Exosomes released from breast cancer carcinomas stimulate cell movement

For metastasis to occur cells must communicate with to their local environment to initiate growth and invasion. Exosomes have emerged as an important mediator of cell-to-cell signalling through the transfer of molecules such as mRNAs, microRNAs, and proteins between cells. Exosomes have been proposed to act as regulators of cancer progression. Here, we study the effect of exosomes on cell migration, an important step in metastasis. We performed cell migration assays, endocytosis assays, and exosome proteomic profiling on exosomes released from three breast cancer cell lines that model progressive stages of metastasis. Results from these experiments suggest: (1) exosomes promote cell migration and (2) the signal is stronger from exosomes isolated from cells with higher metastatic potentials; (3) exosomes are endocytosed at the same rate regardless of the cell type; (4) exosomes released from cells show differential enrichment of proteins with unique protein signatures of both identity and abundance. We conclude that breast cancer cells of increasing metastatic potential secrete exosomes with distinct protein signatures that proportionally increase cell movement and suggest that released exosomes could play an active role in metastasis

Exosomes regulate neurogenesis and circuit assembly.

Exosomes are thought to be released by all cells in the body and to be involved in intercellular communication. We tested whether neural exosomes can regulate the development of neural circuits. We show that exosome treatment increases proliferation in developing neural cultures and in vivo in dentate gyrus of P4 mouse brain. We compared the protein cargo and signaling bioactivity of exosomes released by hiPSC-derived neural cultures lacking MECP2, a model of the neurodevelopmental disorder Rett syndrome, with exosomes released by isogenic rescue control neural cultures. Quantitative proteomic analysis indicates that control exosomes contain multiple functional signaling networks known to be important for neuronal circuit development. Treating MECP2-knockdown human primary neural cultures with control exosomes rescues deficits in neuronal proliferation, differentiation, synaptogenesis, and synchronized firing, whereas exosomes from MECP2-deficient hiPSC neural cultures lack this capability. These data indicate that exosomes carry signaling information required to regulate neural circuit development

Exosomes from Human Adipose Tissue-Derived Mesenchymal Stem Cells Promote Epidermal Barrier Repair by Inducing de Novo Synthesis of Ceramides in Atopic Dermatitis.

Atopic dermatitis (AD) is a multifactorial, heterogeneous disease associated with epidermal barrier disruption and intense systemic inflammation. Previously, we showed that exosomes derived from human adipose tissue-derived mesenchymal stem cells (ASC-exosomes) attenuate AD-like symptoms by reducing multiple inflammatory cytokine levels. Here, we investigated ASC-exosomes' effects on skin barrier restoration by analyzing protein and lipid contents. We found that subcutaneous injection of ASC-exosomes in an oxazolone-induced dermatitis model remarkably reduced trans-epidermal water loss, while enhancing stratum corneum (SC) hydration and markedly decreasing the levels of inflammatory cytokines such as IL-4, IL-5, IL-13, TNF-α, IFN-γ, IL-17, and TSLP, all in a dose-dependent manner. Interestingly, ASC-exosomes induced the production of ceramides and dihydroceramides. Electron microscopic analysis revealed enhanced epidermal lamellar bodies and formation of lamellar layer at the interface of the SC and stratum granulosum with ASC-exosomes treatment. Deep RNA sequencing analysis of skin lesions demonstrated that ASC-exosomes restores the expression of genes involved in skin barrier, lipid metabolism, cell cycle, and inflammatory response in the diseased area. Collectively, our results suggest that ASC-exosomes effectively restore epidermal barrier functions in AD by facilitating the de novo synthesis of ceramides, resulting in a promising cell-free therapeutic option for treating AD

Preclinical translation of exosomes derived from mesenchymal stem/stromal cells.

Exosomes are nanovesicles secreted by virtually all cells. Exosomes mediate the horizontal transfer of various macromolecules previously believed to be cell-autonomous in nature, including nonsecretory proteins, various classes of RNA, metabolites, and lipid membrane-associated factors. Exosomes derived from mesenchymal stem/stromal cells (MSCs) appear to be particularly beneficial for enhancing recovery in various models of disease. To date, there have been more than 200 preclinical studies of exosome-based therapies in a number of different animal models. Despite a growing number of studies reporting the therapeutic properties of MSC-derived exosomes, their underlying mechanism of action, pharmacokinetics, and scalable manufacturing remain largely outstanding questions. Here, we review the global trends associated with preclinical development of MSC-derived exosome-based therapies, including immunogenicity, source of exosomes, isolation methods, biodistribution, and disease categories tested to date. Although the in vivo data assessing the therapeutic properties of MSC-exosomes published to date are promising, several outstanding questions remain to be answered that warrant further preclinical investigation

Lysosomal Delivery of Bioactive Proteins to Living Human Cells via Engineered Exosomes

Exosomes are naturally secreted nanovesicles derived from mammalian cells that are used for intercellular communication in vivo. As a result, they can potentially be used for intracellular delivery of therapeutics for disease treatment. We have developed an exosome pseudotyping approach using vesicular stomatitis virus glycoprotein (VSVG) to produce protein chimeras that optimize production of modified exosomes containing protein therapeutics and facilitate effective delivery to their target cells. To the VSVG transmembrane scaffold, we have fused both fluorescent and luminescent reporters for exosome tracking/visualization and quantification of activity respectively. Through our design, we have shown the biogenesis of VSVG modified exosomes from transfected producer cells through fluorescence imaging and the production of a VSVG-based stable cell line. In addition, we have characterized isolated engineered exosomes and shown that they exhibited the correct size, distribution, and molecular markers, while retaining the bioactivity of their protein cargo. Furthermore, we show that our engineered exosomes and their protein cargo are internalized by multiple cell lines into the endosomal and lysosomal compartments of those cells. Lastly, these modified exosomes can confer their bioactive cargo, either a luminescent reporter or puromycin resistance into these target cells. In summary, this study presents a novel approach to exosome engineering to enhance therapeutic protein loading and delivery, and more importantly, shows the delivery of modified exosomes to intracellular lysosomal compartments. This aspect leads to the assumption that in future studies, these engineered exosomes can be used as a vehicle for delivery of therapeutic proteins for treatment of lysosomal storage diseases

Targeted delivery of lysosomal enzymes to the endocytic compartment in human cells using engineered extracellular vesicles.

Targeted delivery of lysosomal enzymes to the endocytic compartment of human cells represents a transformative technology for treating a large family of lysosomal storage diseases (LSDs). Gaucher disease is one of the most common types of LSDs caused by mutations to the lysosomal β-glucocerebrosidase (GBA). Here, we describe a genetic strategy to produce engineered exosomes loaded with GBA in two different spatial configurations for targeted delivery to the endocytic compartment of recipient cells. By fusing human GBA to an exosome-anchoring protein: vesicular stomatitis virus glycoprotein (VSVG), we demonstrate that the chimeric proteins were successfully integrated into exosomes which were secreted as extracellular vesicles (EVs) by producer cells. Isolation and molecular characterization of EVs confirmed that the fusion proteins were loaded onto exosomes without altering their surface markers, particle size or distribution. Further, enzyme-loaded exosomes/EVs added to cultured medium were taken up by recipient cells. Further, the endocytosed exosomes/EVs targeted to endocytic compartments exhibited a significant increase in GBA activity. Together, we have developed a novel method for targeting and delivery of lysosomal enzymes to their natural location: the endocytic compartment of recipient cells. Since exosomes/EVs have an intrinsic ability to cross the blood-brain-barrier, our technology may provide a new approach to treat severe types of LSDs, including Gaucher disease with neurological complications

Exosomes containing HIV protein Nef reorganize lipid rafts potentiating inflammatory response in bystander cells.

HIV infection has a profound effect on "bystander" cells causing metabolic co-morbidities. This may be mediated by exosomes secreted by HIV-infected cells and containing viral factors. Here we show that exosomes containing HIV-1 protein Nef (exNef) are rapidly taken up by macrophages releasing Nef into the cell interior. This caused down-regulation of ABCA1, reduction of cholesterol efflux and sharp elevation of the abundance of lipid rafts through reduced activation of small GTPase Cdc42 and decreased actin polymerization. Changes in rafts led to re-localization of TLR4 and TREM-1 to rafts, phosphorylation of ERK1/2, activation of NLRP3 inflammasome, and increased secretion of pro-inflammatory cytokines. The effects of exNef on lipid rafts and on inflammation were reversed by overexpression of a constitutively active mutant of Cdc42. Similar effects were observed in macrophages treated with exosomes produced by HIV-infected cells or isolated from plasma of HIV-infected subjects, but not with exosomes from cells and subjects infected with ΔNef-HIV or uninfected subjects. Mice injected with exNef exhibited monocytosis, reduced ABCA1 in macrophages, increased raft abundance in monocytes and augmented inflammation. Thus, Nef-containing exosomes potentiated pro-inflammatory response by inducing changes in cholesterol metabolism and reorganizing lipid rafts. These mechanisms may contribute to HIV-associated metabolic co-morbidities

Exosome-mediated Transfer of αvβ3 Integrin from Tumorigenic to Nontumorigenic Cells Promotes a Migratory Phenotype.

The αvβ3 integrin is known to be highly upregulated during cancer progression and promotes a migratory and metastatic phenotype in many types of tumors. We hypothesized that the αvβ3 integrin is transferred through exosomes and, upon transfer, has the ability to support functional aberrations in recipient cells. Here, for the first time, it is demonstrated that αvβ3 is present in exosomes released from metastatic PC3 and CWR22Pc prostate cancer cells. Exosomal β3 is transferred as a protein from donor to nontumorigenic and tumorigenic cells as β3 protein or mRNA levels remain unaffected upon transcription or translation inhibition in recipient cells. Furthermore, it is shown that upon exosome uptake, de novo expression of an αvβ3 increases adhesion and migration of recipient cells on an αvβ3 ligand, vitronectin. To evaluate the relevance of these findings, exosomes were purified from the blood of TRAMP mice carrying tumors where the expression of αvβ3 is found higher than in exosomes from wild-type mice. In addition, it is demonstrated that αvβ3 is coexpressed with synaptophysin, a biomarker for aggressive neuroendocrine prostate cancer. IMPLICATIONS: Overall this study reveals that the αvβ3 integrin is transferred from tumorigenic to nontumorigenic cells via exosomes, and its de novo expression in recipient cells promotes cell migration on its ligand. The increased expression of αvβ3 in exosomes from mice bearing tumors points to its clinical relevance and potential use as a biomarker. Mol Cancer Res; 14(11); 1136-46. ©2016 AACR

Production of Living Nanoparticles for Blood Cancer Therapy

Current cancer therapies leave much to be desired because they are very harmful to the patient and cause a significant decrease in quality of life. Chimeric Antigen Receptors (CAR) are a promising novel approach for treating specific types of leukemia due to their binding affinity for proteins expressed solely on leukemia B cells. This approach increases specificity of how cells receive treatment, thus allowing for the destruction of cancerous cells while leaving the healthy cells unharmed. In this experiment, we show that production of CAR expressing exosomes (liposome like vesicles produced naturally by human cells) is possible through cell transfection. This finding demonstrates that a new wave of cancer therapeutics, that are more specific and have less harmful side effects, are producible

Digital detection of exosomes by interferometric imaging

Exosomes, which are membranous nanovesicles, are actively released by cells and have been attributed to roles in cell-cell communication, cancer metastasis, and early disease diagnostics. The small size (30–100 nm) along with low refractive index contrast of exosomes makes direct characterization and phenotypical classification very difficult. In this work we present a method based on Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) that allows multiplexed phenotyping and digital counting of various populations of individual exosomes (>50 nm) captured on a microarray-based solid phase chip. We demonstrate these characterization concepts using purified exosomes from a HEK 293 cell culture. As a demonstration of clinical utility, we characterize exosomes directly from human cerebrospinal fluid (hCSF). Our interferometric imaging method could capture, from a very small hCSF volume (20 uL), nanoparticles that have a size compatible with exosomes, using antibodies directed against tetraspanins. With this unprecedented capability, we foresee revolutionary implications in the clinical field with improvements in diagnosis and stratification of patients affected by different disorders.This work was supported by Regione Lombardia and Fondazione Cariplo through POR-FESR, project MINER (ID 46875467); Italian Ministry of Health, Ricerca Corrente. This work was partially supported by The Scientific and Technological Research Council of Turkey (grant #113E643). (Regione Lombardia; 46875467 - Fondazione Cariplo through POR-FESR, project MINER; Italian Ministry of Health, Ricerca Corrente; 113E643 - Scientific and Technological Research Council of Turkey)Published versio