Journal of Extracellular Vesicles最新文献

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.

Extracellular matrix (ECM) stiffness and extracellular vesicles (EVs) are critical regulators of tumour progression, yet their interaction in three-dimensional (3D) microenvironments remains poorly understood. Most studies on ECM stiffness and EV biology rely on 2D cultures, which do not capture the complexity of the tumour microenvironment. Here, a biomimetic 3D nanofibrillar ECM model based on a cellulose nanofibril hydrogel was established to assess stiffness-dependent changes in EV properties and functions. EVs derived from stiff matrices (StEVs) exhibited distinct physicochemical characteristics and carried unique protein and microRNA cargo compared with those from soft matrices (SoEVs). Functionally, StEVs more potently promoted tumour cell proliferation and migration, while in vivo mouse models further demonstrated that StEVs enhanced tumour growth. Multi-omics analyses and pharmacological inhibition studies revealed that StEVs activate the mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (MAPK/ERK1/2) signalling pathway in recipient cells. These findings highlight the mechanobiological regulation of EV-mediated intercellular communication within 3D ECM environments and demonstrate how matrix stiffness shapes EV cargo and pro-tumour activity.

It is increasingly clear that intercellular communication is largely mediated by lipid-bilayer, membrane-bound extracellular vesicles (EVs) and amembranous, non-vesicular extracellular particles (NVEPs), including exomeres and the recently identified supermeres. To elucidate the cargo and functional roles of these carriers, we performed a comprehensive analysis of their lipid, protein and RNA content in the context of colorectal cancer and glioblastoma (GBM). Our results demonstrate that EVs exhibit distinct density profiles correlated with specific biomolecular signatures. Moreover, EVs and NVEPs display notable differences in their protein and RNA composition, which confer distinct functional attributes. Supermeres are notably enriched in components involved in extracellular matrix remodeling and possess the ability to cross the blood–brain barrier, a process dependent on their intact structure and RNA content. Once in the central nervous system (CNS), they preferentially engage with microglia and suppress TGFβ1 expression, suggesting a role in modulating microglial immune activity. Furthermore, systemically administered exogenous supermeres selectively accumulate in GBM tumors in vivo. Together, these findings highlight supermeres as a promising vehicle for delivering therapeutics to the CNS and brain tumors.

Extracellular vesicles (EVs) are biological nanoparticles that play important roles in (patho)physiological processes and are promising new therapeutic and diagnostic tools. Recent evidence suggests that other circulating biological nanoparticles, primarily lipoproteins, bind to EVs, changing their biological identity. Such binding has been demonstrated with complex qualitative techniques, such as cryogenic transmission electron microscopy. There is a need to rapidly and simply quantify EV-lipoprotein binding, as such complexes could have major implications for EV biology and medical applications. This study developed lipoprotein association fluorometry (LAF; based on fluorescent lipophilic indocarbocyanine dyes), as a first-of-its-kind, simple and quick assay to assess EV binding to lipoproteins. The LAF assay was validated with synthetic nanoparticles, small molecules, polymers and proteins that display known interactions with lipoproteins. The LAF assay demonstrates that EVs from various human and non-human (nematode and bacteria) sources bind to very-low-density lipoprotein (VLDL) and low-density lipoprotein (LDL). Notably, EVs derived from cancerous cells displayed substantially increased binding to VLDL, LDL and plasma compared to EVs from normal cells. Additionally, the LAF assay revealed that EVs from metastatic cancer cells bound to VLDL to a greater extent than those from corresponding patient-matched non-metastatic cancer cells. On the contrary, EVs displayed minimal binding to high-density lipoprotein (HDL). Taken together, the LAF assay is capable of measuring EV-lipoprotein binding in a simple, rapid and semi-quantitative manner, leading to new opportunities to probe EV biology and develop novel therapeutics, and diagnostics.

This study investigates a novel approach to overcome Vemurafenib resistance in BRAF-mutant Anaplastic thyroid carcinoma (ATC) using CRISPR/Cas9 gene editing and TMTP1-modified extracellular vesicles (TMTP1-sgBRAF-EVs). By knocking out the BRAF gene, the study elucidates Vemurafenib-induced ferroptosis mechanisms involving lipid peroxidation and reactive oxygen species (ROS) generation in ATC cells. The developed TMTP1-sgBRAF-EVs system demonstrates superior tumour-targeting and drug delivery capabilities, significantly enhancing Vemurafenib efficacy in both in vitro and in vivo models. This innovative combination of gene editing technology with a nanoparticle delivery system shows promising potential as a therapeutic strategy for treating aggressive BRAF-mutant ATC.