Extracellular vesicles in laboratory medicine: a review and outlook

In recent years, the cargo profiles of extracellular vesicles (EVs), which were inherited from their parent cells, have emerged as a reliable biomarker for liquid biopsy (LB) in disease diagnosis, prognosis, and treatment monitoring. EVs secreted by different cells exhibit distinct characteristics, particularly in terms of disease diagnosis and prediction. However, currently available techniques for the quantitative analysis of EV cargoes, including enzyme-linked immunosorbent assay (ELISA), cannot specifically identify the cellular origin of EVs, thus seriously affecting the accuracy of EV-based liquid biopsy. In light of this, we here developed ultrabright fluorescent nanosphere (FNs)-based test strips which have the unique capability to specifically assess the levels of PD-L1-positive EVs (PD-L1+ EVs) derived from both tumor cells and immune cells in bodily fluids. The levels of PD-L1+ EV subpopulations in human saliva were quantified using the ultrabright fluorescent nanosphere-based test strips with more convenience and higher efficiency (detection time <30 min). Results demonstrated that the fluorescence intensity of the test line exhibited a good linear relationship respectively with the PD-L1 levels of tumor cell- (R2 = 0.993) and immune cell-derived EVs (R2 = 0.982) in human saliva. By assessing the levels of PD-L1+ EV subpopulations, our test strips hold immense potential for advancing the application of PD-L1+ EV subpopulation-based predictions in tumor diagnosis and prognosis evaluation. In summary, by integrating the benefits of FNs and lateral flow chromatography, we here provide a strategy to accurately measure the cargo levels of EVs originating from diverse cell sources in bodily fluids.

Extracellular vesicles (EVs) are nanosized vesicles enclosed in a lipid membrane that are sustainably released by nearly all cell types. EVs have been deemed as valuable biomarkers for diagnostics and effective drug carriers, owing to the physiological function of transporting biomolecules for intercellular communication. To investigate their biological properties, efficient labeling strategies have been constructed for EV research, among which fluorescence labeling exerts a powerful function due to the capability of visualizing the nanovesicles with high sensitivity both in vitro and in vivo. In one aspect, with the help of functional fluorescence tags, EVs could be differentiated and categorized in vitro by various analytical techniques, which exert vital roles in disease diagnosis, prognosis, and treatment monitoring. Additionally, innovative EV reporters have been utilized for visualizing EVs, in combination with powerful microscopy techniques, which provide potential tools for investigating the dynamic events of EV release and intercellular communication in suitable animal models. In this feature article, we survey the latest advances regarding EV fluorescence labeling strategies and their application in biomedical application and in vivo biology investigation, highlighting the progresses in individual EV imaging. Finally, the challenges and future perspectives in unravelling EV physiological properties and further biomedical application are discussed.

Purpose/Objective(s): Because Extracellular Vesicles (EVs) carry surface receptors that are characteristic of their cells of origin, EVs have tremendous potential as non-invasive biomarkers for diagnosis, risk-stratification, treatment selection, and treatment monitoring. We developed a first-in-class pipeline to characterize EV heterogeneity and provide high-sensitivity quantification of informative EVs in biofluids before, during, and after treatment. By combining multiplex assays with high-resolution, single EV flow cytometric methods together into a Mutiplex-to-Single EV Analysis (Mt-SEA) pipeline, we are able to characterize a broad range of relevant EV subsets, while also accurately measuring the concentration of specific EV populations. Exploratory studies presented here validate the performance of Mt-SEA method by confirming strong correlations of liquid biopsy EV repertoires with tumor burden and responses to treatment. Furthermore, EV analysis with Mt-SEA may identify previously unrecognized prognostic epitopes or EVs subsets. Materials/Methods: To evaluate the use of this pipeline in an exploratory clinical cohort, we evaluated EVs from plasma samples of Adult T Cell Leukemia/Lymphoma patients receiving palliative radiation. Plasma was obtained before and after treatment (n = 5 treatment courses). A customized multiplex array was used with detection antibodies to identify more than 40 major EV subsets. General exosome and EV detection epitopes included CD63, CD9, and CD81. Tumor-specific epitopes for each patient included CD4, CD5, and CD25, based on available histo-/cyto-pathology results. Next, high-resolution single EV analyses were performed with nanoFACS sorting and a prototype nanoFCM analyzer. Results: The Mt-SEA pipeline provided a broad survey of EV populations, followed by individual EV analysis ATLL-derived EVs were detected in each pre-treatment sample, with reduced specific ATLL-derived EV subsets concentrations at the end of treatment. Furthermore, ATLL-specific EVs from patients with progressive systemic disease prior to treatment were found to carry CD44 and CD133 epitopes, consistent with increasing tumor aggressiveness. Responses to treatment that were clinically evident mirrored changes in the Mt-SEA EV profiles, and Mt-SEA identified new candidate prognostic EV profiles associated with clinical outcomes that would not have been predicted. Conclusions: Optimal leverage the information carried by tumor- and immune cell-derived EVs (to use EVs as clinical biomarkers) will require a combination of methods to 1) broadly characterize EVs and 2) rigorously enumerate specific selected EV subsets using single molecule detection. The Mt-SEA pipeline achieves both of these goals. Our exploratory study with these ATLL cases demonstrates that Mt-SEA provides unexpected insights into tumor biology, along with robust estimations of concentrations of EV subsets of interest. Detection of tumor-associated EVs and detection of EV repertoire changes during treatment paves the way to future evaluation of the Mt-SEA pipeline for personalized, adaptive therapies in a wider range of tumor types. Citation Format: Jennifer Jones, Joshua Welsh, Jason Savage, Jenn Marte, James Gulley, Kevin Conlon, Thomas Waldmann, Kevin Camphausen, Jay Berzofsky. Pipeline for High Throughput Analysis of Exosomes in Clinical Biofluids [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A06.

Optimization of plasma extracellular vesicle enrichment for in‐depth lipidomic profiling in Alzheimer’s disease

Extracellular vesicles (EVs) are carriers of a diverse array of bioactive molecules, making them valuable clinical tools for liquid biopsy in disease diagnosis and prognosis evaluation. These molecules play critical roles in various physiological and pathological conditions, and effective separation of EVs is essential to achieve these objectives. Due to the high heterogeneity of EVs, particularly with regard to their cargo molecules, merely isolating the general EV population is inadequate for liquid biopsy and biological function studies. Therefore, separating EV subpopulations becomes crucial. Traditional separation methods, such as differential ultracentrifugation and size exclusion chromatography, along with burgeoning techniques like classical microfluidic chips and covalent chemistry, often prove time-consuming, yield low purity, and have limited ability to address cargo heterogeneity. Thus, precise separation of EV subpopulations is of utmost importance. Additionally, detecting subpopulation-specific cargo is vital for validating the effectiveness of separation methods and supporting clinical biopsy applications. However, reviews that focus specifically on detection methods for EV subpopulations are limited. This paper provides a comprehensive overview of the methods for separating and detecting EV subpopulations with surface marker heterogeneity, comparing the advantages and limitations of each technique. Furthermore, it discusses challenges and future prospects for these methods in the context of liquid biopsy and downstream research. Collectively, this review aims to offer innovative insights into the separation and detection of EV subpopulations, guiding researchers to avoid common pitfalls and refine their investigative approaches.

Extracellular vesicles (EVs), one of the basic ways of intercellular interactions, carry a large number of biological substances and show great potential in disease diagnosis and treatment. Compared with tissue biopsy, its detection has the advantages of non-invasive, convenient sampling and real-time monitoring. Therefore, the research and clinical application of EVs biomarkers have become an international research hotspot. In order to encourage the development of screening and detection technologies for EVs biomarkers, effectively improve the accuracy of EVs biomarker diagnosis, and promote the transformation of EVs biomarker research results into the clinical application of disease, this review summarizes the significance of EVs in disease diagnosis and its biomarker screening strategy and clinical validation. Key words: Extracellular vesicles; Biomarkers; Clinical laboratory techniques; Validation studies

Extracellular vesicles are commonly found in human body fluids and can reflect current physiological conditions of human body and act as biomarkers of disease. The quality of isolated extracellular vesicles facilitates the early diagnosis of various diseases accompanied by hyperlipidemia. Nonetheless, there are no reports on which special methods are suitable for isolating extracellular vesicles from the plasma of patients with hyperlipidemia. Thus, this study compared three different research-based extracellular vesicle isolation approaches, namely ultracentrifugation (UC), polyethylene glycol (PEG) precipitation, and size exclusion chromatography (SEC), and determined which of them was the most effective method. We selected blood samples from 12 patients with clinically diagnosed hyperlipidemia and isolated plasma-derived extracellular vesicles using three methods. The morphology of the isolated extracellular vesicles was observed using transmission electron microscopy, while the concentration was detected by asymmetric flow field-flow fractionation and multi-angle light scattering. Marker proteins were identified by Western blotting, and protein composition was evaluated by silver staining. Both determined the contaminations in the extracellular vesicle samples. The results showed that the three methods can be successfully used for the isolation of extracellular vesicles. The extracellular vesicles isolated by UC were larger in size, and the yield was much lower. Although the yield of extracellular vesicles isolated by PEG precipitation was greatly improved, the contamination was increased. Of the three methods, only the SEC-isolated extracellular vesicles were characterized by high yield and low contamination. Therefore, our data suggested that the SEC was a more ideal method for isolating extracellular vesicles from the plasma of patients with hyperlipidemia.

CAD increases the long noncoding RNA PUNISHER in small extracellular vesicles and regulates endothelial cell function via vesicular shuttling

Current insights on extracellular vesicle-mediated glioblastoma progression: Implications in drug resistance and epithelial-mesenchymal transition

Extracellular vesicles (EVs) play important roles in cell-to-cell communications and carry high potential as markers targeted in disease diagnosis, prognosis, and therapeutic development. The main obstacles to EV study are their high heterogeneity; low amounts present in samples; and physical similarity to the abundant, interfering matrix components. Multiple rounds of separation and purification are often needed prior to EV characterization and function assessment. Herein, we report the offline coupling of asymmetrical flow field-flow fractionation (AF4) and capillary electrophoresis (CE) for EV analysis. While AF4 provides gentle and fast EV separation by size, CE resolves EVs from contaminants with similar sizes but different surface charges. Employing Western Blotting, ELISA, and SEM, we confirmed that intact EVs were eluted within a stable time window under the optimal AF4 and CE conditions. We also proved that EVs could be resolved from free proteins and high-density lipoproteins by AF4 and be further separated from the low-density lipoproteins co-eluted in AF4. The effectiveness of the coupled AF4-CE system in EV analysis was demonstrated by monitoring the changes in EV secretion from cells and by direct injection of human serum and detection of serum EVs. We believe that coupling AF4 and CE can provide rapid EV quantification in biological samples with much reduced matrix interference and be valuable for the study of total EVs and EV subpopulations produced by cells or present in clinical samples.

Introduction The hunt for new biomarkers – for the diagnosis of subcategories of disease, or for the monitoring of the efficacy of novel therapeutics – is an increasingly relevant challenge in the current era of precision medicine. In neurodegenerative research, the aim is to look for simple tools which can predict cognitive or motor decline early, and to determine whether these can also be used to test the efficacy of new interventions. Extracellular vesicles (EVs) are thought to play an important role in intercellular communication and have been shown to play a vital role in a number of diseases. Areas Covered The aim of this review is to examine what we know about EVs in neurodegeneration and to discuss their potential to be diagnostic and prognostic biomarkers in the future. It will cover the techniques used to isolate and study EVs and what is currently known about their presence in neurodegenerative diseases. In particular, we will discuss what is required for standardization in biomarker research, and the challenges associated with using EVs within this framework. Expert Opinion The technical challenges associated with isolating EVs consistently, combined with the complex techniques required for their efficient analysis, might preclude ‘pure’ EV populations from being used as effective biomarkers. Whilst biomarker discovery is important for more effective diagnosis, monitoring, prediction and prognosis in neurodegenerative disease, reproducibility and ease-of-use should be the priorities.

Diabetic Retinopathy (DR) is a potentially blinding retinal disorder that develops through the pathogenesis of diabetes. The lack of disease predictors implies a poor prognosis with frequent irreversible retinal damage and vision loss. Extracellular Vesicles (EVs) present a novel opportunity for pre-symptomatic disease diagnosis and prognosis, both severely limited in DR. All biological fluids contain EVs, which are currently being studied as disease biomarkers. EV proteins derived from urine have emerged as potential noninvasive biomarkers. In this study, we isolated EVs from DR retinal tissue explants and from DR patients' urine, and characterized the vesicles, finding differences in particle number and size. Next, we performed proteomic analysis on human explanted DR retinal tissue conditioned media, DR retinal EVs and DR urinary EVs and compared to normal human retinal tissue, retinal EVs, and urinary EVs, respectively. Our system biology analysis of DR tissue and EV expression profiles revealed biological pathways related to cell-to-cell junctions, vesicle biology, and degranulation processes. Junction Plakoglobin (JUP), detected in DR tissue-derived EVs and DR urinary EVs, but not in controls, was revealed to be a central node in many identified pathogenic pathways. Proteomic results were validated by western blot. Urinary EVs obtained from healthy donors and diabetic patient without DR did not contain JUP. The absence of JUP in healthy urinary EVs provide the basis for development of a novel Diabetic Retinopathy biomarker, potentially facilitating diagnosis.

Diagnostic methods for Chagas disease rely primarily on serological and molecular tests to detect anti-T cruzi antibodies or parasite-derived DNA. However, these tests do not predict clinical outcomes or treatment responses, as serological results may take years to turn negative, making them impractical for monitoring treatment progress. Molecular tests, though highly sensitive, can identify parasites but cannot rule out the presence of dormant forms or low levels of parasitemia, which may later reactivate. Due to the lack of reliable biomarkers for disease prognosis and treatment monitoring, there has been growing interest in host-derived markers as alternatives. One area of focus is the hypercoagulability observed in T. cruzi-infected individuals, which increases the risk of thrombotic events due to factors like chronic inflammation, dysregulation of immunothrombosis, and vasculitis. In addition, extracellular vesicles (EVs), i.e., double-membrane-enclosed particles found in various biological fluids, have emerged as a potential source of biomarkers for disease detection, prognosis, and monitoring. EVs contain proteins, lipids, and nucleic acids that can provide valuable molecular information about a patient's condition. This chapter outlines procedures for detecting host-derived biomarkers of Chagas disease, particularly those related to hypercoagulability, such as F1+2 and endogenous thrombin potential assays, and the isolation and characterization of EVs as biomarkers for response to treatment and disease progression.

Blood-Based Biomarkers for COVID-19-Associated Neurological Outcomes

Extracellular vesicles (EVs) are released by all kind of cells into the extracellular space, where they shuttle parental cell-derived molecular cargoes (DNA, RNA, proteins) to both adjacent and distant cells, influencing the physiology of target cells. Their specific cargo content and abundance in liquid biopsies make them excellent candidates for biomarker studies. Indeed, EVs isolated from various body fluids, including blood, pleural fluid, urine, cerebrospinal fluid, saliva, milk, ascites, and tears, have been recognized for their potential as biomarkers in diagnosis, monitoring treatment, and predicting outcomes for various diseases. Increasing studies suggest that tears have great promise as a noninvasive liquid biopsy source for EVs. Our aim here is to provide a comprehensive review of the exploration of tears as a noninvasive reservoir of EVs and their contents, evaluating their accessibility and potential utility as a liquid biopsy method. Additionally, the potential of tear EVs in various cancers, including ocular cancer, is discussed. Finally, the advantages and challenges of employing tear-based liquid biopsy for EVs for the disease's biomarker studies are evaluated.