Publications

The similarities between extracellular vesicles (EVs) and viruses make them challenging to distinguish and separate into unique populations. A novel lateral flow test has been developed by utilizing these shared properties to improve at-home testing for HIV. By targeting EVs released from HIV infected cells (HIV-EVs) and HIV virus particles for immunocapture, which share common transmembrane proteins like tetraspanins, HIV proteins can be concentrated from blood samples in a lateral flow device. Others have described immunocapture of EVs by lateral flow; here we describe capture and lysis for downstream detection of specific HIV cargo for a sensitive antigen-only HIV test. We found that HIV antigens p24 and Nef are detected early in infection and may significantly improve the sensitivity of an at-home test format by utilizing EVs as a novel reservoir of HIV antigens.

In this study, we investigated the behavior of extracellular vesicles (EVs), during capillary isoelectric focusing (cIEF). For that, we used different approaches, imaging cIEF with a whole-column imaging detection (WCID) and conventional cIEF as well as different detection methods (LIF after EV labelling, native fluorescence and UV). Our study reveals that EVs exhibit significant aggregation during their migration toward, and upon reaching, their isoelectric point (pI). By optimizing key parameters such as voltage and the addition of solubilizers, we successfully reduced this issue, particularly with bovine milk EVs. Our findings also showed distinct pI regions observed for EVs isolated from different sources: bovine milk EVs shows acidic pI characteristics (4.0–4.1), while pig and human plasma EVs exhibit more basic pI zones (4.7–4.9 and 5.8–6.7, respectively). The study was extended to cIEF coupled to laser induced fluorescence detection (LIF) using intra-vesicular CFDA-labeled EVs, to better understand their susceptibilities. Prolonged mobilization time due to long capillary lengths adversely affected EV’s integrity in conventional cIEF. Our study reveals the necessity to specific cIEF optimization for each EV source due to variations in charge distribution and aggregation behavior across different pI regions. The use of a short capillary length (<10 cm), low electric field and solubilizers such as Tween-20 is recommended to preserve EVs integrity during cIEF-EV studies.

Aim: The miRNA cargo of plasma extracellular vesicles (EVs) is commonly studied for its biomarker potential. However, isolation of EVs from human plasma is challenging. Although size-exclusion chromatography (SEC) is commonly used to isolate plasma EVs, SEC does not completely separate EVs from other miRNA carriers such as cells, lipoproteins, and proteins. Recently, new SEC columns were introduced, but hitherto, no systematic study was performed to compare the recovery and purity of plasma EVs using both traditional and new columns. In this study, we investigated the recovery of EVs and separation efficacy from lipoproteins and proteins using different SEC columns, and how recovery and separation affect miRNA cargo analysis. Methods: EVs were isolated from pooled (n = 5) platelet-depleted plasma using 10 different SEC columns. For each column, three EV-enriched fractions were pooled and concentrations of EVs, lipoproteins, proteins, and miRNAs were measured by flow cytometry, enzyme-linked immunosorbent assay (ELISA), Bradford assay, and qRT-PCR, respectively. Results: Our results show that the resin pore size affects all measured parameters: a small pore size increases recovery of EVs and quantity of miRNA, but decreases the separation efficacy compared to a large pore size. Regression analysis showed that the investigated miRNAs are more strongly associated with EVs than with lipoproteins or proteins. Conclusion: The choice of a SEC column markedly affects the recovery, separation efficacy, and miRNA cargo analysis of human plasma-derived EVs. We recommend either using SEC columns with a 70-nm pore size due to their superior EV purity or studying the effect of non-EV particles on the miRNAs of interest.

Small extracellular vesicles (EVs) play a pivotal role in intercellular communication across various physiological and pathological contexts. Despite their growing significance as disease biomarkers and therapeutic targets in biomedical research, the lack of reliable isolation techniques remains challenging. This study characterizes vesicles that were isolated from conditioned culture media (CCM) sourced from three myeloma cell lines (MM.1S, ANBL-6, and ALMC-1), and from the plasma of healthy donors and multiple myeloma patients. We compared the efficacy, reproducibility, and specificity of isolating small EVs using sucrose cushion ultracentrifugation (sUC) vs. ultrafiltration combined with size-exclusion chromatography (UF-SEC). Our results demonstrate that UF-SEC emerges as a more practical, efficient, and consistent method for EV isolation, outperforming sUC in the yield of EV recovery and exhibiting lower variability. Additionally, the comparison of EV characteristics among the three myeloma cell lines revealed distinct biomarker profiles. Finally, our results suggest that HBS associated with Tween 20 improves EV recovery and preservation over PBS. Standardization of small EV isolation methods is imperative, and our comparative evaluation represents a significant step toward achieving this goal.

Previously, we showed that quantification of lymphoma‐associated miRNAs miR‐155‐5p, ‐127‐3p and let‐7a‐5p levels in plasma extracellular vesicles (EVs) report treatment response in patients with classic Hodgkin lymphoma (cHL). Prior to clinical implementation, quality control (QC) steps and validation are required to meet international regulatory standards. Most published EV‐based diagnostic assays have yet to meet these requirements. In order to advance the assay towards regulatory compliance (e.g., IVDR 2017/746), we incorporated three QC steps in our experimental EV‐miRNA quantitative real‐time reverse‐transcription PCR (q‐RT‐PCR) assay in an ISO‐13485 certified quality‐management system (QMS). Liposomes encapsulated with a synthetic (nematode‐derived) miRNA spike‐in controlled for EV isolation by automated size‐exclusion chromatography (SEC). Additional miRNA spike‐ins controlled for RNA isolation and cDNA conversion efficiency. After deciding on quality criteria, in total 107 out of 120 samples from 46 patients passed QC. Generalized linear mixed‐effect modelling with bootstrapping determined the diagnostic performance of the quality‐controlled data at an area under the curve (AUC) of 0.84 (confidence interval [CI]: 0.76–0.92) compared to an AUC of 0.87 (CI: 0.80–0.94) of the experimental assay. After the inclusion of QC steps, the accuracy of the assay was determined to be 78.5% in predicting active disease status in cHL patients during treatment. We demonstrate that a quality‐controlled plasma EV‐miRNA assay is technically robust, taking EV‐miRNA as liquid biopsy assay an important step closer to clinical evaluation.

Spirulina is an edible cyanobacterium that increasingly gaining recognition for it untapped potential in the biomanufacturing of pharmaceuticals. Despite the rapidly accumulating information on extracellular vesicles (EVs) from most other bacteria, nothing is known about Spirulina extracellular vesicles (SPEVs). This study reports the successful isolation, characterization and visualization of SPEVs for the first time and it further investigates the potential therapeutic benefits of SPEVs using a mouse model. SPEVs were isolated using ultracentrifugation and size‐exclusion‐chromatography. Cryo‐Transmission Electron Microscopy revealed pleomorphic outer‐membrane‐vesicles and outer‐inner‐membrane‐vesicles displaying diverse shapes, sizes and corona densities. To assess short‐ and long‐term immune responses, mice were injected intraperitoneally with SPEVs, which demonstrated a significant increase in neutrophils and M1 macrophages at the injection site, indicating a pro‐inflammatory effect induced by SPEVs without clinical signs of toxicity or hypersensitivity. Furthermore, SPEVs demonstrated potent adjuvanticity by enhancing antigen‐specific IgG responses in mice by over 100‐fold compared to an unadjuvanted model vaccine antigen. Mass‐spectrometry identified 54 proteins within SPEVs, including three protein superfamily members linked to the observed pro‐inflammatory effects. Our findings highlight the potential of SPEVs as a new class of vaccine adjuvant and warrant additional studies to further characterize the nature of the immune response.

Extracellular vesicles (EVs) enable communication between cells and tissues and are implicated in modulation of tumor immunosuppression. Here, we present a protocol for isolating tumor-derived EVs and assessing their functional influence in cultures with different subsets of human T cells. We describe steps for differential ultracentrifugation, size exclusion chromatography, EVs quantification, and fluorescence-activated cell sorting of human T cells. We then detail procedures for culturing T cells with EVs and using high-resolution spectral flow cytometry phenotyping for the analysis thereof. For complete details on the use and execution of this protocol, please refer to Swatler et al.1 and Swatler et al.2

The low incidence of ovarian cancer (OC) dictates that any screening strategy needs to be both highly sensitive and highly specific. This study explored the utility of detecting multiple colocalized proteins or glycosylation epitopes on single tumor-associated extracellular vesicles from blood. The novel Mercy Halo Ovarian Cancer Test (OC Test) uses immunoaffinity capture of tumor-associated extracellular vesicles, followed by proximity-ligation real-time quantitative PCR to detect combinations of up to three biomarkers to maximize specificity and measures multiple combinations to maximize sensitivity. A high-grade serous carcinoma (HGSC) case-control training set of EDTA plasma samples from 397 women was used to lock down the test design, the data interpretation algorithm, and the cutoff between cancer and noncancer. Performance was verified and compared with cancer antigen 125 in an independent blinded case-control set of serum samples from 390 women (132 controls, 66 HGSC, 83 non-HGSC OC, and 109 benign). In the verification study, the OC Test showed a specificity of 97.0% (128/132; 95% CI, 92.4%–99.6%), a HGSC sensitivity of 97.0% (64/66; 95% CI, 87.8%–99.2%), and an area under the curve of 0.97 (95% CI, 0.93–0.99) and detected 73.5% (61/83; 95% CI, 62.7%–82.6%) of the non-HGSC OC cases. This test exhibited fewer false positives in subjects with benign ovarian tumors, nonovarian cancers, and inflammatory conditions when compared with cancer antigen 125. The combined sensitivity and specificity of this new test suggests it may have potential in OC screening.

Abstract Glioblastoma (GBM) is characterized by heterogeneous malignant cells that are functionally integrated within the neuroglial microenvironment. In this study, we model this ecosystem by growing GBM into long-term cultured human cortical organoids that contain the major neuroglial cell types found in the cerebral cortex. Single-cell RNA sequencing analysis suggests that, compared with matched gliomasphere models, GBM cortical organoids more faithfully recapitulate the diversity and expression programs of malignant cell states found in patient tumors. Additionally, we observe widespread transfer of GBM transcripts and GFP to nonmalignant cells in the organoids. Mechanistically, this transfer involves extracellular vesicles and is biased toward defined GBM cell states and astroglia cell types. These results extend previous GBM organoid modeling efforts and suggest widespread intercellular transfer in the GBM neuroglial microenvironment. Significance: Models that recapitulate intercellular communications in GBM are limited. In this study, we leverage GBM cortical organoids to characterize widespread mRNA and GFP transfer from malignant to nonmalignant cells in the GBM neuroglial microenvironment. This transfer involves extracellular vesicles, may contribute to reprogramming the microenvironment, and may extend to other cancer types. See related commentary by Shakya et al., p. 261

Enterotoxigenic Escherichia coli (ETEC) cause hundreds of millions of diarrheal illnesses annually ranging from mildly symptomatic cases to severe, life-threatening cholera-like diarrhea. Although ETEC are associated with long-term sequelae including malnutrition, the acute diarrheal illness is largely self-limited. Recent studies indicate that in addition to causing diarrhea, the ETEC heat-labile toxin (LT) modulates the expression of many genes in intestinal epithelia, including carcinoembryonic cell adhesion molecules (CEACAMs) which ETEC exploit as receptors, enabling toxin delivery. Here, however, we demonstrate that LT also enhances the expression of CEACAMs on extracellular vesicles (EV) shed by intestinal epithelia and that CEACAM-laden EV increase in abundance during human infections, mitigate pathogen–host interactions, scavenge free ETEC toxins, and accelerate ETEC clearance from the gastrointestinal tract. Collectively, these findings indicate that CEACAMs play a multifaceted role in ETEC pathogen–host interactions, transiently favoring the pathogen, but ultimately contributing to innate responses that extinguish these common infections.

Extracellular vesicles (EVs) are amongst the most promising candidates for developing blood-based biomarkers. However, patient sample availability is a key barrier to translational research whilst most biobanks store samples of 1.5mL volume or less. To date, there is no consensus on the most suitable method of EV separation and current techniques frequently require large volumes of biofluids, complicated technology, technical expertise, or significant operating costs, which prevents their widespread adoption by less EV-focussed laboratories. Therefore, there is a need for an easy and reproducible method that separates representative EVs from clinically relevant 1mL volumes of plasma prior to subsequent biomarker identification. Methods In this study, EVs were separated from a clinically relevant 1mL volume of human plasma using four different separation techniques: size exclusion chromatography (SEC), differential ultracentrifugation, precipitation, and immunoaffinity magnetic bead capture. The EVs were characterised using several orthogonal techniques (protein quantification, nanoparticle tracking analysis, transmission electron microscopy, Western blot, single particle interferometric reflectance imaging sensing, and mass spectrometry-based proteomics) to comprehensively compare the separated samples. Results We provide examples of anticipated results highlighting that SEC-processed samples have greater protein quantification yield, greater particle yield of the expected size for EVs, and sufficient EV purity, which facilitates effective EV cargo assessment by proteomics. Moreover, we confirm significant overlap with known EV-related proteins within the Vesiclepedia database. Additionally, using single particle interferometric reflectance imaging sensing (Leprechaun®), we identify that SEC has the most representative surface tetraspanin distribution of the separated EV population compared to unprocessed plasma. Discussion Given that SEC requires minimal expertise, no complicated technology and can separate EVs within 90 min, this comparison reinforces SEC as a clinically relevant EV separation method from 1mL of plasma making it suitable for widespread implementation.

Cancer cells secrete extracellular vesicles (EV) encapsulating bioactive cargoes to facilitate inter-organ communication in vivo and are emerging as critical mediators of tumor progression and metastasis, a condition which is often accompanied by a dysregulated cholesterol metabolism. Whether EVs are involved in the control of cholesterol homeostasis during tumor metastasis is still undefined and warrant further investigation. Here, we find that breast cancer-derived exosomal miR-9-5p induces the expression of HMGCR and CH25H, two enzymes involved in cholesterol synthesis and the conversion of 25-hydroxycholesterol from cholesterol by targeting INSIG1, INSIG2 and ATF3 genes in the liver. Notably, in vivo miR-9-5p antagomir treatment and genetic CH25H ablation prevents tumor metastasis in a mouse model of breast cancer. Thus, our findings reveal the regulatory mechanism of tumor-derived miR-9-5p in liver metastasis by linking oxysterol metabolism and Kupffer cell polarization, shedding light on future applications for cancer diagnosis and treatment.

The continuous emergence of multidrug‐resistant bacterial pathogens poses a major global healthcare challenge, with Klebsiella pneumoniae being a prominent threat. We conducted a comprehensive study on K. pneumoniae’s antibiotic resistance mechanisms, focusing on outer membrane vesicles (OMVs) and polymyxin, a last‐resort antibiotic. Our research demonstrates that OMVs protect bacteria from polymyxins. OMVs derived from Polymyxin B (PB)‐stressed K. pneumoniae exhibited heightened protective efficacy due to increased vesiculation, compared to OMVs from unstressed Klebsiella. OMVs also shield bacteria from different bacterial families. This was validated ex vivo and in vivo using precision cut lung slices (PCLS) and Galleria mellonella. In all models, OMVs protected K. pneumoniae from PB and reduced the associated stress response on protein level. We observed significant changes in the lipid composition of OMVs upon PB treatment, affecting their binding capacity to PB. The altered binding capacity of single OMVs from PB stressed K. pneumoniae could be linked to a reduction in the lipid A amount of their released vesicles. Although the amount of lipid A per vesicle is reduced, the overall increase in the number of vesicles results in an increased protection because the sum of lipid A and therefore PB binding sites have increased. This unravels the mechanism of the altered PB protective efficacy of OMVs from PB stressed K. pneumoniae compared to control OMVs. The lipid A‐dependent protective effect against PB was confirmed in vitro using artificial vesicles. Moreover, artificial vesicles successfully protected Klebsiella from PB ex vivo and in vivo. The findings indicate that OMVs act as protective shields for bacteria by binding to polymyxins, effectively serving as decoys and preventing antibiotic interaction with the cell surface. Our findings provide valuable insights into the mechanisms underlying antibiotic cross‐protection and offer potential avenues for the development of novel therapeutic interventions to address the escalating threat of multidrug‐resistant bacterial infections.

The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) causes coronavirus disease 2019 (COVID‐19) and has been related to more than 7 million deaths globally since 2019. The association of high levels of IL‐6 with severe cases led to the early evaluation of the anti‐IL6 inhibitor tocilizumab as a potential treatment, which unfortunately failed to improve survival in many trials. Moreover, little is known about the development of COVID‐19 sequelae, and biomarkers are needed to understand and anticipate these processes. Because extracellular vesicles (EVs) play an important role in viral infection and immune response, they could potentially serve as predictive and prognostic biomarkers. We isolated EVs from 39 patients with severe COVID‐19, from which 29 received tocilizumab and 10 were considered controls. Blood samples, which were collected at hospitalisation before treatment, at Day 7, and Day 15 during follow‐up, were assessed by immunoblot for longitudinal expression of spike (S) and nucleocapsid (N) proteins. Dynamic expression was calculated and compared with clinicopathological and experimental variables. Expression of EV S was validated by immunogold and imaging flow‐cytometry, revealing an enrichment in CD9+ EVs. As a result, decreasing expression of EV viral proteins was observed in patients treated with tocilizumab. Moreover, higher increase in EV S was observed in patients with lower antibody response, hyperfibrinogenemia, lower respiratory function, higher blood pressure and shorter outcomes. These findings lay the foundation for future studies characterizing the role of EVs in multiorgan assessment and identifying biomarkers in patients with severe COVID‐19 and possible long COVID.

Atherosclerotic lesions mainly form in arterial areas exposed to low shear stress (LSS), where endothelial cells express a senescent and inflammatory phenotype. Conversely, areas exposed to high shear stress (HSS) are protected from plaque development. Endothelial extracellular vesicles (EVs) have been shown to regulate inflammation and senescence, and therefore play a crucial role in vascular homeostasis. Whilst previous studies have shown links between hemodynamic forces and EV release, the effects of shear stress on the release and uptake of endothelial EVs remains elusive. We aim to decipher the interplay between these processes in endothelial cells exposed to atheroprone or atheroprotective shear stress. Confluent HUVECs were exposed to LSS or HSS for 24 h. Large and small EVs were isolated from conditioned medium by centrifugation and size exclusion chromatography. They were characterised by TEM, Western blot, tunable resistive pulse sensing, flow cytometry and proteomics. Uptake experiments were performed using fluorescently‐labelled EVs and differences between groups were assessed by flow cytometry and confocal microscopy. We found that levels of large and small EVs in conditioned media were fifty and five times higher in HSS than in LSS conditions, respectively. In vivo and in vitro uptake experiments revealed greater EV incorporation by cells exposed to LSS conditions. Additionally, endothelial LSS‐EVs have a greater affinity for HUVECs than HSS‐EVs or EVs derived from platelets, erythrocytes and leukocytes. Proteomic analysis revealed that LSS‐EVs were enriched in adhesion proteins (PECAM1, MCAM), participating in EV uptake by endothelial cells. LSS‐EVs also carried mitochondrial material, which may be implicated in elevating ROS levels in recipient cells. These findings suggest that shear stress influences EV biogenesis and uptake. Given the major role of EVs and shear stress in vascular health, deciphering the relation between these processes may yield innovative strategies for the early detection and treatment of endothelial dysfunction.

Extracellular vesicles (EVs) have emerged as important mediators of intercellular communication in the heart under homeostatic and pathological conditions, such as myocardial infarction (MI). However, the basic mechanisms driving cardiomyocyte‐derived EV (CM‐EV) production following stress are poorly understood. In this study, we generated human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) that express NanoLuc‐tetraspanin reporters. These modified hiPSC‐CMs allow for quantification of tetraspanin‐positive CM‐EV secretion from small numbers of cells without the need for time‐consuming EV isolation techniques. We subjected these cells to a panel of small molecules to study their effect on CM‐EV biogenesis and secretion under basal and stress‐associated conditions. We observed that EV biogenesis is context‐dependent in hiPSC‐CMs. Nutrient starvation decreases CM‐EV secretion while hypoxia increases the production of CM‐EVs in a nSmase2‐dependent manner. Moreover, the inflammatory cytokine TNF‐α increased CM‐EV secretion through a process involving NLRP3 inflammasome activation and mTOR signalling. Here, we detailed for the first time the regulatory mechanisms of EV biogenesis in hiPSC‐CMs upon MI‐associated stressors.

The current study analyzed the intersecting biophysical, biochemical, and functional properties of extracellular particles (EPs) with the human immunodeficiency virus type-1 (HIV-1) beyond the currently accepted size range for HIV-1. We isolated five fractions (Frac-A through Frac-E) from HIV-infected cells by sequential differential ultracentrifugation (DUC). All fractions showed a heterogeneous size distribution with median particle sizes greater than 100 nm for Frac-A through Frac-D but not for Frac-E, which contained small EPs with an average size well below 50 nm. Synchronized and released cultures contained large infectious EPs in Frac-A, with markers of amphisomes and viral components. Additionally, Frac-E uniquely contained EPs positive for CD63, HSP70, and HIV-1 proteins. Despite its small average size, Frac-E contained membrane-protected viral integrase, detectable only after SDS treatment, indicating that it is enclosed in vesicles. Single particle analysis with dSTORM further supported these findings as CD63, HIV-1 integrase, and the viral surface envelope (Env) glycoprotein (gp) colocalized on the same Frac-E particles. Surprisingly, Frac-E EPs were infectious, and infectivity was significantly reduced by immunodepleting Frac-E with anti-CD63, indicating the presence of this protein on the surface of infectious small EPs in Frac-E. To our knowledge, this is the first time that extracellular vesicle (EV) isolation methods have identified infectious small HIV-1 particles (smHIV-1) that are under 50 nm. Collectively, our data indicate that the crossroads between EPs and HIV-1 potentially extend beyond the currently accepted biophysical properties of HIV-1, which may have further implications for viral pathogenesis.

Small extracellular vesicles (sEVs) released by acute myeloid leukaemia (AML) cells have been reported to influence the trilineage differentiation of bone marrow-derived mesenchymal stem cells (BM-MSCs). However, it remains elusive which biological cargo from AML-sEVs is responsible for this effect. In this study, sEVs were isolated from cell-conditioned media and blood plasma using size-exclusion chromatography and ultrafiltration and characterized according to MISEV2018 guidelines. Our results demonstrated that AML-sEVs increased the proliferation of BM-MSCs. Conversely, key proteins that are important for normal haematopoiesis were downregulated in BM-MSCs. Additionally, we revealed that AML-sEVs significantly reduced the differentiation of BM-MSCs to osteoblasts without affecting adipogenic or chondrogenic differentiation. Next, LC-MS/MS proteomics elucidated that various proteins, including Y-box-binding protein 1 (YBX1), were upregulated in both AML-sEVs and BM-MSCs treated with AML-sEVs. Clinically relevant, we found that YBX1 is considerably upregulated in most paediatric AML patient-derived sEVs compared to healthy controls. Interestingly, sEVs isolated after the downregulation of YBX1 in AML cells remarkably rescued the osteoblastic differentiation of BM-MSCs. Altogether, our data demonstrate for the first time that YBX1 containing AML-sEVs is one of the key players that disrupt the normal function of bone marrow microenvironment by reducing the osteogenic differentiation of BM-MSCs.

Cardiac fibrosis is a common pathological feature of cardiovascular diseases that arises from the hyperactivation of fibroblasts and excessive extracellular matrix (ECM) deposition, leading to impaired cardiac function and potentially heart failure or arrhythmia. Extracellular vesicles (EVs) released by cardiomyocytes (CMs) regulate various physiological functions essential for myocardial homeostasis, which are disrupted in cardiac disease. Therefore, healthy CM‐derived EVs represent a promising cell‐free therapy for the treatment of cardiac fibrosis.To this end, we optimized the culture conditions of human adult CMs to obtain a large yield of EVs without compromising cellular integrity by using a defined combination of small molecules. EVs were isolated by ultracentrifugation, and their characteristics were analysed. Finally, their effect on fibrosis was tested.Treatment of TGFβ‐activated human cardiac fibroblasts with EVs derived from CMs using our culture system resulted in a decrease in fibroblast activation markers and ECM accumulation. The rescued phenotype was associated with specific EV cargo, including multiple myocyte‐specific and antifibrotic microRNAs, although their effect individually was not as effective as the EV treatment. Notably, pathway analysis showed that EV treatment reverted the transcription of activated fibroblasts and decreased several signalling pathways, including MAPK, mTOR, JAK/STAT, TGFβ, and PI3K/Akt, all of which are involved in fibrosis development. Intracardiac injection of CM‐derived EVs in an animal model of cardiac fibrosis reduced fibrotic area and increased angiogenesis, which correlated with improved cardiac function.These findings suggest that EVs derived from human adult CMs may offer a targeted and effective treatment for cardiac fibrosis, owing to their antifibrotic properties and the specificity of cargo.

Extracellular vesicles (EVs) are emerging as novel therapeutics, particularly in cancer and degenerative diseases. Nevertheless, from both market and clinical viewpoints, high‐yield production methods using minimal cell materials are still needed. Herein, a millifluidic cross‐slot chip is proposed to induce high‐yield release of biologically active EVs from less than three million cells. Depending on the flow rate, a single vortex forms in the outlet channels, exposing transported cellular material to high viscous stresses. Importantly, the chip accommodates producer cells within their physiological environment, such as human mesenchymal stem cells (hMSCs) spheroids, while facilitating their visualization and individual tracking within the vortex. This precise control of viscous stresses at the spheroid level allows for the release of up to 30000 EVs per cell at a Reynolds number of ≈400, without compromising cellular integrity. Additionally, it reveals a threshold initiating EV production, providing evidence for a stress‐dependent mechanism governing vesicle secretion. EVs mass‐produced at high Reynolds displayed pro‐angiogenic and wound healing capabilities, as confirmed by proteomic and cytometric analysis of their cargo. These distinct molecular signatures of these EVs, compared to those derived from monolayers, underscore the critical roles of the production method and the 3D cellular environment in EV generation.

Minimally invasive biomarkers are urgently needed to detect molecular pathology in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Here, we show that plasma extracellular vesicles (EVs) contain quantifiable amounts of TDP-43 and full-length tau, which allow the quantification of 3-repeat (3R) and 4-repeat (4R) tau isoforms. Plasma EV TDP-43 levels and EV 3R/4R tau ratios were determined in a cohort of 704 patients, including 37 genetically and 31 neuropathologically proven cases. Diagnostic groups comprised patients with TDP-43 proteinopathy ALS, 4R tauopathy progressive supranuclear palsy, behavior variant FTD (bvFTD) as a group with either tau or TDP-43 pathology, and healthy controls. EV tau ratios were low in progressive supranuclear palsy and high in bvFTD with tau pathology. EV TDP-43 levels were high in ALS and in bvFTD with TDP-43 pathology. Both markers discriminated between the diagnostic groups with area under the curve values &gt;0.9, and between TDP-43 and tau pathology in bvFTD. Both markers strongly correlated with neurodegeneration, and clinical and neuropsychological markers of disease severity. Findings were replicated in an independent validation cohort of 292 patients including 34 genetically confirmed cases. Taken together, the combination of EV TDP-43 levels and EV 3R/4R tau ratios may aid the molecular diagnosis of FTD, FTD spectrum disorders and ALS, providing a potential biomarker to monitor disease progression and target engagement in clinical trials.

Pancreatic ductal adenocarcinoma (PDAC), a lethal disease, requires a grasp of its biology for effective therapies. Exosomes, implicated in cancer, are poorly understood in living systems. Here we use the genetically engineered mouse model (ExoBow) to map the spatiotemporal distribution of exosomes from healthy and PDAC pancreas in vivo to determine their biological significance. We show that, within the PDAC microenvironment, cancer cells establish preferential communication routes through exosomes with cancer associated fibroblasts and endothelial cells. The latter being a conserved event in the healthy pancreas. Inhibiting exosomes secretion in both scenarios enhances angiogenesis, underscoring their contribution to vascularization and to cancer. Inter-organ communication is significantly increased in PDAC with specific organs as most frequent targets of exosomes communication occurring in health with the thymus, bone-marrow, brain, and intestines, and in PDAC with the kidneys, lungs and thymus. In sum, we find that exosomes mediate an organized intra- and inter- pancreas communication network with modulatory effects in vivo.

Extracellular vesicles (EVs) derived from T cells have been proposed to mediate intercellular communication and orchestrate immune responses. The immunosuppressive drug, tacrolimus (TAC), suppresses T cell activity; however, the impact of TAC on T cell-derived EVs remains primarily unexplored. In this study, human primary T cells purified from healthy donors were used to investigate TAC-mediated regulation of EV secretion by T cells. Using size exclusion chromatography (SEC) to isolate EVs released by T cells, we found that the number of released EVs was increased upon anti-CD3/CD28 bead-mediated activation. Furthermore, pre-treatment with TAC before activation had a potentiating effect on EV release, as evidenced by western blot analysis of EV markers and small particle flow cytometry. In addition, we showed that EVs isolated from the plasma of TAC-treated kidney transplant patients were increased compared to those observed with pre-transplant plasma. Upon examining the mechanism underlying the action of TAC, we found that TAC impaired autophagy-lysosome-mediated degradation by inhibiting the nuclear translocation of transcription factor EB, a master regulator of lysosomal biogenesis. Notably, the addition of trehalose, an autophagy inducer, abrogated the TAC-induced EV release, indicating that TAC regulated EV secretion via the autophagy-lysosomal pathway. At the functional level, we demonstrated that EVs from TAC-treated T cells carried a decreased amount of CD40L, a protein critical for the activation of the adaptive immune response. Collectively, these findings demonstrate that an overall increase in EV production and decreased CD40L levels in EVs are characteristic responses of T cells to TAC.

Extracellular vesicles (EV) are membranous vesicles considered as significant players in cell-to-cell communication. Milk provides adequate nutrition, transfers immunity, and promotes neonatal development, and milk EV are suggested to play a crucial role in these processes. Milk samples were obtained on days 0, 7, and 14 after parturition from sows receiving either a standard diet (ω-6:ω-3 = 13:1) or a test diet enriched in ω-3 (ω-6:ω-3 = 4:1). EV were isolated using ultracentrifugation coupled with size exclusion chromatography, and characterized by nanoparticle tracking analysis, transmission electron microscopy, and assessment of EV markers via Western blotting. The lipidome was determined following a liquid chromatography–quadrupole time-of-flight mass spectrometry approach. Here, we show that different stages of lactation (colostrum vs mature milk) have a distinct extracellular vesicle lipidomic profile. The distinct lipid content can be further explored to understand and regulate milk EV functionalities and primordial for enabling their diagnostic and therapeutic potential.