Journal of Extracellular Vesicles

Extracellular vesicles (EVs) mediate intercellular transfer of lipids, proteins, and nucleic acids between nearly all cell types. We previously showed that astrocyte-derived EVs modulate neuronal mitochondria in vitro, but whether endogenous astrocytic EVs are trafficked to neuronal mitochondria in vivo remained unknown. To address this, we generated an EV reporter mouse, Aldh1l1-Cre; CD9-tGFPfl/fl, in which astrocyte-secreted EVs are labeled with a CD9-turboGFP fusion protein (CD9-tGFP). Astrocyte-specific expression of CD9-tGFP was verified in brain tissue and isolated EVs, comprising 13.2 {+/-} 1.6% of total brain EVs. In primary glial cultures, CD9-tGFP was restricted to astrocytes, localizing to vesicular compartments and cell protrusions (filopodia and cilia), with 89.3 {+/-} 2.2% of astrocyte-derived EVs carrying the label. These EVs were enriched with the sphingolipid ceramide, consistent with its co-distribution with CD9-tGFP in astrocytic cell protrusions. In the cortex, hippocampus, and cerebellum, CD9-tGFP was predominantly detected in astrocytic processes co-labeled with GLAST1 and GFAP, forming contacts with laminin-positive capillaries and parvalbumin-positive neurons. CD9-tGFP-labeled EVs were detected inside capillaries and neurons, and super-resolution STED microscopy revealed partial overlap with neuronal mitochondria. Live-cell spinning disk confocal imaging and AI-assisted proximity analysis confirmed uptake of CD9-tGFP EVs by neuronal cells and trafficking of their cargo to mitochondria in vitro. Biochemical isolation of synaptic and non-synaptic mitochondria confirmed EV-derived cargo on mitochondria in vivo, with 3-fold higher association of CD9-tGFP with synaptic than non-synaptic mitochondria. Together, these findings validate the Aldh1l1-Cre; CD9-tGFPfl/fl reporter mouse as a powerful tool for tracking astrocyte-derived EVs in vivo and provide direct evidence that their cargo is preferentially trafficked to synaptic mitochondria. Graphical AbstractAstrocyte-derived extracellular vesicles target neuronal mitochondria in vivo O_FIG O_LINKSMALLFIG WIDTH=156 HEIGHT=200 SRC="FIGDIR/small/718987v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@174d92aorg.highwire.dtl.DTLVardef@5d8248org.highwire.dtl.DTLVardef@114483borg.highwire.dtl.DTLVardef@924d55_HPS_FORMAT_FIGEXP M_FIG C_FIG

Extracellular vesicles are key intercellular messengers that modulate the function of target cells by carrying effectors, either at their surface or in their lumen. In the latter case, their action depends on the ability to deliver their content into the cytosol of target cells. How efficiently EVs deliver their content upon interaction with their target cell is thus a central question for understanding the functional impact of this mode of action. To address this question, signal-driven bimolecular interactions between two partners located respectively in the EV lumen and the target cell cytosol have become a widely used strategy to detect the cytosolic delivery EV content. However, the detection of cytosolic delivery with these assays was often tributary to the artificial enhancement of the fusion between EV and cell membranes, through for instance VSV-G fusogenic protein expression. Here we provide a robust and quantitative LUCiferase-based complementation assay (HiBiT/LgBiT), to quantify the Internalization and cytosolic Delivery of EV content: LUCID-EV. By optimizing the signal-to-noise ratio of the assay, the method for loading HiBiT fragment into EVs (fusion to a lipid-binding domain rather than to tetraspanins), and the intracellular position of LgBiT (associated to membranes), we could quantify cytosolic delivery from various non-VSV-G-expressing EVs into target immune dendritic cells. Importantly, this delivery did not involve the acidic late endosomes environment required for VSV-G-dependent EV cytosolic delivery. The limited efficacy of the process highlights the need for highly sensitive assays like the one described here. Further development of the LUCID-EV assay could help identifying EV/target cells pairs with enhanced cytosolic delivery properties and characterize the cellular route for delivery.

The intestinal microbiome influences immune recovery and long-term outcomes after allogeneic hematopoietic stem cell transplantation (allo-SCT). While reduced bacterial diversity and depletion of immunomodulatory microbial metabolites during peri-engraftment have been linked to acute graft-versus-host disease (aGvHD) and mortality, it remains unclear whether microbiome recovery after engraftment and immune reconstitution is better reflected by bacterial diversity or by microbial metabolic output. We aimed to define microbiome recovery in the late post-transplant period and test whether a metabolite-based biomarker improves the prediction of clinical outcomes, including overall survival (OS) and chronic (c) GvHD. In this two-center longitudinal observational study, serial stool samples were collected from pre-transplant baseline to day +100 after allo-SCT in a discovery cohort (n = 20, Technical University Munich University Hospital (TUM)) and an independent validation cohort (n = 100, University Hospital Regensburg (UKR)). Gut microbiome composition was assessed by 16S rRNA gene amplicon sequencing, with metagenomic profiling in selected patients, and stool metabolites were quantified using targeted mass spectrometry. Patients were classified as RECOVERY or NO RECOVERY based on changes in bacterial richness between baseline and the post-transplant period. To capture microbial metabolic output, the previously established Immune-Modulatory Metabolite Risk Index (IMM-RI), comprising butyric, propionic, and isovaleric acids, desaminotyrosine and indole-3-carboxaldehyde, was adapted to the late post-transplant period (IMM-RI post-TX). Bacterial alpha diversity frequently improved by day +100; however, this did not consistently indicate restoration of baseline community structure and was not paralleled by recovery of stool metabolite profiles. Accordingly, RECOVERY status showed a limited association with survival or transplant-related mortality (TRM). In contrast, IMM-RI post-TX low-risk identified patients with preserved butyrate-associated biosynthetic capacity and was significantly associated with improved OS in both cohorts (UKR: HR 0.2052, 95% CI 0.07703 - 0.5466, p < 0.0001). In the validation cohort, IMM-RI post-TX low-risk was significantly associated with reduced relapse-related mortality. Interestingly, stool butyric-, propionic and valeric acid concentrations were increased in cGvHD of the skin, indicating context-dependent metabolite effects. These findings suggest that metabolite profiling outperforms bacterial diversity for predicting outcomes after allo-SCT and support microbial metabolites as promising biomarkers for risk stratification and actionable candidates for precision microbiome interventions after allo-SCT.

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.

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) are critical mediators of intercellular communication, carrying molecular cargos such as small noncoding RNAs (ncRNAs) that reflect the physiological and pathological state of their cells of origin. However, studying brain-derived EVs has been challenging due to the blood-brain barrier. Here, we optimized and validated an open-flow microdialysis (OFM) protocol for sampling EVs directly from brain interstitial fluid (ISF) in wild-type and APP/PS1 transgenic mice. Ex-vivo validation using plasma EVs demonstrated that OFM effectively captures the full EV population. In-vivo cerebral OFM (cOFM) enabled successful collection of brain ISF EVs, which were characterized by nanoparticle tracking analysis (NTA), electron microscopy, and western blotting, confirming their similarity to EVs isolated directly from brain tissue and plasma. Identification of small ncRNA cargos revealed that EVs sampled from brain ISF by cOFM were enriched in brain-specific signatures, many of which are associated with neuronal cell populations and biological functions. Furthermore, we observed a unique small ncRNA signature from the brain ISF EVs in the Alzheimers disease preclinical model compared to wild-type mice. These small ncRNAs were associated with genes considered important in biological functions associated with neurodegeneration. Our findings demonstrate that cOFM is a powerful tool for in-vivo sampling of brain EVs and highlight the unique molecular landscape of ISF EV small ncRNA cargos. This study offers new opportunities for biomarker discovery and mechanistic insights into neurodegenerative diseases, such as Alzheimers disease.

BackgroundExtracellular vesicles (EV), secreted membrane particles involved in cell-to-cell communication, carry important information on immunity and its dysregulation. Recent studies have demonstrated the crucial role of peripheral and central inflammation in causing Parkinsons disease (PD), as well as the involvement of EV in mediating neuron-glial interactions during neurodegeneration. However, the underlying mechanism of plasmatic EV in PD pathogenesis remains unknown. MethodsEV were isolated from pool of plasma of PD patients and age- and sex-matched healthy controls (HC) using size-exclusion chromatography and characterized by nanoparticle tracking analysis, western blot, and transmission electron microscopy. SH-SY5Y neurons and HMC3 microglia cells were treated with EV, and their impact was evaluated using flow cytometry and immunofluorescence. Conditioned medium (CM) from EV-treated HMC3 cells was applied to SH-SY5Y neurons to determine indirect neurotoxic effects. Cytokine profiling and senescence-like features of EV-treated HMC3 cells were assessed. Unbiased proteomic analysis of PD-EV and HC-EV were further performed. ResultsPD-EV induced axonal degeneration and cell death in SH-SY5Y neurons and increased levels of TNF-, IL-1{beta}, IFN-{gamma}, IL-8, and CCL11, accompanied by the expression of p16INK4a in HMC3 cells, suggesting a proinflammatory, senescence-associated secretory phenotype (SASP). Enrichment pathway analysis revealed that these changes were mainly related to inflammatory and immune responses. Moreover, CM from PD-EV-HMC3 cells increased apoptotic cell death in SH-SY5Y neurons more than direct PD-EV. Notably, proteomic analysis of PD-EV showed higher expression of proteins involved in complement cascades, immune response, phagocytosis, and post translational protein translation, further supporting the potential of EV to induce inflammatory changes in PD. ConclusionsThis study demonstrates that plasmatic PD-EV contributes to neuronal degeneration by reducing neuronal integrity and indirectly by activating microglia through the secretion of pro-inflammatory, senescence-associated mediators. Circulating EV exerts a role in bridging peripheral inflammation with microglia, modulating neuroinflammatory events.

Matrix-bound nanovesicles (MBVs) are a type of small extracellular vesicle (EV) embedded in the extracellular matrix (ECM) throughout the body. MBVs have been previously isolated from various tissues and in vitro-cultured cell sheets, demonstrating remarkable attributes in regenerative medicine. However, differences between MBVs and conditioned culture medium-derived EVs (liquid-EVs) have yet to be characterized, and the field currently lacks specific protein markers that can identify MBVs from other EV subtypes. Here, we isolate MBVs and liquid-EVs from bone marrow mesenchymal stem cell (MSC) sheets and define differences in size, protein, and zeta potential between these EVs. We show that there is a correlation between cell-driven ECM deposition and MBV and liquid-EV production. We also find that MBVs are smaller, contain less protein per particle, and possess lower zeta potential than liquid-EVs. Interestingly, MBVs also comprise a distinct tetraspanin profile compared to liquid-EVs, with MBVs containing more CD63 and little to no CD81. Finally, we define that CD63, LAMP1, Alix, ITG{beta}1, and GRP94 and their abundance, may be markers specifically used to identify MBVs from liquid-EVs. Our study paves the way for the characteristic differentiation between MBVs from liquid-EVs, elucidates their differences in biogenesis, and reveals a potential connection between EV and ECM production.

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

Mesenchymal stromal cells (MSCs) are multipotent cells with well-established regenerative and immunomodulatory properties, making them promising candidates for the treatment of inflammatory diseases. However, the therapeutic effects of MSCs are largely mediated by their secretome, particularly extracellular vesicles (EVs), which deliver bioactive molecules capable of modulating inflammatory responses. We generated an extracellular vesicle-enriched secretome (EVES) from MSCs under scalable, Good Manufacturing Practice (GMP)-compliant conditions and assessed its therapeutic efficacy in diverse disease models, including lung inflammation and kidney injury induced by distinct innate immune stimuli. EVES was isolated from the secretome of umbilical cord blood-derived MSCs cultured in a chemically defined medium. In vitro, EVES significantly and dose-dependently attenuated cytokine release from airway epithelial cells and macrophages stimulated with inflammatory agents such as lipopolysaccharide or reactive particles. In murine models of lung inflammation, EVES reduced neutrophil infiltration and suppressed multiple cytokines and chemokines in a dose-dependent manner. In models of kidney injury, EVES enhanced tubular epithelial cell proliferation, improved renal histology, and markedly reduced tubular necrosis following ischemia-reperfusion injury. Collectively, these findings demonstrate that MSC-derived EVES exhibits robust and broad-spectrum therapeutic activity across multiple disease contexts driven by innate immune activation, supporting its potential as a scalable, cell-free therapeutic platform.

BackgroundP-glycoprotein (P-gp/ABCB1) is a key efflux transporter that maintains barrier integrity by clearing xenobiotics and toxic metabolites. At the feto-maternal interface, trophoblast-derived extracellular vesicles (CTC-EVs) naturally and transiently transfer functional P-gp to maternal decidual cells, restoring lost and or reduced P-gp function (exofection) to sustain pregnancy homeostasis. A similar loss of P-gp at the blood brain barrier (BBB) contributes to impaired amyloid-{beta} (A{beta}) clearance and neuroinflammation in Alzheimers disease. We investigated whether CTC-EV-mediated exofection could restore P-gp function in human brain endothelial cells (hBECs) and enhance A{beta} clearance under inflammatory and neurodegenerative conditions. MethodsCTC-EVs were isolated and characterized by nanoparticle tracking analysis and western blotting for P-gp and EV markers. Transcriptomic profiling of CTC-EVs identified enrichment of transporter-related genes, including solute carriers and ABC transporters, along with inflammatory mediators. Network analysis revealed coordinated modules linking EV cargo to transporter regulation, endocytosis/trafficking pathways, and inflammatory remodeling processes converging on BBB efflux activity. hBECs were exposed to LPS (500 ng/mL, 48 h) with or without CTC-EVs. P-gp expression was assessed by immunofluorescence (mean fluorescence intensity, MFI) and western blotting, while functional efflux was measured using Calcein-AM assays. A{beta} oligomer transport was evaluated using a transwell hBEC model. In vivo, 3xTg-AD mice received intravenous CTC-EVs (1x10L/day for 5 days), followed by assessment of P-gp expression, A{beta} burden, and neuroinflammatory markers. Pharmacokinetic studies in P-gp knockout mice were conducted to confirm functional transporter recovery. ResultsLPS exposure significantly reduced P-gp expression in hBECs (41.3% decrease in MFI, p=0.0084), which was restored by CTC-EVs (46.7% increase vs. LPS, p=0.0121). Exofection increased P-gp by a 2.1-fold following EV treatment as determined by western blot. Functional assays demonstrated enhanced efflux, with a 38.5% reduction in intracellular Calcein fluorescence (p<0.001). Network-informed mechanisms supported coordinated regulation of transporter and trafficking pathways. CTC-EVs improved A{beta} transport across inflamed hBEC monolayers. In vivo, EV-treated 3xTg-AD mice exhibited increased P-gp expression in the frontal cortex (38.6%) and hippocampus (42.1%), reduced A{beta} plaque burden (27.9%), and decreased inflammatory markers (IL-1{beta} and TNF-, p<0.05). In P-gp knockout mice, EVs reduced brain drug accumulation by 22.4% (p=0.032), confirming restoration of transporter function. ConclusionCTC derived EVs are natural carriers of functional transporter proteins and restore efflux capacity in compromised endothelial barriers. Integration of transcriptomic and network analyses highlights coordinated regulation of transporter, trafficking, and inflammatory pathways underlying exofection. This reproductive biology inspired strategy offers a promising therapeutic approach for enhancing A{beta} clearance and mitigating neuroinflammation in Alzheimers disease.

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.

In root nodule symbiosis, symbiosome compartments accommodate nitrogen-fixing rhizobia inside the plant cell. Differentiated into bacteroids, the rhizobia are surrounded by a peribacteroid space and a plant-derived peribacteroid membrane, which separates them from the plant cytoplasm but allows signal and nutrient exchange between host and microbe. The morphological features of symbiosomes are primarily determined by ultrastructural single focal plane imaging, with limited information about spatial details. This study combines 2D and 3D imaging, using transmission electron microscopy and focused ion beam scanning electron microscopy as complementary techniques to analyse the symbiosome ultrastructure and organisation in Lotus japonicus wild-type plants. The 3D model of a mature colonised root nodule cell region demonstrates a dense, puzzle-like arrangement of symbiosomes relative to one another and adjacent plant organelles. The symbiosome shape and size depends on the orientation and number of bacteroids within the compartment and features connective tubular structures. Furthermore, vesicular structures, some likely of bacterial origin, were present at the interface. The study presents a multi-angled analysis of symbiosome-related structures, highlighting their volumes, spatial distribution, and pronounced compactness. Interface associated vesicles, protrusions and connective structures hint towards a dynamic and flexible system that contributes to the plant-microbe crosstalk.

Extracellular vesicles (EVs) are cell-secreted biological nanoparticles that play a crucial role in intercellular communication and are gaining increasing attention as diagnostic biomarkers, therapeutic agents, and drug delivery vehicles. Consequently, the development of robust and sensitive methods for their characterization is essential. Herein we present the use of a microscope-mounted nanofluidic device for direct size determination and multi-parametric (3-color) fluorescence-based phenotyping of single biological nanoparticles that are in the size range of 20-200 nm in a method we denote Nano-SMF (SMF; size and multiplexed fluorescence). We demonstrate that it is possible to accurately determine the size of nanoparticles by analyzing their one-dimensional Brownian motion during directional flow through nanochannels, achieving size distributions for monodisperse nanoparticle solutions that are on par with TEM analysis, and size discrimination of nanoparticle mixtures that is significantly improved compared to conventional nanoparticle tracking analysis (NTA). Furter, we demonstrate that the method can be applied to analyze EVs directly in minute volumes of cell supernatant, avoiding pre-isolation or concentration steps. The method was applied to phenotype CD63- and CD81-positive EVs from a human embryonic kidney cell model, demonstrating that vesicle sub-populations defined by these two tetraspanin biomarkers differ significantly in size.

Extracellular vesicles are increasingly recognized as important carriers of disease-associated molecular information, yet robust methods for their isolation and molecular characterization from limited clinical samples remain challenging. Here, we present an integrated approach combining standardized EV isolation, label-free Surface-Enhanced Raman Spectroscopy (SERS), and artificial intelligence (AI) for comprehensive molecular profiling of small extracellular vesicles (sEVs) from human plasma. Here, we show systematically isolated and characterized plasma sEVs using ExoTIC in accordance with MISEV2023 guidelines, with SERS analysis revealing quantifiable spectral differences across samples from patients with glioblastoma (n=20) and meningioma (n=23) compared to healthy controls (n=30). Among the evaluated AI models, the convolutional neural network most effectively captured group-level spectral differences in sEVs, achieving accuracies up to 88% in this pilot cohort. Further, an EGFR-based spectral regression model was explored to examine molecular variability across sEV samples. Parallel proteomic analysis presented statistically significant differences in several proteins elevated in glioblastoma or meningioma. This label-free, rapid approach provides a proof-of-concept framework for sEV molecular profiling establishing the basis for broad validation studies across diverse diseases.

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.

Extracellular vesicles (EVs) hold significant promise as biomarkers, but their clinical translation is constrained by variability in pre-analytical handling and isolation. EV isolation methods directly shape which EV populations are captured and characterized, yet systematic method comparisons across multiple analytical dimensions are limited. We comprehensively evaluated eleven EV isolation methods to define their performance and applications. EVs were quantified by NanoFCM, profiled for tetraspanins (CD9, CD63, CD81) via MSD assays, and further characterized by LC-MS/MS proteomics. We show that different EV isolation methods recover different EV populations. Our data provide guidance on method selection based on downstream application needs and serve as a look-up tool if a protein of interest is detected. EV isolation methods broadened proteome coverage but showed divergent performance and recover different EV populations. While all methods captured EVs in the 50-150nm range, centrifugation and ultracentrifugation identified the broadest proteomes (up to 1093 proteins) driven by higher plasma protein carryover. Conversely, ExoEasy and qEV 70 isolated larger EVs and achieved stronger depletion of abundant plasma proteins but showed lower proteome coverage. A total of 117 proteins were detected across all isolation methods. Pre-clearing samples removed contaminants but at the cost of protein identifications. We demonstrate that method selection must align with the specific analytical goal: centrifugation for comprehensive proteome profiling, affinity/size-exclusion methods for contaminant-sensitive assays, and precipitation for high-throughput applications. This systematic characterization provides an evidence-based framework and look-up resource for matching isolation strategies to downstream applications and research questions. Graphical Abstract for Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/710675v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@12ad967org.highwire.dtl.DTLVardef@270e4eorg.highwire.dtl.DTLVardef@1c41bcorg.highwire.dtl.DTLVardef@11fb236_HPS_FORMAT_FIGEXP M_FIG C_FIG This study evaluated 11 extracellular vesicle (EV) isolation methods which enriched distinct EV subpopulations with varying degrees of contaminants. No single approach optimized purity or proteome coverage; in this paper we present an Evidence-Based Framework to select plasma EV isolation methods based on downstream application needs.

LRRK2, the Parkinsons disease-associated kinase, phosphorylates a subset of Rab GTPases and regulates membrane dynamics. We previously reported that lysosomal stress activates LRRK2 and thereby induces the exocytic secretion of lysosomal contents, but the detailed secretion mechanism remained unclear. Here we found that, under lysosomal stress, endolysosomal luminal and membrane components were secreted with extracellular vesicles (EVs) via LRRK2. Bis(monoacylglycerol)phosphate, an endolysosomal lipid and a urinary marker of LRRK2 activity, was similarly secreted via LRRK2, whereas CD9-positive EVs were not involved. Further dissection of the secreted EVs revealed that Alix-positive EVs were secreted via Rab8a as well as the ESCRT component VPS4, whereas LAMP1/cathepsin B-positive EVs were secreted via Rab10/Rab35, and SNARE proteins syntaxin 2 and VAMP8 regulated the secretion of both EV subtypes. These findings suggest a distinctive stress-induced secretory mechanism whereby LRRK2 facilitates the secretion of multiple EV subtypes by controlling Rab GTPases involved in each pathway.

Enhancing targeted delivery of biomedicines improves efficacy and can reduce off-target effects by lowering the effective dose, but achieving targeting is challenging. Extracellular vesicles (EVs) are promising biological nanovesicles which can be targeted by displaying binding proteins and are being developed as therapeutics. Currently, discovering EV targeting constructs is limited by low throughput and resource-intensive EV production and isolation. To accelerate discovery, we developed a screening pipeline to identify EV targeting constructs without requiring EV production. This approach is premised on the hypothesis that cell-cell interactions may predict some cell-EV interactions. Our cell binding assay (CELLISA) quantifies binding of a cell surface-displayed targeting protein to its cognate receptor on a target cell, employing a microscopy-based analysis pipeline. After validating the premise using existing T cell-targeting reagents, we develop CELLISA for either adherent or suspension EV producer cells. Finally, we use CELLISA to evaluate new binders and validate that hits mediate targeting and/or delivery of genetic cargo to natural killer cells and T cells. CELLISA increased throughput > 6-fold and decreased time by 40% compared to standard EV screens, and it identified a T-cell binder conferring efficient gene delivery. CELLISA is easily adaptable to other laboratories and can accelerate EV research.

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.