Publications

A phase-change contrast agent (PCCA) is a liquid nanodroplet (ND) that can phase transition into a gaseous microbubble (MB) when exposed to pulsed ultrasound (US) of sufficient peak negative pressure. Previously, our group has shown that PCCA activation is linearly related to the hydrostatic pressure of the surrounding media. As increases in the hydrostatic pressure requires higher US energies to phase transition, we hypothesize this relationship can be used to allow noninvasive pressure estimation in remote vascular structures. After applying various levels of hydrostatic pressures, the goal of this research project was to establish a more robust quantitative US method to determine the activation threshold of PCCAs in a flow phantom model. Decafluorobutane (DFB) NDs were formulated in house using the popular condensation method. Activation and detection of the resultant MBs was captured using a programmable US system (Vantage 256, Verasonics Inc). A custom scan sequence was applied with interleaved pulsed US transmissions for both PCCA activation and MB detection. Using a progressive increase in peak negative pressures from 3 to 6 MPa, a series of US images were collected at each discrete interval (N = 200). Preliminary results at room temperature revealed that as hydrostatic pressure increased there was a corresponding increase in PCCA activation threshold (R 2 > 0.88, p < 0.001). Overall, this study introduces a more consistent method to determine PCCA activation thresholds and use of sample averaging to help minimize measurement variance during threshold quantification.

Colorectal cancer (CRC) is a leading cause of cancer-related death. There is an urgent need for new methods of early CRC detection and monitoring to improve patient outcomes. Extracellular vesicles (EVs) are secreted, lipid-bilayer bound, nanoparticles that carry biological cargo throughout the body and in turn exhibit cancer-related biomarker potential. RNA binding proteins (RBPs) are post-transcriptional regulators of gene expression that may provide a link between host cell gene expression and EV phenotypes. Insulin-like growth factor 2 RNA binding protein 1 (IGF2BP1/IMP1) is an RBP that is highly expressed in CRC with higher levels of expression correlating with poor prognosis. IMP1 binds and potently regulates tumor-associated transcripts that may impact CRC EV phenotypes. Our objective was to test whether IMP1 expression levels impact EV secretion and/or cargo. We used RNA sequencing, in vitro CRC cell lines, ex vivo colonoid models, and xenograft mice to test the hypothesis that IMP1 influences EV secretion and/or cargo in human CRC. Our data demonstrate that IMP1 modulates the RNA expression of transcripts associated with extracellular vesicle pathway regulation, but it has no effect on EV secretion levels in vitro or in vivo. Rather, IMP1 appears to affect EV regulation by directly entering EVs in a transformation-dependent manner. These findings suggest that IMP1 has the ability to shape EV cargo in human CRC, which could serve as a diagnostic/prognostic circulating tumor biomarker.

Background The protracted preclinical stage of Alzheimer’s disease (AD) provides the opportunity for early intervention to prevent the disease; however, the lack of minimally invasive and easily detectable biomarkers and their measurement technologies remain unresolved. Extracellular vesicles (EVs) are nanosized membrane vesicles released from a variety of cells and play important roles in cell–cell communication. Neuron-derived and ganglioside-enriched EVs capture amyloid-ß protein, a major AD agent, and transport it into glial cells for degradation; this suggests that EVs influence Aß accumulation in the brain. EV heterogeneity, however, requires the use of a highly sensitive technique for measuring specific EVs in biofluid. In this study, immuno-digital invasive cleavage assay (idICA) was developed for quantitating target-intact EVs. Methods EVs were captured onto ganglioside GM1-specific cholera toxin B subunit (CTB)-conjugated magnetic beads and detected with a DNA oligonucleotide-labeled Aß antibody. Fluorescence signals for individual EVs were then counted using an invasive cleavage assay (ICA). This idICA examines the Aß-bound and GM1-containing EVs isolated from the culture supernatant of human APP-overexpressing N2a (APP-N2a) cells and APP transgenic mice sera. Results The idICA quantitatively detected Aß-bound and GM1-containing EVs isolated from culture supernatants of APP-N2a cells and sera of AD model mice. The idICA levels of Aß-associated EVs in blood gradually increased from 3- to 12-month-old mice, corresponding to the progression of Aß accumulations in the brain of AD model mice. Conclusions The present findings suggest that peripheral EVs harboring Aß and GM1 reflect Aß burden in mice. The idICA is a valuable tool for easy quantitative detection of EVs as an accessible biomarker for preclinical AD diagnosis.

Introduction. Cholera is an acute enteric infection caused by Vibrio cholerae , particularly in areas lacking access to clean water. Despite the global effort to improve water quality in these regions, the burden of cholera in recent years has not yet declined. Interest has therefore extended in the use of bicistronic DNA vaccine encoding ctxB and tcpA genes of V. cholerae as a potential vaccine. Hypothesis/Gap Statement. The potential of a bicistronic DNA vaccine, pVAX-ctxB-tcpA has not been determined in vitro and in vivo. Aim. The goal of present study was to evaluate in vitro expression and in vivo potential of pVAX-ctxB-tcpA vaccine against V. cholerae . Methodology. The pVAX-ctxB-tcpA was transiently transfected into mammalian COS-7 cells, and the in vitro expression was assessed using fluorescence and Western blot analyses. Next, the vaccine was encapsulated into sodium alginate using water-in-oil emulsification and evaluated for its efficiency in different pH conditions. Subsequently, oral vaccination using en(pVAX-ctxB-tcpA) was performed in vivo. The animals were challenged with V. cholerae O1 El Tor after 2 weeks of vaccination using the Removable Intestinal Tie-Adult Rabbit Diarrhoea (RITARD) model. Following the infection challenge, the rabbits were monitored for evidence of symptoms, and analysed for systemic cytokine expression level (TNF-α, IFN-γ, IL-6 and IL-10) using quantitative real-time polymerase chain reaction. Results. The in vitro expression of pVAX-ctxB-tcpA was successfully verified via fluorescence and Western blot analyses. Meanwhile, in vivo analysis demonstrated that the en(pVAX-ctxB-tcpA) was able to protect the RITARD model against V. cholerae infection due to a lack of evidence on the clinical manifestations of cholera following bacterial challenge. Furthermore, the bicistronic group showed an upregulation of systemic IFN-γ and IL-10 following 12 days of vaccination, though not significant, suggesting the possible activation of both T-helper 1 and 2 types of response. However, upon bacterial challenge, the gene expression of all cytokines did not change. Conclusion. Our findings suggest that the bicistronic plasmid DNA vaccine, pVAX-ctxB-tcpA, showed a potential role in inducing immune response against cholera through upregulation of in vitro gene and protein expression as well as in vivo cytokine gene expression, particularly IFN-γ and IL-10.

Extracellular vesicles (EVs) obtain properties of immunomodulation and tissue repair from their parental mesenchymal stem cells (MSCs), and upon delivery may be associated with fewer adverse events. EVs derived from adipose-tissue MSCs restored kidney function by attenuating kidney inflammation in a swine model of metabolic syndrome (MetS) and renal artery stenosis via anti-inflammatory pathways. EVs also ameliorated myocardial injury in renovascular hypertension (RVH) secondary to inflammation in cardiorenal disease, but the mechanisms regulating this effect are unknown. We hypothesize that the anti-inflammatory cytokine interleukin (IL)-10 mediates the reparative effects of EVs on cardiovascular complications in a preclinical swine model with coexisting MetS and RVH. Twenty-three pigs established as Lean controls or RVH models were observed for 16 weeks. At 12 weeks RVH subgroups received an intrarenal delivery of 1011 either wildtype (WT) EVs or EVs after IL-10 knockdown (KD) (RVH+WT-EVs or RVH+IL-10-KD-EVs, respectively). Cardiac and renal function were studied in-vivo and myocardial tissue injury in-vitro 4 weeks later. RVH pigs showed myocardial inflammation, fibrosis, and left ventricular diastolic dysfunction. WT-EVs attenuated these impairments, increased capillary density, and decreased myocardial inflammation in-vivo. In-vitro, co-incubation with IL-10-containing WT-EVs decreased activated T-cells proliferation and endothelial cells inflammation and promoted their migration. Contrarily, these cardioprotective effects were largely blunted using IL-10-KD-EVs. Thus, the anti-inflammatory and pro-angiogenic effects of EVs in RVH may be partly attributed to their cargo of anti-inflammatory IL-10. Early intervention of IL-10-containing EVs may be helpful to prevent cardiovascular complications of MetS concurrent with RVH.

(1) Background: Pancreatic ductal adenocarcinoma (PDAC) is expected to be the second-leading cause of cancer deaths by 2030. Imaging techniques are the standard for monitoring the therapy response in PDAC, but these techniques have considerable limits, including delayed disease progression detection and difficulty in distinguishing benign from malignant lesions. Extracellular vesicle (EV) liquid biopsy is an emerging diagnosis modality. Nonetheless, the majority of research for EV-based diagnosis relies on point analyses of EVs at specified times, while longitudinal EV population studies before and during therapeutic interventions remain largely unexplored. (2) Methods: We analyzed plasma EV protein composition at diagnosis and throughout PDAC therapy. (3) Results: We found that IgG is linked with the diagnosis of PDAC and the patient's response to therapy, and that the IgG+ EV population increases with disease progression and reduces with treatment response. Importantly, this covers PDAC patients devoid of the standard PDAC seric marker CA19.9 expression. We also observed that IgG is bound to EVs via the tumor antigen MAGE B1, and that this is independent of the patient's inflammatory condition and IgG seric levels. (4) Conclusions: We here propose that a population analysis of IgG+ EVs in PDAC plasma represents a novel method to supplement the monitoring of the PDAC treatment response.

Extracellular vesicles (EVs) play a key role in many physiological and pathophysiological processes and hold great potential for therapeutic and diagnostic use. Despite significant advances within the last decade, the key issue of EV storage stability remains unresolved and under investigated. Here, we aimed to identify storage conditions stabilizing EVs and comprehensively compared the impact of various storage buffer formulations at different temperatures on EVs derived from different cellular sources for up to 2 years. EV features including concentration, diameter, surface protein profile and nucleic acid contents were assessed by complementary methods, and engineered EVs containing fluorophores or functionalized surface proteins were utilized to compare cellular uptake and ligand binding. We show that storing EVs in PBS over time leads to drastically reduced recovery particularly for pure EV samples at all temperatures tested, starting already within days. We further report that using PBS as diluent was found to result in severely reduced EV recovery rates already within minutes. Several of the tested new buffer conditions largely prevented the observed effects, the lead candidate being PBS supplemented with human albumin and trehalose (PBSHAT). We report that PBS-HAT buffer facilitates clearly improved short-term and long-term EV preservation for samples stored at -80◦C, stability throughout several freeze-thaw cycles, and drastically improved EV recovery when using a diluent for EV samples for downstream applications.

Background: Colorectal cancer (CRC) is currently the fourth most common cancer worldwide. The roles of exosomal competing endogenous RNAs (ceRNAs) in CRC remain unclear. In this study, we constructed an exosomal ceRNA network to identify the core ceRNAs and investigate the diagnostic biomarkers in CRC. Methods and Patients: Serum exosomes were isolated from four CRC patients and two healthy donors by ultracentrifugation, and then subjected to RNA isolation, sequencing and microarray. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) analyses were performed to identify functional enrichment implications of differentially expressed exosomal mRNAs. TargetScan and miRanda were used for identifying the miRNA-mRNA and miRNA-LncRNA interactions. The predicted lncRNAs and mRNAs were intersected with the differentially expressed genes, for which the screening criterion was fold change >1.5 in the microarray. Differentially expressed exosomal miRNAs were identified in the GSE71008 dataset, and differentially expressed mRNAs (DEmRNAs) were further summarized from The Cancer Genome Atlas (TCGA) database. Results: A total of 1186 exosomal DEmRNAs, 2088 exosomal DElncRNAs and 29 exosomal miRNAs were detected in CRC patients compared to the healthy donors. Functional enrichment analysis suggested that exosomal DEmRNAs might participate in pathways related to carcinogenesis and development of cancer. An exosomal ceRNA regulatory network of CRC was constructed based on 40 lncRNAs, two miRNAs, and five mRNAs. Exosomal miR-150-5p and miR-10b-5p expression levels were increased in healthy donors compared with CRC patients in the GSE71008 dataset, and five DEmRNAs (TOMM70A, RBM48, BEND3, RHOBTB1, and ADAMTS2) were significantly upregulated in TCGA database. Two potential exosomal regulatory axes of lncRNA G016261-miR-150-5p-RBM48 and lncRNA XLOC_011677-miR-10b-5p-BEND3 were identified from the network. Conclusion: The current study revealed potential molecular biological regulation pathways and diagnostic biomarkers through the exosomal ceRNA regulatory network.

Stem cells secrete paracrine factors including extracellular vesicles (EVs) which can mediate cellular communication and support the regeneration of injured tissues. Reduced oxygen (hypoxia) as a key regulator in development and regeneration may influence cellular communication via EVs. We asked whether hypoxic conditioning during human induced pluripotent stem cell (iPSC) culture effects their EV quantity, quality or EV-based angiogenic potential. We produced iPSC-EVs from large-scale culture-conditioned media at 1%, 5% and 18% air oxygen using tangential flow filtration (TFF), with or without subsequent concentration by ultracentrifugation (TUCF). EVs were quantified by tunable resistive pulse sensing (TRPS), characterized according to MISEV2018 guidelines, and analyzed for angiogenic potential. We observed superior EV recovery by TFF compared to TUCF. We confirmed hypoxia efficacy by HIF-1α stabilization and pimonidazole hypoxyprobe. EV quantity did not differ significantly at different oxygen conditions. Significantly elevated angiogenic potential was observed for iPSC-EVs derived from 1% oxygen culture by TFF or TUCF as compared to EVs obtained at higher oxygen or the corresponding EV-depleted soluble factor fractions. Data thus demonstrate that cell-culture oxygen conditions and mode of EV preparation affect iPSC-EV function. We conclude that selecting appropriate protocols will further improve production of particularly potent iPSC-EV-based therapeutics.

RESEARCH QUESTION: Does pre-implantation uterine fluid lavage (UFL) of patients undergoing IVF and frozen embryo transfer (FET) affect implantation and clinical pregnancy rates? Which methods among ultracentrifugation, sucrose cushion and qEV column are suitable for isolating UFL extracellular vesicles? DESIGN: First, UFL was collected from 20 patients undergoing IVF and FET 2 days before embryo transfer as the case group. The control group consisted of 20 patients undergoing IVF and FET patients without lavage. All patients were monitored for 6 weeks. In the next step, the UFLs (n = 30) were collected and pooled. The UFL-derived extracellular vesicles were extracted by ultracentrifugation, sucrose cushion and qEV column methods and characterized. RESULTS: Preimplantation uterine lavage sampling did not affect implantation and clinical pregnancy rates. Extracellular vesicles were successfully isolated from UFL by all three methods. Scanning electron microscopy and dynamic light scattering analysis showed that the isolated vesicles were morphologically spherical. The qEV technique showed that they were smaller and homogenized in size. SDS-PAGE of extracellular vesicles showed a weaker albumin band in the qEV column. Western blot analysis indicated that the isolated extracellular vesicles by the qEV column were more immunoreactive for all the common extracellular vesicle markers (CD81, CD9, CD63, and TSG101). Six reference genes were compared by real-time polymerase chain reaction in the isolated extracellular vesicle subpopulations, and lowest cycle threshold value was observed for the 18SrRNA gene. CONCLUSIONS: The isolation of endometrial secretome extracellular vesicles is a minimally invasive procedure for individual assessment of endometrial receptivity and can be carried out during conception cycles along with transvaginal ultrasonography. Molecular analysis of UFL-derived extracellular vesicle components could suggest biomarkers to determine precise extracellular vesicle timing.

Rett syndrome (RTT) is a rare neurodevelopmental disorder that results in multiple disabilities. Exosomal microRNA (miRs) from urine-derived stem cells (USCs) have been shown to induce neurogenesis and aid in functional recovery from brain ischemia. In the present study, we sought to determine whether that exosomal miR-21-5p from USCs could promote early neural formation in a model of RTT. USCs were isolated and evaluated by flow cytometry. Exosomes were analyzed by transmission electron microscopy, tunable resistive pulse sensing (TRPS), and western blotting. PKH26 fluorescent dyes were used to observe intake of exosomes in vivo and in vitro. An RTT mouse model was treated with exosomes for behavioral studies. Dual‐luciferase report gene assays were conducted to evaluate the relationship between miR-21-5p and Eph receptor A4 (EphA4). In vitro, treatment with exosomes from human urine‐derived stem cells (USC-Exos) increased the percentage of neuron-specific class III beta-tubulin (Tuj1)+ nerve cells as well as the transcription levels of β-III tubulin and doublecortin (DCX). A higher level of miR-21-5p was observed in USC-Exos, which promoted differentiation in NSCs by targeting the EPha4/TEK axis. In vivo, exosomal miR-21-5p improved the behavior, motor coordination, and cognitive ability of mice, facilitated the differentiation of NSCs in the subventricular zone of the lateral ventricle and promoted a marked rise in the number of DCX+ cells. Our data provide evidence that exosomal miR-21-5p from human USCs facilitate early nerve formation by regulating the EPha4/TEK axis.

In neurodegenerative diseases, extracellular vesicles (EVs) transfer pathogenic molecules and are consequently involved in disease progression. We have investigated the proteomic profiles of EVs that were isolated from four different humaninduced pluripotent stem cell-derived neural cell types (excitatory neurons, astrocytes, microglia-like cells, and oligodendrocyte-like cells). Novel cell type-specific EV protein markers were then identified for the excitatory neurons (ATP1A3, NCAM1), astrocytes (LRP1, ITGA6), microglia-like cells (ITGAM, LCP1), and oligodendrocytelike cells (LAMP2, FTH1), as well as 16 pan-EV marker candidates, including integrins and annexins. To further demonstrate how cell-type-specific EVs may be involved in Alzheimer’s disease (AD), we performed protein co-expression network analysis and conducted cell type assessments for the proteomes of brain-derived EVs from the control, mild cognitive impairment, and AD cases. A protein module enriched in astrocyte-specific EV markers was most significantly associated with the AD pathology and cognitive impairment, suggesting an important role in AD progression. The hub protein from this module, integrin-β1 (ITGB1), was found to be significantly elevated in astrocyte-specific EVs enriched from the total brain-derived AD EVs and associated with the brain β-amyloid and tau load in independent cohorts. Thus, our study provides a featured framework and rich resource for the future analyses of EV functions in neurodegenerative diseases in a cell type-specific manner.

BACKGROUND AIMS: Acute lung injury (ALI) secondary to sepsis is a complex disease associated with high morbidity and mortality. Mesenchymal stem cells (MSCs) and their conditioned medium have been demonstrated to reduce alveolar inflammation, improve lung endothelial barrier permeability and modulate oxidative stress in vivo and in vitro. Recently, MSCs have been found to release small extracellular vesicles (sEVs) that can deliver functionally active biomolecules into recipient cells. The authors' study was designed to determine whether sEVs released by MSCs would be effective in sepsis-induced ALI mice and to identify the potential mechanisms. METHODS: A total of 6 h after cercal ligation and puncture, the mice received saline, sEV-depleted conditioned medium (sEVD-CM) or MSC sEVs via the tail vein. RESULTS: The administration of MSC sEVs improved pulmonary microvascular permeability and inhibited both histopathological changes and the infiltration of polymorphonuclear neutrophils into lung tissues. In addition, the activities of antioxidant enzymes were significantly increased in the group treated with sEVs compared with the saline and sEVD-CM groups, whereas lipid peroxidation was significantly decreased. Furthermore, sEVs were found to possibly inhibit phosphorylation of the mitogen-activated protein kinase/nuclear factor kappa B (MAPK/NF-κB) pathway and degradation of IκB but increase the activities of nuclear factor erythroid 2-related factor 2 and heme oxygenase 1. CONCLUSIONS: These findings suggest that one of the effective therapeutic mechanisms of sEVs against sepsis-induced ALI may be associated with upregulation of anti-oxidative enzymes and inhibition of MAPK/NF-κB activation.

Acute liver injury (ALI) induced by chemicals or viruses can progress rapidly to acute liver failure (ALF), often resulting in death of patients without liver transplantation. Since liver transplantation is limited due to a paucity of donors, expensive surgical costs, and severe immune rejection, novel therapies are required to treat liver injury. Extracellular vesicles (EVs) are used for cellular communication, carrying RNAs, proteins, and lipids and delivering them intercellularly after being endocytosed by target cells. Recently, it was reported that EVs secreted from human hepatocytes have an ability to modulate the immune responses; however, these roles of EVs secreted from human hepatocytes were studied only with in vitro experiments. In the present study, we evidenced that EVs secreted from human hepatocytes attenuated the CCL4-induced ALI by inhibiting the recruitment of monocytes through downregulation of chemokine receptor in the bone marrow and recruitment of neutrophils through the reduction of C-X-C motif chemokine ligand 1 (CXCL1) and CXCL2 expression levels in the liver.

Introduction Corneal blindness due to scarring is treated with corneal transplantation. However, a global problem is the donor material shortage. Preclinical and clinical studies have shown that cell-based therapy using corneal stromal stem cells (CSSCs) suppresses corneal scarring, potentially mediated by specific microRNAs transported in extracellular vesicles (EVs). However, not every CSSC batch from donors achieves similar anti-scarring effects. Purpose To examine miRNA profiles in EVs from human CSSCs showing “healing” versus “non-healing” effects on corneal scarring and to design a tool to select CSSCs with strong healing potency for clinical applications. Methods Small RNAs from CSSC-EVs were extracted for Nanostring nCounter Human miRNA v3 assay. MicroRNAs expressed > 20 folds in “healing” EVs (P < 0.05) were subject to enriched gene ontology (GO) term analysis. MiRNA groups with predictive regulation on inflammatory and fibrotic signalling were studied by mimic transfection to (1) mouse macrophages (RAW264.7) for M1 phenotype assay; (2) human corneal keratocytes for cytokine-induced fibrosis, and (3) human CSSCs for corneal scar prevention in vivo. The expression of miR-29a was screened in additional CSSC batches and the anti-scarring effect of cells was validated in mouse corneal wounds. Results Twenty-one miRNAs were significantly expressed in “healing” CSSC-EVs and 9 miRNA groups were predicted to associate with inflammatory and fibrotic responses, and tissue regeneration (P <10−6). Overexpression of miR-29a and 381-5p significantly prevented M1 phenotype transition in RAW264.7 cells after lipopolysaccharide treatment, suppressed transforming growth factor β1-induced fibrosis marker expression in keratocytes, and reduced scarring after corneal injury. High miR-29a expression in EV fractions distinguished human CSSCs with strong healing potency, which inhibited corneal scarring in vivo. Conclusion We characterized the anti-inflammatory and fibrotic roles of miR-29a and 381-5p in CSSCs, contributing to scar prevention. MiR-29a expression in EVs distinguished CSSCs with anti-scarring quality, identifying good quality cells for a scarless corneal healing.

After spinal cord injury (SCI), destruction of the blood–spinal cord barrier (BSCB) results in infiltration of blood cells, such as neutrophils and macrophages, leading to permanent neurological dysfunction. Previous studies have shown that human bone marrow mesenchymal stem cell (BMSC)–derived exosomes have a beneficial neuroprotective effect in SCI models. However, whether BMSC-Exos contribute to the integrity of the BSCB has not been clarified. The purpose of this study was to investigate the mechanism of BMSC-Exo-induced changes in the permeability of the BSCB after SCI. Here, we first used BMSC-Exos to treat an SCI rat model, showing that BMSC-Exos can inhibit BSCB permeability damage and improve spontaneous repair. Next, we found that tissue inhibitors of matrix metalloproteinase 2 (TIMP2) have been shown to play an important role in the function of BMSC-Exos by inhibiting the matrix metalloproteinase (MMP) pathway, thereby reducing the reduction of cell junction proteins. Therefore, we constructed siTIMP2 to knock out TIMP2 in BMSC-Exos, which caused the activity of BMSC-Exos to be significantly weakened. Finally, we constructed an in vitro model of BSCB with HBMECs and verified that TIMP2 in BMSC-Exos in vitro can also alleviate BSCB damage. This proof-of-principle study demonstrates that BMSC-Exos can preserve the integrity of the BSCB and improve functional recovery after SCI through the TIMP2/MMP signaling pathway.

Eukaryotic cells, including cancer cells, secrete highly heterogeneous populations of extracellular vesicles (EVs). EVs could have different subcellular origin, composition and functional properties, but tools to distinguish between EV subtypes are scarce. Here, we tagged CD63- or CD9-positive EVs secreted by triple negative breast cancer cells with Nanoluciferase enzyme, to set-up a miniaturized method to quantify secretion of these two EV subtypes directly in the supernatant of cells. We performed a cell-based high-content screening to identify clinically-approved drugs able to affect EV secretion. One of the identified hits is Homosalate, an anti-inflammatory drug found in sunscreens which robustly increased EVs' release. Comparing EVs induced by Homosalate with those induced by Bafilomycin A1, we demonstrate that: (1) the two drugs act on EVs generated in distinct subcellular compartments, and (2) EVs released by Homosalate-, but not by Bafilomycin A1-treated cells enhance resistance to anchorage loss in another recipient epithelial tumour cell line. In conclusion, we identified a new drug modifying EV release and demonstrated that under influence of different drugs, triple negative breast cancer cells release EV subpopulations from different subcellular origins harbouring distinct functional properties.

Abstract Background Crohn’s disease (CD) is a chronic inflammatory gastro-intestinal condition with variable disease course. Impaired barrier function and microbial dysbiosis are associated with disease onset and exacerbations. We hypothesized that perturbed microbial activity may contribute to the impaired barrier function in CD. Therefore, this study aimed to examine the impact of faecal bacterial products of active and remissive CD patients, and healthy controls (HC) on mucin degradation and epithelial barrier function in vitro . Methods Six HC and twelve CD patients were included. Disease activity was determined by endoscopy. Fecal water (FW) and bacterial membrane vesicles (MVs) from fresh fecal samples were applied on mucin agar to determine mucin degradation and on differentiated Caco-2 cell monolayers to assess transepithelial electrical resistance (TEER) and paracellular junction stability. Relative abundances of fecal bacterial genera, which may be associated mucin degradation, were evaluated using 16S rRNA gene amplicon sequencing. Results FW-induced mucin degradation was higher in CD samples as compared to HC (p<0.01), but was not linked to specific bacterial relative abundances. FW resulted in 78-87% decrease of TEER in three of the remissive (p<0.001) but not the active CD or HC samples. MVs did not induce mucin degradation or epithelial barrier disruption. Conclusion The higher mucin degradation capacity of CD-derived FW might indicate contributions of microbial products to CD pathophysiology and warrants further investigation. Moreover, the altered epithelial resistance in some individuals is not due to paracellular disruption. Key Messages What is already known? Intestinal microbial dysbiosis and mucosal barrier dysfunction are important contributors to Crohn’s disease aetiology and disease exacerbations. What is new here? The faecal microbial secretome of Crohn’s disease patients has a higher mucin degradation capacity as compared to the secretome of healthy subjects. How can this study help patient care? The increased mucin degradation based on the microbial secretome may be a new target for the development of complementary, microbiome-based therapy in Crohn’s disease. Summary Microbial dysbiosis and intestinal barrier dysfunction can impact Crohn’s disease course. This translational study found higher mucin degradation, but no epithelial barrier disruption, by the faecal microbial secretome of (active) Crohn’s disease patients, as compared to healthy controls.

Significance Hatching from the zona pellucida is a prerequisite for embryo implantation and is less likely to occur in vitro for reasons unknown. Extracellular vesicles (EVs) are secreted by the embryo into the culture medium. Yet the role that embryonic EVs and their cargo microRNAs (miRNAs) play in blastocyst hatching has not been elucidated, partially due to the difficulties of isolating them from low amounts of culture medium. Here, we optimized EV-miRNA isolation from medium conditioned by individually cultured bovine embryos and subsequently showed that miR-378a-3p, which was up-regulated in EVs secreted by blastocysts, plays a crucial role in promoting blastocyst hatching. This demonstrates the regulatory effect of miR-378-3p on hatching, which is an established embryo quality parameter linked with implantation.

Summary Severe pain is often experienced by patients with head and neck cancer and is associated with a poor prognosis. Despite its frequency and severity, current treatments fail to adequately control cancer-associated pain, because of our lack of mechanistic understanding. Cancer-derived small extracellular vesicles (Cancer-sEVs) are well- positioned to function as mediators of communication between cancer cells and neurons. Inhibition of Cancer-sEV release attenuated pain in tumor-bearing mice. Injection of purified Cancer-sEVs is sufficient to induce pain hypersensitivity in naïve mice. Cancer-sEVs triggered calcium influx in nociceptors and inhibition or ablation of nociceptors protect against cancer pain. Interrogation of published sequencing data of human sensory neurons exposed to human Cancer-sEVs suggested a stimulation of protein translation in neurons. Induction of translation by Cancer-sEVs was validated in our mouse model and its inhibition alleviated cancer pain in mice. These findings define a role of Cancer-sEVs in cancer pain and identify several druggable targets. Graphical abstract

Neutrophils are classically characterized as merely reactive innate effector cells. However, the microbiome is known to shape the education and maturation process of neutrophils, improving their function and immune‐plasticity. Recent reports demonstrate that murine neutrophils possess the ability to exert adaptive responses after exposure to bacterial components such as LPS (Gram‐ negative bacteria) or LTA (Gram‐positive bacteria). We now ask whether small extracellular vesicles (EVs) from the gut may directly mediate adaptive responses in neutrophils in vitro. Murine bone marrow‐derived neutrophils were primed in vitro by small EVs of high purity collected from colon stool samples, followed by a second hit with LPS. We found that low‐dose priming with gut micro‐ biota‐derived small EVs enhanced pro‐inflammatory sensitivity as indicated by elevated levels of TNF‐α, IL‐6, ROS and MCP‐1 and increased migratory and phagocytic activity. In contrast, high‐ dose priming resulted in a tolerant phenotype, marked by increased IL‐10 and decreased transmi‐ gration and phagocytosis. Alterations in TLR2/MyD88 as well as TLR4/MyD88 signaling were cor‐ related with the induction of adaptive cues in neutrophils in vitro. Taken together, our study shows that small EVs from stools can drive adaptive responses in neutrophils in vitro and may represent a missing link in the gut–immune axis.

ABSTRACT Group B Streptococcus (GBS) is a major cause of fetal and neonatal mortality worldwide. Many of the adverse effects associated with invasive GBS are associated with inflammation that leads to chorioamnionitis, preterm birth, sepsis, and meningitis; therefore, understanding bacterial factors that promote inflammation is of critical importance. Membrane vesicles (MVs), which are produced by many pathogenic and non-pathogenic bacteria, may modulate host inflammatory responses. In mice, GBS MVs injected intra-amniotically can induce preterm birth and fetal death. Although it is known that GBS MVs induce large-scale leukocyte recruitment into infected tissues, the immune effectors driving these responses are unclear. Here, we hypothesized that macrophages respond to GBS-derived MVs by producing proinflammatory cytokines and are recognized through one or more pattern recognition receptors. We show that THP-1 macrophage-like cells produce high levels of neutrophil- and monocyte-specific chemokines in response to MVs derived from different clinical isolates of GBS. Interleukin (IL)-1β was significantly upregulated in response to MVs, which was independent of NF-kB signaling but dependent on both caspase-1 and NLRP3. These data indicate that MVs contain one or more pathogen-associated molecular patterns that can be sensed by the immune system. Furthermore, this study identifies the NLRP3 inflammasome as a novel sensor of GBS MVs. Our data additionally indicate that MVs may serve as immune effectors that can be targeted for immunotherapeutics, particularly given that similar responses were observed across this subset of GBS isolates.

Extracellular vesicles (EVs) act as nano-scale molecular messengers owing to their capacity to shuttle functional macromolecular cargo between cells. This intrinsic ability to deliver bioactive cargo has sparked great interest in the use of EVs as novel therapeutic delivery vehicles; investments totaling over $2 billion in 2020 alone were reported for therapeutic EVs. One of the bottlenecks facing the production of EVs is the lack of rapid and high throughput analytics to aid process development. Here CHO cells have been designed and engineered to express GFP-tagged EVs via fusion to CD81. Moreover, this study highlights the importance of parent cell characterization to ensure lack of non-fused GFP for the effective use of this quantitative approach. The fluorescent nature of resulting vesicles allowed for rapid quantification of concentration and yield across the EV purification process. In this manner, the degree of product loss was deduced by mass balance analysis of ultrafiltration processing, reconciled up to 97% of initial feed mass. The use of GFP-tagging allowed for straightforward monitoring of vesicle elution from chromatography separations and detection via western blotting. Collectively, this work illustrates the utility of GFP-tagged EVs as a quantitative and accessible tool for accelerated process development.

Food-derived extracellular vesicles (FDEVs) have attracted increasing attention as potential delivery vehicles for therapeutic agents due to their desirable features such as excellent biocompatibility, easy accessibility and cost effectiveness. However, the intrinsic targeting capability of FDEVs is unsatisfactory compared to artificial nanoparticles or other source-derived EVs, which calls for efficient surface engineering strategies to equip them with specific ligands. Here we report a general and mild modification method via reduction of disulfide groups to maleimide reactive thiols. Taking milk-derived EVs (mEVs) as a model system, we demonstrated the feasibility for tethering various ligands on the surface without compromising the vesicular structures. Building an ultra-sensitive nano-flow cytometer (nFCM), the heterogeneous nature of the functionalized samples was revealed, and a magnetic separation approach was proposed accordingly to remove the as-observed non-EV particles. The cellular uptake and cytotoxicity experiments provided direct evidence showing an enhanced cell targeting and cargo delivery capability of the ligand conjugated mEVs. In addition, the in vivo imaging further proved the applicability of transferrin conjugation for increased tumor enrichment of mEVs. Collectively, this general and mild ligand conjugation method enables an efficient surface functionalization of FDEVs, which is of vital importance for enhanced targeting delivery.