Extracellular Vesicles Markers Research Articles

Extracellular Vesicles from Adipose Tissue-Derived Stromal Cells Stimulate Angiogenesis in a Scaffold-Dependent Fashion.

The extracellular vesicles (EVs) secreted by adipose tissue-derived stromal cells (ASC) are microenvironment modulators in tissue regeneration by releasing their molecular cargo, including miRNAs. However, the influence of ASC-derived extracellular vesicles (ASC-EVs) on endothelial cells (ECs) and vascularisation is poorly understood. The present study aimed to determine the pro-angiogenic effects of ASC-EVs and explore their miRNA profile. EVs were isolated from normoxic and hypoxic cultured ASC conditioned culture medium. The miRNA expression profile was determined by miRseq, and EV markers were determined by Western blot and immunofluorescence staining. The uptake dynamics of fluorescently labelled EVs were monitored for 24h. ASC-EVs' pro-angiogenic effect was assessed by sprouting ex vivo rat aorta rings in left ventricular-decellularized extracellular matrix (LV dECM) hydrogel or basement membrane hydrogel (Geltrex®). ASC-EVs augmented vascular network formation by aorta rings. The vascular network topology and stability were influenced in a hydrogel scaffold-dependent fashion. The ASC-EVs were enriched for several miRNA families/clusters, including Let-7 and miR-23/27/24. The miRNA-1290 was the highest enriched non-clustered miRNA, accounting for almost 20% of all reads in hypoxia EVs. Our study revealed that ASC-EVs augment in vitro and ex vivo vascularisation, likely due to the enriched pro-angiogenic miRNAs in EVs, particularly miR-1290. Our results show promise for regenerative and revascularisation therapies based on ASC-EV-loaded ECM hydrogels.

P-290 Isolation and characterization of extracellular vesicles from human endometrial organoids

Abstract Study question Do human endometrial organoids produce extracellular vesicles (EVs)? Summary answer Endometrial organoids release EVs (ORG-EVs) that we successfully and comprehensively characterize according to guidelines. What is known already EVs play a crucial role in cellular communication, garnering increasing attention in physiological and pathological processes. Although the enormous potential of EVs has been extensively demonstrated across a large variety of research fields, the promise held by EVs derived from human endometrial organoids is still largely unexplored. Indeed, nowadays there is limited research available on the presence, role, and function of EVs derived from organoids of female reproductive tissue samples. This underscores the importance of adopting standardized methods for isolating, characterizing, and reporting findings, which is crucial for advancing in this promising and innovative, yet challenging, research field. Study design, size, duration This study involved establishing organoid cultures from endometrial biopsies of healthy women (n = 10) of reproductive age, who underwent ovarian surgery between January and October 2023. ORG-EVs were isolated using differential ultracentrifugation. Characterization of ORG-EVs, including assessment of their biophysical and biochemical properties, adhered to guidelines established by the International Society for Extracellular Vesicles (ISEV). Participants/materials, setting, methods Endometrial specimens from 10 women were employed to establish organoid cultures. ORG-EVs were isolated from spent medium through differential ultracentrifugation. Morphology, size distribution and concentration analysis were performed using Transmission Electron Microscopy (TEM) and Nanoparticle Tracking Analysis (NTA). Western blotting was used to confirm the presence of EVs markers. Main results and the role of chance TEM analysis confirmed the presence of isolated ORG-EVs, elucidating their morphology and structural integrity. NTA revealed that ORG-EVs had an average size of 160.4 ± 6.2 nm (mean ± SD) and a mean concentration of 5.2E+11 ± 2.1E+10 particles/ml (mean ± SD). Western blot analysis further confirmed the vesicular nature of the assessed particles, demonstrating positivity for standard EV markers such as tetraspanins (CD63 and CD9), and soluble proteins (ALIX and TSG101), while excluding the endoplasmic reticulum-resident protein calnexin (negative control). Additionally, ORG-EVs exhibited a positive signal for mucin1 (MUC1), a glycoprotein present on the surface of endometrial epithelial cells, thereby confirming their cellular origin. Limitations, reasons for caution This is a pilot study, characterized by a small sample size requiring larger cohorts to validate results and, at the same time, to undertake deeper exploration of ORG-EVs’ molecular cargo. Emphasizing the critical need for standardized isolation and characterization methods is paramount. Wider implications of the findings Endometrial organoids enhance EVs production, providing a powerful tool for reproductive aspects studies, and improving regenerative medicine and artificial reproductive technology. These initial findings propel further exploration, unveiling of ORG-EVs’ molecular cargo aiming to elucidate their potential role in human female reproductive tissues and mechanism related to embryo implantation process. Trial registration number not applicable

O-005 The in vitro maturation rate of human oocytes is enhanced following uptake of extracellular vesicles derived from mature follicles

Abstract Study question Could follicular fluid derived extracellular vesicles (ffEVs) impact human oocyte in vitro maturation (IVM)? Summary answer Supplementation of IVM culture with ffEVs isolated from mature follicles enhanced oocyte maturation rates by > 20%, inducing changes in oocyte protein profile and organelle distribution. What is known already IVM involves the culture of immature germinal vesicle (GV) oocytes under set laboratory conditions to allow for their transition to mature metaphase II (MII) stage, which is confirmed by the extrusion of the first polar body. Efficient IVM could circumvent aggressive controlled ovarian stimulation (COS), reduce the cost and broaden the repertoire of infertility treatments. Animal studies suggest that extracellular vesicles (EVs), membranous nanosized vesicles containing different molecular content (e.g. nucleic acids, proteins) and present in the ovarian follicular fluid could facilitate oocyte maturation. The uptake of ffEVs by bovine, equine and feline oocytes, but not human, has been demonstrated. Study design, size, duration Women undergoing transvaginal oocyte retrieval after COS (n = 83) at University Hospital Zurich donated follicular fluid (n = 54 single follicles) and/or immature GV oocytes (n = 95). We aimed to: a) define differences in the protein cargo of ffEVs derived from follicles containing mature oocytes (MII-ffEVs, n = 10) versus (vs.) immature oocytes (GV-ffEVS, n = 5; metaphase I MI-ffEVs, n = 5), b) demonstrate the capacity of GV oocytes to uptake mature MII-ffEVs and c) determine the effect of MII-ffEVs supplementation on oocyte maturation. Participants/materials, setting, methods ffEVs were isolated by ultracentrifugation. The protein content of ffEVs was analysed by mass spectrometry. The uptake of fluorescently-labelled MII-ffEVs by GV oocytes (n = 15) was assessed by confocal microscopy. GVs were cultured for rescue-IVM in a timelapse incubator with MII-ffEVs (n = 45 GVs) or without (n = 40 GVs) and extrusion of polar body denoted maturation. The impact of MII-ffEVs supplementation on IVM-matured oocytes was assessed through single-cell proteomics and intracellular organelles appearance with transmission electron microscopy (TEM). Main results and the role of chance Successful isolation of ffEVs was confirmed by TEM and Western blotting analysis for characteristic protein markers of EVs (TSG101 and CD63). Mass spectrometry identified a total of 1340 proteins across ffEVs samples. Statistical analysis revealed differentially abundant proteins (DAPs) in ffEVs: 61 DAPs in the comparison MII-ffEVs vs. GV-ffEVs, 15 DAPs in MII-ffEVs vs. MI-ffEVs and 14 DAPs in MI-ffEVs vs GV-ffEVs (FDR<0.2). Among the 61 DAPs, 7 proteins in higher abundance in MII-ffEVs (>log2 fold change) were related to oocyte maturation, lipid accumulation, calcium binding and the coagulation cascade (e.g. FABP5, S100A9, F12, SERPINA10). Confocal microscopy confirmed the uptake of MII-ffEVs by GV oocytes after 24 hours of co-incubation. Supplementation of IVM culture with MII-ffEVs for 48 hours significantly increased the oocyte maturation rate compared to control by 22.8±9.4% (77.8% vs 55% mature oocytes respectively; P-value=0.0372). Single oocyte proteomics of IVM-matured oocytes revealed 59 DAPs (FDR 0.1) between ffEVs-supplemented and control groups. Among them, 37 DAPs were in higher abundance in ffEVs-supplemented mature oocytes (>log2 FC) including Hyaluronan Synthase 1 (HAS1) that is associated with oocyte maturation (6.55 fold increase). TEM revealed differences in oocyte organelle distribution and appearance, particularly of endoplasmic reticulum (RE) and RE-mitochondria complexes. Limitations, reasons for caution This study utilised immature oocytes from stimulated cycles, therefore these results should be interpreted within the context of rescue-IVM potential. Wider implications of the findings Our findings pave the way for further mechanistic studies on the role of ffEVs in supporting oocyte maturation and inspire the development of clinical IVM supplements as well as novel complementary regimes for COS based on ffEVs or their associated specific cargo. Trial registration number N/A

P-014 Extracellular vesicles (EVs) from female reproductive tracts positively affect sperm capacitation and progressive motility

Abstract Study question Do EVs from human follicular fluid (FF-EVs), cervicovaginal fluid (CVF-EVs), and endometrial stromal cells spent medium (ESCs-EVs) induce sperm capacitation and enhance sperm motility? Summary answer In humans, EVs from different female reproductive tracts actively contribute to sperm maturation by inducing capacitation and enhancing sperm progressive motility. What is known already In the human body, EVs represent crucial players in cell-to-cell communication. EVs presence has been detected also in the female and male reproductive systems, specifically in seminal, follicular, oviductal, endometrial and vaginal fluid, as well as in embryo secretions. Moreover, EVs have been shown to be of pivotal importance in the fertilization process, as they support the maturation of spermatozoa and oocytes, as well as embryos in their development and endometrial implantation. The majority of these studies, though, have been conducted in animal models, underscoring the necessity to replicate analogous investigations also in humans. Study design, size, duration Follicular fluid (FF, n = 8), cervicovaginal fluid (CVF, n = 20) and endometrial tissue (n = 8) samples were collected from women undergoing assisted reproductive technology (ART) cycles at San Raffaele Hospital Fertility Centre (Milan). CVF were collected 2 and 7 days after the LH peak (CVF LH + 2 and CVF LH + 7, respectively). ESCs-EVs were isolated from both decidualized (dESCs-EVs) and not decidualized (eESCs-EVs) endometrial cells. Semen samples (n = 15) were collected from normozoospermic men. Participants/materials, setting, methods EVs were isolated using differential centrifugations and characterized using Transmission Electron Microscopy (TEM), Nanoparticle Tracking Analysis (NTA), and western blotting (WB). EVs capture by spermatozoa was assessed using flow cytometry. EVs impact on sperm progressive motility was evaluated following WHO guidelines. EVs potential to induce sperm capacitation was examined through sperm acrosome reaction (AR) induction using a Ca2+ ionophore, followed by Coomassie blue staining. All experiments were performed using EVs at 50 and 100 μg/mL. Main results and the role of chance Isolated EVs displayed comparable size, with mean diameters ranging from 147,9±11,6 nm to 168,1±6,2 nm (mean ± SEM) and average concentrations from 9,53E+10 ± 8,11E+09 to 2,70E+11 ± 3,49E+10 particles/ml (mean ± SEM). TEM analysis confirmed their round-shaped morphology and structural integrity, while WB confirmed their positivity for EVs markers (Alix, TSG101, CD63, CD9). Spermatozoa efficiency in capturing these fluorescently-labeled EVs, measured as the percentage of fluorescence-positive spermatozoa, reached up to 3,6±1,1% for FF-EVs, 12,1±3,7% for CVF-EVs and 9,4±1,9% for ESCs-EVs (mean ± SEM). Sperm cells co-incubated with any EVs group maintained unaffected vitality. Following a 4-hour incubation with EVs, a rise in the proportion of progressively motile spermatozoa was observed, with percentages of progressively motile spermatozoa ranging from a minimum of 45,4±3,8% for CVF-EVs LH + 7 to a maximum of 52,7±2,7% for dESCs-EVs (mean ± SEM). This increase resulted to be statistically significant (p < 0.05 and p < 0.01, respectively) compared to the untreated (UT) condition, which exhibited 26,2±0,7% of progressively motile sperm cells. Spermatozoa that completed the AR, indicating capacitation, after 4-hour incubation with EVs resulted to be up to 25,8±3,2% for FF-EVs, 35,0±6,6% for CVF-EVs and 26,9±3,0% for ESCs-EVs, compared to an average of 13% in the UT control. Limitations, reasons for caution This study constitutes a pilot investigation characterized by a limited sample size, necessitating larger cohorts to validate results. Wider implications of the findings Studied EVs may exert beneficial effects on capacitation and progressive motility also on asthenozoospermic (AS) men’s spermatozoa. Therefore, they could be used in ART cycles to prime spermatozoa from AS men and increase the percentage of sperm cells displaying progressive motility, potentially favouring fertilization via classical IVF instead of ICSI. Trial registration number not applicable

O-266 Multi-omics landscape of uterine fluid-derived extracellular vesicles across the menstrual cycle

Abstract Study question Do extracellular vesicles (EVs) mirror the cyclic physiological changes in endometrial tissue, reflecting the endometrial maturation along the entire menstrual cycle? Summary answer Multi-omics data highlights the importance of EVs during the mid-secretory phase and EVs cargo reflects the state of the tissue throughout the menstrual cycle. What is known already Extracellular vesicles isolated from uterine fluid (UF) during the mid-secretory phase have been characterized for their pivotal role in embryo-maternal communication, particularly in the context of microRNAs (miRNAs) and mRNA. EVs have been posited as potential biomarkers in a minimally invasive liquid biopsy approach for assessing endometrial receptivity, hopefully replacing the endometrial tissue biopsy-based testing. However, the dynamics of EVs and their cargo throughout the distinct phases of the menstrual cycle remain unknown. This is the first study to elucidate UF-EV content throughout the maturation of the endometrium. Study design, size, duration Whole-exome RNA and small-RNA sequencing was performed on 15 paired samples, comprising UF and endometrial biopsy (EB), obtained from healthy female subjects across the menstrual cycle. This initial dataset was expanded with 35 additional UF samples. Cumulatively, the analysis encompassed samples (EB and UF) across the menstrual cycle’s four distinct phases: proliferative (n = 10), early-secretory (ES) (n = 17), mid-secretory (MS) (n = 20), and late-secretory (LS) (n = 18). Additionally, 20 UF-EV samples were used for flow cytometry surface proteome analysis. Participants/materials, setting, methods Participants were excluded when infertility-associated conditions were found and included when they had at least one child born, with no hormonal medications used for three months before the sampling. EVs were extracted from UF with size-exclusion chromatography (SEC) and subsequently with glycosaminoglycan-based precipitation. The EVs were characterized with nanoparticle tracking analysis, western blot, transmission electron microscopy and flow-cytometry surface proteome analysis (MACSPlex) for 37 surface markers. Single-end RNA and small-RNA sequencing were performed on NextSeq1000. Main results and the role of chance The transcriptome, miRNome, and protein surface markers of UF-EVs were characterized throughout the menstrual cycle. Transcriptome profiling was performed in comparison to the preceding phase. During the ES phase, 5,507 differentially expressed genes (DEGs) were observed, most of them being downregulated and associated with G-coupled-protein signaling, while in the MS phase, 867 DEGs were identified associated with nucleosome assembly and cell differentiation. Interestingly in the LS phase, the histone genes associated with nucleosome assembly were upregulated compared to MS, highlighting that histone dynamics change corresponding to the menstrual cycle. The exploration of gene ontology revealed consistent enrichment of genes related to extracellular vesicles in UF-EV and EBs across the menstrual cycle. The miRNA analysis of UF-EVs and EB showed significant overlap in miRNA expression level especially in the MS phase. Furthermore, we found 16 unique miRNAs in the mid-secretory phase whose targets were associated with embryo morphogenesis and cell adhesion. MACSPlex analysis revealed that CD9 and CD63, canonical EV markers, were significantly abundant during the MS phase, indicative of an enriched secretion of EVs during the preparation of the endometrium for embryo implantation. These insights underscore that the cargo of UF-EVs reflects the dynamic changes in the endometrial tissue. Limitations, reasons for caution The research on UF-EVs faced notable challenges due to the irregular and non-standardized collection methods of UF in gynecological practice. This variability in UF collection introduces potential confounding factors. The study also operated with a limited sample size, underscoring the need for further validation. Wider implications of the findings Our study indicates that the UF-EVs reflect the menstrual cycle-related molecular changes and UF-EV analysis could be potentially used to follow the menstrual cycle-related molecular processes in the uterine environment. Trial registration number not applicable

Optimized AF4 combined with density cushion ultracentrifugation enables profiling of high-purity human blood extracellular vesicles.

Extracellular vesicles (EVs) have emerged as a promising tool for clinical liquid biopsy. However, the identification of EVs derived from blood samples is hindered by the presence of abundant plasma proteins, which impairs the downstream biochemical analysis of EV-associated proteins and nucleic acids. Here, we employed optimized asymmetric flow field-flow fractionation (AF4) combined with density cushion ultracentrifugation (UC) to obtain high-purity and intact EVs with very low lipoprotein contamination from human plasma and serum. Further proteomic analysis revealed more than 1000 EV-associated proteins, a large proportion of which has not been previously reported. Specifically, we found that cell-line-derived EV markers are incompatible with the identification of plasma-EVs and proposed that the proteins MYCT1, TSPAN14, MPIG6B and MYADM, as well as the traditional EV markers CD63 and CD147, are plasma-EV markers. Benefiting from the high-purity of EVs, we conducted comprehensive miRNA profiling of plasma EVs and nanosized particles (NPs), as well as compared plasma- and serum-derived EVs, which provides a valuable resource for the EV research community. Overall, our findings provide a comprehensive assessment of human blood EVs as a basis for clinical biopsy applications.

Spinal cord injury disrupts plasma extracellular vesicles cargoes leading to neuroinflammation in the brain and neurological dysfunction in aged male mice

Aged individuals with spinal cord injury (SCI) are prevalent with increased mortality and worse outcomes. SCI can cause secondary brain neuroinflammation and neurodegeneration. However, the mechanisms contributing to SCI-induced brain dysfunction are poorly understood. Cell-to-cell signaling through extracellular vesicles (EVs) has emerged as a critical mediator of neuroinflammation, including at a distance through circulation. We have previously shown that SCI in young adult (YA) male mice leads to robust changes in plasma EV count and microRNAs (miRs) content. Here, our goal was to investigate the impact of old age on EVs and brain after SCI. At 24 h post-injury, there was no difference in particle count or size distribution between YA and aged mice. However, aged animals increased expression of EV marker CD63 with SCI. Using the Fireplex® miRs assay, Proteomics, and mass spectrometry-based Lipidomics, circulating EVs analysis identified distinct profiles of miRs, proteins, and lipid components in old and injury animals. In vitro, plasma EVs from aged SCI mice, at a lower concentration comparable to those of YA SCI mice, induced the secretion of pro-inflammatory cytokines and neuronal apoptosis. Systemic administration of plasma EVs from SCI animals was sufficient to impair general physical function and neurological function in intact animals, which is associated with pro-inflammatory changes in the brain. Furthermore, plasma EVs from young animals had rejuvenating effects on naïve aged mice. Collectively, these studies identify the critical changes in circulating EVs cargoes after SCI and in aged animals and support a potential EV-mediated mechanism for SCI-induced brain changes.

Spinal cord injury disrupts plasma extracellular vesicles cargoes leading to neuroinflammation in the brain and neurological dysfunction in aged male mice

Aged individuals with spinal cord injury (SCI) are prevalent with increased mortality and worse outcomes. SCI can cause secondary brain neuroinflammation and neurodegeneration. However, the mechanisms contributing to SCI-induced brain dysfunction are poorly understood. Cell-to-cell signaling through extracellular vesicles (EVs) has emerged as a critical mediator of neuroinflammation, including at a distance through circulation. We have previously shown that SCI in young adult (YA) male mice leads to robust changes in plasma EV count and microRNAs (miRs) content. Here, our goal was to investigate the impact of old age on EVs and brain after SCI. At 24 h post-injury, there was no difference in particle count or size distribution between YA and aged mice. However, aged animals increased expression of EV marker CD63 with SCI. Using the Fireplex® miRs assay, Proteomics, and mass spectrometry-based Lipidomics, circulating EVs analysis identified distinct profiles of miRs, proteins, and lipid components in old and injury animals. In vitro, plasma EVs from aged SCI mice, at a lower concentration comparable to those of YA SCI mice, induced the secretion of pro-inflammatory cytokines and neuronal apoptosis. Systemic administration of plasma EVs from SCI animals was sufficient to impair general physical function and neurological function in intact animals, which is associated with pro-inflammatory changes in the brain. Furthermore, plasma EVs from young animals had rejuvenating effects on naïve aged mice. Collectively, these studies identify the critical changes in circulating EVs cargoes after SCI and in aged animals and support a potential EV-mediated mechanism for SCI-induced brain changes.

An anion exchange membrane sensor detects EGFR and its activity state in plasma CD63 extracellular vesicles from patients with glioblastoma

We present a quantitative sandwich immunoassay for CD63 Extracellular Vesicles (EVs) and a constituent surface cargo, EGFR and its activity state, that provides a sensitive, selective, fluorophore-free and rapid alternative to current EV-based diagnostic methods. Our sensing design utilizes a charge-gating strategy, with a hydrophilic anion exchange membrane functionalized with capture antibodies and a charged silica nanoparticle reporter functionalized with detection antibodies. With sensitivity and robustness enhancement by the ion-depletion action of the membrane, this hydrophilic design with charged reporters minimizes interference from dispersed proteins, thus enabling direct plasma analysis without the need for EV isolation or sensor blocking. With a LOD of 30 EVs/μL and a high relative sensitivity of 0.01% for targeted proteomic subfractions, our assay enables accurate quantification of the EV marker, CD63, with colocalized EGFR by an operator/sample insensitive universal normalized calibration. We analysed untreated clinical samples of Glioblastoma to demonstrate this new platform. Notably, we target both total and “active” EGFR on EVs; with a monoclonal antibody mAb806 that recognizes a normally hidden epitope on overexpressed or mutant variant III EGFR. Analysis of samples yielded an area-under-the-curve (AUC) value of 0.99 and a low p-value of 0.000033, surpassing the performance of existing assays and markers.

Heterogeneous Analysis of Extracellular Vesicles for Osteosarcoma Diagnosis.

Osteosarcoma (OS) is the most prevalent primary tumor of bones, often diagnosed late with a poor prognosis. Currently, few effective biomarkers or diagnostic methods have been developed for early OS detection with high confidence, especially for metastatic OS. Tumor-derived extracellular vesicles (EVs) are emerging as promising biomarkers for early cancer diagnosis through liquid biopsy. Here, we report a plasmonic imaging-based biosensing technique, termed subpopulation protein analysis by single EV counting (SPASEC), for size-dependent EV subpopulation analysis. In our SPASEC platform, EVs are accurately sized and counted on plasmonic sensor chips coated with OS-specific antibodies. Subsequently, EVs are categorized into distinct subpopulations based on their sizes, and the membrane proteins of each size-dependent subpopulation are profiled. We measured the heterogeneous expression levels of the EV markers (CD63, BMP2, GD2, and N-cadherin) in each of the EV subsets from both OS cell lines and clinical plasma samples. Using the linear discriminant analysis (LDA) model, the combination of four markers is applied to classify the healthy donors (n = 37), nonmetastatic OS patients (n = 13), and metastatic patients (n = 12) with an area under the curve of 0.95, 0.92, and 0.99, respectively. SPASEC provides accurate EV sensing technology for early OS diagnosis.

#2382 Renal tubule-derived EVs carrying complement 3 aggravate CKD vascular calcification by downregulating autophagy in vascular smooth muscle cells

Abstract Background and Aims Vascular calcification (VC) is a serious complication of chronic kidney disease (CKD) patients, recognized in KDIGO guidelines as a leading risk factor for cardiovascular diseases. Despite acknowledgment, the mechanisms of VC in CKD remain unclear. The kidneys intricately regulate blood vessel pathophysiology. Clinical studies suggest a correlation between elevated blood complement levels in CKD and arterial calcification, hinting at complement involvement in CKD-associated VC. Renal-origin complement, a significant source of circulating complement, activates in CKD. Extracellular vesicles (EVs), mediating intercellular communication, transport complement-related proteins in various physiological and pathological processes. Studies suggest that plasma EV rather than EV-depleted plasma causes pathogenic calcium salt deposition in smooth muscle cells. This study explores how renal-origin EVs carrying complement mediate CKD-associated VC. Method From October 2021 to August 2023, data from 110 clinical patients were gathered, encompassing both blood C3 levels and CT images of the thoracic aorta. Subsequently, we conducted a scoring analysis of the CT images and carried out correlation analyses. A CKD-associated VC model was induced by feeding C57BL/6j mice an adenine and high-phosphate diet for 16 weeks. Techniques, including WB, PCR, and immunofluorescence, detected complement activation and localization in renal tissues. EVs were isolated from kidney tissue and plasma and identified by NTA, electron microscopy, WB, etc. WB examined C3 and tubular markers of EVs. At the same time, the Elisa method was used to detect C3 in plasma, plasma EV, and EV-depleted plasma. Immunofluorescence investigated tubular-derived EVs uptake in calcified arteries. Tubular-derived EVs, C3a, C3aR receptor inhibitors (SB290157), and autophagy inhibitors (3-MA) intervened in high-phosphate-induced vascular smooth muscle cells (VSMCs). PCR and WB assessed calcification indicators, complement receptors, and autophagy-related indicators. Alizarin Red staining observed calcification nodules. Transcriptome sequencing was performed on control, high-phosphate, and renal tubular-derived EV intervention groups of VSMCs. An in vivo model infused renal tubular-derived EVs and injected C3aR inhibitors and autophagy inhibitors, measuring complement activation and VC indicators. Results There is a correlation between C3 and thoracic aorta calcium score in CKD patients, with a correlation coefficient of 0.3750 (P < 0.01). The CKD-associated VC mice model revealed complement C3 activation in renal tubules, secreted into the blood through EVs. The C3 proportion of plasma EV in CKD patients was significantly higher than that in the control group, but there was no statistical difference between the EV-depleted plasma control group and the CKD group. Immunofluorescence indicated tubular-derived EV uptake by the calcified vascular wall. In vitro, tubular-derived EVs enhanced VSMC osteogenic differentiation and upregulated C3aR, mimicking C3a intervention effects. C3aR receptor inhibitors produced opposing effects. Transcriptome sequencing suggested renal tubular-derived EVs downregulated VSMC autophagy-related pathways; in vitro autophagy inhibition reversed osteogenic differentiation. In vivo, reinfusing renal tubular-derived EVs exacerbated complement activation and VC, while injecting C3aR inhibitors and autophagy inhibitors alleviated CKD-associated VC. Conclusion This study confirms that tubular-derived EVs carrying C3 downregulate autophagy in VSMCs, mediating CKD-associated VC. These findings provide insights into understanding CKD-associated VC initiation and progression, revealing potential therapeutic targets.

High throughput Surface Epitope Immunoaffinity Isolation of Extracellular Vesicles and Downstream Analysis

Abstract Extracellular vesicles (EVs), including exosomes, have significant potential for diagnostic and therapeutic applications. The lack of standardized methods for efficient and high throughput isolation and analysis of EVs, however, has limited their widespread use in clinical practice. Surface epitope immunoaffinity (SEI) isolation utilises affinity ligands, including antibodies, aptamers, or lectins, that target specific surface proteins present on EVs. Paramagnetic bead-SEI isolation represents a fit-for-purpose solution for the reproducible, high throughput isolation of EVs from biofluids and downstream analysis of RNA, protein and lipid biomarkers that is compatible with clinical laboratory workflows. This study evaluates a new surface epitope immunoaffinity isolation method for enriching subpopulations of EVs. EVs were isolated from human plasma using a bead-based SEI method designed for on-bead and downstream analysis of EV-associated RNA and protein biomarkers. Western blot analysis confirmed the presence of EV markers in the captured nanoparticles. Mass spectrometry analysis of the SEI lysate identified over 1500 proteins, with the top 100 including known EV-associated proteins. miRNA sequencing followed by RT-qPCR analysis identified EV-associated miRNA transcripts. Using SEI, EVs were isolated using automated high throughput particle moving instruments, demonstrating equal or higher protein and miRNA yield and recovery compared to manual processing. SEI is a rapid, efficient, and high throughput method for isolating enriched populations of EVs; effectively reducing contamination and enabling the isolation of a specific subpopulation of EVs. In this study, high throughput EV isolation and RNA extraction have been successfully implemented. This technology holds great promise for advancing the field of EV research and facilitating their application for biomarker discovery and clinical research.

Arf GTPase-Activating proteins ADAP1 and ARAP1 regulate incorporation of CD63 in multivesicular bodies.

Arf GTPase-activating proteins (ArfGAPs) mediate the hydrolysis of GTP bound to ADP-ribosylation factors. ArfGAPs are critical for cargo sorting in the Golgi-to-ER traffic. However, the role of ArfGAPs in sorting into intralumenal vesicles (ILVs) in multivesicular bodies (MVBs) in post-Golgi traffic remains unclear. Exosomes are extracellular vesicles (EVs) of endosomal origin. CD63 is an EV marker. CD63 is enriched ILVs in MVBs of cells. However, the secretion of CD63 positive EVs has not been consistent with the data on CD63 localization in MVBs, and how CD63-containing EVs are formed is yet to be understood. To elucidate the mechanism of CD63 transport to ILVs, we focused on CD63 localization in MVBs and searched for the ArfGAPs involved in CD63 localization. We observed that ADAP1 and ARAP1 depletion inhibited CD63 localization to enlarged endosomes after Rab5Q79L overexpression. We tested epidermal growth factor (EGF) and CD9 localization in MVBs. We observed that ADAP1 and ARAP1 depletion inhibited CD9 localization in enlarged endosomes but not EGF. Our results indicate ADAP1 and ARAP1, regulate incorporation of CD63 and CD9, but not EGF, in overlapped and different MVBs. Our work will contribute to distinguish heterogenous ILVs and exosomes by ArfGAPs.

Abstract PO3-24-11: Impact of Circulating Extracellular Vesicles derived from Obese Women on Pro-Tumor Functions of Stromal Fibroblasts in Breast Cancer

Abstract The incidence of obesity has risen sharply worldwide, and growing evidence shows its impact on several malignancies, including breast cancer (BC). Excessive adiposity and high body mass index (BMI) significantly affect BC incidence, prognosis, and therapeutic response of patients. Indeed, obese women exhibit larger tumor size, limph node involvement, metastatic spread, and poorer survival outcomes. Preclinical and clinical studies indicated that the obese setting provides local and systemic modifications that may stimulate BC progression and influence tumor microenvironment, particularly composed of stromal fibroblasts (SF). The mechanisms behind this interplay are likely to be multifactorial and may involve important adipocyte-derived mediators, such as adipokines and growth factors. Aside from these soluble factors, extracellular vesicles (EVs), nanoscale lipid-bilayer enclosed vesicles released by a variety of cells, are emerging as powerful regulators of cell-to-cell communication able to promote BC initiation, growth, metastatic dissemination and drug resistance. However, the potential impact of obesity-derived EVs in affecting tumor-stimulatory features of SF has not been yet investigated. To this aim, circulating EVs were isolated from the serum of women categorized as normal weight (NW; 18 > BMI < 24.9 Kg/m2) and overweight/obese (OW/Ob; BMI > 25 Kg/m2). Immortalised human mammary control fibroblasts (CF), their cancer-associated counterparts (exp-CAFs), and human CAFs, isolated from biopsies of primary BC, were used as experimental models for stromal fibroblasts. Human MCF-7 and T47D BC cells were employed as experimental cell models for mammary carcinoma in co-culture experiments. Circulating EVs were successfully extracted from the serum of NW and OW/Ob women, and characterized by their shapes, size and protein markers. Transmission Electron Microscopy (TEM), and Nanoparticle Tracking Analysis (NTA) showed that EVs in the isolated fractions were oval or bowl-shaped with a size range between 50-200 nm. In accordance with the Minimal Information for Studies of Extracellular Vesicles (MISEV) 2018, enrichment of the EV markers TSG101, CD81, and CD63 were all detected in the EV isolated fractions. Interestingly, elevated levels of circulating EVs were detected in OW/Ob women compared to NW subjects. Treatment of CF with OW/Ob-EVs resulted in an increased cell motility, invasiveness, contractility along with an up-regulation of CF markers. BC cells cocultured with conditioned medium (CM) derived from OW/Ob-EV-treated CF exhibited higher anchorage-independent growth and migratory capacity than BC cells cocultured with CM derived from NW-EV-treated CF. Moreover, OW/Ob-EVs enhanced tumor-stimulatory activities of exp-CAFs and CAFs, further highlighting how obese-related EVs may have the potential to modulate fibroblast phenotype in BC. Finally, cytokine arrays revealed that treatment of CF with OW/Ob EVs was associated with an enhanced release of Fibroblast Growth Factor 19 and Cystatin C, factors that are closely related to BC growth and development. In conclusion, our data provide first evidence for the functional importance of EVs isolated from obese women in impacting tumor-stroma cross-talk and drive BC progression. Since obesity and its pathophysiological sequelae are on the rise, this new knowledge may help to identify specific biomarkers and innovative targets (i.e. FGF-19, and Cystatin C) that may allow a personalized management of patients affected by BC and obesity. Citation Format: Ines Barone, Piercarlo Del Console, Felice Maria Accattatis, Luca Gelsomino, Giuseppina Augimeri, Ennio Avolio, Sebastiano Andò, Daniela Bonofiglio, Cinzia Giordano, Stefania Catalano. Impact of Circulating Extracellular Vesicles derived from Obese Women on Pro-Tumor Functions of Stromal Fibroblasts in Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-11.

Neonatal Human Enteroids Take Up Human Milk Extracellular Vesicles That Survive Neonatal Human Digestion

Background: Human milk (HM) is the ideal infant nutrition and reduces infant death and disease. HM contains thousands of bioactive components, including extracellular vesicles (HMEVs). HMEVs are lipid bilayer-encased nanoparticles released from mammary epithelial cells that carry biological cargo to the infant. Pre-clinical models suggest that HMEVs may enhance intestinal function and limit inflammation; however, it is unknown if HMEVs or their cargo survive neonatal human digestion. This limits the ability to leverage HMEV cargo as additives to infant nutrition or as therapeutics. This study aims to develop a neonatal enteroid model and EV isolation pipeline to study HMEV-intestinal epithelial cell (IEC) interactions with the ultimate goal of therapy development. Hypothesis: HMEVs survive in vivo neonatal digestion to be taken up by infant IECs. Methods: All studies were conducted under approved IRB protocols. Neonatal intestinal contents (digesta) were collected after gastric feeding from post-pyloric tubes. HMEVs were isolated from raw and pasteurized human milk and digesta (n=3 each) by density-gradient ultracentrifugation following two-step skimming, acid precipitation of caseins, and multi-step filtration. EVs were validated by electron microscopy, nanoparticle tracking analysis (NTA), and western blotting. HMEV sub-populations were examined by super-resolution microscopy. HMEV uptake was tested using apical-out neonatal human enteroids isolated from a 2-month-old patient undergoing surgery for ileal atresia and validated by qPCR for proliferation and differentiation markers (mean ± SEM). Enteroid uptake was assessed with CMTPX dye-labeled-HMEVs, dynamin inhibitor, and confocal microscopy. Results: Our HMEV isolation pipeline reliably isolates HMEVs exhibiting classic EV morphology and enriched in extracellular vesicle markers CD81 and CD9, but depleted of b-casein and lactalbumin from HM and digesta. NTA data indicates that only 17.8 ± 2.9% (p>0.0001) of input HMEVs survive to reach the neonatal intestine. Human mammary gland-derived protein BTN1A1 is present in digesta EVs. Neonatal apical-out human enteroids represent a less proliferative and more differentiated epithelium, e.g. down-regulated KI67 (0.05 ± 0.03-fold relative to basal-out, p<0.0001, n=4-6) and elevated CHGA (14.22 ± 9.30-fold relative to basal-out, p=0.0072, n=4-6) as measured by qRT-PCR and normalized to housekeeping gene RPLP0. Neonatal enteroids take up digested HMEVs largely via dynamin-mediated endocytosis, as measured by CMTPX dye intensity normalized to enteroid area using Fiji (2.05 x 105 ± 2.95 x 104 vs 5.38 x 104 ± 6.08 x 103, p<0.0001, n=62-115 enteroids, HMEV-treatment alone and HMEVs + dynamin inhibitor, respectively).Conclusion: Our data suggest that HMEVs survive to the neonatal intestine and can be absorbed by neonatal enteroids largely via dynamin-mediated endocytosis. These data are an important first step to leveraging HMEVs and their cargo to treat neonatal intestinal inflammation. Funding: NIH K01DK129401, NIH R01HD097367, USDA NIFA, Collins Medical Trust, Medical Research Foundation, OHSU Exploratory Research Seed Grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Possible Roles of Extracellular Vesicles in the Pathogenesis and Interventions of Immune-Mediated Central Demyelinating Diseases.

Multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD) are two of the most devastating immune-mediated central demyelinating disorders. NMOSD was once considered as a variant of MS until the discovery of an antibody specific to the condition. Despite both MS and NMOSD being considered central demyelinating disorders, their pathogenesis and clinical manifestations are distinct, however the exact mechanisms associated with each disease remain unclear. Extracellular vesicles (EVs) are nano-sized vesicles originating in various cells which serve as intercellular communicators. There is a large body of evidence to show the possible roles of EVs in the pathogenesis of several diseases, including the immune-mediated central demyelinating disorders. Various types of EVs are found across disease stages and could potentially be used as a surrogate marker, as well as acting by carrying a cargo of biochemical molecules. The possibility for EVs to be used as a next-generation targeted treatment for the immune-mediated central demyelinating disorders has been investigated. The aim of this review was to comprehensively identify, compile and discuss key findings from in vitro, in vivo and clinical studies. A summary of all findings shows that: 1) the EV profiles of MS and NMOSD differ from those of healthy individuals, 2) the use of EV markers as liquid biopsy diagnostic tools appears to be promising biomarkers for both MS and NMOSD, and 3) EVs are being studied as a potential targeted therapy for MS and NMOSD. Any controversial findings are also discussed in this review.

Optimization of protocols for blood-derived extracellular vesicles for studies in liver diseases

Aim: Extracellular vesicles (EVs) have gained significant attention for their diagnostic and therapeutic potential in various diseases, including liver disorders. This study focuses on optimizing the isolation and characterization of small EVs from plasma and serum samples in patients with liver diseases, aiming to advance our understanding and potential clinical applications of EVs. Methods: Blood samples were collected from patients with end-stage liver disease (ESLD) enlisted in the TransplantLines Cohort and Biobank Study, and healthy donors. We employed differential ultracentrifugation (DUC) to evaluate three distinct protocols: a 3-step DUC, a washing step omitted [samples without washing (WW)], and a contaminant-depleted plasma (CDP) protocol. RNA isolation methodologies were compared, involving the use of TRI-reagent or the commercial AllPrep DNA/RNA kit. Further insights into EV composition were obtained through proteomic analyses, comparing samples subjected to traditional cell lysis (L) with those processed without lysis (NL). Results: We successfully isolated EVs from both plasma and serum samples as confirmed by the presence of specific EV markers, including CD9, CD63, CD81, and tumor susceptibility gene 101 (TSG-101). While some contaminants remained, such as albumin and lipoproteins, the protocol selected to continue EVs analysis was the 3-step protocol. Transmission electron microscopy (TEM) and nanotracking analysis (NTA) further confirmed EVs presence. RNA extraction was achieved using TRI-reagent, but not with the commercial kit highlighting the importance of selecting an appropriate method for RNA isolation. Finally, proteomics analysis showed that lysed samples were significantly more enriched in proteins compared to non-lysed samples, although protein variability was still present in both groups. Conclusions: Optimizing EV isolation techniques is essential for harnessing their potential in liver disease diagnosis and therapy. Further refinement of purification methods, a deep characterization of our cohort and understanding the variability and cargo within EVs will be crucial for future biomarker discovery and therapeutic applications in liver-related diseases.

The tumour-derived extracellular vesicle proteome varies by endometrial cancer histology and is confounded by an obesogenic environment.

Endometrial cancer, the most common gynaecological cancer worldwide, is closely linked to obesity and metabolic diseases, particularly in younger women. New circulating biomarkers have the potential to improve diagnosis and treatment selections, which could significantly improve outcomes. Our approach focuses on extracellular vesicle (EV) biomarker discovery by directly profiling the proteome of EVs enriched from frozen biobanked endometrial tumours. We analysed nine tissue samples to compare three clinical subgroups-low BMI (Body Mass Index) Endometrioid, high BMI Endometrioid, and Serous (any BMI)-identifying proteins related to histological subtype, BMI, and shared secreted proteins. Using collagenase digestion and size exclusion chromatography, we successfully enriched generous quantities of EVs (range 204.8-1291.0µg protein: 1.38×1011-1.10×1012 particles), characterised by their size (∼150nm), expression of EV markers (CD63/81), and proposed endometrial cancer markers (L1CAM, ANXA2). Mass spectrometry-based proteomic profiling identified 2075 proteins present in at least one of the 18 samples. Compared to cell lysates, EVs were successfully depleted for mitochondrial and blood proteins and enriched for common EV markers and large secreted proteins. Further analysis highlighted significant differences in EV protein profiles between the high BMI subgroup and others, underlining the impact of comorbidities on the EV secretome. Interestingly, proteins differentially abundant in tissue subgroups were largely not also differential in matched EVs. This research identified secreted proteins known to be involved in endometrial cancer pathophysiology and proposed novel diagnostic biomarkers (EIF6, MUC16, PROM1, SLC26A2).

Extracellular vesicles mediate inflammasome signaling in the brain and heart of Alzheimer's disease mice.

Alzheimer's disease (AD) is an inflammatory neurodegenerative disease characterized by memory loss and cognitive impairment that worsens over time. AD is associated with many comorbidities, including cardiovascular disease that are associated with poorer outcomes. Comorbidities, especially heart disease and stroke, play a significant role in the demise of AD patients. Thus, it is important to understand how comorbidities are linked to AD. We have previously shown that extracellular vesicle (EV)-mediated inflammasome signaling plays an important role in the pathogenesis of brain injury and acute lung injury after traumatic brain injury. We analyzed the cortical, hippocampal, ventricular, and atrial protein lysates from APP/PS1 mice and their respective controls for inflammasome signaling activation. Additionally, we analyzed serum-derived EV for size, concentration, and content of inflammasome proteins as well as the EV marker CD63. Finally, we performed conditioned media experiments of EV from AD patients and healthy age-matched controls delivered to cardiovascular cells in culture to assess EV-induced inflammation. We show a significant increase in Pyrin, NLRP1, caspase-1, and ASC in the brain cortex whereas caspase-8, ASC, and IL-1β were significantly elevated in the heart ventricles of AD mice when compared to controls. We did not find significant differences in the size or concentration of EV between groups, but there was a significant increase of caspase-1 and IL-1β in EV from AD mice compared to controls. In addition, conditioned media experiments of serum-derived EV from AD patients and age-matched controls delivered to cardiovascular cells in culture resulted in inflammasome activation, and significant increases in TNF-α and IL-2. These results indicate that EV-mediated inflammasome signaling in the heart may play a role in the development of cardiovascular diseases in AD patients.

Process development for the production of mesenchymal stromal cell-derived extracellular vesicles in conventional 2D systems

BackgroundIn recent years, the importance of extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) has increased significantly. For their widespread use, a standardized EV manufacturing is needed which often includes conventional, static 2D systems. For these system critical process parameters need to be determined. MethodsWe studied the impact of process parameters on MSC proliferation, MSC-derived particle production including EVs, EV- and MSC-specific marker expression, and particle functionality in a HaCaT cell migration assay. ResultsWe found that cell culture growth surface and media affected MSCs and their secretory behavior. Interestingly, the materials that promoted MSC proliferation did not necessarily result in the most functional MSC-derived particles. In addition, we found that MSCs seeded at 4 × 103 cells cm−2 produced particles with improved functional properties compared to higher seeding densities. MSCs in a highly proliferative state did not produce the most particles, although these particles were significantly more effective in promoting HaCaT cell migration. The same correlation was found when investigating the cultivation temperature. A physiological temperature of 37°C was not optimal for particle yield, although it resulted in the most functional particles. We observed a proliferation-associated particle production and found potential correlations between particle production and glucose consumption, enabling the estimation of final particle yields. ConclusionsOur findings suggest that parameters, which must be defined prior to each individual cultivation and do not require complex and expensive equipment, can significantly increase MSC-derived particle production including EVs. Integrating these parameters into a standardized EV process development paves the way for robust and efficient EV manufacturing for early clinical phases.