Journal of Extracellular Vesicles最新文献

Extracellular vesicles (EVs) are biological nanovectors that retain molecular signatures of their cells of origin and mediate intercellular communication, resulting in ideal platforms for the development of diagnostic tools and bio-inspired drug delivery technologies. Despite their potential, the mechanisms underlying EV release, uptake and distribution remain unclear. Here, we leverage high-resolution live-cell imaging, quantitative analytical methods and in vivo mouse models to define the major determinants of EV diffusion. Our findings reveal that cell density plays a crucial role in EV dissemination. Specifically, sparsely distributed cells exhibit higher EV release rates into the supernatant due to increased surface exposure in vitro. In contrast, densely packed cells promote EV internalization and degradation by adjacent cells, effectively restricting EV diffusion in vivo and in vitro. As a result, EVs travel only limited distances, with the majority being internalized by adjacent cells. These findings challenge the prevailing assumption that any EV can act as long-range messengers and instead highlight their function as short-range communication agents primarily confined within their tissue or organ of origin. This study provides fundamental insights into EV-mediated intercellular signalling and has important implications for their use in diagnostic and therapeutic applications.

Extracellular vesicles (EVs) are critical mediators of intercellular communication within the tumour microenvironment and play a significant role in drug resistance. We aimed to investigate the mechanisms underlying gemcitabine (GCB) resistance in bladder cancer. GCB-resistant bladder cancer cells exhibited dysregulation of nucleoside-metabolizing enzymes and transporters. Characterization of EV subpopulations derived from GCB-resistant cells revealed their ability to transfer drug-resistant phenotypes to naïve cancer cells by modulating intracellular levels of nucleoside metabolic proteins and transporters. Proteomic and transcriptomic analyses identified the histone protein H3.2 and its corresponding transcript, H3C14, as key regulators in the transmission of GCB resistance. Notably, H3C14 overexpression in resistant cells restored GCB sensitivity, whereas its knockdown induced GCB resistance. Rab27A-mediated biogenesis and secretion emerged as a crucial mechanism regulating EV release and H3C14 excretion in GCB-resistant cells. A specific EV subpopulation enriched in CD147 and LAMB1—referred to as Excretion EVs—carried H3.2 (H3C14) but did not induce GCB resistance in recipient cells, suggesting their primary role in eliminating proteins associated with tumour progression and drug resistance. These findings highlight the role of EV-mediated H3C14 excretion in regulating GCB resistance and suggest potential therapeutic strategies targeting EV pathways to overcome drug resistance in bladder cancer.

With a membership of nearly 2000 individuals spanning the globe, the International Society for Extracellular Vesicles (ISEV) stands as the premier professional organization for scientists and researchers engaged in the exploration of extracellular vesicles (EVs). Established in 2012 in Sweden, ISEV subsequently relocated its headquarters to New Jersey, USA. ISEV is dedicated to fostering global consistency and robustness in EV research, as underscored by the MISEV guidelines of 2014 and 2018 and the update in 2023. The society facilitates this mission through an array of initiatives, including educational offerings, task forces, special interest groups, workshops, and summer schools, while also managing two peer-reviewed, gold open access journals—the Journal of Extracellular Vesicles and the Journal of Extracellular Biology. A cornerstone of ISEV's activities is its flagship annual gathering, a focal point that provides a crucial avenue for knowledge exchange. By means of its comprehensive programmes and services, ISEV plays an indispensable role in delivering vital training and research prospects for those immersed in the realm of EV research.

The International Society for Extracellular Vesicles is the premier international conference of extracellular vesicle research, covering the latest in exosomes, microvesicles and more. With more than 1500 attendees, the meeting has achieved a new level of recognition in the global community and features presentations from the top researchers in the field, as well as providing opportunities for talks from students and early-career researchers.

IOC Chairs: Eva Rohde (Austria), Eva-Maria Krämer-Albers (Germany)

IOC Members: An Hendrix (Belgium), Andreas Moeller (China), Bo Li (China), Edit Buzas (Hungary), Johannes Grillari (Austria), Lucia Languino (USA), Mario Gimona (Austria), Sun Young Lee (USA), Wolf Holnthoner (Austria), Cristóbal Cerda-Troncoso (Belgium), Madhusudhan Bobbili (Austria).

Plenary Presenter: Nicole Meisner-Kober

Ludwig Boltzmann Institute for Nanovesicular Precision Medicine, Vienna, Austria

Presenter: Shili Yao

Tianjin University, Tianjin, China

Introduction: The advent of HER2-targeted antibody drug conjugates (HER2-ADCs) has marked a revolutionary advancement in targeted breast cancer therapy. However, 60%–70% of patients who are completely HER2 negative and do not meet the HER2-low criterion are unable to benefit from these treatments. Engineered extracellular vesicles (EVs), which are naturally occurring nanocarriers, possess the distinctive attributes of high biocompatibility and low immunogenicity. This study aims to utilize engineered EVs to facilitate the continuous and safe presentation of the HER2 antigen to breast tumour cell membranes via membrane fusion, thereby exploring the potential for enhancing HER2-ADCs' efficacy.

Methods: Firstly, magnetic bead-assisted high-speed dispersion technology was employed

Extracellular vesicles (EVs) have been investigated as nanotherapeutics and drug delivery systems for a wide range of disease indications. However, translational application of EVs is challenging due to their physical heterogeneity and variation in functional potency. The current study generated a novel hybrid EV formulation by membrane fusion of mesenchymal stem/stromal cell-derived EVs with astrocyte-derived EVs and defined its physicochemical and functional properties. Both EV populations have translational potential for neurodegenerative disease applications—a disease area with limited treatment strategies due to its complex disease biology requiring multi-faceted therapeutic approaches. However, individual EV sources lack the full set of therapeutic properties needed for comprehensive treatment. Stem cell-derived EVs possess general neuroprotective and anti-inflammatory effects but lack widespread blood–brain barrier penetration and specific neurodegenerative pathology targeting. Astrocytes are uniquely involved in key neuronal processes but are prone to adopting neuroinflammatory phenotypes in disease states. Using super resolution microscopy and quantitative proteomic analysis, we characterized, optimized, and validated a hybrid EV formulation for its brain cell targeting, neuroprotective function, and immunomodulatory capability. These results establish a platform for EV engineering through EV-EV hybridization and demonstrate the potential of one formulation for neurodegenerative applications.

This study investigates the distribution of extracellular vesicles (EVs) in superficial articular cartilage, hypothesizing that EVs (a) are unevenly distributed in this zonally organized tissue and (b) share a pattern similar to tissue autofluorescence. Fresh unfixed superficial cartilage from the femoropatellar groove of bovine knees was analysed using multiphoton microscopy (second harmonic generation, SHG, and multiphoton-autofluorescence, MPAF). Transmission electron microscopy (TEM) and immunostaining (CD9, membrane marker; collagen type VI, pericellular matrix marker) were performed on fixed tissue. Cartilage-bone cylinders were analysed in a simulated endomicroscopic setting. Additionally, EVs were isolated from synovial fluid and chondrocyte cell culture medium to demonstrate autofluorescence and staining properties. MPAF revealed a specific spatial distribution around superficial chondrocytes: lateral ring-like accumulations inside the cell lacunae and snow cap-like formations above cells outside the lacunae. CD9 staining was found outside the collagen type VI-positive matrix in MPAF-correlating locations. TEM confirmed a similar EV distribution. The endomicroscopic setting also visualized the lateral MPAF accumulations. In tissue with early osteoarthritic degeneration these patterns were not found. In conclusion, EVs/CD9 exhibit a specific spatial distribution, suggesting guided EV transport or binding within the extracellular matrix, which changes with tissue degeneration. These findings provide insight into the spatial relation between EVs and superficial cartilage architecture in health and disease and indicate a potential link between extracellular MPAF and EVs as a basis for the development of diagnostic methods and in vivo EV tracking.

Surface functionalization is an effective approach for enhancing the cancer-targeting efficiency of extracellular vesicles (EVs). However, the lack of direct comparisons between functionalization strategies has hindered the rational design of EV delivery systems. To address this gap, we developed a nano-flow cytometry-based methodology to quantitatively evaluate ligand conjugation and its relationship to targeting efficiency across three representative post‑production EV engineering strategies: lipid modification, protein modification, and membrane insertion. All three strategies achieved high conjugation efficiencies (>90%) with ligand densities ranging from several to tens of ligands per 100 nm² under optimized conditions. Beyond ligand density, functionalization strategies resulted in varying degrees of ligand clustering and reduced accessibility of endogenous cell-binding proteins on EVs, such as MFGE8, leading to differences in targeting performance. For milk-derived EVs, lipid modification achieved the highest ligand density, the most uniform conjugation, and minimal disruption to surface protein accessibility, yielding superior cancer-targeting efficiency. These findings highlight the importance of precise quantification of ligand conjugation performance and provide a robust methodology for optimizing surface functionalization to advance EV-based drug delivery.

Prostate cancer is the most common non-cutaneous cancer among men in the United States. Most prostate cancers are driven by androgen receptor (AR) signalling, but there are an increasing number of cases that lose AR and gain neuroendocrine (NE) features (AR−/NE+) or lack both (AR−/NE−). These latter subtypes are particularly aggressive and lethal. Extracellular vesicles (EVs) have shown great potential as biomarkers for non-invasive liquid biopsy assays, as EVs contain biomolecules from their cells of origin. Here, we used a shotgun proteomics approach with mass spectrometry to interrogate the global proteome of EVs isolated from prostate cancer cell lines reflecting diverse clinical subtypes, including AR−/NE+ and AR−/NE− models. We identified 3952 EV proteins, which clustered largely by tumour subtype and provided enough proteomic coverage to derive classic gene signatures of AR or NE identity that are of high relevance for prostate cancer prognostication. EVs isolated from AR+ cells displayed high levels of proteins regulated by AR and mTOR signalling. EVs isolated from AR−/NE+ cells contained known NE markers such as SYP and CHGA, whereas EVs from AR−/NE− models were enriched in basal cell markers and proteins that regulate epithelial-to-mesenchymal transition (EMT). We integrated our cell line data with recently published EV proteomics data from 27 advanced prostate cancer patients and found 2733 overlapping proteins, including cell surface markers relevant to prostate cancer, AR activity indicators, and proteins enriched in specific subtypes (AR+, AR−/NE−, AR−/NE+). This approach may be useful for rare cancer subtypes, such as prostate cancers that lose AR-related features and gain NE features, to optimise the use of these liquid biopsy samples for clinical decision making.

Immunofluorescence (IF) staining represents a convenient and cost-effective approach to analysing single extracellular vesicles (EVs) and identifying subpopulations with specific roles or biological functions. However, the application of the method is challenged by the weak and unstable signals generated by the low abundant markers carried by the vesicles. In this study, we report the development of an IF strategy based on tyramide signal amplification (TSA) that employs tyramide probes for signal enhancement. The technique is first validated on glioblastoma circulating tumour cells (GBM CTCs) and systematically compared with conventional approaches using fluorescently labelled primary and secondary antibodies. Thereafter, the proposed method is adapted, tested and optimised for the multiplexed fluorescent staining of single EVs isolated from the parental GBM CTCs. The results demonstrate specific staining of single EVs by the developed TSA method, highlighting its advantages of amplified (>6×) signal intensities, more stable signals and broader (∼3×) signal dynamic ranges as compared to the conventional fluorescence methods. The developed protocol also supports multiplexing by incorporating a quenching buffer between the different staining colours. Finally, the protocol demonstrates its applicability to CTCs and EVs derived from plasma samples of GBM patients, with easy adaptation to other cancers or proteins of interest.

Tumour-derived extracellular vesicles (tdEVs) have emerged as a promising representative of cancer manifestation that can be accessed non-invasively through liquid biopsy. Selective examination of tdEVs requires their isolation, which relies on tumour-specific surface markers. These markers are often identified using cancer cell lines cultured in EV-depleted serum or serum-free conditions to avoid interference by exogenous EVs in serum. However, these nutrient-deprived media can alter gene expression and the proteomic composition of EVs. This study aims to develop a method to identify potential EV surface markers for paediatric neuroblastoma from tumour cell lines grown in native serum. Our methodology enables distinguishing tumour-specific EVs from the exogenous serum EVs, without prior knowledge of any tumour-specific surface markers. By metabolically incorporating an azide-tagged sugar analogue into nascent glycoproteins, we differentially marked only tumour-derived EVs and captured them using copper-catalysed click chemistry-mediated biotinylation and affinity enrichment. Subsequent analysis through mass spectrometry and western blotting led to the identification of gap junction protein GJC1 (connexin 45) as a potential surface marker for neuroblastoma EVs. This methodology not only aids in EV surface profiling but also has significant implications for time-resolved and spatial EV studies in various biological contexts, including disease development, progression, therapy resistance, and cellular communication.

Stem cell-derived extracellular vesicles (EVs) offer a promising cell-free approach for cardiovascular regenerative medicine. In this study, we developed magnetically labelled induced pluripotent stem cell-derived EVs (magneto-iPSC-EVs) encapsulated with superparamagnetic iron oxide (SPIO) nanoparticles for image-guided regenerative treatment of myocardial infarction, in which EVs that can be detected by both magnetic resonance imaging (MRI) and magnetic particle imaging (MPI). iPSC-EVs were isolated, characterized per MISEV2023 guidelines, and loaded with SuperSPIO20 nanoparticles using optimized electroporation conditions (300 V, 2 × 10 ms pulses), achieving a high loading efficiency of 1.77 ng Fe/10⁶ EVs. In vitro results show that magneto-iPSC-EVs can be sensitively detected by MPI and MRI, with a detectability of approximately 10⁷ EVs. In a mouse myocardial ischemia-reperfusion model, intramyocardially injected magneto-iPSC-EVs (2 × 10⁹) were imaged non-invasively by in vivo MPI for 7 days and ex vivo MRI, with the presence of magneto-iPSC-EVs confirmed by Prussian blue staining. Therapeutically, both native and magneto- iPSC-EVs significantly improved cardiac function, with a 37.3% increase in left ventricular ejection fraction and 61.0% reduction in scar size. This study highlights the potential of magneto-iPSC-EVs as a cell-free approach for cardiovascular regenerative medicine, offering both non-invasive imaging capabilities and therapeutic benefits for myocardial repair.