Aggressive human brain tumours (gliomas) often express a truncated and oncogenic form of the epidermal growth factor receptor, known as EGFRvIII. Within each tumour only a small percentage of glioma cells may actually express EGFRvIII; however, most of the cells exhibit a transformed phenotype. Here we show that EGFRvIII can be 'shared' between glioma cells by intercellular transfer of membrane-derived microvesicles ('oncosomes'). EGFRvIII expression in indolent glioma cells stimulates formation of lipid-raft related microvesicles containing EGFRvIII. Microvesicles containing this receptor are then released to cellular surroundings and blood of tumour-bearing mice, and can merge with the plasma membranes of cancer cells lacking EGFRvIII. This event leads to the transfer of oncogenic activity, including activation of transforming signalling pathways (MAPK and Akt), changes in expression of EGFRvIII-regulated genes (VEGF, Bcl-x(L), p27), morphological transformation and increase in anchorage-independent growth capacity. Thus, membrane microvesicles of cancer cells can contribute to a horizontal propagation of oncogenes and their associated transforming phenotype among subsets of cancer cells.

Activated EGF receptor (EGFR) plays an oncogenic role in several human malignancies. Although the intracellular effects of EGFR are well studied, its ability to induce and modulate tumor angiogenesis is less understood. We found previously that oncogenic EGFR can be shed from cancer cells as cargo of membrane microvesicles (MVs), which can interact with surfaces of other cells. Here we report that MVs produced by human cancer cells harboring activated EGFR (A431, A549, DLD-1) can be taken up by cultured endothelial cells, in which they elicit EGFR-dependent responses, including activation of MAPK and Akt pathways. These responses can be blocked by annexin V and its homodimer, Diannexin, both of which cloak phosphatidylserine residues on the surfaces of MVs. Interestingly, the intercellular EGFR transfer is also accompanied by the onset of VEGF expression in endothelial cells and by autocrine activation of its key signaling receptor (VEGF receptor-2). In A431 human tumor xenografts in mice, angiogenic endothelial cells stain positively for human EGFR and phospho-EGFR, while treatment with Diannexin leads to a reduction of tumor growth rate and microvascular density. Thus, we propose that oncogene-containing tumor cell-derived MVs could act as a unique form of angiogenesismodulating stimuli and are capable of switching endothelial cells to act in an autocrine mode.tumor angiogenesis ͉ antiangiogenesis ͉ exosomes ͉ annexin V ͉ oncogenes

Cancer cells emit a heterogeneous mixture of vesicular, organelle-like structures (microvesicles, MVs) into their surroundings including blood and body fluids. MVs are generated via diverse biological mechanisms triggered by pathways involved in oncogenic transformation, microenvironmental stimulation, cellular activation, stress, or death. Vesiculation events occur either at the plasma membrane (ectosomes, shed vesicles) or within endosomal structures (exosomes). MVs are increasingly recognized as mediators of intercellular communication due to their capacity to merge with and transfer a repertoire of bioactive molecular content (cargo) to recipient cells. Such processes may occur both locally and systemically, contributing to the formation of microenvironmental fields and niches. The bioactive cargo of MVs may include growth factors and their receptors, proteases, adhesion molecules, signalling molecules, as well as DNA, mRNA, and microRNA (miRs) sequences. Tumour cells emit large quantities of MVs containing procoagulant, growth regulatory and oncogenic cargo (oncosomes), which can be transferred throughout the cancer cell population and to non-transformed stromal cells, endothelial cells and possibly to the inflammatory infiltrates (oncogenic field effect). These events likely impact tumour invasion, angiogenesis, metastasis, drug resistance, and cancer stem cell hierarchy. Ongoing studies explore the molecular mechanisms and mediators of MV-based intercellular communication (cancer vesiculome) with the hope of using this information as a possible source of therapeutic targets and disease biomarkers in cancer.

Cellular interactions play a crucial role in progression, angiogenesis and invasiveness of tumors, including glioma. The traditionally accepted view is that medium and long-range cellular communications occur primarily through gradients of soluble ligands, recognizable by the cell-associated receptors. Recent findings, however, suggest the existence of another mode of intercellular communication, where the 'units' of information are microvesicles containing a multitude of biologically active protein and RNA species, including oncogenic receptors, such as EGFRvIII. Moreover, microvesicles can be retrieved from the circulating blood of cancer patients, and reveal the presence of oncogenes in their tumors, thereby potentially serving as information-rich prognostic and predictive biomarkers.

Ingestion of a commensal bacteria, Lactobacillus rhamnosus JB-1, has potent immunoregulatory effects, and changes nerve-dependent colon migrating motor complexes (MMCs), enteric nerve function, and behavior. How these alterations occur is unknown. JB-1 microvesicles (MVs) are enriched for heat shock protein components such as chaperonin 60 heat-shock protein isolated from Escherichia coli (GroEL) and reproduce regulatory and neuronal effects in vitro and in vivo. Ingested labeled MVs were detected in murine Peyer's patch (PP) dendritic cells (DCs) within 18 h. After 3 d, PP and mesenteric lymph node DCs assumed a regulatory phenotype and increased functional regulatory CD4 + 25 + Foxp3 + T cells. JB-1, MVs, and GroEL similarly induced phenotypic change in cocultured DCs via multiple pathways including C-type lectin receptors specific intercellular adhesion molecule-3 grabbing non-integrin-related 1 and Dectin-1, as well as TLR-2 and -9. JB-1 and MVs also decreased the amplitude of neuronally dependent MMCs in an ex vivo model of peristalsis. Gut epithelial, but not direct neuronal application of, MVs, replicated functional effects of JB-1 on in situ patch-clamped enteric neurons. GroEL and anti-TLR-2 were without effect in this system, suggesting the importance of epithelium neuron signaling and discrimination between pathways for bacteria-neuron and -immune communication. Together these results offer a mechanistic explanation of how Gram-positive commensals and probiotics may influence the host's immune and nervous

Background: Cargo of extracellular vesicles (EVs) may reveal responses to targeted anticancer drugs. Results: Kinase inhibitors of the oncogenic epidermal growth factor receptor (EGFR) activate emission of exosome-like EVs containing EGFR protein and DNA. Conclusion: Co-detection of EGFR and DNA in tumor-related EVs reflects the responses to kinase inhibitors. Significance: EV cargo may serve as a biomarker for targeted therapy.

PTEN is a potent tumor-suppressor protein. Aggressive and metastatic prostate cancer (PC) is associated with a reduction or loss of PTEN expression. PTEN reduction often occurs without gene mutations, and its downregulation is not fully understood. Herein, we show that PTEN is incorporated in the cargo of exosomes derived from cancer cells. PTEN is not detected in exosomes derived from normal, noncancerous cells. We found that PTEN can be transferred to other cells through exosomes. In cells that have a reduction or complete loss of PTEN expression, the transferred PTEN is competent to confer tumor-suppression activity to acceptor cells. In PC patients, we show that PTEN is incorporated in the cargo of exosomes that circulate in their blood. Interestingly, normal subjects have no PTEN expression in their blood exosomes. Further, we found that the prostate-specific antigen (PSA) is incorporated in PC patients’ and normal subjects’ blood exosomes. These data suggest that exosomal PTEN can compensate for PTEN loss in PTEN deficient cells, and may have diagnostic value for prostate cancer.

Objective-Previous studies supported the contribution of exosomes to an acellular mode of communication, leading to intercellular transfer of molecules. In this study we provide evidence that mast cell-derived exosomes induce plasminogen activator inhibitor type 1 (PAI-1) expression in endothelial cells, detectable at the level of PAI-1 mRNA and protein synthesis. The stimulating effect was also measured at the level of PAI-1 promoter activity. Methods and Results-To identify components responsible for this activity, exosome proteins were separated by 2-dimensional PAGE, and protein spots were identified by microsequencing using electrospray (ISI-Q-TOF-Micromass) spectrometer. Components of 3 independent systems that can be involved in activation of endothelial cells, namely the prothrombinase complex, tumor necrosis factor-␣, and angiotensinogen precursors were identified. Procoagulant activity of exosomes was confirmed by a thrombin generation assay using a specific chromogenic substrate. Because the potential of mast cell-derived exosomes to induce PAI-1 expression was completely abolished by hirudin, thrombin generated on exosomes seems to be responsible for this activity. Conclusions-It can be concluded that mast cell-derived exosomes via significant upregulation of PAI-1 secretion from endothelial cells may provide feedback between the characteristically increased PAI-1 levels and procoagulant states, both observed in diverse syndromes manifesting as endothelial cell dysfunction. 5,6 or diabetes mellitus, 7 and contributes to procoagulant state in these and other conditions. Mast cells exert profound pleiotropic effects on immune cell reactions at inflammatory sites, where they are most likely influenced not only by the extracellular matrix and inflammatory mediators but also by the proximity of activated T lymphocytes. These cells have been implicated in 2 contrasting types of immune responses, the immediate hypersensitivity reactions associated with allergic phenomena and their acute activation by bacterial products during infection. 8 Mast cells are localized near blood vessels and are involved in the activation of the clotting system during inflammation to contain the injury and initiate tissue repair. This concept is supported by studies of the reverse passive Arthus reaction in mast cell-deficient mice cells, which showed that these cells contributed to the exudation of clotting factors resulting in fibrin deposition and enhancement of fibrin cross-linkage. 9,10 In view of the role of mast cells in deposition of fibrin during inflammation near the site of injury, the aim of the present study was to examine the effect of mast cells-derived exosomes on expression and secretion of plasminogen activator inhibitor type 1 (PAI-1) from endothelial cells. Because exocytosis is the process by which stimulation of plasma membrane receptors on secretory cells results in the release of proteins and/or peptides from the intracellular stores into the extracellular space, 11 we attempted to identify active compone...