Receptor activator of nuclear factor-kappa B is enriched in CD9-positive extracellular vesicles released by osteoclasts.

Aim: Receptor activator of nuclear factor-kappa B (RANK)-containing extracellular vesicles (EVs) bind RANK-Ligand (RANKL) on osteoblasts, and thereby simultaneously inhibit bone resorption and promote bone formation. Because of this, they are attractive candidates for therapeutic bone anabolic agents. Previously, RANK was detected in 1 in every 36 EVs from osteoclasts by immunogold electron microscopy.

PDZ scaffolds regulate extracellular vesicle production, composition, and uptake.

Extracellular vesicles (EVs) are membrane-limited organelles mediating cell-to-cell communication in health and disease. EVs are of high medical interest, but their rational use for diagnostics or therapies is restricted by our limited understanding of the molecular mechanisms governing EV biology. Here, we tested whether PDZ proteins, molecular scaffolds that support the formation, transport, and function of signal transduction complexes and that coevolved with multicellularity, may represent important EV regulators.

Extracellular Vesicle-Encapsulated Adeno-Associated Viruses for Therapeutic Gene Delivery to the Heart.

Background: Adeno-associated virus (AAV) has emerged as one of the best tools for cardiac gene delivery due to its cardiotropism, long-term expression, and safety. However, a significant challenge to its successful clinical use is preexisting neutralizing antibodies (NAbs), which bind to free AAVs, prevent efficient gene transduction, and reduce or negate therapeutic effects. Here we describe extracellular vesicle-encapsulated AAVs (EV-AAVs), secreted naturally by AAV-producing cells, as a superior cardiac gene delivery vector that delivers more genes and offers higher NAb resistance.

Plasma extracellular vesicle transcriptome as a dynamic liquid biopsy in acute heart failure.

Background: Acute decompensation is associated with increased mortality in heart failure (HF) patients, though the underlying etiology remains unclear. Extracellular vesicles (EVs) and their cargo may mark specific cardiovascular physiologic states. We hypothesized that EV transcriptomic cargo, including long non-coding RNAs (lncRNAs) and mRNAs, is dynamic from the decompensated to recompensated HF state, reflecting molecular pathways relevant to adverse remodeling.

Single vesicle analysis of CD47 association with integrins and tetraspanins on extracellular vesicles released by T lymphoblast and prostate carcinoma cells.

CD47 regulates the trafficking of specific coding and noncoding RNAs into extracellular vesicles (EVs), and the RNA contents of CD47 EVs differ from that of CD63 EVs released by the same cells. Single particle interferometric reflectance imaging sensing combined with immunofluorescent imaging was used to analyse the colocalization of tetraspanins, integrins, and CD47 on EVs produced by wild type and CD47-deficient Jurkat T lymphoblast and PC3 prostate carcinoma cell lines. On Jurkat cell-derived EVs, β1 and α4 integrin subunits colocalized predominantly with CD47 and CD81 but not with CD63 and CD9, conserving the known lateral interactions between these proteins in the plasma membrane.

A shared, stochastic pathway mediates exosome protein budding along plasma and endosome membranes.

Exosomes are small extracellular vesicles of ∼30 to 150 nm that are secreted by all cells, abundant in all biofluids, and play important roles in health and disease. However, details about the mechanism of exosome biogenesis are unclear. Here, we carried out a cargo-based analysis of exosome cargo protein biogenesis in which we identified the most highly enriched exosomal cargo proteins and then followed their biogenesis, trafficking, and exosomal secretion to test different hypotheses for how cells make exosomes.

ICAM-1-mediated adhesion is a prerequisite for exosome-induced T cell suppression.

Tumor-derived extracellular vesicles (TEVs) suppress the proliferation and cytotoxicity of CD8 T cells, thereby contributing to tumor immune evasion. Here, we report that the adhesion molecule intercellular adhesion molecule 1 (ICAM-1) co-localizes with programmed death ligand 1 (PD-L1) on the exosomes; both ICAM-1 and PD-L1 are upregulated by interferon-γ. Exosomal ICAM-1 interacts with LFA-1, which is upregulated in activated T cells.

Single-Particle Interferometric Reflectance Imaging Characterization of Individual Extracellular Vesicles and Population Dynamics.

Extracellular vesicles (EVs) are nanometer-sized vesicles with a lipid bilayer that are secreted by most cells. EVs carry a multitude of different biological molecules, including protein, lipid, DNA, and RNA, and are postulated to facilitate cell-to-cell communication in diverse tissues and organs. Recently, EVs have attracted significant attention as biomarkers for diagnostics and therapeutic agents for various diseases.

The membrane associated accessory protein is an adeno-associated viral egress factor.

Adeno-associated viruses (AAV) rely on helper viruses to transition from latency to lytic infection. Some AAV serotypes are secreted in a pre-lytic manner as free or extracellular vesicle (EV)-associated particles, although mechanisms underlying such are unknown. Here, we discover that the membrane-associated accessory protein (MAAP), expressed from a frameshifted open reading frame in the AAV cap gene, is a novel viral egress factor.

Quantification of protein cargo loading into engineered extracellular vesicles at single-vesicle and single-molecule resolution.

Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single-vesicle and single-molecule level to accurately quantify the efficiency of different EV-sorting proteins in promoting cargo loading into EVs.

Multiplexed Affinity Measurements of Extracellular Vesicles Binding Kinetics.

Extracellular vesicles (EVs) have attracted significant attention as impactful diagnostic biomarkers, since their properties are closely related to specific clinical conditions. However, designing experiments that involve EVs phenotyping is usually highly challenging and time-consuming, due to laborious optimization steps that require very long or even overnight incubation durations. In this work, we demonstrate label-free, real-time detection, and phenotyping of extracellular vesicles binding to a multiplexed surface.

PDX-derived organoids model in vivo drug response and secrete biomarkers.

Patient-derived organoid models are proving to be a powerful platform for both basic and translational studies. Here we conduct a methodical analysis of pancreatic ductal adenocarcinoma (PDAC) tumor organoid drug response in paired patient-derived xenograft (PDX) and PDX-derived organoid (PXO) models grown under WNT-free culture conditions. We report a specific relationship between area under the curve value of organoid drug dose response and in vivo tumor growth, irrespective of the drug treatment.

Annexin A1-dependent tethering promotes extracellular vesicle aggregation revealed with single-extracellular vesicle analysis.

Extracellular vesicles (EVs) including plasma membrane-derived microvesicles and endosomal-derived exosomes aggregate by unknown mechanisms, forming microcalcifications that promote cardiovascular disease, the leading cause of death worldwide. Here, we show a framework for assessing cell-independent EV mechanisms in disease by suggesting that annexin A1 (ANXA1)-dependent tethering induces EV aggregation and microcalcification. We present single-EV microarray, a method to distinguish microvesicles from exosomes and assess heterogeneity at a single-EV level.

Small extracellular vesicles modulated by αVβ3 integrin induce neuroendocrine differentiation in recipient cancer cells.

The ability of small extracellular vesicles (sEVs) to reprogram cancer cells is well established. However, the specific sEV components able to mediate aberrant effects in cancer cells have not been characterized. Integrins are major players in mediating sEV functions.

Reporter mice for isolating and auditing cell type-specific extracellular vesicles in vivo.

Extracellular vesicles (EVs) are abundant, lipid-enclosed vectors that contain nucleic acids and proteins, they can be secreted from donor cells and freely circulate, and they can be engulfed by recipient cells thus enabling systemic communication between heterotypic cell types. However, genetic tools for labeling, isolating, and auditing cell type-specific EVs in vivo, without prior in vitro manipulation, are lacking. We have used CRISPR-Cas9-mediated genome editing to generate mice bearing a CD63-emGFP knock-in cassette that enables the specific labeling of circulating CD63 vesicles from any cell type when crossed with lineage-specific Cre recombinase driver mice.

Membrane-binding peptides for extracellular vesicles on-chip analysis.

Small extracellular vesicles (sEVs) present fairly distinctive lipid membrane features in the extracellular environment. These include high curvature, lipid-packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. sEV membrane could be then considered as a "universal" marker, alternative or complementary to traditional, characteristic, surface-associated proteins.

Summary of the ISEV workshop on extracellular vesicles as disease biomarkers, held in Birmingham, UK, during December 2017.

This report summarises the presentations and activities of the ISEV Workshop on extracellular vesicle biomarkers held in Birmingham, UK during December 2017. Among the key messages was broad agreement about the importance of biospecimen science. Much greater attention needs to be paid towards the provenance of collected samples.

Small RNA Sequencing across Diverse Biofluids Identifies Optimal Methods for exRNA Isolation.

Poor reproducibility within and across studies arising from lack of knowledge regarding the performance of extracellular RNA (exRNA) isolation methods has hindered progress in the exRNA field. A systematic comparison of 10 exRNA isolation methods across 5 biofluids revealed marked differences in the complexity and reproducibility of the resulting small RNA-seq profiles. The relative efficiency with which each method accessed different exRNA carrier subclasses was determined by estimating the proportions of extracellular vesicle (EV)-, ribonucleoprotein (RNP)-, and high-density lipoprotein (HDL)-specific miRNA signatures in each profile.

CD44s Assembles Hyaluronan Coat on Filopodia and Extracellular Vesicles and Induces Tumorigenicity of MKN74 Gastric Carcinoma Cells.

CD44 is a multifunctional adhesion molecule typically upregulated in malignant, inflamed and injured tissues. Due to its ability to bind multiple ligands present in the tumor microenvironment, it promotes multiple cellular functions related to tumorigenesis. Recent data has shown that CD44 and its principal ligand hyaluronan (HA) are carried by extracellular vesicles (EV) derived from stem and tumor cells, but the role of CD44 in EV shedding has not been studied so far.

Digital Microarrays: Single-Molecule Readout with Interferometric Detection of Plasmonic Nanorod Labels.

DNA and protein microarrays are a high-throughput technology that allow the simultaneous quantification of tens of thousands of different biomolecular species. The mediocre sensitivity and limited dynamic range of traditional fluorescence microarrays compared to other detection techniques have been the technology's Achilles' heel and prevented their adoption for many biomedical and clinical diagnostic applications. Previous work to enhance the sensitivity of microarray readout to the single-molecule ("digital") regime have either required signal amplifying chemistry or sacrificed throughput, nixing the platform's primary advantages.

Enhanced light microscopy visualization of virus particles from Zika virus to filamentous ebolaviruses.

Light microscopy is a powerful tool in the detection and analysis of parasites, fungi, and prokaryotes, but has been challenging to use for the detection of individual virus particles. Unlabeled virus particles are too small to be visualized using standard visible light microscopy. Characterization of virus particles is typically performed using higher resolution approaches such as electron microscopy or atomic force microscopy.

Visualizing Ebolavirus Particles Using Single-Particle Interferometric Reflectance Imaging Sensor (SP-IRIS).

This chapter describes an approach for the label-free imaging and quantification of intact Ebola virus (EBOV) and EBOV viruslike particles (VLPs) using a light microscopy technique. In this technique, individual virus particles are captured onto a silicon chip that has been printed with spots of virus-specific capture antibodies. These captured virions are then detected using an optical approach called interference reflectance imaging.

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.