Sybren L. N. Maas

Extracellular Vesicles: Unique Intercellular Delivery Vehicles

Extracellular vesicles (EVs) are a heterogeneous collection of membrane-bound carriers with complex cargoes including proteins, lipids, and nucleic acids. While the release of EVs was previously thought to be only a mechanism to discard nonfunctional cellular components, increasing evidence implicates EVs as key players in intercellular and even interorganismal communication. EVs confer stability and can direct their cargoes to specific cell types. EV cargoes also appear to act in a combinatorial manner to communicate directives to other cells. This review focuses on recent findings and knowledge gaps in the area of EV biogenesis, release, and uptake. In addition, we highlight examples whereby EV cargoes control basic cellular functions, including motility and polarization, immune responses, and development, and contribute to diseases such as cancer and neurodegeneration.

Possibilities and limitations of current technologies for quantification of biological extracellular vesicles and synthetic mimics

Nano-sized extracelullar vesicles (EVs) released by various cell types play important roles in a plethora of (patho)physiological processes and are increasingly recognized as biomarkers for disease. In addition, engineered EV and EV-inspired liposomes hold great potential as drug delivery systems. Major technologies developed for high-throughput analysis of individual EV include nanoparticle tracking analysis (NTA), tunable resistive pulse sensing (tRPS) and high-resolution flow cytometry (hFC). Currently, there is a need for comparative studies on the available technologies to improve standardization of vesicle analysis in diagnostic or therapeutic settings. We investigated the possibilities, limitations and comparability of NTA, tRPS and hFC for analysis of tumor cell-derived EVs and synthetic mimics (i.e. differently sized liposomes). NTA and tRPS instrument settings were identified that significantly affected the quantification of these particles. Furthermore, we detailed the differences in absolute quantification of EVs and liposomes using the three technologies. This study increases our understanding of possibilities and pitfalls of NTA, tRPS and hFC, which will benefit standardized and large-scale clinical application of (engineered) EVs and EV-mimics in the future.

Obstacles and opportunities in the functional analysis of extracellular vesicle RNA – an ISEV position paper

ABSTRACT The release of RNA-containing extracellular vesicles (EV) into the extracellular milieu has been demonstrated in a multitude of different in vitro cell systems and in a variety of body fluids. RNA-containing EV are in the limelight for their capacity to communicate genetically encoded messages to other cells, their suitability as candidate biomarkers for diseases, and their use as therapeutic agents. Although EV-RNA has attracted enormous interest from basic researchers, clinicians, and industry, we currently have limited knowledge on which mechanisms drive and regulate RNA incorporation into EV and on how RNA-encoded messages affect signalling processes in EV-targeted cells. Moreover, EV-RNA research faces various technical challenges, such as standardisation of EV isolation methods, optimisation of methodologies to isolate and characterise minute quantities of RNA found in EV, and development of approaches to demonstrate functional transfer of EV-RNA in vivo. These topics were discussed at the 2015 EV-RNA workshop of the International Society for Extracellular Vesicles. This position paper was written by the participants of the workshop not only to give an overview of the current state of knowledge in the field, but also to clarify that our incomplete knowledge – of the nature of EV(-RNA)s and of how to effectively and reliably study them – currently prohibits the implementation of gold standards in EV-RNA research. In addition, this paper creates awareness of possibilities and limitations of currently used strategies to investigate EV-RNA and calls for caution in interpretation of the obtained data.

Quantification and size-profiling of extracellular vesicles using tunable resistive pulse sensing.

Extracellular vesicles (EVs), including ‘microvesicles’ and ‘exosomes’, are highly abundant in bodily fluids. Recent years have witnessed a tremendous increase in interest in EVs. EVs have been shown to play important roles in various physiological and pathological processes, including coagulation, immune responses, and cancer. In addition, EVs have potential as therapeutic agents, for instance as drug delivery vehicles or as regenerative medicine. Because of their small size (50 to 1,000 nm) accurate quantification and size profiling of EVs is technically challenging. This protocol describes how tunable resistive pulse sensing (tRPS) technology, using the qNano system, can be used to determine the concentration and size of EVs. The method, which relies on the detection of EVs upon their transfer through a nano sized pore, is relatively fast, suffices the use of small sample volumes and does not require the purification and concentration of EVs. Next to the regular operation protocol an alternative approach is described using samples spiked with polystyrene beads of known size and concentration. This real-time calibration technique can be used to overcome technical hurdles encountered when measuring EVs directly in biological fluids.

A standardized method to determine the concentration of extracellular vesicles using tunable resistive pulse sensing

Background Understanding the pathogenic role of extracellular vesicles (EVs) in disease and their potential diagnostic and therapeutic utility is extremely reliant on in-depth quantification, measurement and identification of EV sub-populations. Quantification of EVs has presented several challenges, predominantly due to the small size of vesicles such as exosomes and the availability of various technologies to measure nanosized particles, each technology having its own limitations. Materials and Methods A standardized methodology to measure the concentration of extracellular vesicles (EVs) has been developed and tested. The method is based on measuring the EV concentration as a function of a defined size range. Blood plasma EVs are isolated and purified using size exclusion columns (qEV) and consecutively measured with tunable resistive pulse sensing (TRPS). Six independent research groups measured liposome and EV samples with the aim to evaluate the developed methodology. Each group measured identical samples using up to 5 nanopores with 3 repeat measurements per pore. Descriptive statistics and unsupervised multivariate data analysis with principal component analysis (PCA) were used to evaluate reproducibility across the groups and to explore and visualise possible patterns and outliers in EV and liposome data sets. Results PCA revealed good reproducibility within and between laboratories, with few minor outlying samples. Measured mean liposome (not filtered with qEV) and EV (filtered with qEV) concentrations had coefficients of variance of 23.9% and 52.5%, respectively. The increased variance of the EV concentration measurements could be attributed to the use of qEVs and the polydisperse nature of EVs. Conclusion The results of this study demonstrate the feasibility of this standardized methodology to facilitate comparable and reproducible EV concentration measurements.

Tunable Resistive Pulse Sensing for the Characterization of Extracellular Vesicles.

Accurate characterization of extracellular vesicles (EVs), including exosomes and microvesicles, is essential to obtain further knowledge on the biological relevance of EVs. Tunable resistive pulse sensing (tRPS) has shown promise as a method for single particle-based quantification and size profiling of EVs. Here, we describe the technical background of tRPS and its applications for EV characterization. Besides the standard protocol, we describe an alternative protocol, in which samples are spiked with polystyrene beads of known size and concentration. This alternative protocol can be used to overcome some of the challenges of direct EV characterization in biological fluids.

Heparin in malignant glioma: review of preclinical studies and clinical results

Glioblastoma multiforme (GBM) is the most common primary brain tumor that is invariably lethal. Novel treatments are desperately needed. In various cancers, heparin and its low molecular weight derivatives (LMWHs), commonly used for the prevention and treatment of thrombosis, have shown therapeutic potential. Here we systematically review preclinical and clinical studies of heparin and LMWHs as anti-tumor agents in GBM. Even though the number of studies is limited, there is suggestive evidence that heparin may have various effects on GBM. These effects include the inhibition of tumor growth and angiogenesis in vitro and in vivo, and the blocking of uptake of extracellular vesicles. However, heparin can also block the uptake of (potential) anti-tumor agents. Clinical studies suggest a non-significant trend of prolonged survival of LMWH treated GBM patients, with some evidence of increased major bleedings. Heparin mimetics lacking anticoagulant effect are therefore a potential alternative to heparin/LMWH and are discussed as well.

Multidimensional communication in the microenvirons of glioblastoma

Glioblastomas are heterogeneous and invariably lethal tumours. They are characterized by genetic and epigenetic variations among tumour cells, which makes the development of therapies that eradicate all tumour cells challenging and currently impossible. An important component of glioblastoma growth is communication with and manipulation of other cells in the brain environs, which supports tumour progression and resistance to therapy. Glioblastoma cells recruit innate immune cells and change their phenotype to support tumour growth. Tumour cells also suppress adaptive immune responses, and our increasing understanding of how T cells access the brain and how the tumour thwarts the immune response offers new strategies for mobilizing an antitumour response. Tumours also subvert normal brain cells — including endothelial cells, neurons and astrocytes — to create a microenviron that favours tumour success. Overall, after glioblastoma-induced phenotypic modifications, normal cells cooperate with tumour cells to promote tumour proliferation, invasion of the brain, immune suppression and angiogenesis. This glioblastoma takeover of the brain involves multiple modes of communication, including soluble factors such as chemokines and cytokines, direct cell–cell contact, extracellular vesicles (including exosomes and microvesicles) and connecting nanotubes and microtubes. Understanding these multidimensional communications between the tumour and the cells in its environs could open new avenues for therapy.Glioblastomas remain one of the most aggressive and lethal tumours, with no effective treatments available. Here, Xandra Breakefield and colleagues examine the ways in which glioblastomas manipulate brain cells and immune cells in their environment to support tumour growth and the opportunities available for new therapies that disrupt these interactions.Key PointsGlioblastomas use numerous forms of communication to hijack many different cell types in the brain environs to support tumour progression.Communication routes include secreted proteins and molecules, gap junctions between cells, extracellular vesicles, tunnelling nanotubes and microtubes.Tumour cells co-opt microglia and infiltrating macrophages for their own benefit through the release of cytokines and extracellular vesicles.Glioblastomas and pericytes generate a state of reduced T cell effector function that is commonly referred to as T cell exhaustion or dysfunction.The interaction of tumour cells with normal brain cells, such as neurons, is not unidirectional, and neuronal activity is subverted to promote glioblastoma progression.Comprehension and disruption of tumour directives in the glioblastoma microenvironment could improve therapeutic intervention for these lethal tumours.

A methodology for comparable measurements and characterization of extracellular vesicles in plasma as an international standardization collaboration

Introduction: Besides providing nutrition, breast milk delivers important signals that stimulate the infants developing immune system. It has been postulated that extracellular vesicles (EV) in milk support the instruction and/or development of neonatal immunity. However, little is known about the composition of milk-derived EV, partly due to the difficulty to purify EV from other components in milk. Methods: In this study, an extensive LC-MS/MS proteomic analysis was performed, whereby EV were isolated from breast milk of 7 individual donors using our recently established optimized density gradient-based isolation protocol [1]. High-density, non-floating complexes were included to compare the contents of EV to other macromolecular structures in milk. A comprehensive protein network was composed tracing the possible cellular origins of milk-derived EV and the potential targets in the gut. Results: An average of 579 proteins was identified in EV, compared to 205 proteins in the non-floating fraction. Interestingly, EV associated proteins like ANXA5 and Flotillin were exclusively identified in EV, while CD9, CD63 and CD81 were also present in non-floating protein complexes. Additionally, MHC-II was identified in the EV fraction only, suggesting that antigenic epitopes may be delivered via EV released from antigen-presenting cells. Besides MHC-I, the mammary epithelial cell marker beta-1,4-galactosyltransferase (lactose subunit) was identified in the EV fraction only, demonstrating EV of epithelial origin. Furthermore, several adhesion molecules (ICAM-1, CEACAM-1) were associated to EV which could allow EV binding to gut epithelial cells and gut resident immune cells. Summary/conclusion: In-depth proteomic analysis and compilation of an extensive network of EV proteins involved in immunity demonstrates that milk-derived EV originate from multiple cellular sources and have the ability to target various cell types in the gut.ISEV 2015 is organized by The Local Organizing Committee: Kenneth Witwer (Chair, Baltimore), Shilpa Buch (Omaha), Prasun Datta (Philadelphia), Dolores Di Vizio (Los Angeles), Uta Erdbrügger (Charlottesville), Steven Jay (College Park), Dimitrios Kapogiannis (Baltimore), Leonid Margolis (Bethesda) & Susmita Sahoo (New York) Together with the Executive ISEV Board (2014 – 2016) President: Jan Lötvall Secretary General: Clotilde Théry Treasurer: Fred Hochberg Executive Chair Science / Meetings: Marca Wauben Executive Chair Education: Yong Song Gho Executive Chair Communication: Andrew Hill Members at Large: Peter Quesenberry, Kenneth Witwer, Susmita Sahoo, Dolores Di Vizio, Chris Gardiner, Edit Buzas, Hidetoshi Tahara, Suresh Mathivanan, Igor Kurochkin