Extracellular Vesicles Concentration Research Articles

Extracellular vesicles secreted by Brugia malayi microfilariae modulate the melanization pathway in the mosquito host

Vector-borne, filarial nematode diseases cause significant disease burdens in humans and domestic animals worldwide. Although there is strong direct evidence of parasite-driven immunomodulation of mammalian host responses, there is less evidence of parasite immunomodulation of the vector host. We have previously reported that all life stages of Brugia malayi, a filarial nematode and causative agent of Lymphatic filariasis, secrete extracellular vesicles (EVs). Here we investigate the immunomodulatory effects of microfilariae-derived EVs on the vector host Aedes aegypti. RNA-seq analysis of an Ae. aegypti cell line treated with B. malayi microfilariae EVs showed differential expression of both mRNAs and miRNAs. AAEL002590, an Ae. aegypti gene encoding a serine protease, was shown to be downregulated when cells were treated with biologically relevant EV concentrations in vitro. Injection of adult female mosquitoes with biologically relevant concentrations of EVs validated these results in vivo, recapitulating the downregulation of AAEL002590 transcript. This gene was predicted to be involved in the mosquito phenoloxidase (PO) cascade leading to the canonical melanization response and correspondingly, both suppression of this gene using RNAi and parasite EV treatment reduced PO activity in vivo. Our data indicate that parasite-derived EVs interfere with critical immune responses in the vector host, including melanization.

Low extracellular vesicle concentrations predict survival in patients with heart failure.

Heart disease is of worldwide importance due to high morbidity and mortality. Extracellular vesicle (EV) concentration and size represent novel diagnostic and prognostic biomarkers, e.g. in patients with liver cancer, but data on their prognostic relevance in heart disease are lacking. Here, we investigated the role of EV concentration, size and zeta potential in patients with heart disease. Vesicle size distribution, concentration and zeta potential were measured by nanoparticle tracking analysis (NTA) in 28 intensive care unit (ICU) and 20 standard care (SC) patients and 20 healthy controls. Patients with any disease had a lower zeta potential compared to the healthy controls. Vesicle size (X50) was significantly higher in ICU patients (245 nm) with heart disease as compared to those patients with heart disease receiving standard care (195 nm), or healthy controls (215 nm) (p = 0.001). Notably, EV concentration was lower in ICU patients with heart disease (4.68 × 1010 particles/ml) compared to SC patients with heart disease (7,62 × 1010 particles/ml) and healthy controls (1.50 × 1011 particles/ml) (p = 0.002). Extracellular vesicle concentration is prognostic for overall survival in patients with heart disease. Overall survival is significantly reduced when the vesicle concentration is below 5.55 × 1010 particles/ml. Median overall survival was only 140 days in patients with vesicle concentrations below 5.55 × 1010 particles/ml compared to 211 days in patients with vesicle concentrations above 5.55 × 1010 particles/ml (p = 0.032). Concentration of EVs is a novel prognostic marker in ICU and SC patients with heart disease.

Extracellular vesicles in systemic juvenile idiopathic arthritis.

Systemic juvenile idiopathic arthritis is a chronic pediatric inflammatory disease of unknown etiology, characterized by fever, rash, hepatosplenomegaly, serositis, and arthritis. We hypothesized that intercellular communication, mediated by extracellular vesicles, contributes to systemic juvenile idiopathic arthritis pathogenesis and that the number and cellular sources of extracellular vesicles would differ between inactive and active states of systemic juvenile idiopathic arthritis and healthy controls. We evaluated plasma from healthy pediatric controls and patients with systemic juvenile idiopathic arthritis with active systemic flare or inactive disease. We isolated extracellular vesicles by size exclusion chromatography and determined total extracellular vesicle abundance and size distribution using microfluidic resistive pulse sensing. Cell-specific extracellular vesicle subpopulations were measured by nanoscale flow cytometry. Isolated extracellular vesicles were validated using a variety of ways, including nanotracking and cryo-electron microscopy. Extracellular vesicle protein content was analyzed in pooled samples using mass spectrometry. Total extracellular vesicle concentration did not significantly differ between controls and patients with systemic juvenile idiopathic arthritis. Extracellular vesicles with diameters <200 nm were the most abundant, including the majority of cell-specific extracellular vesicle subpopulations. Patients with systemic juvenile idiopathic arthritis had significantly higher levels of extracellular vesicles from activated platelets, intermediate monocytes, and chronically activated endothelial cells, with the latter significantly more elevated in active systemic juvenile idiopathic arthritis relative to inactive disease and controls. Protein analysis of isolated extracellular vesicles from active patients showed a proinflammatory profile, uniquely expressing heat shock protein 47, a stress-inducible protein. Our findings indicate that multiple cell types contribute to altered extracellular vesicle profiles in systemic juvenile idiopathic arthritis. The extracellular vesicle differences between systemic juvenile idiopathic arthritis disease states and healthy controls implicate extracellular vesicle-mediated cellular crosstalk as a potential driver of systemic juvenile idiopathic arthritis disease activity.

Proteomics and cytokine analyses distinguish myalgic encephalomyelitis/chronic fatigue syndrome cases from controls

BackgroundMyalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex, heterogenous disease characterized by unexplained persistent fatigue and other features including cognitive impairment, myalgias, post-exertional malaise, and immune system dysfunction. Cytokines are present in plasma and encapsulated in extracellular vesicles (EVs), but there have been only a few reports of EV characteristics and cargo in ME/CFS. Several small studies have previously described plasma proteins or protein pathways that are associated with ME/CFS.MethodsWe prepared extracellular vesicles (EVs) from frozen plasma samples from a cohort of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) cases and controls with prior published plasma cytokine and plasma proteomics data. The cytokine content of the plasma-derived extracellular vesicles was determined by a multiplex assay and differences between patients and controls were assessed. We then performed multi-omic statistical analyses that considered not only this new data, but extensive clinical data describing the health of the subjects.ResultsME/CFS cases exhibited greater size and concentration of EVs in plasma. Assays of cytokine content in EVs revealed IL2 was significantly higher in cases. We observed numerous correlations among EV cytokines, among plasma cytokines, and among plasma proteins from mass spectrometry proteomics. Significant correlations between clinical data and protein levels suggest roles of particular proteins and pathways in the disease. For example, higher levels of the pro-inflammatory cytokines Granulocyte-Monocyte Colony-Stimulating Factor (CSF2) and Tumor Necrosis Factor (TNFα) were correlated with greater physical and fatigue symptoms in ME/CFS cases. Higher serine protease SERPINA5, which is involved in hemostasis, was correlated with higher SF-36 general health scores in ME/CFS. Machine learning classifiers were able to identify a list of 20 proteins that could discriminate between cases and controls, with XGBoost providing the best classification with 86.1% accuracy and a cross-validated AUROC value of 0.947. Random Forest distinguished cases from controls with 79.1% accuracy and an AUROC value of 0.891 using only 7 proteins.ConclusionsThese findings add to the substantial number of objective differences in biomolecules that have been identified in individuals with ME/CFS. The observed correlations of proteins important in immune responses and hemostasis with clinical data further implicates a disturbance of these functions in ME/CFS.

Extracellular Vesicles from Candida haemulonii var. vulnera Modulate Macrophage Oxidative Burst

Members of the Candida haemulonii species complex are multidrug-resistant emergent yeast pathogens able to cause superficial and invasive infections in risk populations. Fungal extracellular vesicles (EVs) play a critical role in the pathogenicity and virulence of several species and may perform essential functions during infections, such as carrying virulence factors that behave in two-way communications with the host, affecting survival and fungal resistance. Our study aimed to describe EV production from Candida haemulonii var. vulnera and evaluate whether murine macrophage RAW 264.7 cells respond to their stimuli by generating an oxidative response after 24 h. For this purpose, reactive oxygen species detection assays demonstrated that high concentrations of yeast and EVs (1010 particles/mL) of Candida haemulonii did not change macrophage viability. However, the macrophages recognized these EVs and triggered an oxidative response through the classical NOX-2 pathway, increasing O2•- and H2O2 levels. However, this stress did not cause lipid peroxidation in the RAW 264.7 cells and neither lead to the activation of the COX-2-PGE2 pathway. Thus, our data suggest that low concentrations of C. haemulonii EVs are not recognized by the classical pathway of the oxidative burst generated by macrophages, which might be an advantage allowing the transport of virulence factors via EVs, not identified by the host immune system that could work as fine tube regulators during infections caused by C. haemulonii. In contrast, C. haemulonii var. vulnera and high EV concentrations activated microbicidal actions in macrophages. Therefore, we propose that EVs could participate in the virulence of the species and that these particles could be a source of antigens to be exploited as new therapeutic targets.

Analysis of EVs from patients with advanced pancreatic cancer identifies antigens and miRNAs with predictive value

The identification of predictive factors for treatment of pancreatic cancer (PC) is an unmet clinical need. In the present work, we analyzed blood-derived extracellular vesicles (EVs) from patients with advanced PC in order to find a molecular signature predictive of response to therapy. We analyzed samples from 21 patients with advanced PC, all receiving first-line treatment with gemcitabine+ nab-paclitaxel. Isolated EVs have been analyzed, and the results of laboratory have been matched with clinical data in order to investigate possible predictive factors. EV concentration and size were similar between responder and non-responder patients. Analysis of 37 EV surface epitopes showed a decreased expression of SSEA4 and CD81 in responder patients. We detected more than 450 expressed miRNAs in EVs. A comparative survey between responder and non-responder patients showed that at least 44 miRNAs were differently expressed. Some of these miRNAs have already been observed in relation to the survival and gemcitabine sensitivity of tumor cells. In conclusion, we showed the ability of our approach to identify EV-derived biomarkers with predictive value for therapy response in PC. Our findings are worthy of further investigation, including the analysis of samples from patients treated with different schedules and in different settings.

Differential expression of adipocyte and myotube extracellular vesicle miRNA cargo in chronic binge alcohol-administered SIV-infected male macaques.

Our studies in chronic binge alcohol (CBA) -treated simian immunodeficiency virus (SIV)-infected macaques and in people living with HIV (PLWH) show significant alterations in metabolic homeostasis. CBA promotes a profibrotic phenotype in adipose tissue and skeletal muscle (SKM) and decreases adipose-derived stem cell and myoblast differentiation, making adipose and SKM potential drivers in metabolic dysregulation. Furthermore, we have shown that the differential expression of microRNAs (miRs) in SKM contributes to impaired myoblast differentiation potential. Beyond modulation of intracellular responses, miRs can be transported in extracellular vesicles (EVs) to mediate numerous cellular responses through intercellular and interorgan communication. This study tested the hypothesis that CBA alters concentration and miR cargo of EVs derived from adipocytes and myotubes isolated from SIV-infected male macaques. Fourteen male rhesus macaques received either CBA (2.5g/kg/day) or sucrose (VEH) for 14.5 months. Three months following the initiation of CBA/VEH, all animals were infected with SIVmac251 and 2.5 months later were initiated on antiretroviral therapy. SKM and adipose tissue samples were collected at the study endpoint (blood alcohol concentration=0mM). EVs were isolated by ultracentrifugation of myotube and adipocyte cell culture supernatant. Nanoparticle tracking revealed no differences in concentration or size of particles between VEH and CBA groups. Adipocyte-derived EVs from CBA animals showed decreased miR-let-7a expression (p=0.03). Myotube-derived EVs from CBA animals had decreased miR-16 (p=0.04) and increased miR-133a and miR-133b (both p=0.04) expression. These results indicate that CBA administration differentially regulates EV miR content but does not alter the number of EVs from adipocytes or myotubes. Future studies are warranted to determine the functional relevance of CBA-altered EV miR cargo and their role in intercellular and interorgan communication and metabolic dysregulation.

Assessment of circulating extracellular vesicles from calorie‐restricted mice and humans in ischaemic injury models

AbstractCalorie restriction (CR) and fasting affect lifespan, disease susceptibility and response to acute injury across multiple animal models, including ischaemic injuries such as myocardial infarction or kidney hypoxia. The cargo and function of circulating extracellular vesicles (EV) respond to changes in host physiology, including exercise, injury, and other interventions. Thus, we hypothesised that EVs induced following CR may reflect some of the beneficial properties of CR itself. In a pilot study, EVs were isolated from mice following 21 days of 30 % CR, and from eight human donors after 72 h water‐only fasting. EV size, concentration and morphology were profiled by NTA, western blot and cryoEM, and their function was assessed using multiple assays related to ischaemic diseases. We found that EVs from post‐fasting samples better protected cardiac cells from hypoxia/reperfusion (H/R) injury compared to pre‐fasting EVs. However, there was no difference when used to treat H/R‐injured kidney epithelial cells. Post‐fasting derived EVs slowed the rate of fibroblast migration and slightly reduced macrophage inflammatory gene expression compared to pre‐fasting derived EVs. Lastly, we compared miRNA cargos of pre‐ and post‐fasting human serum EVs and found significant changes in a small number of miRNAs. We conclude that fasting appears to influence EV cargo and function, with varied effects worthy of further exploration.

Dual-Wave Acoustofluidic Centrifuge for Ultrafast Concentration of Nanoparticles and Extracellular Vesicles.

Extracellular vesicles (EVs) are secreted nanostructures that play various roles in critical cancer processes. They operate as an intercellular communication system, transferring complex sets of biomolecules from cell to cell. The concentration of EVs is difficult to decipher, and there is an unmet technological need for improved (faster, simpler, and gentler) approaches to isolate EVs from complex matrices. Herein, an acoustofluidic concentration of extracellular vesicles (ACEV) is presented, based on a thin-film printed circuit board with interdigital electrodes mounted on a piezoelectric substrate. An angle of 120° is identified between the electrodes and the reference flat of the piezoelectric substrate for simultaneous generation of Rayleigh and shear horizontal waves. The dual waves create a complex acoustic field in a droplet, resulting in effective concentration of nanoparticles and EVs. The ACEV is able to concentrate 20nm nanospheres within 105 s and four EV dilutions derived from the human prostate cancer (Du145) cell line in approximately 30 s. Cryo-electron microscopy confirmed the preservation of EV integrity. The ACEV device holds great potential to revolutionize investigations of EVs. Its faster, simpler, and gentler approach to EV isolation and concentration can save time and effort in phenotypic and functional studies of EVs.

A standardized method for plasma extracellular vesicle isolation and size distribution analysis.

The following protocol describes our workflow for isolation and quantification of plasma extracellular vesicles (EVs). It requires limited sample volume so that the scientific value of specimens is maximized. These steps include isolation of vesicles by automated size exclusion chromatography and quantification by tunable resistive pulse sensing. This workflow optimizes reproducibility by minimizing variations in processing, handling, and storage of EVs. EVs have significant diagnostic and therapeutic potential, but clinical application is limited by disparate methods of data collection. This standardized protocol is scalable and ensures efficient recovery of physiologically intact EVs that may be used in a variety of downstream biochemical and functional analyses. Simultaneous measurement quantifies EV concentration and size distribution absolutely. Absolute quantification corrects for variations in EV number and size, offering a novel method of standardization in downstream applications.

Toward a Biomarker Panel measured in CNS-originating Extracellular Vesicles for Differential Diagnosis of Parkinson’s Disease and Multiple System Atrophy (S37.005)

Toward a Biomarker Panel measured in CNS-originating Extracellular Vesicles for Differential Diagnosis of Parkinson’s Disease and Multiple System Atrophy (S37.005)

Epigenetic Reprogramming via Synergistic Hypomethylation and Hypoxia Enhances the Therapeutic Efficacy of Mesenchymal Stem Cell Extracellular Vesicles for Bone Repair.

Mesenchymal stem cells (MSCs) are a promising cell population for regenerative medicine applications, where paracrine signalling through the extracellular vesicles (EVs) regulates bone tissue homeostasis and development. MSCs are known to reside in low oxygen tension, which promotes osteogenic differentiation via hypoxia-inducible factor-1α activation. Epigenetic reprogramming has emerged as a promising bioengineering strategy to enhance MSC differentiation. Particularly, the process of hypomethylation may enhance osteogenesis through gene activation. Therefore, this study aimed to investigate the synergistic effects of inducing hypomethylation and hypoxia on improving the therapeutic efficacy of EVs derived from human bone marrow MSCs (hBMSCs). The effects of the hypoxia mimetic agent deferoxamine (DFO) and the DNA methyltransferase inhibitor 5-azacytidine (AZT) on hBMSC viability was assessed by quantifying the DNA content. The epigenetic functionality was evaluated by assessing histone acetylation and histone methylation. hBMSC mineralisation was determined by quantifying alkaline phosphate activity, collagen production and calcium deposition. EVs were procured from AZT, DFO or AZT/DFO-treated hBMSCs over a two-week period, with EV size and concentration defined using transmission electron microscopy, nanoflow cytometry and dynamic light scattering. The effects of AZT-EVs, DFO-EVs or AZT/DFO-EVs on the epigenetic functionality and mineralisation of hBMSCs were evaluated. Moreover, the effects of hBMSC-EVs on human umbilical cord vein endothelial cells (HUVECs) angiogenesis was assessed by quantifying pro-angiogenic cytokine release. DFO and AZT caused a time-dose dependent reduction in hBMSC viability. Pre-treatment with AZT, DFO or AZT/DFO augmented the epigenetic functionality of the MSCs through increases in histone acetylation and hypomethylation. AZT, DFO and AZT/DFO pre-treatment significantly enhanced extracellular matrix collagen production and mineralisation in hBMSCs. EVs derived from AZT/DFO-preconditioned hBMSCs (AZT/DFO-EVs) enhanced the hBMSC proliferation, histone acetylation and hypomethylation when compared to EVs derived from AZT-treated, DFO-treated and untreated hBMSCs. Importantly, AZT/DFO-EVs significantly increased osteogenic differentiation and mineralisation of a secondary hBMSC population. Furthermore, AZT/DFO-EVs enhanced the pro-angiogenic cytokine release of HUVECs. Taken together, our findings demonstrate the considerable utility of synergistically inducing hypomethylation and hypoxia to improve the therapeutic efficacy of the MSC-EVs as a cell-free approach for bone regeneration.

Isolation and Characterization of Extracellular Vesicles in Human Bowel Lavage Fluid.

Colorectal cancer (CRC) is the third most common cancer worldwide and is detected in late stages because of a lack of early and specific biomarkers. Tumors can release extracellular vesicles (EVs), which participate in different functions, such as carrying nucleic acids to target cells; promoting angiogenesis, invasion, and metastasis; and preparing an adequate tumor microenvironment. Finally, bowel lavage fluid (BLF) is a rarely used sample that is obtained during colonoscopy. It presents low variability and protein degradation, is easy to handle, and is representative of EVs from tumor cells due to proximity of the sample collection. This sample has potential as a research tool and possible biomarker source for CRC prognosis and monitoring. In this study, EVs were isolated from human BLF by ultracentrifugation, then characterized by transmission electron microscopy and atomic force microscopy. EV concentration was determined by nanoparticle tracking analysis, and tetraspanins were determined by Western blot, confirming correct EV isolation. RNA, DNA, and proteins were isolated from these EVs; RNA was used in real-time PCR, and proteins were used in an immunoblotting analysis, indicating that EV cargo is optimal for use and study. These results indicate that EVs from BLF can be a useful tool for CRC study and could be a source of biomarkers for the diagnosis and monitoring of CRC.

Characteristics of size-exclusion chromatography enriched porcine follicular fluid extracellular vesicles

Extracellular vesicles (EVs) are membrane-bound nanoparticles that are released by different cell types and play a crucial role in the intercellular communication. They carry various biomolecular compounds such as DNA, RNA, proteins, and lipids. Given that EVs are a new element of the communication within the ovarian follicle, extensive research is needed to optimize method of their isolation. The aim of the study was to assess size-exclusion chromatography (SEC) as a tool for effective EVs isolation from porcine ovarian follicular fluid. The characterization of EVs was performed by nanoparticle tracking analysis, transmission electron microscopy, atomic force microscopy, mass spectrometry and Western blot. We determined EVs concentration, size distribution, zeta potential, morphology, purity, and marker proteins. Our results show that SEC is an effective method for isolation of EVs from porcine follicular fluid. They displayed predominantly exosome properties with sufficient purity and possibility for further functional analyses, including proteomics.

CHARACTERIZATION OF MESENCHYMAL STROMAL CELL EXTRACELLULAR VESICLE RELEASE PROFILES AND CARGO: RELEVANCE FOR BONE REGENERATIVE THERAPIES

Recent studies suggested that both the soluble protein of the mesenchymal stromal cell (MSC) secretome, as well as the secreted extracellular vesicles (EVs) promote bone regeneration. However, there is limited knowledge of the changes in MSC secretome vesicular fraction during aging. We therefore aimed to characterize the release profiles and cargo of EVs from MSCs of different chronological ages.Conditioned medium (CM) was collected from 13 bone marrow MSC strains (20-89 years) and from one MSC strain derived from human induced pluripotent stem cells (iPSCs). The EV-containing fraction was enriched with ultracentrifugation. The number of particles in the CM was evaluated by nanoparticle tracking analysis (NTA), and the number of EVs was evaluated by flow cytometry (FC) after staining with cell-mask-green and anti-CD81 antibody. EV cargo analysis was conducted using next-generation sequencing (NGS).Our data confirmed the release of EVs from all MSC strains used in the study. There were no correlations between the number of particles and the number of EVs released in the CM, and between the number of EVs released and the strain age. Nevertheless, some of the lowest concentrations of EVs were found in the CM of strains over 70 years of age, which exhibited a low/absent chondrogenic and osteogenic differentiation potential. In contrast, iPSC-MSCs, which exhibited a high growth and three-lineage differentiation potential, released a similar amount of EVs as the best performing bone marrow MSC strain. NGS analysis identified several microRNAs that were significantly enriched in EVs of young MSC strains exhibiting low senescence, and those that were enriched in EVs of strains exhibiting high differentiation potentials. Gender had no influence on microRNA profiles in EVs or releasing MSCs.Taken together, our data provides new insights into the properties of MSC vesicular secretome and its therapeutic potential during aging.

Abstract 324: Nickel exposure alters extracellular vesicles biogenesis to carry NUPR1 and mediate cancer progression

Abstract Nickel (Ni), a group I human carcinogen by the International Agency for Research on Cancer (IARC) alters expression of various oncogenic molecules in cells resulting in adoption of cancer cell phenotypes, such as anchorage-independent growth, migration, and invasion. Cancer cell phenotypes are altered by internal genetic or epigenetic changes or in response to external signals mediated by soluble proteins or extracellular vesicles. However, few studies have linked extracellular vesicles (EVs) in response to environmental pollutants, such as Ni. Nuclear Protein 1 (NUPR1) is a stress response protein that is upregulated in cancer cells and it is also upregulated in cells following chronic Ni exposure. However, the role of NUPR1 in EV biogenesis or EV-mediated cancer progression is unknown. To explore the potential role of EVs in Ni-induced cancer progression, this study examined the effect of Ni-exposure on EV signature. Sizes and concentration of EVs from BEAS-2B cells, 6 weeks-Ni-treated BEAS-2B cells (Ni-6W) and Ni-transformed BEAS-2B cells (Ni-3) were identified by nanoparticle-tracking analysis (NTA) and transmission electron microscopy (TEM). In the results, showed that Ni-6W and Ni-3 released more EVs compared to the control. The transcription expression levels of EVs biogenesis markers, CD63, CD82 and Flotillin-1 (FLOT) were coincidentally increased in Ni-6W and Ni-3 compared to the BEAS-2B. We further identified that inhibition of NUPR1 by using the shRNA or NUPR1 inhibitor ZZW-130 can rescue the increased releasing of EVs induced by Ni exposure. Here we demonstrated that Ni exposure elevated EV release and that these Ni-altered EVs contributed to a pro-inflammatory environment associated with tumorigenesis and cancer progression. Moreover, we provided a new method of regulating EVs biogenesis by targeting NUPR1, this would be crucial for EV-based cancer therapy. Citation Format: Shan Liu, Angelica Ortiz, Juan Iovanna, Max Costa. Nickel exposure alters extracellular vesicles biogenesis to carry NUPR1 and mediate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 324.

Abstract 697: Extracellular vesicles (EV) - mediators of therapeutic vaccination? In vivo and in vitro characterization of EVs generated after infection of human and murine cells with therapeutic poxviruses

Abstract Virus-based therapies show promise in inducing systemic anti-tumor immunity. Over the past years, modified poxviruses were developed to induce a sustained antitumoral immune response. Several studies confirm this hypothesis and report induction of T cell response against the antigen targeted by these vaccines. We previously reported MVA induced T-cell response against HPV oncoviral antigens E6 and E7 in HPV anogenital cancers (NCT03260023) and against private tumor neoantigen in ovarian and head and neck cancer. Despite these promising results the molecular and cellular mechanisms involved in vaccine response remains to be elucidated. In this work, we inquired whether EVs, well known for mediating intercellular communication, play a role in these therapeutic vaccination process. We first asked whether poxviral infection of human primary cells modifies their EV secretion properties. To this end, peripheral blood mononuclear cells (PBMCs) from healthy donors were infected with poxviral vectors, cell culture supernatants were harvested, and different isolation methods were compared for their ability to separate EVs from viral particles: ultracentrifugation, immunocapture, density gradient, velocity gradient, size exclusion chromatography and filtration. EVs size distribution and concentration were determined by nanoparticle tracking analysis (NTA) and the presence of infectious viral particles was titrated by plaque assays. Our data showed that 0.1µm filtration allowed the complete separation of EVs from infectious viral particles. All other methods showed co-isolation of EVs and infectious viral particles. Using this method, we demonstrate that infection of PBMCs with the poxviruses VACV, MVA and PCPV significantly increased the amount of secreted EVs. Next, we investigated the molecular content of EVs isolated from MVA-infected PBMCs by mass spectrometry. We observed differences in the abundance of at least 57 human proteins between EVs from infected and non-infected cells. In addition, several viral proteins, including virus-encoded payloads were detected in EVs from infected cells. We are now confirming those data with flow cytometry to detect virus-encoded payloads like tumor antigens, antigen-presenting molecules, and EV markers such as tetraspanins. The RNA content of EVs from infected and non-infected cells will be compared. Finally, EVs were isolated from murine dendritic cells infected with therapeutic pox vaccines. We are now evaluating the effects of local and systemic application of EVs in murine tumor models. Citation Format: Lucas Walther, Caroline Tosch, Jules Deforges, Christine Carapito, Magali Rompais, Marie-Christine Claudepierre, Nathalie Silvestre, Kaïdre Bendjama, Eric Quemeneur, Jacky Goetz, Vincent Hyenne, Karola Rittner. Extracellular vesicles (EV) - mediators of therapeutic vaccination? In vivo and in vitro characterization of EVs generated after infection of human and murine cells with therapeutic poxviruses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 697.

MP20-08 RADIOTHERAPY-INDUCED URINARY EXTRACELLULAR VESICLES PREDICT LATE BLADDER TOXICITY IN PROSTATE CANCER PATIENTS

You have accessJournal of UrologyCME1 Apr 2023MP20-08 RADIOTHERAPY-INDUCED URINARY EXTRACELLULAR VESICLES PREDICT LATE BLADDER TOXICITY IN PROSTATE CANCER PATIENTS Ryan Molony, Sarah Kerns, Brian Marples, Emmanuel Oshodi, YuhChyau Chen, and Yi-Fen Lee Ryan MolonyRyan Molony More articles by this author , Sarah KernsSarah Kerns More articles by this author , Brian MarplesBrian Marples More articles by this author , Emmanuel OshodiEmmanuel Oshodi More articles by this author , YuhChyau ChenYuhChyau Chen More articles by this author , and Yi-Fen LeeYi-Fen Lee More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003245.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Radiotherapy (RT) is the standard of care for over 50% of prostate cancer (PCa) patients, but up to 25% develop delayed bladder toxicities months to years after treatment. Late radiation cystitis is a dose-limiting toxicity that can cause hematuria and other potentially severe symptoms, leading to further morbidity and decisional regret. Additional work is needed to define early biomarkers of late radiation cystitis in order to better guide patient care. Extracellular vesicles (EVs) are stable membrane-enclosed particles that are released from diverse cell types and present at high levels in biofluid samples, but the relationship between EV biogenesis and the development of late radiation cystitis has not been examined. This study was developed to explore the relationships between late hematuria incidence and urinary and serum EV concentrations in a cohort of PCa patients undergoing RT. METHODS: PCa patients scheduled to receive curative-intent external beam RT with or without brachytherapy boost were enrolled for this study. Longitudinal urine and serum samples were collected on enrollment (pre-treatment) and within 1 week after RT. Patients completed outcome questionnaires containing questions regarding radiation-related symptoms and quality of life at the time of sample collection, 6 months post-RT, and annually after RT for up to 5 years. Demographic, clinical, and treatment data were obtained from electronic medical records. EVs in collected urine and serum samples were quantified through a nanoparticle tracking analysis approach, and EV concentrations at different collection time points were then compared between patients that did and did not develop hematuria. RESULTS: RT was found to significantly increase post-RT urinary EV concentrations relative to pre-RT levels in patients who subsequently developed late hematuria, whereas this induction of enhanced EV biogenesis was not observed in patients who did not develop this toxicity. The same effect was evident but less robust in patient serum samples. Re-examination of prior GWAS data additionally revealed that RT-related hematuria risk was strongly associated with the EV biogenesis-related genes SYTL3 and COG. CONCLUSIONS: Post-treatment EV concentrations in urine samples collected from PCa patients undergoing RT are associated with the future onset of radiation cystitis, providing a promising biomarker that may enable the more-timely treatment of at-risk patients to mitigate the development of late bladder toxicities. Source of Funding: University of Rochester departmental research funding © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e278 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ryan Molony More articles by this author Sarah Kerns More articles by this author Brian Marples More articles by this author Emmanuel Oshodi More articles by this author YuhChyau Chen More articles by this author Yi-Fen Lee More articles by this author Expand All Advertisement PDF downloadLoading ...

Pharmacokinetic Approach for the Elucidation of Elevated Plasma Small Extracellular Vesicle (sEV) Concentration in Cancer

The abundance of circulating plasma small extracellular vesicles (sEVs) has been reported to be elevated in cancer; however, the underlying mechanism remains unclear. In this study, a pharmacokinetic approach was used to determine the factors contributing to elevated plasma sEV levels during cancer in a tumor-bearing mouse model. Mouse plasma-derived sEVs (MP-sEVs) isolated from tumor-bearing mice showed increased protein concentrations and physicochemical characteristics comparable to MP-sEVs isolated from healthy mice. The steady-state concentration of sEVs is determined by the balance between the MP-sEV production and clearance. Thus, to determine whether tumorigenesis influences sEV clearance, isolated MP-sEVs were intravenously administered to either tumor-bearing or healthy mice. The results showed minimal differences in sEV clearance rates, suggesting that sEV production is the driving force of elevated MP-sEV concentrations. Lastly, CD63-gLuc stably expressing B16BL6-bearing mice were used to estimate the contribution of tumor cell-derived sEVs in the plasma. The gLuc activity of the MP-sEVs isolated was below the limit of detection, and it was estimated that the tumor cell-derived sEVs comprised at most 0.5% of the total MP-sEVs. Taken together, these results suggest that cells other than tumor cells contribute to elevated plasma sEV levels in cancer.

Extracellular vesicles are dynamic regulators of maternal glucose homeostasis during pregnancy

Homeostatic regulation of the maternal milieu during pregnancy is critical for maternal and fetal health. The placenta facilitates critical communication between maternal and fetal compartments, in part, through the production of extracellular vesicles (EVs). EVs enable tissue synchrony via cell–cell and long-distance communication and are at their highest circulating concentration during pregnancy. While much work has been done investigating how physiological challenges in pregnancy affect the fetus, the role of placental communication in maternal health has not been well examined. We previously identified placental O-glycosyl transferase (OGT), a glucose-sensing enzyme, as a target of maternal stress where OGT levels and activity affected the O-glycosylation of proteins critical for EV cargo loading and secretion. Here, we hypothesized that placental OGT plays an essential role in maternal homeostatic regulation during pregnancy via its regulation of maternal circulating EV concentrations. Our studies found that changes to key metabolic factors over the circadian cycle, including glucocorticoids, insulin, and glucose, were significantly associated with changes in circulating EV concentration. Targeting placental OGT in mice, we found a novel significant positive relationship between placental OGT and maternal circulating EV concentration that was associated with improving maternal glucose tolerance during pregnancy. Finally, an intravenous elevation in EVs, matching the concentration of EVs during pregnancy, shifted non-pregnant female glucose sensitivity, blunted glucose variance, and improved synchrony of glucose uptake. These data suggest an important and novel role for circulating EVs as homeostatic regulators important in maternal health during pregnancy.