RAB14. From the quantitative proteomic analysis, we demonstrated that hADSC-derived exosome protein expression varied, with lipopolysaccharide (LPS) treatment, in the different isolation methods. Pathway analysis and proliferation, migration and endothelial tube formation assays showed varying effects in cells stimulated with hADSC-derived exosomes from different isolation methods. Our study revealed that different isolation methods might introduce variations in the protein composition in exosomes, which reflects their effects on biological function. The pros and cons of these methods are important points to consider for downstream research applications.     

Induction of exosome release in primary B cells stimulated via CD40 and the IL-4 receptor

Exosomes are lipid-bound nanovesicles formed by inward budding of the endosomal membrane and released following fusion of the endosomal limiting membrane with the plasma membrane. We show here that primary leukocytes do not release exosomes unless subjected to potent activation signals, such as cytokine or mitogen stimulation. In particular, high levels of exosomes were released when murine splenic B cells were stimulated via CD40 and the IL-4 receptor. This property was shared by B cells from different anatomic locations, as newly formed marginal zone and follicular B cells were capable of secreting exosomes upon CD40/IL-4 triggering. B cell exosomes expressed high levels of MHC class I, MHC class II, and CD45RA (B220), as well as components of the BCR complex, namely, surface Ig, CD19, and the tetraspanins CD9 and CD81. Ig on the plasma membrane of primary B cells was targeted to the exosome pathway, demonstrating a link between the BCR and this exocytic pathway. IgD and IgM were the predominant Ig isotypes associated with CD40/IL-4 elicited exosomes, though other isotypes (IgA, IgG1, IgG2a/2b, and IgG3) were also detected. Together, these results suggest that exosome release is not constitutive activity of B cells, but may be induced following cell: cell signaling.     

Targeting endothelial exosomes for the prevention of cardiovascular disease

Exosomes are small lipid bilayer-enclosed 30–140 nm diameter vesicles formed from endosomes. Exosomes are secreted by various cell types including endothelial cells, immune cells and other cardiovascular tissues, and they can be detected in plasma, urine, cerebrospinal fluid, as well as tissues. Exosomes were initially regarded as a disposal mechanism to discard unwanted materials from cells. Recent studies suggest that exosomes play an important role in mediating of intercellular communication through the delivery and transport of cellular components such as nucleic acids, lipids, and proteins and thus regulate cardiovascular disease. Further, the underlying mechanisms by which abnormally released exosomes promote cardiovascular disease are not well understood. This review highlights recent studies involving endothelial exosomes, gives a brief overview of exosome biogenesis and release, isolation and identification of exosomes, and provides a contemporary understanding of the endothelial exosome pathophysiology and potential therapeutic strategies.     

Physical exosome:exosome interactions

Exosomes are extracellular nanovesicles that mediate a number of cellular processes, including intracellular signalling. There are many published examples of exosome–exosome dimers; however, their relevance has not been explored. Here, we propose that cells release exosomes to physically interact with incoming exosomes, forming dimers that we hypothesize attenuate incoming exosome‐mediated signalling. We discuss experiments to test this hypothesis and potential relevance in health and disease.     

Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions

Exosomes regulate cell behavior by binding to and delivering their cargo to target cells; however, the mechanisms mediating exosome-cell interactions are poorly understood. Heparan sulfates on target cell surfaces can act as receptors for exosome uptake, but the ligand for heparan sulfate on exosomes has not been identified. Using exosomes isolated from myeloma cell lines and from myeloma patients, we identify exosomal fibronectin as a key heparan sulfate-binding ligand and mediator of exosome-cell interactions. We discovered that heparan sulfate plays a dual role in exosome-cell interaction; heparan sulfate on exosomes captures fibronectin, and on target cells it acts as a receptor for fibronectin. Removal of heparan sulfate from the exosome surface releases fibronectin and dramatically inhibits exosome-target cell interaction. Antibody specific for the Hep-II heparin-binding domain of fibronectin blocks exosome interaction with tumor cells or with marrow stromal cells. Regarding exosome function, fibronectin-mediated binding of exosomes to myeloma cells activated p38 and pERK signaling and expression of downstream target genes DKK1 and MMP-9, two molecules that promote myeloma progression. Antibody against fibronectin inhibited the ability of myeloma-derived exosomes to stimulate endothelial cell invasion. Heparin or heparin mimetics including Roneparstat, a modified heparin in phase I trials in myeloma patients, significantly inhibited exosome-cell interactions. These studies provide the first evidence that fibronectin binding to heparan sulfate mediates exosome-cell interactions, revealing a fundamental mechanism important for exosome-mediated cross-talk within tumor microenvironments. Moreover, these results imply that therapeutic disruption of fibronectin-heparan sulfate interactions will negatively impact myeloma tumor growth and progression.     

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

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 radiation response.     

Regulation of exosome production and cargo sorting

Cellular communication can be mediated by the exchange of biological information, mainly in the form of proteins and RNAs. This can occur when extracellular vesicles, such as exosomes, secreted by a donor cell are internalized by an acceptor cell. Exosomes bear specific repertoires of proteins and RNAs, indicating the existence of mechanisms that control the sorting of molecules into them. Knowledge about loadings and processes and mechanisms of cargo sorting of exosomes is essential to shed light on the physiological and pathological functions of these vesicles as well as on clinical applications involving their use and/or analysis. In this review, we will discuss the molecular mechanisms associated with exosome secretion and their specific cargo sorting, with special attention to the sorting of RNAs and proteins, and thus the outcome and the emerging therapeutic opportunities of the communication between the exosome-producer and recipient cells.     

外泌体介导的靶向基因运送及在心血管疾病中的应用

外泌体是体内几乎所有细胞分泌的具有双层脂质膜结构的纳米级小囊泡。外泌体大小均匀,平均直径为40～120 nm,存在于所有体液中。外泌体曾一度被认为是细胞成熟过程中清除废弃细胞器的‘垃圾袋’。但近年研究显示：外泌体含有丰富的来源于‘供体细胞’的信号分子,如蛋白质、DNA、mRNA、miRNA以及lncRNA等。当外泌体与‘受体细胞’融合时,这些信号分子便被运送到‘受体细胞’,从而实现细胞 细胞之间的通讯,影响‘受体细胞’的生理病理过程。虽然外泌体的研究目前主要集中在癌症等疾病的预防、诊断与治疗中,但是越来越多的研究显示,外泌体在心血管系统的生理及病理过程中同样发挥着重要作用。本文将对外泌体的起源、分离与纯化方法及外泌体介导的‘细胞 细胞’之间的通讯机制进行综述,并重点论述利用基因工程技术对外泌体进行靶向运输的方法及靶向外泌体运送在心血管疾病治疗中的应用。     

Heparanase activates the syndecan-syntenin-ALIX exosome pathway

Exosomes are secreted vesicles of endosomal origin involved in signaling processes. We recently showed that the syndecan heparan sulfate proteoglycans control the biogenesis of exosomes through their interaction with syntenin-1 and the endosomal-sorting complex required for transport accessory component ALIX. Here we investigated the role of heparanase, the only mammalian enzyme able to cleave heparan sulfate internally, in the syndecan-syntenin-ALIX exosome biogenesis pathway. We show that heparanase stimulates the exosomal secretion of syntenin-1, syndecan and certain other exosomal cargo, such as CD63, in a concentration-dependent manner. In contrast, exosomal CD9, CD81 and flotillin-1 are not affected. Conversely, reduction of endogenous heparanase reduces the secretion of syntenin-1-containing exosomes. The ability of heparanase to stimulate exosome production depends on syntenin-1 and ALIX. Syndecans, but not glypicans, support exosome biogenesis in heparanase-exposed cells. Finally, heparanase stimulates intraluminal budding of syndecan and syntenin-1 in endosomes, depending on the syntenin-ALIX interaction. Taken together, our findings identify heparanase as a modulator of the syndecan-syntenin-ALIX pathway, fostering endosomal membrane budding and the biogenesis of exosomes by trimming the heparan sulfate chains on syndecans. In addition, our data suggest that this mechanism controls the selection of specific cargo to exosomes.     

Exosomes containing glycoprotein 350 released by EBV-transformed B cells selectively target B cells through CD21 and block EBV infection in vitro

Exosomes are nano-sized membrane vesicles released from a wide variety of cells, formed in endosomes by inward budding of the endosomal limiting membrane. They have immune stimulatory-, inhibitory-, or tolerance-inducing effects, depending on their cellular origin, which is why they are investigated for use in vaccine and immune therapeutic strategies. In this study, we explored whether exosomes of different origins and functions can selectively target different immune cells in human peripheral blood. Flow cytometry, confocal laser scanning microscopy, and multispectral imaging flow cytometry (ImageStream) revealed that exosomes derived from human monocyte-derived dendritic cells and breast milk preferably associated with monocytes. In contrast, exosomes from an EBV-transformed B cell line (LCL1) preferentially targeted B cells. This was not observed for an EBV(-) B cell line (BJAB). Electron microscopy, size-distribution analysis (NanoSight), and a cord blood transformation assay excluded the presence of virions in our LCL1 exosome preparations. The interaction between LCL1-derived exosomes and peripheral blood B cells could be blocked efficiently by anti-CD21 or anti-gp350, indicating an interaction between CD21 on B cells and the EBV glycoprotein gp350 on exosomes. The targeting of LCL1-derived exosomes through gp350-CD21 interaction strongly inhibited EBV infection in B cells isolated from umbilical cord blood, suggesting a protective role for exosomes in regulating EBV infection. Our finding also suggests that exosome-based vaccines can be engineered for specific B cell targeting by inducing gp350 expression.     

Cancer-associated fibroblasts–derived exosomes-mediated transfer of LINC00355 regulates bladder cancer cell proliferation and invasion

The aim of this study was to investigate the regulatory mechanism of cancer-associated fibroblasts (CAFs) exosome in bladder cancer (BC) cell proliferation and invasion. CAFs and normal fibroblasts (NFs) were isolated from tumor tissues and adjacent normal tissues of BC patients, and examined by immunocytochemistry for the expression of fibroblast activation protein alpha (FAP) and α-smooth muscle actin (α-SMA). Exosomes were extracted from CAFs and NFs and observed under a transmission electron microscope, and expression of the exosome markers CD9 and CD63 was confirmed by western blotting. The distribution and intensity of fluorescence were observed by confocal laser microscopy to analyze exosomes uptake by BC cell lines T24 or 5367. BC cell proliferation and invasion were detected by MTT and Transwell assays, respectively. LINC00355 levels in CAFs, NFs, CAFs exosome, NFs exosome, and BC cells were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Results showed that CAFs exosome significantly promoted BC cell proliferation and invasion relative to NFs exosome. LINC00355 expression was significantly elevated in CAFs exosome when compared with that in NFs-exosome. Up-regulated LINC00355 expression was observed both in T24 and 5367 cells co-incubated with CAFs exosome. Exosomes derived from LINC00355 siRNA-transfected CAFs observably repressed BC cell proliferation and invasion when compared with control siRNA-CAFs exosome. In conclusion, CAFs exosome–mediated transfer of LINC00355 regulates BC cell proliferation and invasion. Significance of the study. In this study, our data suggest that the exosomes released from CAFs promote BC cell proliferation and invasion. The mechanism of this effect is, at least in part, related to the increased LINC00355. Regulation of LINC00355 expression in exosomes released from CAFs might be a putative therapeutic strategy against the pathogenesis of BC.     

CD8+ dendritic cells use LFA-1 to capture MHC-peptide complexes from exosomes in vivo

Exosomes are secreted vesicles formed in late endocytic compartments. Mature dendritic cells (DCs) secrete exosomes bearing functional MHC-peptide complexes and high levels of ICAM-1. Such exosomes can activate Ag-specific naive T cells but only after recapture by recipient APCs. In this study, we addressed the molecular mechanisms of interaction between exosomes and recipient DCs. We show that exosomes can be presented by mouse DCs without the need for internalization and processing. Exosomes interact with DCs through a specific saturable receptor. Although the two major ligands of ICAM-1, LFA-1 and Mac-1, are expressed by lymphoid organ DCs, only LFA-1 is required for exosome capture by these cells. Accordingly, we show that CD8(+) DCs express higher levels of LFA-1 than CD8(-) DCs, and that they are the main recipients of exosomes in vivo. We propose a new role for LFA-1 on DCs, as a receptor for exosomes to favor Ag transfer between DCs in vivo.     

Ionizing Radiation and Glioblastoma Exosomes: Implications in Tumor Biology and Cell Migration

Exosomes are nanometer-sized lipid vesicles released ubiquitously by cells, which have been shown to have a normal physiological role, as well as influence the tumor microenvironment and aid metastasis. Recent studies highlight the ability of exosomes to convey tumor-suppressive and oncogenic mRNAs, microRNAs, and proteins to a receiving cell, subsequently activating downstream signaling pathways and influencing cellular phenotype. Here, we show that radiation increases the abundance of exosomes released by glioblastoma cells and normal astrocytes. Exosomes derived from irradiated cells enhanced the migration of recipient cells, and their molecular profiling revealed an abundance of molecules related to signaling pathways important for cell migration. In particular, connective tissue growth factor (CTGF) mRNA and insulin-like growth factor binding protein 2 (IGFBP2) protein levels were elevated, and coculture of nonirradiated cells with exosomes isolated from irradiated cells increased CTGF protein expression in the recipient cells. Additionally, these exosomes enhanced the activation of neurotrophic tyrosine kinase receptor type 1 (TrkA), focal adhesion kinase, Paxillin, and proto-oncogene tyrosine-protein kinase Src (Src) in recipient cells, molecules involved in cell migration. Collectively, our data suggest that radiation influences exosome abundance, specifically alters their molecular composition, and on uptake, promotes a migratory phenotype.     

Towards analyzing the potential of exosomes to deliver microRNA therapeutics

Exosome selectivity mechanisms underlying exosome–target cell interactions and the specific traits affecting their capability to communicate still remain unclear. Moreover, the capacity of exosomes to efficiently deliver their molecular cargos intracellularly needs precise investigation towards establishing functional exosome‐based delivery platforms exploitable in the clinical practice. The current study focuses on: (a) exosome production from normal MRC‐5 and Vero cells growing in culture, (b) physicochemical characterization by dynamic light scattering (DLS) and cryo‐transmission electron microscopy; (c) cellular uptake studies of rhodamine‐labeled exosomes in normal and cancer cells, providing to exosomes either “autologous” or “heterologous” cellular delivery environments; and (d) loading exogenous Alexa Fluor 488‐labeled siRNA into exosomes for the assessment of their delivering capacity by immunofluorescence in a panel of recipient cells. The data obtained thus far indicate that MRC‐5 and Vero exosomes, indeed exhibit an interesting delivering profile, as promising “bio‐shuttles,” being pharmacologically exploitable in the context of theranostic applications.     

The role of exosomes in hepatitis,liver cirrhosis and hepatocellular carcinoma

Exosomes are small vesicles that were initially thought to be a mechanism for discarding unneeded membrane proteins from reticulocytes. Their mediation of intercellular communication appears to be associated with several biological functions. Current studies have shown that most mammalian cells undergo the process of exosome formation and utilize exosome‐mediated cell communication. Exosomes contain various microRNAs, mRNAs and proteins. They have been reported to mediate multiple functions, such as antigen presentation, immune escape and tumour progression. This concise review highlights the findings regarding the roles of exosomes in liver diseases, particularly hepatitis B, hepatitis C, liver cirrhosis and hepatocellular carcinoma. However, further elucidation of the contributions of exosomes to intercellular information transmission is needed. The potential medical applications of exosomes in liver diseases seem practical and will depend on the ingenuity of future investigators and their insights into exosome‐mediated biological processes.     

Cancer exosomes express CD39 and CD73, which suppress T cells through adenosine production

Extracellular adenosine is elevated in cancer tissue, and it negatively regulates local immune responses. Adenosine production from extracellular ATP has attracted attention as a mechanism of regulatory T cell-mediated immune regulation. In this study, we examined whether small vesicles secreted by cancer cells, called exosomes, contribute to extracellular adenosine production and hence modulate immune effector cells indirectly. We found exosomes from diverse cancer cell types exhibit potent ATP- and 5'AMP-phosphohydrolytic activity, partly attributed to exosomally expressed CD39 and CD73, respectively. Comparable levels of activity were seen with exosomes from pleural effusions of mesothelioma patients. In such fluids, exosomes accounted for 20% of the total ATP-hydrolytic activity. Exosomes can perform both hydrolytic steps sequentially to form adenosine from ATP. This exosome-generated adenosine can trigger a cAMP response in adenosine A(2A) receptor-positive but not A(2A) receptor-negative cells. Similarly, significantly elevated cAMP was also triggered in Jurkat cells by adding exosomes with ATP but not by adding exosomes or ATP alone. A proportion of healthy donor T cells constitutively express CD39 and/or CD73. Activation of T cells by CD3/CD28 cross-linking could be inhibited by exogenously added 5'AMP in a CD73-dependent manner. However, 5'AMP converted to adenosine by exosomes inhibits T cell activation independently of T cell CD73 expression. This T cell inhibition was mediated through the adenosine A(2A) receptor. In summary, the data highlight exosome enzymic activity in the production of extracellular adenosine, and this may play a contributory role in negative modulation of T cells in the tumor environment.     

Detachment of breast tumor cells induces rapid secretion of exosomes which subsequently mediate cellular adhesion and spreading

Exosomes are nano-vesicles secreted by a wide range of mammalian cell types. These vesicles are abundant in serum and other extracellular fluids and contain a large repertoire of proteins, mRNA and microRNA. Exosomes have been implicated in cell to cell communication, the transfer of infectious agents, and neurodegenerative diseases as well as tumor progression. However, the precise mechanisms by which they are internalized and/or secreted remain poorly understood. In order to follow their release and uptake in breast tumor cells in real time, cell-derived exosomes were tagged with green fluorescent protein (GFP)-CD63 while human serum exosomes were rhodamine isothiocynate-labeled. We show that detachment of adherent cells from various substrata induces a rapid and substantial secretion of exosomes, which then concentrate on the cell surfaces and mediate adhesion to various extracellular matrix proteins. We also demonstrate that disruption of lipid rafts with methyl-beta-cyclodextrin (MβCD) inhibits the internalization of exosomes and that annexins are essential for the exosomal uptake mechanisms. Taken together, these data suggest that cellular detachment is accompanied by significant release of exosomes while cellular adhesion and spreading are enhanced by rapid uptake and disposition of exosomes on the cell surface.