An Hendrix

Biological properties of extracellular vesicles and their physiological functions.

In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.

Vesiclepedia: A Compendium for Extracellular Vesicles with Continuous Community Annotation

Vesiclepedia is a community-annotated compendium of molecular data on extracellular vesicles.

Applying extracellular vesicles based therapeutics in clinical trials - an ISEV position paper.

Extracellular vesicles (EVs), such as exosomes and microvesicles, are released by different cell types and participate in physiological and pathophysiological processes. EVs mediate intercellular communication as cell-derived extracellular signalling organelles that transmit specific information from their cell of origin to their target cells. As a result of these properties, EVs of defined cell types may serve as novel tools for various therapeutic approaches, including (a) anti-tumour therapy, (b) pathogen vaccination, (c) immune-modulatory and regenerative therapies and (d) drug delivery. The translation of EVs into clinical therapies requires the categorization of EV-based therapeutics in compliance with existing regulatory frameworks. As the classification defines subsequent requirements for manufacturing, quality control and clinical investigation, it is of major importance to define whether EVs are considered the active drug components or primarily serve as drug delivery vehicles. For an effective and particularly safe translation of EV-based therapies into clinical practice, a high level of cooperation between researchers, clinicians and competent authorities is essential. In this position statement, basic and clinical scientists, as members of the International Society for Extracellular Vesicles (ISEV) and of the European Cooperation in Science and Technology (COST) program of the European Union, namely European Network on Microvesicles and Exosomes in Health and Disease (ME-HaD), summarize recent developments and the current knowledge of EV-based therapies. Aspects of safety and regulatory requirements that must be considered for pharmaceutical manufacturing and clinical application are highlighted. Production and quality control processes are discussed. Strategies to promote the therapeutic application of EVs in future clinical studies are addressed.

The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling

Despite an enormous interest in the role of extracellular vesicles, including exosomes, in cancer and their use as biomarkers for diagnosis, prognosis, drug response and recurrence, there is no consensus on dependable isolation protocols. We provide a comparative evaluation of 4 exosome isolation protocols for their usability, yield and purity, and their impact on downstream omics approaches for biomarker discovery. OptiPrep density gradient centrifugation outperforms ultracentrifugation and ExoQuick and Total Exosome Isolation precipitation in terms of purity, as illustrated by the highest number of CD63-positive nanovesicles, the highest enrichment in exosomal marker proteins and a lack of contaminating proteins such as extracellular Argonaute-2 complexes. The purest exosome fractions reveal a unique mRNA profile enriched for translation, ribosome, mitochondrion and nuclear lumen function. Our results demonstrate that implementation of high purification techniques is a prerequisite to obtain reliable omics data and identify exosome-specific functions and biomarkers.

Electroporation-induced siRNA precipitation obscures the efficiency of siRNA loading into extracellular vesicles

Extracellular vesicles (EVs) are specialised endogenous carriers of proteins and nucleic acids and are involved in intercellular communication. EVs are therefore proposed as candidate drug delivery systems for the delivery of nucleic acids and other macromolecules. However, the preparation of EV-based drug delivery systems is hampered by the lack of techniques to load the vesicles with nucleic acids. In this work we have now characterised in detail the use of an electroporation method for this purpose. When EVs were electroporated with fluorescently labelled siRNA, siRNA retention was comparable with previously published results (20-25% based on fluorescence spectroscopy and fluorescence fluctuation spectroscopy), and electroporation with unlabelled siRNA resulted in significant siRNA retention in the EV pellet as measured by RT-PCR. Remarkably, when siRNA was electroporated in the absence of EVs, a similar or even greater siRNA retention was measured. Nanoparticle tracking analysis and confocal microscopy showed extensive formation of insoluble siRNA aggregates after electroporation, which could be dramatically reduced by addition of EDTA. Other strategies to reduce aggregate formation, including the use of cuvettes with conductive polymer electrodes and the use of an acidic citrate electroporation buffer, resulted in a more efficient reduction of siRNA precipitation than EDTA. However, under these conditions, siRNA retention was below 0.05% and no significant differences in siRNA retention could be measured between samples electroporated in the presence or absence of EVs. Our results show that electroporation of EVs with siRNA is accompanied by extensive siRNA aggregate formation, which may cause overestimation of the amount of siRNA actually loaded into EVs. Moreover, our data clearly illustrate that electroporation is far less efficient than previously described, and highlight the necessity for alternative methods to prepare siRNA-loaded EVs.

EVpedia: a community web portal for extracellular vesicles research

MOTIVATION Extracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging. RESULTS We present an improved version of EVpedia, a public database for EVs research. This community web portal contains a database of publications and vesicular components, identification of orthologous vesicular components, bioinformatic tools and a personalized function. EVpedia includes 6879 publications, 172 080 vesicular components from 263 high-throughput datasets, and has been accessed more than 65 000 times from more than 750 cities. In addition, about 350 members from 73 international research groups have participated in developing EVpedia. This free web-based database might serve as a useful resource to stimulate the emerging field of EV research. AVAILABILITY AND IMPLEMENTATION The web site was implemented in PHP, Java, MySQL and Apache, and is freely available at http://evpedia.info.

Bone marrow stromal cell–derived exosomes as communicators in drug resistance in multiple myeloma cells

The interplay between bone marrow stromal cells (BMSCs) and multiple myeloma (MM) cells performs a crucial role in MM pathogenesis by secreting growth factors, cytokines, and extracellular vesicles. Exosomes are membranous vesicles 40 to 100 nm in diameter constitutively released by almost all cell types, and they mediate local cell-to-cell communication by transferring mRNAs, miRNAs, and proteins. Although BMSC-induced growth and drug resistance of MM cells has been studied, the role of BMSC-derived exosomes in this action remains unclear. Here we investigate the effect of BMSC-derived exosomes on the viability, proliferation, survival, migration, and drug resistance of MM cells, using the murine 5T33MM model and human MM samples. BMSCs and MM cells could mutually exchange exosomes carrying certain cytokines. Both naive and 5T33 BMSC-derived exosomes increased MM cell growth and induced drug resistance to bortezomib. BMSC-derived exosomes also influenced the activation of several survival relevant pathways, including c-Jun N-terminal kinase, p38, p53, and Akt. Exosomes obtained from normal donor and MM patient BMSCs also induced survival and drug resistance of human MM cells. Taken together, our results demonstrate the involvement of exosome-mediated communication in BMSC-induced proliferation, migration, survival, and drug resistance of MM cells.

Effect of the Secretory Small GTPase Rab27B on Breast Cancer Growth, Invasion, and Metastasis

BACKGROUND Secretory GTPases like Rab27B control vesicle exocytosis and deliver critical proinvasive growth regulators into the tumor microenvironment. The expression and role of Rab27B in breast cancer were unknown. METHODS Expression of green fluorescent protein (GFP) fused with wild-type Rab3D, Rab27A, or Rab27B, or Rab27B point mutants defective in GTP/GDP binding or geranylgeranylation, or transient silencing RNA to the same proteins was used to study Rab27B in estrogen receptor (ER)-positive human breast cancer cell lines (MCF-7, T47D, and ZR75.1). Cell cycle progression was evaluated by flow cytometry, western blotting, and measurement of cell proliferation rates, and invasion was assessed using Matrigel and native type I collagen substrates. Orthotopic tumor growth, local invasion, and metastasis were analyzed in mouse xenograft models. Mass spectrometry identified proinvasive growth regulators that were secreted in the presence of Rab27B. Rab27B protein levels were evaluated by immunohistochemistry in 59 clinical breast cancer specimens, and Rab3D, Rab27A, and Rab27B mRNA levels were analyzed by quantitative real-time polymerase chain reaction in 20 specimens. Statistical tests were two-sided. RESULTS Increased expression of Rab27B promoted G(1) to S phase cell cycle transition, proliferation and invasiveness of cells in culture, and invasive tumor growth and hemorrhagic ascites production in a xenograft mouse model (n = 10; at 10 weeks, survival of MCF-7 GFP- vs GFP-Rab27B-injected mice was 100% vs 62.5%, hazard ratio = 0.26, 95% confidence interval = 0.08 to 0.88, P = .03). Mass spectrometric analysis of purified Rab27B-secretory vesicles identified heat-shock protein 90alpha as key proinvasive growth regulator. Heat-shock protein 90alpha secretion was Rab27B-dependent and was required for matrix metalloproteinase-2 activation. All Rab27B-mediated functional responses were GTP- and geranylgeranyl-dependent. Presence of endogenous Rab27B mRNA and protein, but not of Rab3D or Rab27A mRNA, was associated with lymph node metastasis (P < .001) and differentiation grade (P = .001) in ER-positive human breast tumors. CONCLUSIONS Rab27B regulates invasive growth and metastasis in ER-positive breast cancer cell lines, and increased expression is associated with poor prognosis in humans.

Cellular disposal of miR23b by RAB27-dependent exosome release is linked to acquisition of metastatic properties.

Exosomes are small secreted vesicles that can transfer their content to recipient cells. In cancer, exosome secretion has been implicated in tumor growth and metastatic spread. In this study, we explored the possibility that exosomal pathways might discard tumor-suppressor miRNA that restricts metastatic progression. Secreted miRNA characterized from isogenic bladder carcinoma cell lines with differing metastatic potential were uncoupled from binding to target transcripts or the AGO2-miRISC complex. In metastatic cells, we observed a relative increase in secretion of miRNA with tumor-suppressor functions, including miR23b, miR224, and miR921. Ectopic expression of miR23b inhibited invasion, anoikis, angiogenesis, and pulmonary metastasis. Silencing of the exocytotic RAB family members RAB27A or RAB27B halted miR23b and miR921 secretion and reduced cellular invasion. Clinically, elevated levels of RAB27B expression were linked to poor prognosis in two independent cohorts of patients with bladder cancer. Moreover, highly exocytosed miRNA from metastatic cells, such as miR23b, were reduced in lymph node metastases compared with patient-matched primary tumors and were correlated with increments in miRNA-targeted RNA. Taken together, our results suggested that exosome-mediated secretion of tumor-suppressor miRNA is selected during tumor progression as a mechanism to coordinate activation of a metastatic cascade.

Evidence-Based Clinical Use of Nanoscale Extracellular Vesicles in Nanomedicine

Recent research has demonstrated that all body fluids assessed contain substantial amounts of vesicles that range in size from 30 to 1000 nm and that are surrounded by phospholipid membranes containing different membrane microdomains such as lipid rafts and caveolae. The most prominent representatives of these so-called extracellular vesicles (EVs) are nanosized exosomes (70-150 nm), which are derivatives of the endosomal system, and microvesicles (100-1000 nm), which are produced by outward budding of the plasma membrane. Nanosized EVs are released by almost all cell types and mediate targeted intercellular communication under physiological and pathophysiological conditions. Containing cell-type-specific signatures, EVs have been proposed as biomarkers in a variety of diseases. Furthermore, according to their physical functions, EVs of selected cell types have been used as therapeutic agents in immune therapy, vaccination trials, regenerative medicine, and drug delivery. Undoubtedly, the rapidly emerging field of basic and applied EV research will significantly influence the biomedicinal landscape in the future. In this Perspective, we, a network of European scientists from clinical, academic, and industry settings collaborating through the H2020 European Cooperation in Science and Technology (COST) program European Network on Microvesicles and Exosomes in Health and Disease (ME-HAD), demonstrate the high potential of nanosized EVs for both diagnostic and therapeutic (i.e., theranostic) areas of nanomedicine.