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

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

Exosome-Related Multi-Pass Transmembrane Protein TSAP6 Is a Target of Rhomboid Protease RHBDD1-Induced Proteolysis

We have previously reported that rhomboid domain containing 1 (RHBDD1), a mammalian rhomboid protease highly expressed in the testis, can cleave the Bcl-2 protein Bik. In this study, we identified a multi-pass transmembrane protein, tumor suppressor activated pathway-6 (TSAP6) as a potential substrate of RHBDD1. RHBDD1 was found to induce the proteolysis of TSAP6 in a dose- and activity-dependent manner. The cleavage of TSAP6 was not restricted to its glycosylated form and occurred in three different regions. In addition, mass spectrometry and mutagenesis analyses both indicated that the major cleavage site laid in the C-terminal of the third transmembrane domain of TSAP6. A somatic cell knock-in approach was used to genetically inactivate the endogenous RHBDD1 in HCT116 and RKO colon cancer cells. Exosome secretion was significantly elevated when RHBDD1 was inactivated in the two cells lines. The increased exosome secretion was verfied through the detection of certain exosomal components, including Tsg101, Tf-R, FasL and Trail. In addition, the elevation of exosome secretion by RHBDD1 inactivation was reduced when TSAP6 was knocked down, indicating that the role of RHBDD1 in regulating exosomal trafficking is very likely to be TSAP6-dependent. We found that the increase in FasL and Trail increased exosome-induced apoptosis in Jurkat cells. Taken together, our findings suggest that RHBDD1 is involved in the regulation of a nonclassical exosomal secretion pathway through the restriction of TSAP6

Contribution by Polymorphonucleate Granulocytes to Elevated Gamma-Glutamyltransferase in Cystic Fibrosis Sputum

Background: Cystic fibrosis (CF) is an autosomal recessive disorder characterized by a chronic neutrophilic airways inflammation, increasing levels of oxidative stress and reduced levels of antioxidants such as glutathione (GSH). Gammaglutamyltransferase (GGT), an enzyme induced by oxidative stress and involved in the catabolism of GSH and its derivatives, is increased in the airways of CF patients with inflammation, but the possible implications of its increase have not yet been investigated in detail. Principal Findings: The present study was aimed to evaluate the origin and the biochemical characteristics of the GGT detectable in CF sputum. We found GGT activity both in neutrophils and in the fluid, the latter significantly correlating with myeloperoxidase expression. In neutrophils, GGT was associated with intracellular granules. In the fluid, gel-filtration chromatography showed the presence of two distinct GGT fractions, the first corresponding to the human plasma b-GGT fraction, the other to the free enzyme. The same fractions were also observed in the supernatant of ionomycin and fMLPactivated neutrophils. Western blot analysis confirmed the presence of a single band of GGT immunoreactive peptide in the CF sputum samples and in isolated neutrophils. Conclusions: In conclusion, our data indicate that neutrophils are able to transport and release GGT, thus increasing GGT activity in CF sputum. The prompt release of GGT may have consequences on all GGT substrates, including major inflammatory mediators such as S-nitrosoglutathione and leukotrienes, and could participate in early modulation of inflammatory response

Tumor-Derived Microvesicles Induce Proangiogenic Phenotype in Endothelial Cells via Endocytosis

Background: Increasing evidence indicates that tumor endothelial cells (TEC) differ from normal endothelial cells (NEC). Our previous reports also showed that TEC were different from NEC. For example, TEC have chromosomal abnormality and proangiogenic properties such as high motility and proliferative activity. However, the mechanism by which TEC acquire a specific character remains unclear. To investigate this mechanism, we focused on tumor-derived microvesicles (TMV). Recent studies have shown that TMV contain numerous types of bioactive molecules and affect normal stromal cells in the tumor microenvironment. However, most of the functional mechanisms of TMV remain unclear. Methodology/Principal Findings: Here we showed that TMV isolated from tumor cells were taken up by NEC through endocytosis. In addition, we found that TMV promoted random motility and tube formation through the activation of the phosphoinositide 3-kinase/Akt pathway in NEC. Moreover, the effects induced by TMV were inhibited by the endocytosis inhibitor dynasore. Our results indicate that TMV could confer proangiogenic properties to NEC partly via endocytosis. Conclusion: We for the first time showed that endocytosis of TMV contributes to tumor angiogenesis. These findings offer new insights into cancer therapies and the crosstalk between tumor and endothelial cells mediated by TMV in the tumor microenvironment

Biological membranes in EV biogenesis, stability, uptake, and cargo transfer: an ISEV position paper arising from the ISEV membranes and EVs workshop

Paracrine and endocrine roles have increasingly been ascribed to extracellular vesicles (EVs) generated by multicellular organisms. Central to the biogenesis, content, and function of EVs are their delimiting lipid bilayer membranes. To evaluate research progress on membranes and EVs, the International Society for Extracellular Vesicles (ISEV) conducted a workshop in March 2018 in Baltimore, Maryland, USA, bringing together key opinion leaders and hands-on researchers who were selected on the basis of submitted applications. The workshop was accompanied by two scientific surveys and covered four broad topics: EV biogenesis and release; EV uptake and fusion; technologies and strategies used to study EV membranes; and EV transfer and functional assays. In this ISEV position paper, we synthesize the results of the workshop and the related surveys to outline important outstanding questions about EV membranes and describe areas of consensus. The workshop discussions and survey responses reveal that while much progress has been made in the field, there are still several concepts that divide opinion. Good consensus exists in some areas, including particular aspects of EV biogenesis, uptake and downstream signalling. Areas with little to no consensus include EV storage and stability, as well as whether and how EVs fuse with target cells. Further research is needed in these key areas, as a better understanding of membrane biology will contribute substantially towards advancing the field of extracellular vesicles.Fil: Russell, Ashley E.. University Johns Hopkins; Estados UnidosFil: Sneider, Alexandra. University Johns Hopkins; Estados UnidosFil: Witwer, Kenneth W.. University Johns Hopkins; Estados UnidosFil: Bergese, Paolo. Università Degli Studi Di Brescia; ItaliaFil: Bhattacharyya, Suvendra N.. Indian Institute of Chemical Biology; IndiaFil: Cocks, Alexander. Cardiff University; Reino UnidoFil: Cocucci, Emanuele. Ohio State University; Estados UnidosFil: Erdbrügger, Uta. University of Virginia; Estados UnidosFil: Falcon Perez, Juan M.. Ikerbasque Basque Foundation for Science; EspañaFil: Freeman, David W.. National Institute On Aging National Institute for Helth ; Estados UnidosFil: Gallagher, Thomas M.. Loyola University Of Chicago; Estados UnidosFil: Hu, Shuaishuai. Technological University Dublin; IrlandaFil: Huang, Yiyao. University Johns Hopkins; Estados Unidos. Southern Medical University; ChinaFil: Jay, Steven M.. University of Maryland; Estados UnidosFil: Kano, Shin-ichi. The University of Alabama at Birmingham School of Medicine; Estados UnidosFil: Lavieu, Gregory. Institut Curie; FranciaFil: Leszczynska, Aleksandra. University of California at San Diego; Estados UnidosFil: Llorente, Alicia M.. Oslo University Hospital; NoruegaFil: Lu, Quan. Harvard University. Harvard School of Public Health; Estados UnidosFil: Mahairaki, Vasiliki. University Johns Hopkins; Estados UnidosFil: Muth, Dillon C.. University Johns Hopkins; Estados UnidosFil: Noren Hooten, Nicole. National Institute On Aging National Institute for Helth ; Estados UnidosFil: Ostrowski, Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; ArgentinaFil: Prada, Ilaria. Consiglio Nazionale delle Ricerche; ItaliaFil: Sahoo, Susmita. Icahn School of Medicine at Mount Sinai ; Estados UnidosFil: Schøyen, Tine Hiorth. Uit The Arctic University Of Norway; Noruega. University Johns Hopkins; Estados UnidosFil: Sheng, Lifuy. University of Washington. School of Medicine; Estados UnidosFil: Tesch, Deanna. Shaw University; Estados UnidosFil: Van Niel, Guillaume. No especifíca;Fil: Vandenbroucke, Roosmarijn E.. University of Ghent; BélgicaFil: Verweij, Frederik J.. No especifíca;Fil: Villar, Ana V.. Universidad de Cantabria; EspañaFil: Wauben, Marca. University of Utrecht; Países BajosFil: Wehman, Ann M.. Universität Würzburg; AlemaniaFil: Ardavan, Arzhang. Peking University; ; ChinaFil: Carter, David Raul Francisco. Oxford Brookes University; Reino UnidoFil: Vader, Pieter. University Medical Center Utrecht; Países Bajo

Lipid rafts are essential for release of phosphatidylserine-exposing extracellular vesicles from platelets.

Platelets protect the vascular system during damage or inflammation, but platelet activation can result in pathological thrombosis. Activated platelets release a variety of extracellular vesicles (EVs). EVs shed from the plasma membrane often expose phosphatidylserine (PS). These EVs are pro-thrombotic and increased in number in many cardiovascular and metabolic diseases. The mechanisms by which PS-exposing EVs are shed from activated platelets are not well characterised. Cholesterol-rich lipid rafts provide a platform for coordinating signalling through receptors and Ca2+ channels in platelets. We show that cholesterol depletion with methyl-β-cyclodextrin or sequestration with filipin prevented the Ca2+-triggered release of PS-exposing EVs. Although calpain activity was required for release of PS-exposing, calpain-dependent cleavage of talin was not affected by cholesterol depletion. P2Y12 and TPα, receptors for ADP and thromboxane A2, respectively, have been reported to be in platelet lipid rafts. However, the P2Y12 antagonist, AR-C69931MX, or the cyclooxygenase inhibitor, aspirin, had no effect on A23187-induced release of PS-exposing EVs. Together, these data show that lipid rafts are required for release of PS-exposing EVs from platelets.Isaac Newton Trust/ Wellcome Trust ISSF/University of Cambridge Joint Research Grant British Heart Foundation grant SP/15/7/3156

Distinct Dynamics of Endocytic Clathrin-Coated Pits and Coated Plaques

Here we classify endocytic structures at the adherent (bottom) surface of many cells in culture into shorter-lived coated pits and longer-lived coated plaques which internalize by different mechanisms

Glia-to-neuron transfer of miRNAs via extracellular vesicles: a new mechanism underlying inflammation-induced synaptic alterations

Recent evidence indicates synaptic dysfunction as an early mechanism affected in neuroinflammatory diseases, such as multiple sclerosis, which are characterized by chronic microglia activation. However, the mode(s) of action of reactive microglia in causing synaptic defects are not fully understood. In this study, we show that inflammatory microglia produce extracellular vesicles (EVs) which are enriched in a set of miRNAs that regulate the expression of key synaptic proteins. Among them, miR-146a-5p, a microglia-specific miRNA not present in hippocampal neurons, controls the expression of presynaptic synaptotagmin1 (Syt1) and postsynaptic neuroligin1 (Nlg1), an adhesion protein which play a crucial role in dendritic spine formation and synaptic stability. Using a Renilla-based sensor, we provide formal proof that inflammatory EVs transfer their miR-146a-5p cargo to neuron. By western blot and immunofluorescence analysis we show that vesicular miR-146a-5p suppresses Syt1 and Nlg1 expression in receiving neurons. Microglia-to-neuron miR-146a-5p transfer and Syt1 and Nlg1 downregulation do not occur when EV\ue2\u80\u93neuron contact is inhibited by cloaking vesicular phosphatidylserine residues and when neurons are exposed to EVs either depleted of miR-146a-5p, produced by pro-regenerative microglia, or storing inactive miR-146a-5p, produced by cells transfected with an anti-miR-146a-5p. Morphological analysis reveals that prolonged exposure to inflammatory EVs leads to significant decrease in dendritic spine density in hippocampal neurons in vivo and in primary culture, which is rescued in vitro by transfection of a miR-insensitive Nlg1 form. Dendritic spine loss is accompanied by a decrease in the density and strength of excitatory synapses, as indicated by reduced mEPSC frequency and amplitude. These findings link inflammatory microglia and enhanced EV production to loss of excitatory synapses, uncovering a previously unrecognized role for microglia-enriched miRNAs, released in association to EVs, in silencing of key synaptic genes

Secretion of Novel SEL1L Endogenous Variants Is Promoted by ER Stress/UPR via Endosomes and Shed Vesicles in Human Cancer Cells

We describe here two novel endogenous variants of the human endoplasmic reticulum (ER) cargo receptor SEL1LA, designated p38 and p28. Biochemical and RNA interference studies in tumorigenic and non-tumorigenic cells indicate that p38 and p28 are N-terminal, ER-anchorless and more stable relative to the canonical transmembrane SEL1LA. P38 is expressed and constitutively secreted, with increase after ER stress, in the KMS11 myeloma line and in the breast cancer lines MCF7 and SKBr3, but not in the non-tumorigenic breast epithelial MCF10A line. P28 is detected only in the poorly differentiated SKBr3 cell line, where it is secreted after ER stress. Consistently with the presence of p38 and p28 in culture media, morphological studies of SKBr3 and KMS11 cells detect N-terminal SEL1L immunolabeling in secretory/degradative compartments and extracellularly-released membrane vesicles. Our findings suggest that the two new SEL1L variants are engaged in endosomal trafficking and secretion via vesicles, which could contribute to relieve ER stress in tumorigenic cells. P38 and p28 could therefore be relevant as diagnostic markers and/or therapeutic targets in cancer