Extracellular Vesicles Populations Research Articles

Analyses of single extracellular vesicles from non-small lung cancer cells to reveal effects of epidermal growth factor receptor inhibitor treatments

Precision cancer medicine has changed the treatment landscape of non-small cell lung cancer (NSCLC) as illustrated by the introduction of tyrosine kinase inhibitors (TKIs) towards mutated epidermal growth factor receptor (EGFR). However, as responses to EGFR-TKIs are heterogenous among NSCLC patients, there is a need for ways to early monitor changes in treatment response in a non-invasive way e.g., in patient's blood samples. Recently, extracellular vesicles (EVs) have been identified as a source of tumor biomarkers which could improve on non-invasive liquid biopsy-based diagnosis of cancer. However, the heterogeneity in EVs is high. Putative biomarker candidates may be hidden in the differential expression of membrane proteins in a subset of EVs hard to identify using bulk techniques. Using a fluorescence-based approach, we demonstrate that a single-EV technique can detect alterations in EV surface protein profiles. We analyzed EVs isolated from an EGFR-mutant NSCLC cell line, which is refractory to EGFR-TKIs erlotinib and responsive to osimertinib, before and after treatment with these drugs and after cisplatin chemotherapy. We studied expression level of five proteins; two tetraspanins (CD9, CD81), and three markers of interest in lung cancer (EGFR, programmed death-ligand 1 (PD-L1), human epidermal growth factor receptor 2 (HER2)). The data reveal alterations induced by the osimertinib treatment compared to the other two treatments. These include the growth of the PD-L1/HER2-positive EV population, with the largest increase in vesicles exclusively expressing one of the two proteins. The expression level per EV decreased for these markers. On the other hand, both the TKIs had a similar effect on the EGFR-positive EV population.

Abstract 6717: Extracellular vesicles in diagnostic retinoblastoma aqueous humor correlate with disease stage and clinical outcome: a pilot study

Abstract Introduction: Retinoblastoma (RB), a pediatric retinal malignancy, is unique in that it cannot be biopsied, impeding biomarker discovery. Aqueous humor (AH) is an ocular fluid that is an established source of tumor information, serving as a surrogate liquid biopsy. Extracellular vesicles (EVs) are promising biomarker candidates, and were recently established in RB AH. EVs are membrane-enclosed secretory vesicles carrying bioactive molecular information, which can be phenotyped using validated tetraspanin protein markers. Currently, relationships between EVs and RB clinical features are unknown, but establishing such relationships is necessary to determine the utility of EVs as RB biomarkers. Methods: We quantified and phenotyped EVs in 37 AH samples from 18 RB eyes with varying International Intraocular Retinoblastoma Classification (IIRC) stages and explored clinical correlations. Samples were collected from 10 eyes at diagnosis (DX) and 27 eyes during treatment (Tx). By outcome, 6 DX eyes were enucleated and 4 were salvaged. DX samples were collected from one A, one B, six D and two E eyes by IIRC group. 10μL of unprocessed AH underwent Single Particle-Interferometric Reflectance Imaging Sensor (SP-IRIS) (Exoview R100) analysis for fluorescent-based particle count and immunophenotyping of tetraspanin expression (CD63/81/9). Counts were converted to percentages. Statistics were done with Mann-Whitney U, ANOVA, and Tukey’s tests. Results: Samples from DX eyes (n=10) contained a higher number of EVs than samples from Tx eyes (n=27) (1.68 x 107 ± 4.28 x 107 per mL vs. 2.10x 106 ± 1.33 x 106 per mL) by SP-IRIS analysis. By percentage, DX samples were enriched with CD63/81+ EVs (P<0.001) while Tx samples had more homogenous mono-CD63+ dominant EV populations (P=0.007). Enrichment of CD63/81+ EVs was significantly higher in enucleated AH (n=6) than salvaged AH (n=4) when compared by count (P=0.038) and marginally higher when compared by percentage (P=0.067). The CD9/81+ EV population percentage was significant in salvaged eyes (P=0.01). By IIRC, CD63/81+ EVs were most abundant in Group E eyes (n= 2) when compared to Group D eyes (n=6) by count (P<0.001), and when compared to Group A+B eyes (n=2) by count (P=0.01) and percentage (P=0.019). Group A+B eyes had a larger percentage of CD9/81+ EVs than did Group E eyes (P=0.043). Conclusions: EV quantity in RB AH decreases with treatment, and phenotypic expression profiles vary by treatment status and clinical outcome. TheCD63/81+ EV subpopulation is higher in eyes with advanced Group E disease and those requiring enucleation, while the CD9/81+ subpopulation is higher in eyes with less advanced Group A+B disease and those salvaged with treatment. Our data suggests that EV phenotypes correlate with RB clinical features, and further research into these subpopulations offers the prospect of establishing critical RB biomarkers. Citation Format: Sarah Pike, Chen-Ching Peng, Paolo Neviani, Jesse L. Berry, Liya Xu. Extracellular vesicles in diagnostic retinoblastoma aqueous humor correlate with disease stage and clinical outcome: a pilot study [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 6717.

Abstract 2370: The role of extracellular vesicles in the glioblastoma tumor microenvironment

Abstract Glioblastoma (GBM) is the most malignant form of gliomas and is the most common and lethal primary brain tumor in adults. Within the tumor, there is a subtype of cells deemed brain tumor initiating cells (BTICs), which contribute to tumor progression, therapy resistance, and tumoral heterogeneity. The tumor microenvironment, surrounding tumors, contains an ecosystem of cells in which BTICs can interact with and further promote tumor progression. In the lateral wall of the lateral ventricles, the neurogenic niche of the subventricular zone (SVZ), contains neural stem cells (NSCs) that self-renew and generate highly migratory neural progenitor cells (NPCs). Interestingly, GBM tumors proximal to the SVZ are more proliferative, migratory, and negatively impact the survival of patients. We have previously observed that the presence of GBM induces reciprocal changes to the SVZ altering its neurogenic capacity and resulting in a more proliferative tumor. BTICs share the similar self-renewal capacity to NPCs and may exert intercellular communication resulting in increase malignancy features. However, the exact mechanisms of how GSCs communicate with NPCs is not well understood. Cancer cells release higher amounts of extracellular vesicles (EVs) than non-malignant cells. Cancer EVs help communicate with other nearby cells, leading to promotion of tumorigenesis. EVs are a heterogenous group of cell-derived membranous structures that serve as means of intercellular communication, allowing for cells to exchange proteins, lipids, RNA, and other genetic material. They can be further characterized into two main subtypes, small EVs, that range from 30-150nm, and large EVs, that range from 50-1000nm. EVs derived from gliomas or non-glioma cells in the tumor microenvironment are involved in tumor cell proliferation, invasion, malignancy, and drug resistance. The overarching goal of this project is to characterize BTIC-derived small EV cargo to identify proteins and RNAs that modify the GBM tumor microenvironment. We hypothesize that GBM-derived small extracellular vesicles alter the phenotype of non-cancer cells in the tumor microenvironment through distinct cargo contained in small extracellular vesicles. The results of these experiments will help us to elucidate what exact small EV cargo impacts the tumor microenvironment, with the potential of revealing possible therapeutic targets for GBM.The optimized protocol gives us the ability to enrich our desired EV population of small EVs from BTICs. BTIC-derived small EVs influence recipient NPCs by significantly upregulating markers for stemness, tumor aggressiveness, and invasion, as well as increasing NPC migration. Future experiments will include trying to establish a cell-specific labeling system in vitro to label EVs from BTICs, to allow for direct co-culture of BTICs with NPCs and other cells in the tumor microenvironment. Citation Format: Marissa Russo. The role of extracellular vesicles in the glioblastoma tumor microenvironment [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 2370.

Abstract 371: Integrin-mediated lipolysis by pancreatic cancer-derived extracellular vesicles in cancer-associated cachexia

Abstract Introduction: Cancer-associated cachexia (CAC) is a life-threating condition frequently observed in cancer patients, especially those with pancreatic cancer. CAC is a multifactorial disease, with the notable characteristic of systemic lipolysis. Although humoral factors have been implicated in its pathogenesis, critical factors remain to be determined. Since even localized pancreatic cancer frequently induce lipolysis systemically, we examined the molecular mechanisms in vitro and in vivo based on the hypothesis that extracellular vesicles (EVs) systemically released from pancreatic cancer cells induce lipolysis. Procedures: EVs derived from the supernatants of cultured pancreatic cancer cell lines and from the sera of pancreatic cancer patients were characterized. The effects of EVs on adipocytes were determined using mature human adipocytes induced from human adipose-derived mesenchymal stem cells. To confirm the in vitro findings, lipolysis induction was examined in mice by intravenous injection of EVs. EV characteristics inducing lipolysis were determined. Those characteristics were confirmed by knockout studies and examining specifically isolated pancreatic cancer-derived EVs from heterogenous EV population in patients’ sera. Results: Lipolysis was triggered by specific EVs from pancreatic cancer cell line via the cAMP inside and uptake of more EVs into adipocytes was crucial for higher levels of lipolysis. In vivo studies, database search, and knockout studies revealed that integrin β1 and integrin α6 on EVs were the determinants for the attachment and uptake of the EVs to adipocytes, resulting in inducing more lipolysis. There was no difference in the percentage of these integrins positive EVs between heterogenous EV population in cancer patients’ sera and EVs in cancer-free controls’ sera. However, pancreatic cancer cell-derived EVs specifically isolated by targeting CA19-9, an aberrant glycosylation, from heterogenous EV population in patients’ sera, confirmed that pancreatic cancer-derived EVs have more such integrins. Conclusions: Cancer cell-derived EVs in the sera of pancreatic cancer patients have more ITGB1 and ITGA6 levels, which may explain the systemic induction of lipolysis in cancer cachexia from the early stages and could be novel therapeutic targets against CAC. In addition, specific isolation of cancer-derived EVs in sera from heterogenous EV population is crucial for elucidating cancer-related pathogenesis. Citation Format: Chikako Shibata, Motoyuki Otsuka, Mitsuhiro Fujishiro. Integrin-mediated lipolysis by pancreatic cancer-derived extracellular vesicles in cancer-associated cachexia [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 371.

Apoptotic vesicles: emerging concepts and research progress in physiology and therapy

Abstract Apoptosis represents the dominant form of programmed cell death and plays critical roles in maintaining tissue and organ homeostasis. A notable population of extracellular vesicles (EVs) is generated during apoptosis, known as apoptotic vesicles (apoVs). These apoVs are increasingly the subject of studies concerning their identity and mechanisms of production, which have been revealed unique biological and functional characteristics that are emerging as crucial regulators for diverse processes. Furthermore, apoVs have been gradually noticed for their essential role in regulating the physiology of various organ systems in vivo, and growing evidence suggests that apoV dysregulation contributes to age- and pathology-associated tissue alterations. Importantly, apoVs can be therapeutically harnessed to unleash their potential in treating several diseases such as immune disorders, osteoporosis, cutaneous wound and acute liver failure; these vesicles, mainly derived from cultured mesenchymal stem cells, hold great translational promise. Here we review the current landscape of scientific knowledge about apoVs, with emphasis on mechanistic insights into how apoVs contribute to organismal health and disease, which also provide novel cell-free strategies for EV-based regenerative therapeutics.

Interaction network of extracellular vesicles building universal analysis via eye tears: iNEBULA

Discovering the secrets of diseases from tear extracellular vesicles (EVs) is well-recognized and appreciated. However, a precise understanding of the interaction network between EV populations and their biogenesis from our body requires more in-depth and systematic analysis. Here, we report the biological profiles of different-size tear EV subsets from healthy individuals and the origins of EV proteins. We have identified about 1800 proteins and revealed the preferential differences in the biogenesis among distinct subsets. We observe that eye-related proteins that maintain retinal homeostasis and regulate inflammation are preferentially enriched in medium-size EVs (100 to 200 nm) fractions. Using universal analysis in combination with the Human Protein Atlas consensus dataset, we found the genesis of tear EV proteins with 37 tissues and 79 cell types. The proteins related to retinal neuronal cells, glial cells, and blood and immune cells are selectively enriched among EV subsets. Our studies in heterogeneous tear EVs provide building blocks for future transformative precision molecular diagnostics and therapeutics.

Comprehensive isolation of extracellular vesicles and nanoparticles.

There is an increasing appreciation for the heterogeneous nature of extracellular vesicles (EVs). In addition, two nonvesicular extracellular nanoparticles (NVEPs), exomeres and supermeres, have been discovered recently that are enriched in many cargo previously ascribed to EVs. The EV field has largely focused on EV isolation and characterization, while studies on NVEPs are limited. At this juncture, it is critically important to have robust and reliable methods to separate distinct populations of EVs and NVEPs to assign cargo to their correct carrier. Here, we provide a comprehensive step-by-step protocol for sequential isolation of large and small EVs, nonvesicular fractions, exomeres and supermeres from the same starting material. We describe in detail the use of differential ultracentrifugation, filtration, concentration and high-resolution density-gradient fractionation to obtain purified fractions of distinct populations of EVs and NVEPs. This protocol allows assignment and enrichment of a biomolecule of interest to its specific extracellular compartment. Compared to other isolation methods, our protocol has unique advantages, including high purity and reproducibility, with minimal expertise required. The protocol can be applied to purification of EVs and NVEPs from cell culture medium and human plasma and requires ~72 h to complete. Adoption of this protocol will help translational investigators identify potential circulating biomarkers and therapeutic targets for a host of human diseases and allow basic scientists to better understand EV and NVEP biogenesis and function. Overall, this protocol will allow those interested in isolating EVs and extracellular particles to advance scientific inquiry to answer outstanding questions in the field.

Comparison of Different Extracellular Vesicles Populations Derived from Adipose Tissue Derived Stromal Cells Depending on Conditioning Period

Introduction: Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSC) became an important player in regenerative medicine over past years. The most common protocol to isolate EVs from cell culture, includes a period of fetal calf serum (FCS)-free conditioning. Different conditioning periods are common ranging from 12h up to 96h. Recent guidelines(MISEV 2018) only paid little attention to this variable without any recommendations. The aim of this study was to investigate EVs released from adipose tissue stromal cells (ADSCs) in different periods. Materials and Methods: For EV preparation, condition medium from primary ADSC culture was collected each 24h by replacing it with fresh FCS-free GM for 72h. Thus, we obtained EVs released within first 24h (24h), 48h-72h (48h), and 48h-72h (72h). The groups were compared by their size, number, phenotype, and amount of protein. The proliferation potential was assessed by a coculture with primary Schwann cells. Results: EVs from all the groups (24h, 48h, and 72h) fulfilled the characterization criteria according to MISEV 2018. However, the properties of EVs differed significantly by size, number, EV to protein ratio and the expression profiles CD9, CD63, CD81. Further, Schwann cells treated with “24h” EVs exhibited a significant increase in proliferation. The proliferative effect of EVs was diminished with longer conditioning period. Conclusion: For groups working with EVs it might be enticing to prefer longer FCS-free conditions for a higher yield. More, as EVs from all groups fulfilled the minimal criteria, which generates an erroneous belief of a coherent population. With our approach, we identified distinct populations of EVs with significantly different proliferative potential. For the sake of reproducibility, our data demonstrate the urgent need for a regulation of the FCS-free period for at least EVs from primary ADSCs. Future experiments are needed to validate our results for EVs from immortalized cells, or for further cell lines.

Tissue factor activity of small and large extracellular vesicles in different diseases

BackgroundCirculating procoagulant extracellular vesicles (EVs) are increased in diseases, such as cancer, sepsis, and COVID-19. EV tissue factor (TF) activity is associated with disseminated intravascular coagulation in sepsis and venous thrombosis in patients with pancreatic cancer and COVID-19. EVs are commonly isolated by centrifugation at ∼20,000 g. ObjectivesIn this study, we analyzed the TF activity of 2 EV populations enriched for large and small EVs in patients with either sepsis, pancreatic cancer, or COVID-19. MethodsEVs were isolated from plasma by sequential centrifugation at 20,000 g (large EVs, LEVs) and then 100,000 g (small EVs, SEVs). We analyzed EVs from plasma prepared from whole blood samples from healthy individuals with or without lipopolysaccharide (LPS) stimulation as well as EVs from plasma samples from patients with either sepsis, pancreatic cancer, or COVID-19. TF-dependent (EV-TF activity) and TF-independent factor Xa (FXa) generation of the EVs was measured. ResultsLPS increased EV-TF activity in LEVs but not SEVs. Similarly, in 2 patients with sepsis who had EV-TF activity above the background of the assay we observed EV-TF activity in LEVs but not SEVs. Patients with pancreatic cancer or COVID-19 had circulating EV-TF activity in both LEVs and SEVs. ConclusionWe recommend that EVs are isolated from plasma from patients by centrifugation at 100,000 g rather than 20,000 g to obtain a more accurate measure of levels of circulating EV-TF activity.

Cryo-electron microscopy of adipose tissue extracellular vesicles in obesity and type 2 diabetes mellitus.

Extracellular vesicles (EVs) are cell-derived membrane vesicles which play an important role in cell-to-cell communication and physiology. EVs deliver biological information from producing to recipient cells by transport of different cargo such as proteins, mRNAs, microRNAs, non-coding RNAs and lipids. Adipose tissue EVs could regulate metabolic and inflammatory interactions inside adipose tissue depots as well as distal tissues. Thus, adipose tissue EVs are assumed to be implicated in obesity-associated pathologies, notably in insulin resistance and type 2 diabetes mellitus (T2DM). In this study we for the first time characterize EVs secreted by visceral (VAT) and subcutaneous adipose tissue (SAT) of patients with obesity and T2DM with standard methods as well as analyze their morphology with cryo-electron microscopy. Cryo-electron microscopy allowed us to visualize heterogeneous population of EVs of various size and morphology including single EVs and EVs with internal membrane structures in samples from obese patients as well from the control group. Single vesicles prevailed (up to 85% for SAT, up to 75% for VAT) and higher proportion of EVs with internal membrane structures compared to SAT was typical for VAT. Decreased size of single and double SAT EVs compared to VAT EVs, large proportion of multilayered EVs and all EVs with internal membrane structures secreted by VAT distinguished obese patients with/without T2DM from the control group. These findings could support the idea of modified biogenesis of EVs during obesity and T2DM.

Membrane Protein Modification Modulates Big and Small Extracellular Vesicle Biodistribution and Tumorigenic Potential in Breast Cancers In Vivo.

Extracellular vesicles (EVs) are released by cells to mediate intercellular communication under pathological and physiological conditions. While small EVs (sEVs; <100-200nm, exosomes) are intensely investigated, the properties and functions of medium and large EVs (big EVs (bEVs); >200nm, microvesicles) are less well explored. Here, bEVs and sEVs are identified as distinct EV populations, and it is determined that bEVs are released in a greater bEV:sEV ratio in the aggressive human triple-negative breast cancer (TNBC) subtype. PalmGRET, bioluminescence-resonance-energy-transfer (BRET)-based EV reporter, reveals dose-dependent EV biodistribution at nonlethal and physiological EV dosages, as compared to lipophilic fluorescent dyes. Remarkably, the bEVs and sEVs exhibit unique biodistribution profiles, yet individually promote in vivo tumor growth in a syngeneic immunocompetent TNBC breast tumor murine model. The bEVs and sEVs share mass-spectrometry-identified tumor-progression-associated EV surface membrane proteins (tpEVSurfMEMs), which include solute carrier family 29 member 1, Cd9, and Cd44. tpEVSurfMEM depletion attenuates EV lung organotropism, alters biodistribution, and reduces protumorigenic potential. This study identifies distinct in vivo property and function of bEVs and sEVs in breast cancer, which suggest the significant role of bEVs in diseases, diagnostic and therapeutic applications.

Non-contact microfluidic analysis of the stiffness of single large extracellular vesicles from IDH1-mutated glioblastoma cells.

In preparation for leveraging extracellular vesicles (EVs) for disease diagnostics and therapeutics, fundamental research is being done to understand EV biological, chemical, and physical properties. Most published studies have investigated nanoscale EVs and focused on EV biochemical content. There is much less understanding of large microscale EV characteristics and EV mechanical properties. We recently introduced a non-contact microfluidic technique that measures the stiffness of large EVs (>1 μm diameter). This pilot study probes the robustness of the microfluidic technique to distinguish between EV populations by comparing stiffness distributions of large EVs derived from glioblastoma cell lines. EVs derived from cells expressing the IDH1 mutation, a common glioblastoma mutation known to disrupt lipid metabolism, were stiffer than those expressed from wild-type cells in a statistical comparison of sample medians. A supporting lipidomics analysis showed that the IDH1 mutation increased the amount of saturated lipids in EVs. Taken together, these data encourage further investigation into the potential of high-throughput microfluidics to distinguish between large EV populations that differ in biomolecular composition. These findings contribute to the understanding of EV biomechanics, in particular for the less studied microscale EVs.

Neuronal deletion of nSMase2 reduces the production of Aβ and directly protects neurons

Extracellular vesicles (EVs) have been proposed to regulate the deposition of Aβ. Multiple publications have shown that APP, amyloid processing enzymes and Aβ peptides are associated with EVs. However, very little Aβ is associated with EVs compared with the total amount Aβ present in human plasma, CSF, or supernatants from cultured neurons. The involvement of EVs has largely been inferred by pharmacological inhibition or whole body deletion of the sphingomyelin hydrolase neutral sphingomyelinase-2 (nSMase2) that is a key regulator for the biogenesis of at-least one population of EVs. Here we used a Cre-Lox system to selectively delete nSMase2 from pyramidal neurons in APP/PS1 mice (APP/PS1-SMPD3-Nex1) and found a ∼ 70% reduction in Aβ deposition at 6 months of age and ∼ 35% reduction at 12 months of age in both cortex and hippocampus. Brain ceramides were increased in APP/PS1 compared with Wt mice, but were similar to Wt in APP/PS1-SMPD3-Nex1 mice suggesting that elevated brain ceramides in this model involves neuronally expressed nSMase2. Reduced levels of PSD95 and deficits of long-term potentiation in APP/PS1 mice were normalized in APP/PS1-SMPD3-Nex1 mice. In contrast, elevated levels of IL-1β, IL-8 and TNFα in APP/PS1 mice were not normalized in APP/PS1-SMPD3-Nex1 mice compared with APP/PS1 mice. Mechanistic studies showed that the size of liquid ordered membrane microdomains was increased in APP/PS1 mice, as were the amounts of APP and BACE1 localized to these microdomains. Pharmacological inhibition of nSMase2 activity with PDDC reduced the size of the liquid ordered membrane microdomains, reduced the localization of APP with BACE1 and reduced the production of Aβ1–40 and Aβ1–42. Although inhibition of nSMase2 reduced the release and increased the size of EVs, very little Aβ was associated with EVs in all conditions tested. We also found that nSMase2 directly protected neurons from the toxic effects of oligomerized Aβ and preserved neural network connectivity despite considerable Aβ deposition. These data demonstrate that nSMase2 plays a role in the production of Aβ by stabilizing the interaction of APP with BACE1 in liquid ordered membrane microdomains, and directly protects neurons from the toxic effects of Aβ. The effects of inhibiting nSMase2 on EV biogenesis may be independent from effects on Aβ production and neuronal protection.

ЭНДОТЕЛИАЛЬНАЯ ДИСФУНКЦИЯ ПРИ ХРОНИЧЕСКОМ ОПИСТОРХОЗЕ

Relevance. The basis of the complex, in some cases, of interrelated pathological processes in opisthorchiasis invasion may be endothelial dysfunction, which provokes the risks of cardiovascular events, and is an important aspect in establishing pathogenetic connections between diseases of the liver and the cardiovascular system. When assessing the inflammatory process in the pathogenesis of invasion, there is increasing interest in the contribution of not only cellular elements and circulating inflammatory markers, but also in intercellular communication and regulation through microvesicles. Purpose of the study. To study the circulation of extracellular vesicles, which are associated with blood and endothelial cells, in patients with chronic opisthorchiasis. Materials and methods. The study was conducted on two groups of people (men and women aged 18 to 55 years (34.3±1.3 years)). The experimental group included patients with chronic opisthorchiasis (n=26). There was the control group (n=14) matched by gender and age with experimental group. The qualitative composition and quantity of extracellular vesicles (EVs) was determined by flow cytometry. Results. In the experimental group, there was a significant increase in the number of CD41+ events (platelet EVs) 2 times (from 120 to 245 events/μl (p = 0.030), CD45+ events (panleukocyte EVs) - more than 120 times (from 95 to 11713 events/μl (p=0. 0330), as well as a trend towards an increase in the rate for CD31+ events by 19.8 times (from 495 to 9818 events/μl (p=0.133) in comparison with control group. Screening assessment of the CD9+ population of platelet EVs (CD41+31+CD9+), endothelial EVs (CD41-31+9+) and leukocyte EVs (CD45+CD9+) showed a significant (p=0.0330) increase in events, while their content corresponded to the data obtained by CD31+41+ events in quantitative terms. For endothelial EVs, there was a significant increase in the proportion of CD9+ events from 23.3% in the control to 46.2% in the experimental group, while for leukocyte EVs there was no statistically significant change in the percentage ratio, and it was 9.9% in the control and 12.9% in the experimental group. Conclusions. These results can be considered from the perspective of platelet activation, as well as the development of the inflammatory process and endothelial dysfunction, where microvesicles can act as potential highly sensitive markers for opisthorchiasis invasion.

Receptor activator of nuclear factor-kappa B is enriched in CD9-positive extracellular vesicles released by osteoclasts.

Receptor activator of nuclear factor-kappa B (RANK)-containing extracellular vesicles (EVs) bind RANK-Ligand (RANKL) on osteoblasts, and thereby simultaneously inhibit bone resorption and promote bone formation. Because of this, they are attractive candidates for therapeutic bone anabolic agents. Previously, RANK was detected in 1 in every 36 EVs from osteoclasts by immunogold electron microscopy. Here, we have sought to characterize the subpopulation of EVs from osteoclasts that contains RANK in more detail. The tetraspanins CD9 and CD81 were localized in osteoclasts by immunofluorescence. EVs were visualized by transmission electron microscopy. A Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) and immunoaffinity isolations examined whether RANK is enriched in specific types of EVs. Immunofluorescence showed CD9 was mostly on or near the plasma membrane of osteoclasts. In contrast, CD81 was localized deeper in the osteoclast's cytosolic vesicular network. By interferometry, both CD9 and CD81 positive EVs from osteoclasts were small (56-83 nm in diameter), consistent with electron microscopy. The CD9 and CD81 EV populations were mostly distinct, and only 22% of the EVs contained both markers. RANK was detected by SP-IRIS in 2%-4% of the CD9-containing EVs, but not in CD81-positive EVs, from mature osteoclasts. Immunomagnetic isolation of CD9-containing EVs from conditioned media of osteoclasts removed most of the RANK. A trace amount of RANK was isolated with CD81. RANK was enriched in a subset of the CD9-positive EVs. The current study provides the first report of selective localization of RANK in subsets of EVs.

Plasma extracellular vesicle phenotyping for the differentiation of early-stage lung cancer and benign lung diseases.

The development of a minimally invasive technique for early-stage lung cancer detection is crucial to reducing mortality. Phenotyping of tumor-associated extracellular vesicles (EVs) has the potential for early-stage lung cancer detection, yet remains challenging due to the lack of sensitive, integrated techniques that can accurately detect rare tumor-associated EV populations in blood. Here, we integrated gold core-silver shell nanoparticles and nanoscopic mixing in a microfluidic assay for sensitive phenotypic analysis of EVs directly in plasma without EV pre-isolation. The assay enabled multiplex detection of lung cancer-associated markers PTX3 and THBS1 and canonical EV marker CD63 by surface-enhanced Raman spectroscopy, providing a squared correlation coefficient of 0.97 in the range of 103-107 EVs mL-1 and a limit of detection of 19 EVs mL-1. Significantly, our machine learning-based nanostrategy provided 92.3% sensitivity and 100% specificity in differentiating early-stage lung cancer from benign lung diseases, superior to the CT scan-based lung cancer diagnosis (92.3% sensitivity and 71.4% specificity). Overall, our integrated nanostrategy achieved an AUC value of 0.978 in differentiating between early-stage lung cancer patients (n = 28) and controls consisting of patients with benign lung diseases (n = 23) and healthy controls (n = 26), which showed remarkable diagnostic performance and great clinical potential for detecting the early occurrence of lung cancer.

87 Characterisation of extracellular vesicle populations secreted by bovine female reproductive tissues and embryos

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Extracellular Vesicles from Human Umbilical Cord-Derived MSCs Affect Vessel Formation In Vitro and Promote VEGFR2-Mediated Cell Survival

Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) have emerged as novel tools in regenerative medicine. Angiogenesis modulation is widely studied for the treatment of ischaemic diseases, wound healing, and tissue regeneration. Here, we have shown that EVs from human umbilical cord-derived MSCs can affect VEGFR2 signalling, a master regulator of angiogenesis homeostasis, via altering the phosphorylation of AKT. This translates into an inhibition of apoptosis, promoting exclusively cell survival, but not proliferation, in human microvascular endothelial cells. Interestingly, when comparing EVs from normoxic cells to those obtained from hypoxia (1% O2) preconditioned cells, hypoxia-derived EVs appear to have a slightly enhanced effect. Furthermore, when studied in a longer term endothelial-fibroblast co-culture angiogenesis model in vitro, both EV populations demonstrated a positive effect on vessel formation, evidenced by increased vessel networks with tubes of significantly larger diameters. Our data reveals that EVs selectively target components of the angiogenic pathway, promoting VEGFR2-mediated cell survival via enhancement of AKT activation. Our data show that EVs are able to enhance specific components of the VEGF signalling pathway and may have therapeutic potential to support endothelial cell survival.

Extracellular Vesicles from Bothrops jararaca Venom Are Diverse in Structure and Protein Composition and Interact with Mammalian Cells.

Snake venoms are complex cocktails of non-toxic and toxic molecules that work synergistically for the envenoming outcome. Alongside the immediate consequences, chronic manifestations and long-term sequelae can occur. Recently, extracellular vesicles (EVs) were found in snake venom. EVs mediate cellular communication through long distances, delivering proteins and nucleic acids that modulate the recipient cell's function. However, the biological roles of snake venom EVs, including possible cross-organism communication, are still unknown. This knowledge may expand the understanding of envenoming mechanisms. In the present study, we isolated and characterized the EVs from Bothrops jararaca venom (Bj-EVs), giving insights into their biological roles. Fresh venom was submitted to differential centrifugation, resulting in two EV populations with typical morphology and size range. Several conserved EV markers and a subset of venom related EV markers, represented mainly by processing enzymes, were identified by proteomic analysis. The most abundant protein family observed in Bj-EVs was 5'-nucleotidase, known to be immunosuppressive and a low abundant and ubiquitous toxin in snake venoms. Additionally, we demonstrated that mammalian cells efficiently internalize Bj-EVs. The commercial antibothropic antivenom partially recognizes Bj-EVs and inhibits cellular EV uptake. Based on the proteomic results and the in vitro interaction assays using macrophages and muscle cells, we propose that Bj-EVs may be involved not only in venom production and processing but also in host immune modulation and long-term effects of envenoming.

Smaller extracellular vesicles are released from pancreatic cancer cells by the alteration of the lipid composition under low glucose conditions

Extracellular vesicles (EVs) released from cells into the blood facilitate intercellular communication and serve as new biomarkers to understand the pathophysiology of several conditions. Although the importance of the cargo inside EVs has been extensively studied, the sizes of EVs that vary with different types of cancers are relatively poorly explored. Here, we show that pancreatic cancer cell-derived EVs are significantly smaller than non-cancer cell-derived EVs. The smaller size distribution of these EVs was confirmed by specifically isolating and examining tumor-derived EVs from the heterogeneous EV population isolated from the sera of patients with pancreatic ductal adenocarcinoma. In vitro analyses mimicking tumor microenvironment conditions revealed that low glucose conditions reduced the size distribution and increased the level of unsaturated fatty acids in the tumor-derived EVs. Because the lipid composition defines the fluidity of the membrane, the results suggest that the alterations in the size of EVs could be due to the alteration of the fluidity and stability of the membrane covering the EVs. Furthermore, the uptake of smaller EVs by recipient cells was increased, which may lead to enhanced functional results. These results provide fundamental insights into the factors defining the size of EVs, which may be important for developing cancer screening methods and understanding cancer-related pathophysiology.