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PubMed2026

Bovine Colostrum-Derived Extracellular Vesicles Impair Cancer Cell Proliferation Through Transcriptional Dysregulation.

Milk-derived extracellular vesicles (EVs) are a promising source of molecules with therapeutic potential. Bovine colostrum is particularly enriched in EVs, which carry cargo of proteins involved in immune regulation, development and cellular signalling. Some studies have explored their role as bioactive anti-cancer agents, however, their mechanistic effects remain underexplored. Here, we show that colostrum-derived EVs (Col-EVs) exert anti-proliferative effects in gastrointestinal cancer models, including cell lines and patient-derived organoids, which is independent of apoptosis induction. Using a multi-modal approach combining proteomics, imaging and functional assays, we demonstrate that Col-EVs induce a reversible growth-arrest state, characterized by widespread transcriptional and RNA-processing dysregulation, chromatin compaction, nuclear reorganization and cytoskeletal remodelling. Proteomic analyses reveal that Col-EV treatment disrupts key components of the transcriptional machinery and cell cycle regulatory pathways, effects that are reversible upon EV withdrawal and can be rescued pharmacologically using an EZH2 inhibitor. Col-EVs enhance the sensitivity of cancer cells as well to DNA-targeting chemotherapies such as 5-fluorouracil, indicating their potential as modulatory adjuvants rather than cytotoxic agents. Overall, our findings reveal that Col-EVs can reversibly suppress cancer cell proliferation by reprogramming transcriptional and nuclear architecture, offering a natural, biocompatible strategy for modulating tumour growth and sensitizing cancer cells to conventional therapies.

Extracellular Vesicles↗

PubMed2025

High-Frequency Irreversible Electroporation Alters Proteomic Profiles and Tropism of Small Tumor-Derived Extracellular Vesicles to Promote Immune Cell Infiltration.

High-frequency irreversible electroporation (H-FIRE) is a nonthermal tumor ablation technique that disrupts the blood-brain barrier (BBB) in a focal and reversible manner. However, the mechanisms underlying this disruption remain poorly understood, particularly the role of small tumor-derived extracellular vesicles (sTDEVs) released from ablated tumor cells. In this study, we investigate the proteomic and functional alterations of sTDEVs released from F98 glioma and LL/2 Lewis lung carcinoma cells following H-FIRE ablation. Mass spectrometry analysis revealed 108 unique proteins in sTDEVs derived from ablative doses of H-FIRE, which are capable of disrupting the BBB in an in vitro model. Proteomic analysis of TDEVs highlights key changes in pathways related to integrin signaling, Platelet-derived growth factor receptor (PDGFR) signaling, and ubiquitination, which may underline their interactions with brain endothelial cells. These "disruptive" sTDEVs exhibit enhanced tropism for cerebral endothelial cells both in vitro and in vivo, where they persist in the brain longer than sTDEVs released after non-ablative H-FIRE doses. Notably, when introduced into a healthy Fischer rat model, disruptive sTDEVs are associated with increased recruitment of Iba1+ immune cells, suggesting a potential role in modulating post-ablation immune responses. However, despite their altered protein composition, these vesicles do not directly increase BBB permeability in vivo. This study is the first to demonstrate that electroporation-based tumor ablation significantly alters the composition and functionality of tumor-derived extracellular vesicles, potentially influencing the tumor microenvironment post-ablation. These findings have important implications for developing multimodal treatment strategies that combine H-FIRE with systemic therapies to enhance efficacy while managing the peritumoral microenvironment.

Animals↗

PubMed2026

Enrichment Performance Assessment of Extracellular Vesicles Using Different Functionalized Magnetic Materials and Application in Urinary Proteomics of Prostate Cancer.

Extracellular vesicles (EVs) are lipid bilayer nanovesicles that mediate intercellular communication and hold significant potential for clinical applications. Although material-based isolation strategies offer promising alternatives to conventional methods, their relative performances have not been systematically evaluated. In this study, we conducted a comparative assessment of magnetic nanomaterials with distinct surface functionalities, including metal oxides (TiO2), metal-organic frameworks (UiO-66), biopolymeric materials (chitosan), and lipid probes (DSPE-PEG, DOPE-PEG, and CLS-PEG). A comprehensive evaluation across multiple dimensions including capture capacity, capture rate, sample volume, and product purity reveals that the bifunctional magnetic nanomaterial Fe3O4@UiO-66@DSPE material exhibits superior EV capture performance. This material enables the efficient and stable enrichment of high-purity EVs by synergizing Zr4+-phosphate coordination with lipid bilayer anchoring, and preserves EV biological integrity and activity. Meanwhile, this method could be highly compatible with proteomics, and over 1000 proteins are identified by proteomic analysis of urinary EVs, while 34 proteins are upregulated and 25 proteins are downregulated in prostate cancer patients relative to healthy donors. Notably, the differentially expressed proteins, such as AGT, ITIH4, and PGLYRP2, are associated with disease progression. Overall, this work highlights the superior performance of the Fe3O4@UiO-66@DSPE material for efficient and selective EV isolation. It provides a powerful tool for clinical liquid biopsy and proteomic biomarker discovery, enabling early diagnosis, prognostic evaluation, and precision therapy.

Humans↗

PubMed2025

Correlation of extracellular vesicle Alu RNA with brain aging and neuronal injury: a potential biomarker for brain aging.

BACKGROUND: Extracellular vesicles (EVs) are promising biomarkers for neurodegeneration. Alu elements are retrotransposons increasingly expressed with age and may be involved in aging-related diseases. OBJECTIVE: To determine the potential of Alu RNA in plasma-derived EVs as a biomarker for brain aging and neuronal injury. METHODS: EVs were isolated from plasma samples across different age groups. EV Alu RNA levels were measured and their associations with biomarkers of brain aging, including plasma neurofilament light chain (NfL), plasma amyloid-beta (Aβ42 and Aβ40), and plasma phosphorylated tau (p-Tau181), were analyzed. RESULTS: EV Alu RNA levels were increased significantly with age and were strongly correlated with plasma NfL, suggesting a strong association between EV Alu RNA and neuronal injury. Significant correlations were also found between EV Alu RNA and plasma amyloid-beta levels, while no significant association was observed with tau pathology. CONCLUSIONS: EV Alu RNA levels are elevated with age and associated with neuronal injury, highlighting their potential as a novel, non-invasive biomarker for brain aging and neurodegeneration.

Humans↗

PubMed2024

Proteomic profiles of peritoneal fluid-derived small extracellular vesicles correlate with patient outcome in ovarian cancer.

Cancer-derived small extracellular vesicles (sEVs) are capable of modifying the tumor microenvironment and promoting tumor progression. Ovarian cancer (OvCa) is a lethal malignancy that preferentially spreads through the abdominal cavity. Thus, the secretion of such vesicles into the peritoneal fluid could be a determinant factor in the dissemination and behavior of this disease. We designed a prospective observational study to assess the impact of peritoneal fluid-derived sEVs (PFD-sEVs) in OvCa clinical outcome. For this purpose, 2 patient cohorts were enrolled: patients with OvCa who underwent a diagnostic or cytoreductive surgery and nononcological patients, who underwent abdominal surgery for benign gynecological conditions and acted as the control group. Systematic extraction of PFD-sEVs from surgical samples enabled us to observe significant quantitative and qualitative differences associated with cancer diagnosis, disease stage, and platinum chemosensitivity. Proteomic profiling of PFD-sEVs led to the identification of molecular pathways and proteins of interest and to the biological validation of S100A4 and STX5. In addition, unsupervised analysis of PFD-sEV proteomic profiles in high-grade serous ovarian carcinomas (HGSOCs) revealed 2 clusters with different outcomes in terms of overall survival. In conclusion, comprehensive characterization of PFD-sEV content provided a prognostic value with potential implications in HGSOC clinical management.

Humans↗

PubMed1992

Extracellular vesicles of calcifying turkey leg tendon characterized by immunocytochemistry and high voltage electron microscopic tomography and 3-D graphic image reconstruction.

To gain insight into the structure and possible function of extracellular vesicles in certain calcifying vertebrate tissues, normally mineralizing leg tendons from the domestic turkey, Meleagris gallopavo, have been studied in two separate investigations, one concerning the electron microscopic immunolocalization of the 66 kDa phosphoprotein, osteopontin, and the other detailing the organization and distribution of mineral crystals associated with the vesicles as determined by high voltage microscopic tomography and 3-D graphic image reconstruction. Immunolabeling shows that osteopontin is related to extracellular vesicles of the tendon in the sense that its initial presence appears coincident with the development of mineral associated with the vesicle loci. By high voltage electron microscopy and 3-D imaging techniques, mineral crystals are found to consist of small irregularly shaped particles somewhat randomly oriented throughout individual vesicles sites. Their appearance is different from that found for the mineral observed within calcifying tendon collagen, and their 3-D disposition is not regularly ordered. Possible spatial and temporal relationships of vesicles, osteopontin, mineral, and collagen are being examined further by these approaches.

Animals↗

PubMed2026

Probiotic-derived extracellular vesicles as food-based nanocarriers: Mechanisms, functional applications, and future perspectives in food systems.

Probiotic-derived extracellular vesicles (PDEVs) are a promising type of postbiotic nanoparticle derived by fermentation of probiotics, and have gained growing interest as a potential application in food science and nutrition. These are lipid bilayer vesicles of nanoscale, which are naturally released by probiotic cells and contain a wide variety of bioactive molecules, such as proteins, nucleic acids, and metabolites. Moreover, PDEVs are highly stable, biocompatible, and can be easily engineered to have surfaces with high functionality, which makes them good candidates in functional engineering. In contrast to traditional live probiotics, PDEVs overcome the difficulties of preserving microbial viability during processing and storage, thus providing superior safety, stability, and predictable biological performance. This is a systematic review of the various functions of PDEVs in food systems. We conclude on the processes through which PDEVs control intestinal barrier integrity, alter gut microbiota composition, and alter host immune responses, and their potential to enhance gut health when added to functional foods. In addition to their health-promoting effects, PDEVs have shown significant potential as natural antimicrobial agents to preserve food and as effective nanocarriers of hydrophobic bioactive compounds, including fucoxanthin, to improve their stability, bioavailability, and targeted delivery. Moreover, PDEVs can be used as new regulators of microbial fermentation. However, it should be noted that a lot of the evidence that is available is still preliminary and the effectiveness of these applications in real food-processing and storage conditions has not been fully proven. Although they have potential, there are a number of challenges that still hinder the widespread use of PDEVs in the food industry. These involve the creation of scalable and cost-effective production processes, batch-to-batch consistency, vesicle stability in a variety of food matrices, and regulatory and safety considerations. Other emerging engineering approaches, such as surface functionalization and cargo loading, are also discussed in this review and could further increase the specificity, functionality, and application versatility of PDEVs in food systems. Moving forward, the incorporation of PDEVs into the next generation functional foods, novel food preservation methods, and customized nutrition plans should be prioritized in future studies. Further developments in these fields can make PDEVs useful platforms at the interface of food microbiology, nanotechnology, and human health.

Probiotics↗

PubMed2025

Multicompartment Quantitative Proteomics Revealing Potential Mechanisms Underlying the Treatment Effects of Mesenchymal-Derived Extracellular Vesicles in a Monkey Model of Cortical Injury.

Previous studies have demonstrated that mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) enhance functional recovery after cortical injury in rhesus monkeys by reducing chronic microglial inflammation, neuronal damage, and myelination deficits. However, the signaling pathways underlying these therapeutic effects remain largely unexplored. In this study, employing a reliable quantitative proteomics platform UHR-IonStar, we identified the protein cargo of MSC-EVs infused intravenously to rhesus monkeys 24 h and 2 weeks post-injury to the motor cortex. We then analyzed global protein expression changes across cerebrospinal fluid (CSF), plasma, and brain tissue of MSC-EV-treated versus vehicle-treated female, aged rhesus monkeys. A total of 1241/431/4124 monkey proteins were reliably quantified in CSF/plasma/tissue samples, respectively. Longitudinal analysis of CSF and plasma samples highlighted a shift from MSC-EV modulation of inflammatory and metabolic proteins in plasma at early recovery (2 weeks), toward modulation of plasticity-related proteins in CSF and brain tissue at later stages (4 weeks). Further protein-protein interaction analysis identified potential MSC-EV targets related to complementary signaling, proteolysis, and aminoglycan stability, which aligned with our previous findings. This comprehensive, multicompartment monkey proteomics study advances understanding of MSC-EV contents and treatment effects, paving the way for novel treatment of cortical injury.

Animals↗

PubMed2025

The extracellular vesicle transcriptome provides tissue-specific functional genomic annotation relevant to disease susceptibility in obesity.

We characterized circulating extracellular vesicles (EVs) in obese and lean humans, identifying transcriptional cargo differentially expressed in obesity (277 unique genes; false discovery rate < 10%). Since circulating EVs may have broad origin, we compared this obesity EV transcriptome with expression from human visceral-adipose-tissue-derived EVs from freshly collected and cultured biopsies from the same obese individuals, observing high concordance. Using a comprehensive set of adipose-specific epigenomic and chromatin conformation assays, we found that the differentially expressed transcripts from the EVs were those regulated in adipose by body mass index-associated SNPs (p < 5 × 10-8) from a large-scale genome-wide association study (GWAS). Using a phenome-wide association study of the regulatory SNPs for the EV-derived transcripts, we identified a substantial enrichment for inflammatory phenotypes, including type 2 diabetes. Collectively, these findings represent the convergence of the GWAS (genetics), epigenomics (transcript regulation), and EV (liquid biopsy) fields, enabling powerful future genomic studies of complex diseases.

Humans↗

PubMed2026

Enzyme-Metabolite Network Analysis of Endometrial Cancer-Derived Extracellular Vesicles Through Integrated Proteomics and Metabolomics.

Endometrial cancer (EC) is the most common gynecological malignancy in high-income countries. Extracellular vesicles (EVs) are key mediators of intercellular communication and metabolic reprogramming, but their molecular cargo in EC remains poorly characterized. EVs were isolated from four EC cell lines representing Type I and Type II subtypes (AN3CA, ISHIKAWA, HEC1A, and KLE). Untargeted metabolomics was performed by HILIC-LC-MS/MS, proteomics by data-independent acquisition (DIA) mass spectrometry, and multi-omics integration using MetaboAnalyst and OmicsNet. Metabolomic profiling identified 1463 annotated features and revealed significant differences among EC cell lines (PERMANOVA, p = 0.002). Twenty-eight differentially abundant metabolites, including lactic acid, succinic acid, and uric acid, were identified. Proteomic analysis quantified 8513 proteins with subtype-specific expression patterns. Integrated analysis revealed seven significantly enriched pathways, including glycolysis/gluconeogenesis, central carbon metabolism in cancer, and the pentose phosphate pathway. Increased LDHA abundance in metastatic AN3CA-derived EVs was confirmed by Western blot (p = 0.047). EC-derived EVs display subtype- and metastatic-status-specific metabolo-proteomic signatures, with glycolysis, TCA cycle remodeling, and central carbon metabolism as convergent pathway signatures of molecular reprogramming. These findings establish a multi-omics framework for characterizing EV cargo in EC and identify candidate enzyme-metabolite nodes for future biomarker validation in patient-derived specimens.

Female↗

PubMed2026

Bacterial extracellular vesicles exhibit distinct functional potential across biogeographic provinces of the South Pacific Ocean.

Bacterial extracellular vesicles (BEVs) are nanoscale membranous structures released by diverse types of bacteria, and are capable of transporting and delivering biological compounds between cells. Experimental investigation of BEVs in laboratory model systems indicates that these nanoparticles may play a number of roles in the ecophysiology of marine bacterial communities, but their functional potential in the environment remains unclear. Here we describe the proteomic composition of BEV populations over more than 5000 nautical miles of surface waters in the South Pacific, linking BEV cargoes to the bacterial communities producing them. The presence of marine BEVs was consistently observed across a range of biogeochemical conditions, with an overall abundance comparable to that of bacterial cells (up to 108 BEVs L-1). The protein cargo of marine BEVs, however, differed significantly among ocean regions. The BEV populations were enriched in carbohydrate transporters under phytoplankton bloom conditions, and contained iron uptake-related proteins in nutrient-limited waters. These data suggest that BEVs could enable cells to perform key extracellular functions in the marine environment. Our observations highlight the ubiquity of marine BEVs and biogeographic patterns in their ecological potential across oceanic scales.

Extracellular Vesicles↗

PubMed2026

CircExor enables interpretable prediction of circRNA localization into extracellular vesicles.

Certain circular RNAs (circRNAs) are selectively enriched in extracellular vesicles (EVs), in which they contribute to intercellular communication and represent promising biomarkers, yet the sequence determinants of their sorting remain unclear. Existing computational predictors are optimized mainly for linear RNAs and rarely address circRNA localization into EVs. Here we introduce circExor, the first framework specifically designed for circRNA EV localization. We curate a dedicated benchmark data set of 2102 circRNAs and implement a variable-length end-to-end concatenation strategy together with k-mer frequency encoding to accommodate circular topology, long sequence length, and length heterogeneity. Using a tree-based classifier, circExor achieves superior performance compared with RNAlocate-v3 and ExoGRU, reaching an AUROC of 0.743 on the internal test set and an average AUROC of 0.680 on the held-out test set. SHAP-based analysis, sequence perturbation analysis, motif mapping, and cell-based experimental validation support the predicted EV tendency and identify YBX1, HNRNPK, HNRNPL, and NOVA2 as candidate RBPs potentially associated with circRNA sorting. CircExor therefore provides a predictive and interpretable framework that links in silico modeling to mechanistic hypotheses, and supports biomarker discovery and candidate prioritization for downstream studies of EV-associated circRNAs.

Journal Article↗

PubMed2025

Parkin Induces Ubiquitination and Large Extracellular Vesicle Release of HMGB1 to Activate Antitumor Immunity.

UNLABELLED: Parkin (PRKN) is a mitochondria-associated E3 ubiquitin ligase that mediates mitophagy and organelle quality control. More recently, PRKN has been implicated in stimulating antitumor immunity and reprogramming the tumor immune microenvironment. In this study, we showed that PRKN ubiquitinates the alarmin molecule, high-mobility group box-1 (HMGB1) on Lys146 (K146) using predominantly K48 linkages. By molecular modeling, the in-between-ring domain of PRKN (Gln326-Leu358) made extensive contacts with the amino-terminus A-box of HMGB1 (Met1-Ser42), forming a mitochondria-associated PRKN-HMGB1 complex that juxtaposes K146 to ubiquitin active site residues Gly76 and Arg74. Instead of proteasomal degradation, PRKN ubiquitination of K146 enabled the loading of HMGB1 but not HMGB1 K146A mutant, onto autophagy- and mitochondria-derived large extracellular vesicles (LEV). In turn, released PRKN-HMGB1-LEV stimulated a potent IFN and cytokine response in recipient cells, expanding CD8+ T-cell subsets with effector (CD69+/KLRG1+), self-renewal (TCF1+/PD-1+), and cytotoxic (KLRG1+/GrzB+) properties. Conditional expression of PRKN induced HMGB1 release, activated intratumoral CD8+ T cells, and suppressed syngeneic tumor growth in vivo in a response that was abolished by HMGB1 silencing. These data identify that PRKN-LEV-regulated release of HMGB1 reprograms antitumor immunity via stimulation of IFN signaling and expansion of specialized CD8+ T-cell subsets. SIGNIFICANCE: Parkin ubiquitinates the alarmin molecule HMGB1 to enable its regulated release in large extracellular vesicles that activate interferon signaling, expand specialized CD8+ T-cell subsets, and promote antitumor immunity.

HMGB1 Protein↗

PubMed1998

Biologically active Fas antigen and its cognate ligand are expressed on plasma membrane-derived extracellular vesicles.

Exfoliation of plasma membrane components is a directed process that consumes energy and requires active cell metabolism. Proteins involved in regulating the survival and proliferation of eukaryotic cells are released on exfoliated vesicles. We examine here whether the Fas receptor and its cognate ligand (FasL) are present on vesicles shed from high metastatic potential CX-1 cells and low metastatic potential MIP-101 cells and from HuT 78 cells, respectively. Rates of exfoliation at 2 hours and cumulative levels of extracellular vesicles in serum-free medium conditioned by CX-1 cells are increased by 1.8-fold and 1.6-fold, respectively, relative to that in medium conditioned by MIP-101 cells. Although vesicles shed from both cancer cell lines contain Fas antigen, the amount of Fas per vesicle and the percentage of vesicles containing Fas are increased for vesicles isolated from MIP-101 cells, relative to those from CX-1 cells, as determined by immunogold particle labeling and electron microscopy and by immunofluorescence microscopy and flow cytometry. Results of metabolic labeling with 35S-methionine indicate that Fas biosynthesis is reduced by up to 3.3-fold for CX-1 cells, relative to that of MIP-101 cells, consistent with the finding of decreased Fas on vesicles shed from the plasma membrane of CX-1 cells. Although mRNA for soluble Fas receptor is detectable in both cell lines, depletion of shed vesicles from serum-free medium by ultracentrifugation removes all detectable biological activity. FasL is detected on vesicles exfoliated from HuT 78 cells by immunoelectron microscopy and Western blot analysis. FasL-bearing vesicles induce apoptosis of Fas-expressing cancer cells at the same level as observed by treatment with monoclonal anti-Fas antibody. Furthermore, Fas-bearing extracellular vesicles from MIP-101 but not from CX-1 cells protect the CX-1 cell line from FasL-induced and anti-Fas-mediated apoptosis, indicating that Fas present on shed vesicles is biologically active. We conclude that the Fas antigen and its cognate ligand are exfoliated from the cell surface in a bioactive configuration. Exfoliation may provide a mechanism for long-range signal-directed apoptosis while maintaining Fas/FasL on a membrane surface.

Adenocarcinoma↗

PubMed2026

Proteomic Analysis of Extracellular Vesicles Reveals Vitronectin and Laminin Subunit Alpha-3 as Candidate Biomarkers for Gastric Cancer.

BACKGROUND/AIMS: Clinically useful noninvasive biomarkers for gastric cancer remain limited. Extracellular vesicles (EVs) carry a molecular cargo reflective of their cells of origin and have emerged as promising candidates for blood-based cancer biomarkers. We aimed to identify EV-associated protein biomarkers for gastric cancer via a proteomic approach. METHODS: Proteomic profiling of EVs was performed using one normal gastric cell line (Hs738st/int) and two gastric cancer cell lines (AGS and NCI-N87). Selected proteins were validated in blood-derived EVs isolated from plasma samples of 10 healthy controls and 36 patients with gastric cancer. RESULTS: Proteomic analysis identified 224 differentially expressed proteins whose expression was consistently altered in gastric cancer cell line-derived EVs. Among these, vitronectin (VTN) and laminin subunit alpha-3 (LAMA3) were selected based on their consistent upregulation. EV-associated LAMA3 levels were significantly higher in patients with gastric cancer than in healthy controls (p=0.003), with significant elevations observed from stage II onward (p=0.041, p=0.017, and p=0.004 for stages II, III, and IV, respectively). EV-associated VTN levels were not significantly different overall (p=0.089); however, stage-specific analysis demonstrated significant increases in VTN levels in patients with stage III (p=0.036) and stage IV (p=0.005) gastric cancer. Both EV-associated VTN and LAMA3 levels showed significant positive correlations with the cancer stage (ρ=0.564 and ρ=0.611, respectively; both p<0.001). CONCLUSIONS: The levels of EV-associated VTN and LAMA3 appear to be more closely associated with disease progression than with early-stage detection of gastric cancer. These findings suggest that EV-based proteomic biomarkers may have clinical utility for monitoring tumor progression in patients with clinically advanced gastric cancer.

Humans↗

PubMed2024

Extracellular Vesicle- MDM2 -DNA as a Potential Liquid Biopsy Biomarker for Disease Identification in Retroperitoneal Liposarcoma.

OBJECTIVE: We aimed to assess the levels of MDM2 -DNA within extracellular vesicles (EVs) isolated from the serum of retroperitoneal liposarcoma (RLS) patients versus healthy donors, as well as within the same patients at the time of surgery versus postoperative surveillance visits. To determine whether EV- MDM2 may serve as a possible first-ever biomarker of liposarcoma recurrence. BACKGROUND: A hallmark of well-differentiated and dedifferentiated (WD/DD) retroperitoneal liposarcoma is elevated MDM2 due to genome amplification, with recurrence rates of >50% even after complete resection. Imaging technologies frequently cannot resolve recurrent WD/DD-RLS versus postoperative scarring. Early detection of recurrent lesions, for which biomarkers are lacking, would guide surveillance and treatment decisions. METHODS: WD/DD-RLS serum samples were collected both at the time of surgery and during follow-up visits from 42 patients, along with sera from healthy donors (n=14). EVs were isolated, DNA purified, and MDM2 -DNA levels determined through q-PCR analysis. Nonparametric tests were employed to compare EV- MDM2 DNA levels from patients versus the control group, as well as the time of surgery versus postsurgery conditions. RESULTS: EV -MDM2 levels were significantly higher in WD/DD-RLS than controls ( P =0.00085). Moreover, EV- MDM2 levels were remarkably decreased in WD/DD-RLS patients after resection ( P =0.00036), reaching values comparable to control group ( P =0.124). During postoperative surveillance, significant increases of EV- MDM2 were observed in some patients, correlating with computed tomography scan evidence of recurrent or persistent postresection disease. CONCLUSIONS: Serum EV- MDM2 may serve as a potential biomarker of early recurrent or postoperatively persistent WD/DD-RLS, a disease currently lacking such determinants.

Humans↗

PubMed2025

FACS-Proteomics strategy toward extracellular vesicles single-phenotype characterization in biological fluids: exploring the role of leukocyte-derived EVs in multiple sclerosis.

BACKGROUND: The isolation and proteomics characterization of extracellular vesicles (EVs) from body fluids is challenging due to their vast heterogeneity. We have recently demonstrated that Fluorescence-activated Cell Sorting (FACS) efficiently isolates the whole EV circulating compartment directly from untouched body fluids enabling a comprehensive EV proteomics analysis. RESULTS: Here, we characterized, for the first time, a single-phenotype EV subset by sorting leukocyte-derived EVs (Leuko EVs) from peripheral blood and tears of healthy volunteers. Using an optimized and patented staining protocol of the whole EV compartment we identified and excluded non-EV particles, debris and damaged EVs. We further isolated, using an anti-CD45 antibody, Leuko EVs (CD45+ EVs), reaching a high level of purity (> 90%). Purified Leuko EVs were characterized using atomic force microscopy, nanoparticle tracking, and shotgun proteomics analysis revealing a similar coded protein cargo in both biological fluids. Subsequently, the same workflow was applied to tears from Relapsing-Remitting Multiple Sclerosis (RRMS) patients, revealing a Leuko EVs protein cargo enrichment that reflects the neuroinflammatory condition characteristics of RRMS. This enrichment was evidenced by the activation of upstream regulators TGFB1 and NFE2L2, which are associated with inflammatory responses. Additionally, the analysis identified markers indicative of endothelial cell proliferation and the development of enhanced vascular networks, with AGNPT2 and VEGF emerging as activated upstream regulators. These findings indicate the complex interplay between inflammation and angiogenesis in RRMS. CONCLUSIONS: In conclusion, our combined FACS-Proteomics strategy offers a promising approach for biomarker discovery, analysing cell-specific EV phenotypes directly from untouched body fluids, advancing the clinical value of tears EVs and improving the understanding of EV-mediated processes in vivo. Data are available via ProteomeXchange with the identifier PXD049036 and in EV-TRACK knowledgebase with ID: EV240150.

Humans↗

PubMed2026

Downregulated lysyl oxidase in plasma extracellular vesicles: a biomarker linked to brain metastasis risk in lung adenocarcinoma.

BACKGROUND: Brain metastasis (BrM) is a leading cause of mortality in patients with lung adenocarcinoma (LUAD). Extracellular vesicles (EVs), which carry bioactive molecules, play a critical role in tumor microenvironment remodeling and exhibit metastatic organotropism, holding promise as liquid biopsy biomarkers. This study aims to identify plasma EV-derived proteins associated with LUAD-BrM. METHODS: A multi-omics framework was applied. Plasma EVs from 59 stage IV LUAD patients (30 BrM vs 29 non-BrM) were profiled using data-independent acquisition mass spectrometry proteomics. Candidate proteins were screened via bioinformatics and machine learning (LASSO/RF/SVM). Initial validation included tissue proteomics (n = 13), single-cell transcriptomics (TISCH2), and Western blot analysis of a subset of the discovery samples. Functional experiments were conducted in vitro. The lead candidate was ultimately validated in an independent plasma cohort (n = 158) through ELISA. RESULTS: Proteomic analysis implicated collagen-containing extracellular matrix (ECM) pathways. Lysyl oxidase (LOX), a key ECM cross-linking enzyme, was identified as a lead candidate. LOX and its family member LOXL1 were consistently downregulated in BrM tissues and plasma EVs. Single-cell analysis revealed decreased LOX expression specifically in BrM-associated fibroblasts, which showed suppressed ECM-related pathways. In vitro experiments supported a PI3K/AKT-LOX-ECM regulatory axis. Plasma EV-derived LOX demonstrated strong diagnostic performance in the independent cohort, with an AUC of 0.786 (95% CI 0.713iated fi. CONCLUSIONS: Our study establishes plasma EV-derived LOX as a promising non-invasive biomarker for LUAD-BrM through a comprehensive multi-omics validation strategy. We propose a model wherein downregulation of LOX, potentially driven by PI3K/AKT signaling in tumor-associated fibroblasts, contributes to ECM degradation and may promote brain-tropic metastasis. This finding offers new insights for risk stratification and timely intervention in LUAD patients.

Humans↗