Single Extracellular Vesicles Research Articles

EVAnalyzer: High content imaging for rigorous characterisation of single extracellular vesicles using standard laboratory equipment and a new open‐source ImageJ/Fiji plugin

EVAnalyzer: High content imaging for rigorous characterisation of single extracellular vesicles using standard laboratory equipment and a new open‐source ImageJ/Fiji plugin

Microfluidic Platform for Profiling of Extracellular Vesicles from Single Breast Cancer Cells.

Extracellular vesicles (EVs) are considered as valuable biomarkers to discriminate healthy from diseased cells such as cancer. Passing cytosolic and plasma membranes before their release, EVs inherit the biochemical properties of the cell. Here, we determine protein profiles of single EVs to understand how much they represent their cell of origin. We use a microfluidic platform which allows to immobilize EVs from completely isolated single cells, reducing heterogeneity of EVs as strongly seen in cell populations. After immunostaining, we employ four-color total internal reflection fluorescence microscopy to enumerate EVs and determine their biochemical fingerprint encoded in membranous or cytosolic proteins. Analyzing single cells derived from pleural effusions of two different human adenocarcinoma as well as from human embryonic kidney (SkBr3, MCF-7 and HEK293, respectively), we observed that a single cell secretes enough EVs to extract the respective tissue fingerprint. We show that overexpressed integral plasma membrane proteins are also found in EV membranes, which together with populations of colocalized proteins, provide a cell-specific, characteristic pattern. Our method highlights the potential of EVs as a diagnostic marker and can be directly employed for fundamental studies of EV biogenesis.

Dual Imaging Single Vesicle Surface Protein Profiling and Early Cancer Detection.

Single vesicle molecular profiling has the potential to transform cancer detection and monitoring by precisely probing cancer-associated extracellular vesicles (EVs) in the presence of normal EVs in body fluids, but it is challenging due to the small EV size, low abundance of antigens on individual vesicles, and a complex biological matrix. Here, we report a facile dual imaging single vesicle technology (DISVT) for surface protein profiling of individual EVs and quantification of target-specific EV subtypes based on direct molecular capture of EVs from diluted biofluids, dual EV-protein fluorescence-light scattering imaging, and fast image analysis using Bash scripts, Python, and ImageJ. Plasmonic gold nanoparticles (AuNPs) were used to label and detect targeted surface protein markers on individual EVs with dark-field light scattering imaging at the single particle level. Monte Carlo calculations estimated that the AuNPs could detect EVs down to 40 nm in diameter. Using the DISVT, we profiled surface protein markers of interest across individual EVs derived from several breast cancer cell lines, which reflected the parental cells. Studies with plasma EVs from healthy donors and breast cancer patients revealed that the DISVT, but not the traditional bulk enzyme-linked immunosorbent assay, detected human epidermal growth factor receptor 2 (HER2)-positive breast cancer at an early stage. The DISVT also precisely differentiated HER2-positive breast cancer from HER2-negative breast cancer. We additionally showed that the amount of tumor-associated EVs was tripled in locally advanced patients compared to that in early-stage patients. These studies suggest that single EV surface protein profiling with DISVT can provide a facile and high-sensitivity method for early cancer detection and quantitative monitoring.

Plasmon-Enhanced Characterization of Single Extracellular Vesicles.

Extracellular vesicles (EVs) represent heterogeneous populations of membrane-bound vesicles shed from almost all kinds of cells. Although superior to conventional methods, most newly developed EV sensing platforms still require a certain number of EVs, measuring bulk signals from a group of vesicles. A new analytical approach that enables single EV analysis could be extremely valuable for understanding EVs' subtypes, heterogeneity, and production dynamics during disease development and progression. Here, we describe a new nanoplasmonic sensing platform for sensitive single EV analysis. Termed nPLEX-FL (nano-plasmonic EV analysis with enhanced fluorescence detection), the system amplifies EVs' fluorescence signals using periodic gold nanohole structures, enabling sensitive, multiplexed analysis of single EVs.

Direct detection of circulating donor-derived extracellular vesicles in kidney transplant recipients

Extracellular vesicles (EVs) are tissue-specific particles containing valuable diagnostic information. However, single EV analysis in blood is challenging due to their physical properties, the molecular complexity of plasma, and a lack of robust data interpretation methods. We assess the applicability of our recently-developed calibrated Imaging Flow Cytometry (IFCM)-based methodology to detect/characterize circulating tissue-specific EV subsets in the clinical setting of kidney transplantation. Platelet-poor plasma was generated from 36 HLA-A3 mismatched donor (HLA-A3 +) and kidney transplant recipients (KTRs; HLA-A3-). Samples taken before transplantation, 3 days, 7 days, and 6 months after transplantation as well as before ‘for-cause’ kidney transplant biopsies were stained with anti-CD9 (plasma EV-marker) and anti-HLA-A3. Before transplantation, no significant differences in total CD9 + EV concentrations were detected between donor and KTR samples. Tissue-specific EVs were identified as CD9 + HLA-A3 + . Serial dilution experiments of HLA-A3 + in HLA-A3- PPP showed that single CD9 + HLA-A3 + EVs were detectable down to ~ 1% above the recipient ‘self-signal’. After transplantation, CD9 + HLA-A3 + EVs were detected above pre-transplantation concentrations in individuals with stable allograft function, but not in individuals with allograft dysfunction. These results demonstrate the applicability of our calibrated IFCM-based methodology in the direct detection of tissue-specific EV subsets in clinical samples. We believe that this EV methodology is applicable in a variety of clinical contexts.

Isolation-free measurement of single urinary extracellular vesicles by imaging flow cytometry

Urinary extracellular vesicles (uEVs) are promising biomarkers for various diseases. However, many tools measuring uEVs rely on time-consuming uEV isolation methods, which could induce sample bias. This study demonstrates the detection of single uEVs without isolation using imaging flow cytometry (IFCM). Unstained urine samples contained auto-fluorescent (A-F) particles when characterized with IFCM. Centrifugation successfully removed A-F particles from the unprocessed urine. Based on the disappearance of A-F particles, a gate was defined to distinguish uEVs from A-F particles. The final readouts of IFCM were verified as single EVs based on detergent treatment and serial dilutions. When developing this protocol to measure urine samples with abnormally high protein levels, 25 mg/mL dithiothreitol (DTT) showed improved uEV recovery over 200 mg/mL DTT. This study provides an isolation-free protocol using IFCM to quantify and phenotype single uEVs, eliminating the hindrance and influence of A-F particles, protein aggregates, and coincidence events.

EVAnalyzer: High content imaging for rigorous characterisation of single extracellular vesicles using standard laboratory equipment and a new open-source ImageJ/Fiji plugin.

Extracellular vesicle (EV) research increasingly demands for quantitative characterisation at the single vesicle level to address heterogeneity and complexity of EV subpopulations. Emerging, commercialised technologies for single EV analysis based on, for example, imaging flow cytometry or imaging after capture on chips generally require dedicated instrumentation and proprietary software not readily accessible to every lab. This limits their implementation for routine EV characterisation in the rapidly growing EV field. We and others have shown that single vesicles can be detected as light diffraction limited fluorescent spots using standard confocal and widefield fluorescence microscopes. Advancing this simple strategy into a process for routine EV quantitation, we developed 'EVAnalyzer', an ImageJ/Fiji (Fiji is just ImageJ) plugin for automated, quantitative single vesicle analysis from imaging data. Using EVAnalyzer, we established a robust protocol for capture, (immuno-)labelling and fluorescent imaging of EVs. To exemplify the application scope, the process was optimised and systematically tested for (i) quantification of EV subpopulations, (ii) validation of EV labelling reagents, (iii) in situ determination of antibody specificity, sensitivity and species cross-reactivity for EV markers and (iv) optimisation of genetic EV engineering. Additionally, we show that the process can be applied to synthetic nanoparticles, allowing to determine siRNA encapsulation efficiencies of lipid-based nanoparticles (LNPs) and protein loading of SiO2 nanoparticles. EVAnalyzer further provides a pipeline for automated quantification of cell uptake at the single cell-single vesicle level, thereby enabling high content EV cell uptake assays and plate-based screens. Notably, the entire procedure from sample preparation to the final data output is entirely based on standard reagents, materials, laboratory equipment and open access software. In summary, we show that EVAnalyzer enables rigorous characterisation of EVs with generally accessible tools. Since we further provide the plugin as open-source code, we expect EVAnalyzer to not only be a resource of immediate impact, but an open innovation platform for the EV and nanoparticle research communities.

Characterizing single extracellular vesicles by droplet barcode sequencing for protein analysis.

Small extracellular vesicles (sEVs) have in recent years evolved as a source of biomarkers for disease diagnosis and therapeutic follow up. sEV samples derived from multicellular organisms exhibit a high heterogeneous repertoire of vesicles which current methods based on ensemble measurements cannot capture. In this work we present droplet barcode sequencing for protein analysis (DBS-Pro) to profile surface proteins on individual sEVs, facilitating identification of sEV-subtypes within and between samples. The method allows for analysis of multiple proteins through use of DNA barcoded affinity reagents and sequencing as readout. High throughput single vesicle profiling is enabled through compartmentalization of individual sEVs in emulsion droplets followed by droplet barcoding through PCR. In this proof-of-concept study we demonstrate that DBS-Pro allows for analysis of single sEVs, with a mixing rate below 2%. A total of over 120,000 individual sEVs obtained from a NSCLC cell line and from malignant pleural effusion (MPE) fluid of NSCLC patients have been analyzed based on their surface proteins. We also show that the method enables single vesicle surface protein profiling and by extension characterization of sEV-subtypes, which is essential to identify the cellular origin of vesicles in heterogenous samples.

Upconversion nanoparticles for super-resolution quantification of single small extracellular vesicles

Although small EVs (sEVs) have been used widely as biomarkers in disease diagnosis, their heterogeneity at single EV level has rarely been revealed. This is because high-resolution characterization of sEV presents a major challenge, as their sizes are below the optical diffraction limit. Here, we report that upconversion nanoparticles (UCNPs) can be used for super-resolution profiling the molecular heterogeneity of sEVs. We show that Er3+-doped UCNPs has better brightness and Tm3+-doped UCNPs resulting in better resolution beyond diffraction limit. Through an orthogonal experimental design, the specific targeting of UCNPs to the tumour epitope on single EV has been cross validated, resulting in the Pearson’s R-value of 0.83 for large EVs and ~ 65% co-localization double-positive spots for sEVs. Furthermore, super-resolution nanoscopy can distinguish adjacent UCNPs on single sEV with a resolution of as high as 41.9 nm. When decreasing the size of UCNPs from 40 to 27 nm and 18 nm, we observed that the maximum UCNPs number on single sEV increased from 3 to 9 and 21, respectively. This work suggests the great potentials of UCNPs approach “digitally” quantify the surface antigens on single EVs, therefore providing a solution to monitor the EV heterogeneity changes along with the tumour progression progress.

Wrapping of a vesicle nanoparticle with variable bending stiffness by membrane

Cellular uptake of nanoparticle (NP) is an important biological process involving mechanically and structurally heterogeneous environments, such as biophysical heterogeneity of single extracellular vesicles and biomechanical heterogeneity of small viral capsids. Despite of these heterogeneous environments, poor understanding of membrane interacting with vesicle NP of non-uniform mechanical properties still exists. To address this issue, combining the continuum Canham–Helfrich theory of membrane and the stochastic Markov method on state transition, we establish a theoretical model of the wrapping of a NP with any variable bending stiffness. As NPs are hypothesized with two typical types of non-uniform bending stiffness distributions (Gaussian-like and piecewise), through both minimum energy and stochastic dynamic approaches, it is identified that the membrane tends to encapsulate the NP with a sealing near soft region of NP. Because complete wrapping of NP with a sealing near its stiff region requires large elastic deformation of membrane. In addition, during cellular uptake, rotation phenomenon of the NP with variable bending stiffness is found, which is reminiscent of previous observations on homogeneous NP as reported in literature. These predictions are verified by relevant Monte Carlo simulations. Our findings are of interest to not only the fundamental understanding of cellular uptake but also the applications in the design of nanocarriers for drug delivery.

Electrochemical Resistive-Pulse Sensing of Extracellular Vesicles.

Extracellular vesicles (EVs) released from biological cells have attracted considerable interest due to their potential for cancer diagnostics and important role in cell signaling. Most previously reported studies have been concerned with the detection of EVs in biofluids and analysis of proteins and nucleic acids they contain. Electrochemical resistive-pulse (ERP) sensing enables direct detection of single EVs released from a specific cell and analysis of reactive oxygen and nitrogen species in such vesicles. Here, we demonstrate the applicability of ERP sensing to distinguish between nontransformed and cancerous breast cell lines as well as between breast cancer cell lines with different metastatic potential. Another application of ERP sensing is in real-time monitoring of changes in a single cell induced by a chemical agent. This approach is potentially useful for evaluating the efficacy of therapeutic agents, including those that trigger breast cancer cell death by inducing intense oxidative stress.

A Mechanoelastic Glimpse on Hyaluronan-Coated Extracellular Vesicles.

Cancer cells secrete extracellular vesicles (EVs) covered with a carbohydrate polymer, hyaluronan (HA), linked to tumor malignancy. Herein, we have unravelled the contour lengths of HA on a single cancer cell-derived EV surface using single-molecule force spectroscopy (SMFS), which divulges the presence of low molecular weight HA (LMW-HA < 200 kDa). We also discovered that these LMW-HA-EVs are significantly more elastic than the normal cell-derived EVs. This intrinsic elasticity of cancer EVs could be directly allied to the LMW-HA abundance and associated labile water network on EV surface as revealed by correlative SMFS, hydration dynamics with fluorescence spectroscopy, and molecular dynamics simulations. This method emerges as a molecular biosensor of the cancer microenvironment.

Transcriptomic Features in a Single Extracellular Vesicle via Single-Cell RNA Sequencing.

Although many studies have investigated functional molecules in extracellular vesicles (EVs), the exact number of ribonucleic acid molecules in a single-EV is unknown. Therefore, it is critical to explore the transcriptomic features and heterogeneity at the level of a single-EV. Here, using the 10x Genomics platform, the RNA cargos are profiled in single EVs derived from human K562 and mesenchymal stem cells. The key steps are labeling intact EVs using calcein-AM, detecting the EV concentration via flow cytometry, and using the CB2 algorithm with adaptive thresholds to effectively distinguish real EVs from background. The gene number in a single-EV varied from 6 to 148, with a mean of 52. Ribosomal genes, mitochondrial genes, and eukaryotic translation elongation factor 1 alpha has a high EV percentage in all EV samples. Hemoglobin genes are uniquely highly expressed in K562-EVs, and cytoskeleton genes are enriched in MSC-EVs. Ten or more clusters with different marker genes in each single-EV dataset demonstrated EV heterogeneity. Moreover, integrating EVs and their parental cells reveal both EVs and cells in each cluster, indicating different cell origins of various EVs. To the best of the author's knowledge, this study provides the first high-throughput transcriptome at the single-EV level and improves the understanding of EVs.

Single-particle assessment of six different drug-loading strategies for incorporating doxorubicin into small extracellular vesicles.

Extracellular vesicles (EVs) have emerged as an attractive drug delivery system owing to their natural roles in intercellular communication. On account of the large intrinsic heterogeneity of EVs, it is highly desirable to evaluate not only the encapsulation efficiency but also the alteration of biological functionality after the drug-loading process at the single-particle level. However, the nanoscale size of EVs poses a great challenge. Taking advantage of nano-flow cytometry (nFCM) in the multiparameter analysis of single EVs as small as 40nm, six commonly used drug-loading strategies (coincubation, electroporation, extrusion, freeze-thawing, sonication, and surfactant treatment) were exploited by employing doxorubicin (Dox) as the model drug. Encapsulation ratio, EV concentration, drug content, and membrane proteins of Dox-loaded EVs were measured at the single-particle level. Our data indicated that coincubation and electroporation outperformed other methods with an encapsulation ratio of approximately 45% and a higher Dox content in single EVs. Interestingly, the labeling ratios of membrane proteins indicated that varying degrees of damage to the surface proteins of EVs occurred upon extrusion, freeze-thawing, sonication, and surfactant treatment. Confocal fluorescence microscopy and flow cytometry analysis revealed that Dox-loaded EVs prepared by electroporation induced the strongest apoptosis followed by coincubation. These results correlated well with their cellular uptake rate and fundamentally with the Dox encapsulation efficiency of single EVs. nFCM provides a rapid and sensitive platform for single-particle assessment of drug-loading strategies for incorporating drugs into EVs.

Quantitative, in situ visualization of intracellular insulin vesicles in pancreatic beta cells

Characterizing relationships between Zn2+, insulin, and insulin vesicles is of vital importance to the study of pancreatic beta cells. However, the precise content of Zn2+ and the specific location of insulin inside insulin vesicles are not clear, which hinders a thorough understanding of the insulin secretion process and diseases caused by blood sugar dysregulation. Here, we demonstrated the colocalization of Zn2+ and insulin in both single extracellular insulin vesicles and pancreatic beta cells by using an X-ray scanning coherent diffraction imaging (ptychography) technique. We also analyzed the elemental Zn2+ and Ca2+ contents of insulin vesicles using electron microscopy and energy dispersive spectroscopy (EDS) mapping. We found that the presence of Zn2+ is an important characteristic that can be used to distinguish insulin vesicles from other types of vesicles in pancreatic beta cells and that the content of Zn2+ is proportional to the size of insulin vesicles. By using dual-energy contrast X-ray microscopy and scanning transmission X-ray microscopy (STXM) image stacks, we observed that insulin accumulates in the off-center position of extracellular insulin vesicles. Furthermore, the spatial distribution of insulin vesicles and their colocalization with other organelles inside pancreatic beta cells were demonstrated using three-dimensional (3D) imaging by combining X-ray ptychography and an equally sloped tomography (EST) algorithm. This study describes a powerful method to univocally describe the location and quantitative analysis of intracellular insulin, which will be of great significance to the study of diabetes and other blood sugar diseases.

Single extracellular vesicle analysis for early cancer detection.

Extracellular vesicles (EVs) are actively shed into the circulation from both cancer and host cells. EVs are emerging as one of the diagnostic frontrunners for early cancer detection, disease monitoring, and treatment evaluation. The advantages of EVs rely on the fact that vesicles are being shed by dividing tumor cells, with indications that human and viral oncogenes, cellular metabolic rate, and tumor characteristics such as pH and hypoxia contribute to the high shed rates in cancer. In this review, we provide an overview of EVs and the rationale for using them for early cancer detection. We examine emerging technologies for single EV analysis (sEVA) and why these technologies will be necessary for early cancer detection.

Future of Digital Assays to Resolve Clinical Heterogeneity of Single Extracellular Vesicles.

Extracellular vesicles (EVs) are complex lipid membrane vehicles with variable expressions of molecular cargo, composed of diverse subpopulations that participate in the intercellular signaling of biological responses in disease. EV-based liquid biopsies demonstrate invaluable clinical potential for overhauling current practices of disease management. Yet, EV heterogeneity is a major needle-in-a-haystack challenge to translate their use into clinical practice. In this review, existing digital assays will be discussed to analyze EVs at a single vesicle resolution, and future opportunities to optimize the throughput, multiplexing, and sensitivity of current digital EV assays will be highlighted. Furthermore, this review will outline the challenges and opportunities that impact the clinical translation of single EV technologies for disease diagnostics and treatment monitoring.

Recent Advances in Molecular Diagnosis of Pulmonary Fibrosis for Precision Medicine.

Pulmonary fibrosis is a serious, progressive lung disease characterized by scarring and stiffening lung tissues, affecting the respiratory system and leading to organ failure. It is a complex disease consisting of alveolar damage, chronic inflammation, and a varying degree of lung fibrosis. Significant challenges with pulmonary fibrosis include the lack of effective means to diagnose the disease at early stages, identify patients at higher risks of progress, and assess disease progression and treatment response. Precision medicine powered by accurate molecular profiling and phenotyping could significantly improve our understanding of the disease's heterogeneity, potential biomarkers for diagnosis and prognosis, and molecular targets for treatment development. This Review discusses various translational model systems, including organoids and lung-on-a-chip systems, biomarkers in single cells and extracellular vesicles, and functional pharmacodynamic markers. We also highlight emerging sensing technologies for molecular characterization of pulmonary fibrosis and biomarker detection.

An imaging flow cytometry-based methodology for the analysis of single extracellular vesicles in unprocessed human plasma

Extracellular vesicles (EVs) are tissue-specific particles released by cells containing valuable diagnostic information in the form of various biomolecules. To rule out selection bias or introduction of artefacts caused by EV isolation techniques, we present a clinically feasible, imaging flow cytometry (IFCM)–based methodology to phenotype and determine the concentration of EVs with a diameter ≤400 nm in human platelet-poor plasma (PPP) without prior isolation of EVs. Instrument calibration (both size and fluorescence) were performed with commercial polystyrene beads. Detergent treatment of EVs was performed to discriminate true vesicular events from artefacts. Using a combination of markers (CFSE & Tetraspanins, or CD9 & CD31) we found that >90% of double-positive fluorescent events represented single EVs. Through this work, we provide a framework that will allow the application of IFCM for EV analysis in peripheral blood plasma in a plethora of experimental and potentially diagnostic settings. Additionally, this direct approach for EV analysis will enable researchers to explore corners of EVs as cellular messengers in healthy and pathological conditions.

Mesenchymal Stem Cell-derived Extracellular Vesicles Prevent Experimental Bronchopulmonary Dysplasia Complicated By Pulmonary Hypertension.

Mesenchymal stem cell (MSC) extracellular vesicles (EVs) have beneficial effects in preclinical bronchopulmonary dysplasia and pulmonary hypertension (BPD-PH) models. The optimal source, dosing, route, and duration of effects are however unknown. The objectives of this study were to (a) compare the efficacy of GMP-grade EVs obtained from Wharton’s Jelly MSCs (WJ-MSCs) and bone marrow (BM-MSCs), (b) determine the optimal dosing and route of administration, (c) evaluate its long-term effects, and (d) determine how MSC EVs alter the lung transcriptome. Newborn rats exposed to normoxia or hyperoxia (85% O2) from postnatal day (P)1-P14 were given (a) intra-tracheal (IT) BM or WJ-MSC EVs or placebo, (b) varying doses of IT WJ-MSC EVs, or (c) IT or intravenous (IV) WJ-MSC EVs on P3. Rats were evaluated at P14 or 3 months. Early administration of IT BM-MSC or WJ-MSC EVs had similar beneficial effects on lung structure and PH in hyperoxia-exposed rats. WJ-MSC EVs however had superior effects on cardiac remodeling. Low, medium, and high dose WJ-MSC EVs had similar cardiopulmonary regenerative effects. IT and IV WJ-MSC EVs similarly improved vascular density and reduced PH in hyperoxic rats. Gene-set enrichment analysis of transcripts differentially expressed in WJ-MSC EV-treated rats showed that induced transcripts were associated with angiogenesis. Long-term studies demonstrated that a single early MSC EV dose has pulmonary vascular protective effects 3 months after administration. Together, our findings have significant translational implications as it provides critical insight into the optimal source, dosing, route, mechanisms of action, and duration of effects of MSC-EVs for BPD-PH.