Journal of Extracellular Vesicles

Extracellular vesicles (EVs) carry microRNAs (miRNAs) that mediate intercellular communication and have strong potential as disease biomarkers, yet the roles of miRNA isoforms (isomiRs) in EVs remain poorly understood. Here, we analyzed 96 human EV and corresponding source samples from nine public datasets. We found that EV samples consistently contained substantially higher proportions of isomiR reads than their corresponding source samples, indicating widespread isomiR enrichment in EVs. Although individual isomiRs showed limited reproducibility across biological replicates and limited sharing between EVs and their corresponding source samples, the parent miRNAs that generated these isomiRs remained highly reproducible across replicates and strongly shared between EV-source pairs. Despite extensive isomiR diversification, EV-source pairs retained highly correlated miRNA expression profiles. Using integrated miRNA- and isomiR-related features, we further developed a random forest model that successfully associated EV samples with their corresponding source samples, with improved performance when isomiR information was included. Together, our results demonstrate that EVs are enriched for biologically meaningful isomiRs while preserving source-associated miRNA landscapes, highlighting the importance of incorporating isomiRs into future EV studies.

Microvascular endothelial cell-derived extracellular vesicles (EVs) mediate local intercellular communication relevant to wound healing and inflammation, yet their proteomic cargo and functional properties remain poorly characterized. Here, EVs were isolated from human microvascular endothelial cells (HMEC-1) by standard ultracentrifugation (UC Bulk) or ultracentrifugation combined with size exclusion chromatography (UC+SEC) and characterized by nanoparticle tracking analysis, immunoblotting, cryogenic electron microscopy, and label-free mass spectrometry. UC+SEC achieved a 77-fold improvement in particle-to-protein ratio with 70-93% depletion of serum and extracellular matrix contaminants while preserving canonical EV markers (ALIX, CD9). Mass spectrometry identified 673 proteins in UC+SEC versus 336 in UC Bulk, with both preparations enriched in wound healing, hemostasis, and angiogenesis pathways. Despite dramatic purity differences, both isolation methods produced functionally comparable EVs that significantly enhanced dermal fibroblast wound closure. Functional assays on primary human dermal microvascular endothelial cells (HDMECs) revealed that HMEC-1-derived EVs exert inflammation-dependent dual effects on TNF- pre-treated endothelium: upregulating VCAM-1 expression while simultaneously preserving VE-cadherin-mediated junction integrity. These effects were strictly inflammatory-dependent, with no detectable activity on healthy endothelial cells. This research uncovers a paradoxical phenotype in which microvascular endothelial EVs enhance immune cell recruitment signals while protecting barrier function exclusively under inflammatory conditions, suggesting a regulatory mechanism that may contribute to vascular homeostasis during inflammatory challenges.

Melanoma is the deadliest form of skin cancer, and improved non-invasive approaches to monitor tumor burden and immune dynamics are needed. Although extracellular vesicles (EVs) are increasingly explored as cancer biomarkers, how distinct EV subpopulations reflect dynamic tumor states induced by immune pressure remains insufficiently understood. Using proteomic analyses, we identified the melanoma-associated antigens gp100 (PMEL) and GPNMB in EVs derived from B16F10 melanoma cells and incorporated them into sandwich enzyme-linked immunosorbent assays (ELISAs) that capture total EVs while selectively detecting gp100 and GPNMB EV subpopulations. We subsequently evaluated these EV populations in murine models of anti-tumor vaccination and in plasma samples from melanoma patients. In vivo, melanoma-associated EV subpopulations increased in the serum of tumor-bearing mice and were further augmented following antigen-specific anti-tumor vaccination, whereas total CD81 EVs accumulated more gradually. Notably, gp100 EV levels, but not GPNMB EVs, correlated with tumor-infiltrating lymphocyte densities across multiple time points and treatment conditions. In vitro, TNF/IFN{gamma} stimulation preferentially increased total CD81 EV release, whereas gp100 EVs were promoted by IL-1{beta} stimulation. In contrast, GPNMB EVs accumulated more gradually and broadly across inflammatory, hypoxic, and cytotoxic stress conditions. Together, these findings indicate that immune and stress signals differentially remodel melanoma-associated EV composition. The assay translated to humans, revealing elevated gp100 EV levels in the plasma of melanoma patients compared with healthy donors, whereas GPNMB EVs identified a subset of melanoma patients. We describe a clinically feasible approach that enables direct detection of melanoma-associated EV subpopulations from blood without prior EV isolation. Conceptually, our findings suggest that immune and stress signals dynamically shape circulating EV composition, generating distinct EV signatures that reflect tumor state.

Cancer-associated fibroblasts (CAFs) secrete small extracellular vesicles (sEVs) that mediate stromal remodelling, tumour progression, and pre-metastatic niche formation. A foundational assumption in EV research is that MISEV-compliant preparations from the same conditioned medium are biologically equivalent. Here, we directly challenged this assumption through a side-by-side comparison of ultracentrifugation (UC), size exclusion chromatography (SEC), and the EXODUS nanofiltration platform using breast CAF-conditioned media, characterised in accordance with MISEV2023 guidelines using nanoparticle tracking analysis, cryogenic transmission electron microscopy (Cryo-TEM), and quantitative proteomics. EXODUS and SEC recovered approximately 7-fold more particles per mL than UC. While Cryo-TEM confirmed intact vesicle morphology across all methods, UC preparations exhibited substantial non-vesicular background, with gene ontology analysis revealing significant enrichment of ribosomal, mitochondrial, and ER-derived proteins absent from EXODUS and SEC. Matrisome profiling further uncovered method-dependent divergence in the composition of core versus matrisome-associated proteins, highlighting differences extending beyond standard purity metrics. These findings demonstrate that MISEV2023 compliance is necessary but insufficient for methodological equivalence. The isolation method should be treated as a biological variable and selected according to the EV subpopulation or cargo class under investigation.

Extracellular vesicles (EVs) are emerging mediators of oncogenic communication within the neuroblastoma (NB) tumour microenvironment (TEM). Here, we investigated how constitutive MYCN overexpression influences the proteomic and functional properties of small EVs (sEVs) derived from SKNAS-MYCN-GFP (SK-M) cells and assessed their impact on non-cancerous immune cells. SK-M cells exhibited robust MYCN upregulation at both the mRNA and protein levels and produced sEVs that were selectively enriched in MYCN. Transwell co-culture revealed transfer of MYCN-GFP to recipient DC2.4 nuclei, indicating intercellular transport of functional transcription factor cargo. LC-MS/MS profiling showed that SK-M sEVs incorporated oncogenic cargo non-randomly, displaying significant enrichment of metabolic and MYC/MYCN-regulated pathways, including glycolysis, mTORC1 signalling, and suppression of oxidative phosphorylation (OXPHOS). These observations are consistent with emerging evidence that MYC family proteins can regulate metabolism through vesicular transfer of glycolytic kinases to neighbouring cells. Functionally, SK-M cells displayed elevated lactate secretion and reduced acetyl-CoA, and their sEVs induced a glycolytic shift in recipient immune cells, increasing lactate output in DC2.4, RAW264.7, BMDCs, and splenocytes. sEV-treated BMDCs and splenocytes acquired immunoregulatory phenotypes characterised by increased IL-10, reduced IL-12, expansion of regulatory T cells (Tregs), and macrophage polarization toward an M2-like state. These findings demonstrate that MYCN-driven NB cells disseminate metabolic and immunosuppressive cues via sEVs, reshaping the local immune landscape to favour tumour tolerance. This study provides mechanistic insight into how MYCN-amplified NB cells exploit EV-based communication to coordinate metabolic rewiring and immune escape.

Alzheimers disease (AD) is a neurodegenerative disorder affecting millions of patients globally. Despite significant efforts from researchers in recent decades, there are still many unanswered questions about AD pathogenesis. AD patient brains manifest changes in extracellular vesicles (EVs) secreted from diseased neurons, and the effect of this phenomenon remains poorly understood. EVs contain a variety of biomolecules and play a critical role in cell-to-cell communication in all eukaryotic organisms. Here, we report a thorough characterization of small EVs purified from cultures of human cerebrocortical organoids. These organoids are differentiated from human patient-derived stem cells that bear a familial AD mutation in the presenilin 1 (PSEN1) gene, or from an isogenic wildtype (WT) control. The organoid conditioned media was aspirated from cultures and processed for EV enrichment using a non-invasive technique that requires no cellular disruption. EVs purified from AD organoid conditioned media have a wider size distribution and show differential expression of tetraspanins CD63, CD9, and CD81 when compared to WT organoid-derived EVs. AD organoid-derived EVs can have single, double, and even triple membranes and display luminal fibrillar material. A deep proteomic profiling of the EVs reveals several statistically significant differences, including evidence for modifications in secretory autophagy. EV isolates from both WT and AD organoids show strong binding to amyloid detecting dyes, both in bulk fluorescence and fluorescence microscopy assays. After a 1-week co-culture of AD organoids with WT organoids, there is evidence of endosomal membrane transfer between the isogenic cultures with an increase in amyloid-{beta} peptides in the WT organoids. These observations support the notion that non-cell-autonomous spread of amyloid-containing EVs in human AD brains can be modeled in a cerebral organoid system.

Extracellular vesicles (EVs) circulate in biofluids and carry tissue-specific molecular cargo, offering significant potential for the discovery of minimally invasive biomarkers. However, translation in neurodegenerative diseases has been hindered by the lack of validated neuronal EV surface markers that enable selective isolation from plasma. We hypothesized that proteomic profiling of EVs released from human induced pluripotent stem cell (hiPSC)-derived neurons would identify 1. robust Alzheimers disease (AD)-associated signatures that reflect disease pathogenesis, and 2. surface-accessible neuronal markers capable of enriching disease-relevant cargo. Neurons differentiated from AD patients and age-matched cognitively normal (CN) individuals were used to isolate EVs, which were characterized and analyzed by LC-MS proteomics in both total and membrane-enriched fractions. Proteomic profiling identified numerous dysregulated proteins, with a subset validated across independent AD datasets. We identified CNTNAP2 and STX1B as neuronal, brain-enriched EV surface proteins accessible for selective capture and confirmed their presence in EVs from post-mortem human brain, supporting them as bona fide brain-derived EV markers. Immuno-isolation of plasma EVs showed that CNTNAP2-positive EVs had a robust AD-associated increase in phosphorylated tau, identifying CNTNAP2 as a highly discriminative brain-derived EV marker and supporting its potential for blood-based AD diagnostics. Graphical AbstractPrevious studies indicate that extracellular vesicles (EVs) released from neurons carry disease-relevant cargo, yet the search for the optimal neuronal surface markers for the selective isolation of EVs pertinent to Alzheimers disease (AD) from blood is ongoing. To address this need, we performed proteomic profiling of EVs derived from hiPSC-neurons (iNEVs) of AD patients and cognitively normal individuals (CN). LC-MS analysis of whole and membrane-enriched EV fractions revealed robust protein dysregulation and identified CNTNAP2 and STX1B as surface-exposed neuronal EV proteins, confirmed in human brain-derived EVs (BDEVs). Immuno-isolation of plasma EVs demonstrated that CNTNAP2-positive EVs are enriched for phosphorylated tau in AD, whereas the previously used markers; NrCAM and ATP1A3-positive EVs showed limited discrimination. These findings establish a robust framework for proteomic discovery and nominate CNTNAP2 as a promising EV selection marker for blood-based AD diagnostics. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/720870v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@754b45org.highwire.dtl.DTLVardef@a0a566org.highwire.dtl.DTLVardef@cb0134org.highwire.dtl.DTLVardef@1bc0275_HPS_FORMAT_FIGEXP M_FIG C_FIG

Lipophilic dyes are widely used to track extracellular vesicles (EVs), yet their labeling efficiency toward bona fide small EVs (sEVs) remains poorly defined. Here, we critically reassess this efficiency using a serum-free HEK293F system that generates endogenously fluorescent protein-tagged sEVs (sEVs-FPT) as an unambiguous positive reference, thereby minimizing interference from co-isolated, dye-labelable non-vesicular extracellular particles (NVEPs). Two orthogonal methods, nanoflow cytometry and fluorescence microscopy, were employed for cross-validation. We found that PKH26, PKH67, and DiD labeled <0.5% of sEVs-FPT, regardless of vesicle heterogeneity. In vivo tracking confirmed that dye-derived signals were far weaker than FPT signals and strikingly failed to colocalize with them. Preliminary mechanistic evidence indicates that this failure is due to an inability of sEVs to actively internalize dye aggregates. Our findings raise serious concerns about the validity of lipophilic dye-based EV tracking and call for a critical reevaluation of the relevant literature.

This protocol provides a standardized workflow for the isolation of extracellular vesicles (EVs) from mouse retinal tissue, and includes an assessment of EV size and concentration, marker expression, and EV visualization in accordance with the International Society for Extracellular Vesicles Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines. Most retinal EV studies rely on cell culture, which may not fully capture in vivo biology. Our approach more accurately reflects physiological and pathological EV states in vivo by enabling the extraction of EVs from intact retinal tissue. This method addresses a key gap in the field by providing a reproducible and rigorous protocol for studying retinal EVs in a biologically relevant context.

Extracellular vesicles (EVs) derived from activated dendritic cells (DCs) are promising cell-free mediators capable of shaping CD8+ T-cell responses. However, their early molecular and functional effects on CD8+ T cells remain incompletely characterized, and whether engineering activated DC-derived EVs with immunomodulatory cargo can fine-tune these responses remains largely unexplored. Here, we investigated whether curcumin loading into EVs derived from CpG-activated and peptide-pulsed DC2.4 cells (EV-ACT) modulates early activation of primary CD8+ T cells. EVs were isolated by ultrafiltration coupled with size-exclusion chromatography (UF-SEC) and characterized physicochemically and molecularly. Exploratory proteomic profiling identified an activation-associated EV protein signature enriched in antigen-processing and immune-related pathways. Curcumin loading achieved an encapsulation efficiency of 16.4% while preserving EV properties, and spectral confocal fluorescence microscopy revealed heterogeneous fluorescence emission patterns consistent with distinct EV-associated curcumin microenvironments. Following rapid cellular association, EV-ACT promoted early CD8+ T-cell activation, inducing an effector-like phenotype characterized by increased CD69 expression, TNF- and Granzyme B production, and reduced Bcl-2 levels without compromising cell viability. Unlike free curcumin, EV-mediated curcumin delivery selectively reinforced these immunostimulatory responses by significantly increasing CD69 expression and STAT3 phosphorylation, sustaining early activation-associated functional and molecular reprogramming of primary CD8+ T cells.

Extracellular vesicles (EVs) are versatile therapeutic candidates due to biological roles in intercellular communication and amenability to bioengineering. Compared with lipid nanoparticles (LNPs), native or surface-modified EVs may have favorable immunogenicity and biodistribution profiles. However, when administered intravenously (IV), EVs are rapidly cleared and accumulate mostly in the liver and spleen. With the goal of modifying EV biodistribution, we engineered EVs to display the human endogenous retrovirus (HERV) envelope glycoprotein Syncytin-1, an SLC1A5-binding fusogenic viral protein essential for syncytiotrophoblast formation in pregnancy. Here, we comprehensively characterize engineered Syncytin-1+ EVs, examine their interactions with cells in vitro, and assay biodistribution, immunogenicity, and pharmacokinetics ex vivo and in vivo in non-human primates. IV-administered Syncytin-1+ EVs are well tolerated, persist in the blood stream, and have altered organ biodistribution compared with unmodified EVs, suggesting therapeutic potential of Syncytin-1+ EVs at specific sites.

In this work we show that small extracellular vesicles (sEVs) from embryonic mouse cortex or from cultured embryonic cortical neurons contain high levels of BDNF and sustain TrkB-dependent neuroprotective signaling. By contrast, sEVs from aged cortex are depleted of BDNF, and cells lacking active TrkB fail to mount a protective response when exposed to the same sEVs. Biochemical fractionation and trypsin sensitivity assay indicate that BDNF is a constitutive EV component and is exposed on or tightly associated with the vesicle surface--an arrangement that likely increases local ligand density. In a stability assay, EV-associated BDNF retained activity longer than soluble BDNF. Together, our findings suggest that many developmental effects of BDNF may be mediated by EVs, that impaired stress responses in the aged brain could reflect reduced formation of BDNF-containing EVs, and that embryonic sEVs may provide a more efficient vehicle for BDNF delivery than current therapeutic approaches.

The development of lung-directed therapeutics is limited by poor translational fidelity between preclinical models and early-phase clinical trials. We report a first-in-human Phase 0 intratarget microdosing study demonstrating the feasibility of intrapulmonary delivery and pharmacological interrogation of a novel inflammasome inhibitor. A 100 g microdose of ADS032, a dual NLRP1/NLRP3 inhibitor, was administered to distal airways via bronchoscopy in patients with interstitial lung disease, informed by optimisation in ex vivo human lung perfusion and ventilation systems. Clinical-grade manufacture, formulation, stability, and toxicology enabled intrapulmonary administration. Using liquid chromatography-mass spectrometry, ADS032 was detected in plasma, bronchoalveolar lavage fluid, distal airway micro-aspirates, and recovered cells, with spatially resolved sampling achieved without cross-contamination. Fluorescent labelling enabled direct visualisation of alveolar drug uptake ex vivo. These findings establish intrapulmonary intratarget microdosing as a human-relevant platform for early pharmacological evaluation of lung therapeutics prior to Phase 1 trials.

Ovarian cancer (OC) remains a leading cause of gynecologic cancer mortality due to late-stage diagnosis and limited early detection strategies. Ascites fluid, a pathological hallmark of OC, is a rich source of tumor-derived extracellular vesicles (EVs) that reflect the tumor microenvironment and hold promise for biomarker discovery. However, isolating EVs from minimal ascites volumes (<100 {micro}L) poses technical challenges using conventional methods like ultracentrifugation or size-exclusion chromatography (SEC). This study explores the application of strong anion exchange (SAX) magnetic beads (Mag-Net) for efficient EV isolation from as little as 2 {micro}L of ascites fluid from both murine models and a human patient with mucinous borderline tumor. We demonstrate that SAX achieves robust EV capture at 10{micro}l of input volume, enabling comprehensive proteomic profiling and single-EV surface-enhanced Raman spectroscopy (SERS) with a >2-fold increase in proteomic depth compared to raw ascites. Notably, this study was able to identify 1000 proteins not previously annotated in Vesiclepedia for OC-derived EVs, alongside distinct SERS signatures, highlighting the potential for multiomic analysis. Comparative analysis with UC revealed enhanced proteomic depth obtained with SAX beads, albeit we also observed differential detection of canonical markers (e.g., CD9, CD81) between input volumes of ascites fluid. These findings establish SAX as a scalable, low-input platform for EV-based biomarker discovery, paving the way for improved early detection and molecular insights into OC progression.

Harnessing the immunomodulatory capacity of commensal bacteria is an emerging avenue in cancer therapy. Bacterial extracellular vesicles (BEVs) provide a non-replicating, nanoscale alternative to live microbes with the potential for safer systemic delivery. Here, we investigated BEVs from a novel Gram-positive strain of Bifidobacterium pseudocatenulatum (Bif-210). Intravenous administration of Bif-210 BEVs reduced B16-F10 melanoma growth in C57BL/6J mice. Mechanistically, BEVs increased tumour-infiltrating Ly6G+ granulocytes in vivo, increased CD11b+Ly6G+ and ICAM-1+Ly6G+ bone marrow populations, and induced production of the neutrophil-attracting chemokines KC/CXCL1 (mouse) and IL-8 (human). Although Ly6G+ depletion independently inhibited tumour growth, it did not combine additively with BEVs, supporting a model in which Bif-210 BEVs alter Ly6G+ granulocyte function rather than simply expanding a conventional pro-tumour granulocyte pool. BEVs activated TLR2, did not activate TLR4, and upregulated TLR2 on Ly6G+ cells, while proxy assays provided no evidence of NETosis-associated activation. Repeated intravenous BEV administration produced no overt toxicity by tissue histology, body temperature, or body weight monitoring. These findings position B. pseudocatenulatum BEVs as a promising systemic immunotherapy that recruits and re-educates granulocytes via a TLR2-centred pathway to restrain melanoma progression. HIGHLIGHTSO_LIIntravenous Bif-210 BEVs reduce established B16-F10 melanoma growth in mice. C_LIO_LIBif-210 BEVs selectively increase tumour-associated Ly6G+ granulocytes. C_LIO_LIBEV treatment and Ly6G depletion are non-additive, linking BEV activity to granulocyte biology. C_LIO_LIBif-210 BEVs expand Ly6G+ bone marrow populations and induce granulocyte-recruiting chemokines. C_LIO_LIBif-210 BEVs engage TLR2 and enhance granulocyte fitness without NETosis-associated activation. C_LI

Immunoglobulin (Ig) replacement therapies (IgRT) including intravenous (IVIg) and subcutaneous (SCIg), are pooled IgG preparations widely used to restore humoral immunity and to suppress pathological inflammation in autoimmune and inflammatory disorders. Despite broad clinical use, the mechanisms underlying their immunomodulatory effects remain incompletely defined. Here, we identify extracellular vesicle (EV)-associated cytokines as mediators of IVIg activity. Multiplex bead-based flow cytometry revealed that EVs isolated by size exclusion followed by ultracentrifugation from IVIg were CD63 positive but depleted of platelet-derived and HLA markers relative to EVs from unprocessed human plasma. Luminex profiling demonstrated substantial reduction of pro-inflammatory cytokines in IVIg EVs. Notably, although IVIg EVs contained abundant IFN{gamma}, they failed to activate IFNGR/JAK/STAT1 signaling. Instead, prolonged exposure to IVIg EVs suppressed subsequent IFN{gamma}-induced STAT1 activation. Engineered IFN{gamma}-coated EVs (IFN{gamma}-eEVs) recapitulated both activating and inhibitory effects indicating context-dependent signaling bias. Critically, cold ethanol precipitation, a key step in IVIg manufacturing, selectively abrogated the activating function of IFN{gamma}-eEVs while preserving their inhibitory capacity. These findings define a previously unrecognized mechanism where IVIg processing generates EVs that bias IFN{gamma} signaling toward suppression. EV-associated cytokines therefore represent a generalizable pathway through which IVIg exerts anti-inflammatory effects across immune-mediated diseases.

A subset of extracellular vehicles (EVs) with particle diameters >200 nm, large vesicles (lEVs) contain mitochondria that increase recipient cell bioenergetics. To date, sequential centrifugation (SC) is the most reported protocol to separate lEVs from the smaller EVs (<200 nm)/exosomes. We have previously demonstrated that lEVs derived from brain endothelial cells (BECs) using the standard SC method transferred their innate mitochondria to recipient BECs, increased recipient BEC bioenergetics, reduced brain infarct volume, and improved behavioral outcomes in a mouse model of transient ischemic stroke. Despite their promising therapeutic activity, SC-isolated lEVs are likely a mixture of mitochondria-containing lEVs and non-mitochondria-containing lEVs. We hypothesized that subsequent purification of SC-isolated lEVs using density-gradient centrifugation (DGC) may yield a purer sample of mitochondria-containing lEVs. We established a DGC protocol to purify lEVs. In this pilot study, lEVs isolated using SC and DGC protocols were compared to determine their physicochemical characteristics and their effects on recipient BEC bioenergetics. SC-lEVs and DGC-lEVs both significantly restored ATP levels in OGD-injured BECs with no difference between groups. However, a Seahorse mitochondrial function assay revealed distinct functional effects: SC-lEVs did not significantly alter respiration, whereas DGC-lEVs induced a dose-dependent increase in oxygen consumption rate, indicating enhanced oxidative phosphorylation. These findings demonstrate that DGC purification yields a more mitochondria-enriched and functionally potent lEV preparation with an enhanced capacity to restore oxidative phosphorylation in ischemic BECs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/732469v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@12d5e90org.highwire.dtl.DTLVardef@19b44a5org.highwire.dtl.DTLVardef@b7ad75org.highwire.dtl.DTLVardef@dd1a3d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Sarcoidosis is a multisystem disorder that primarily affects the lungs and is characterizedby granulomatous inflammation. However, much of the underlying disease mechanisms remain poorly understood. Extracellular vesicles (EVs) are small membrane-bound particles released by all cells and carry various cargos including metabolites. They are involved in intercellular communication that can be dysregulated in diseases.This study characterizes the metabolic cargo of EVs isolated from bronchoalveolar lavage fluid (BALF), using liquid chromatography-mass spectrometry (LC-MS)-based metabolomic analysis, in patients with sarcoidosis (n=37), compared to healthy controls (n=10). Additionally, the sarcoidosis signature was compared to another pulmonary disorder, anti-synthetase syndrome (ASyS, n=10). Arachidonic acid (AA) results were verified by ELISA. A total of 1202 metabolites were detected, with 111 annotated ones further analyzed. EVs from sarcoidosis patients showed distinct metabolomic profiles compared to both ASyS patients and healthy controls, with 38 annotated metabolites differentially expressed in any of the groups. In both annotated and non-annotated data, sarcoidosis patients clustered separately from ASyS patients and healthy individuals. Furthermore, sarcoidosis patients clustered in 3 subgroups, whereof one was similar to ASyS patients and one stood out as showing higher cell counts in BALF. Higher AA levels were found in sarcoidosis patient EVs by LC-MS, and AA results were verified by ELISA. Our data show that BALF EV metabolites are disease-dependent and support the notion thatsarcoidosis patients should be further subgrouped for better diagnosis and treatment.

Breast cancer is the most common malignancy in women, with triple-negative breast cancer (TNBC) representing the most aggressive subtype and carrying a poor metastatic prognosis. Metastasis requires tumor cells to cross the endothelial barrier, a process facilitated by tumor-derived extracellular vesicles (EVs), which can disrupt vascular integrity. Fluid shear stress (FSS), generated by blood flow, shapes endothelial physiology and may influence EV uptake, yet the mechanisms underlying TNBC-derived small EV (sEV) internalization remain unclear. Here, we investigated TNBC sEV-endothelial interactions using combined in silico and in vitro approaches. Human umbilical vein endothelial cells (HUVECs) were cultured under static or FSS conditions (20 dyn/cm{superscript 2}), followed by proteomic profiling and protein-protein interaction analyses with sEV proteomes. Uptake assays employed pharmacological inhibition (Dynasore, M{beta}CD, Pitstop2), Caveolin-1 (CAV-1) and Clathrin Heavy Chain (CLHC), siRNA-mediated knockdown, and junctional interaction analyses via confocal microscopy and co-immunoprecipitation. FSS downregulated proliferation- and angiogenesis-associated proteins while upregulating adhesion and cytoskeletal regulators assessed by proteomics. Network analysis identified clathrin- and caveolin-mediated endocytosis (CME and CavME), integrins, and early endosomes as central mediators of sEV uptake. Functionally, uptake was reduced by Pitstop2, M{beta}CD, and CAV-1/CLHC knockdown under static conditions, but silencing paradoxically enhanced uptake under FSS, suggesting compensatory flow-dependent pathways. Notably, under FSS, sEVs accumulated at endothelial junctions, colocalizing with VE-CAD and associating with CLDN5, indicating a potential disruption mechanism of adherens and tight junctions and consequent endothelial permeability. These findings identify CME and CavME as key uptake routes while underscoring FSS as a critical determinant of endothelial-tumor EV interactions. By revealing junctional targeting of sEVs, this work provides new mechanistic insight into vascular remodeling during metastasis and highlights EV pathways as potential therapeutic targets in TNBC. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/721946v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@f91c5org.highwire.dtl.DTLVardef@2b4dc8org.highwire.dtl.DTLVardef@ff94f1org.highwire.dtl.DTLVardef@18b714b_HPS_FORMAT_FIGEXP M_FIG C_FIG Uptake and localization of sEVs on HUVEC under (a) static and (b) fluid shear-stress conditions. sEVs: Small Extracellular Vesicles. CME: Clathrin-mediated Endocytosis. CavME: Caveolin-mediated Endocytosis. CLDN5: Claudin-5. VE-CAD: Vascular Endothelial Cadherin. FSS: Fluid shear-stress.

BACKGROUNDMesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) exert neuroprotective effects in ischemic stroke largely through immunomodulatory mechanisms. Monocytes are first-line responders to MSC-EVs. Their contribution to MSC-EV-induced neuroprotection remains poorly understood. This study investigated the role of monocytes in shaping neuroprotective responses to MSC-EVs after ischemic stroke. METHODSMale C57BL/6J mice were exposed to transient middle cerebral artery occlusion (MCAO). Monocytes were depleted using pharmacological (clodronate liposomes), immunological (anti-CCR2), or genetic (Mrp8-Cre+/- Nr4a1fl/fl) approaches removing total, CCR2+, or Ly6Clow monocytes, respectively. In additional cohorts, neutrophils and T cells were simultaneously depleted by anti-Ly6G or anti-CD4/CD8 antibodies. Small EVs from clonally expanded immortalized MSCs were administered intravenously. Neurological deficits, ischemic injury, and immune responses were analyzed up to 72 hours post-MCAO. Complementary ex vivo studies were performed, in which MSC-EVs were administered to monocyte-depleted or non-depleted peripheral blood mononuclear cells (PBMCs) obtained from acute ischemic stroke patients. RESULTSIn ischemic mice with intact monocyte compartment, MSC-EVs reduced neurological deficits, infarct volume, neuronal injury, and brain leukocyte infiltrates. These protective effects were abolished in monocyte-depleted mice, particularly following CCR2+ monocyte depletion. Under these conditions, MSC-EV treatment exacerbated neurological deficits, ischemic injury, and leukocyte infiltration, accompanied by neutrophil and T cell expansion and overactivation. Depletion of neutrophils or T cells prevented the EV-induced worsening of stroke outcome in monocyte-deficient mice. Ly6Clow monocytes played a crucial role in orchestrating immune responses to MSC-EVs. Their depletion abolished EV-induced neuroprotection. In stroke patient PBMCs, MSC-EVs induced phenotypic reprogramming of monocytes, whereas they promoted CD4+ and CD8+ T cell activation in the absence of monocytes. CONCLUSIONSMonocytes shape the immunomodulatory actions of MSC-EVs. In their absence, MSC-EVs trigger neutrophil and T cell overactivation that worsens stroke outcome. These findings highlight the importance of monocyte- and T cell-related potency assays for the clinical translation of MSC-EV therapies.