Journal of Extracellular Vesicles最新文献

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.

Excessive activation of NF-κB is implicated in the pathogenesis of numerous inflammatory and autoimmune diseases; however, conventional NF-κB inhibitors often cause widespread immunosuppression. In contrast, extracellular vesicles (EVs) are promising vehicles for therapeutic cargo delivery with advantages including reduced risk of replication. In this single-centre, randomized, double-blind, placebo-controlled phase 1 trial, we evaluated ILB-202, an engineered, allogeneic EV derived from HEK293 cells and loaded with a super-repressor IκBα. A single ascending intravenous dose of ILB-202 was administered to 18 healthy volunteers, and the short-term safety, tolerability, and preliminary pharmacodynamic effects were assessed. ILB-202 was well tolerated at all dose levels with no serious or dose-limiting toxicities; only minor adverse events, including a mild decrease in NK cell counts and one case of grade 1 neutropenia, were observed. The laboratory parameters, vital signs and cytokine profiles remained stable, indicating no systemic immunogenicity. Single-cell RNA sequencing revealed subtle, time-dependent modulation of NF-κB-associated pathways, enhanced TGF-β and visfatin signalling and reduced TNF signalling—suggesting a shift towards an anti-inflammatory state. These findings support the safety and immunomodulatory activity of ILB-202 and pave the way for future trials in diseases characterized by dysregulated NF-κB activation.

Trial Registration: ClinicalTrials.gov identifier: NCT05843799

Extracellular vesicles (EVs) hold immense potential in therapeutic delivery, warranting a comprehensive investigation of the mechanisms that regulate their uptake by target cells. To identify key molecular regulators of EV internalization, we conducted a genome-wide CRISPR (GWC) screen aimed to pinpoint candidate genes that influence EV uptake. We employed a GWC library spanning the entire human genome in K562 cells. 3.6 × 10¹² EVs isolated from the SKMEL147 human melanoma cell line were labelled with Alexa633-C5-Maleimide and incubated for 2 h with 500 × 10⁶ K562 cells, providing a 2000× coverage of the library. The top 5% of high and low fluorescence populations were sorted. Next-generation sequencing (NGS) was performed to quantify sgRNA enrichment in the sorted populations compared to the unsorted control. Remarkably, among other genes, several members of the COMMANDER complex emerged as significant hits in our screen. We validated the hits in knockout (KO) cell lines of both K562 and HeLa cells using EVs derived either from melanoma or breast cancer cell lines. Kinetic follow-up of EV cargo, including surface or luminal proteins, suggests that the COMMANDER complex plays a pivotal role in the early stages of EV uptake but also in the final fate of EV components in the target cell.

Extracellular vesicles (EVs) are released from cells by unconventional secretion, but little is known about the biogenesis routes, composition or cargoes of EVs from fungal or oomycete plant pathogens. We investigated the proteome of EV-associated proteins secreted by the oomycete Phytophthora infestans, cause of potato late blight disease. We found that vesicle-associated proteins, transmembrane proteins and RxLR effectors, which are delivered into host cells to suppress immunity, were enriched in the EV proteome. By contrast, the EV-independent secreted proteome was enriched in cell wall modifying enzymes and apoplastic effectors which act outside plant cells. Two proteins, each containing two tetraspanning MARVEL domains, PiMDP1 and PiMDP2, were associated with P. infestans EVs. PiMDP1 and PiMDP2 were co-buoyant with RxLR effectors in sucrose density fractions containing EVs and co-localised frequently with each other and with RxLRs at vesicles within pathogen hyphae grown in vitro and during infection. Interestingly, PiMDP2, which is up-regulated during the early biotrophic phase of infection, accumulates at the haustorial interface, a major site of effector secretion during infection. We argue that PiMDP1 and PiMDP2 are molecular markers that will facilitate studies of the biogenesis and secretion of infection-associated P. infestans EVs.

Acute myeloid leukaemia (AML) is a haematologic malignancy with high relapse incidence and mortality. Approximately one-third of AML patients carry an fms-like tyrosine kinase 3 (FLT3) mutation, often associated with GLI expression and Hedgehog signalling. AML cells shape their microenvironment into a leukaemia-permissive space by releasing extracellular vesicles (EVs). EVs can transfer chemoresistance and thereby play an important role in refractory and relapsing diseases. Here, we discovered a synergistic effect of combined treatment with the FLT3 inhibitor Crenolanib and the Hedgehog pathway inhibitor HPI-1 in the AML cell lines MOLM-14 and MV4-11. In-depth comparative proteomics revealed alterations in the cellular and the EV proteome upon single or combined inhibition of FLT3 and GLI, highlighting affected pathways. By comparing cellular and EV proteomes, we found that transport of ribosomal proteins, such as RPS26 and RPL27A, and ErbB pathway members such as GAB1, GRB2 and SHC1 to EVs, is selectively avoided upon treatment with Crenolanib. These findings were corroborated by comparative proteomics of EVs derived from AML patients and healthy donors. Ribosomal and ErbB signalling pathway proteins may play an important role in microenvironmental modulation by EVs, and Crenolanib treatment potentially acts by interfering with leukaemia niche formation.