Extracellular Vesicles Biomarkers Research Articles

Single step capture and assessment of multiple plasma extracellular vesicle biomarkers in Alzheimer's disease detection.

Blood tests for Alzheimer's disease (AD) that measure biomarkers related to neuropathology have demonstrated to be useful, minimally-invasive ways to identify patients for screening into clinical trials. While some AD biomarkers can be detected in plasma, greater sensitivity is needed to make plasma AD tests more effective. Extracellular vesicles (EVs) in plasma carry AD-related biomarkers from the brain and could offer a concentrated source of brain-related biomarkers, though the methodological complexities involved in isolating plasma EVs have hampered its validation for clinical use. To explore the feasibility and effectiveness of developing blood tests for AD utilizing extracellular vesicle-bound protein biomarkers. We developed a simplified method for isolating EVs directly from plasma using an alternating current electrokinetic (ACE) microchip. No sample pretreatment steps were needed. Protein biomarkers on the EVs were detected by adding fluorescent antibodies to the plasma samples before capture by the chip. This allowed measurement of EV biomarker levels directly on the chip. AD or non-AD control plasma was measured for ten different AD-related biomarkers. EV-associated NCAM1, pTau231, α-synuclein, and TDP-43 levels were able to distinguish a group of 10 AD, 10 mild cognitive impairment (MCI), and 10 non-AD subjects. pTau231 was different between AD and non-AD (p = 0.0300) and α-synuclein differentiated AD from MCI (p = 0.0148). This study shows how ACE microfluidic chip technology can help differentiate AD and MCI patients from non-AD controls with clinical relevance. This work also highlights the important diagnostic role of plasma EV biomarkers in neurodegenerative disease.

Abstract PR013: Highly accurate detection of early-stage colorectal cancer using tumor and immune extracellular vesicles biomarkers

Abstract Introduction: Colorectal cancer (CRC) is one of the most common cancers worldwide, and early detection is critical for successful treatment and improved survival rates. However, precancerous and early-stage CRC present significant diagnostic challenges due to the small size of lesions and the very low expression of tumor-specific biomarkers in the bloodstream. Current genomics-based diagnostic methods struggle to detect these lesions, making it imperative to develop more sensitive and specific approaches. Circulating extracellular vesicles (EVs) are emerging as a promising solution for early-stage CRC detection, since EVs are produced by tumor cells as well as the tumor microenviroment and host immune cells. Thus, EVs may offer the means to detect and monitor small early-stage tumors through both direct detection of tumor- associated biomarkers as well through tumor specific host response and tumor microenvironment biomarkers. Methods: To identify novel early-stage CRC specific biomarkers, we performed proteomics analysis on EVs purified from patient plasma using size exclusion chromatography and a proprietary buffer system that enhances EV and corona protein recovery. TrueDiscovery™ Data-independent acquisition (DIA) mass spectrometry (MS) analysis was conducted on 24 pre- cancer/stage 0 and 25 stage 1 CRC patients and 75 normal patient plasma samples. An in-house developed machine learning pipeline was used to identify differentially expressed proteins and model candidate multiplexes to identify those with extremely high diagnostic accuracy (> 0.99). Results: An average of ∼2,500 proteins were identified per sample and included in bioinformatics analysis. Comparative analysis between control patient EVs and either pre- cancerous/Stage 0, or Stage 1 colon cancer EVs using an in-house Machine Learning pipeline identified 336 and 493 differentially expressed proteins for each group (FDR adjusted p-value < 0.001). Of these, the best 17 pre/stage 0 proteins and 53 stage 1 proteins were trained and tested using Support Vector Machine to assess all potential 3-plexes in order to identify those with mean diagnostic accuracy > 99%. This yielded 5 3-plexes in precancer/stage 0 and 34 3-plexes in stage 1 with near perfect diagnostic accuracy. These plexes are currently being assessed in single analyte and multiplex immunoassays with the goal of translating candidate 3-plexes to the clinical. Conclusions: Key biomarkers from CRC patient EVs indicate the potential to detect and diagnose early-stage and low tumor burden cancers using our methods. Interestingly, the most accurate 3-plexes consist of proteins from immune, inflammatory and metabolic processes, suggesting that for the detection of early stage cancer it may be critical to include both tumor and host specific biomarkers. Citation Format: Poorva Mudgal, Cheryl Bandoski, Zachary Opheim, Martin Mehnert, Valesca Anschau, Issa Isaac, Alan Ezrin, Todd Hembrough. Highly accurate detection of early-stage colorectal cancer using tumor and immune extracellular vesicles biomarkers [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr PR013.

Abstract A063: Highly accurate detection of early-stage colorectal cancer using tumor and immune extracellular vesicles biomarkers

Abstract This abstract is being presented as a short talk in the scientific program. A full abstract is printed in the Proffered Abstracts section (PR013) of the Conference Program/Proceedings. Citation Format: Poorva Mudgal, Cheryl Bandoski, Zachary Opheim, Martin Mehnert, Valesca Anschau, Issa Isaac, Alan Ezrin, Todd Hembrough. Highly accurate detection of early-stage colorectal cancer using tumor and immune extracellular vesicles biomarkers [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A063.

A novel multiplexed immunoassay for surface-exposed proteins in plasma extracellular vesicles.

Small membranous extracellular vesicles (EV) incorporate proteins and nucleic acids from the parent cell. Proteins exposed on EV surface are dictated by cellular origin and biogenesis pathway. To better understand the EV origin and function, it is important to develop methods that reveal surface protein composition of heterogeneous EV populations in culture supernatants and in biofluids. Tetraspanins CD9, CD63, and CD81 are common and abundant EV markers. However, their relative enrichment (profile) on EVs of specific cellular origins is not fully elucidated. We introduce LuminEV, a novel version of the Luminex assay for the multiplexed analysis of EV surface proteins. Optimized LuminEV reagents enable direct, specific, and sensitive measurements of EV markers in biofluids and in culture supernatants, bypassing EV isolation step. LuminEV assay for CD9, CD63, and CD81 was validated by comparing simplex and multiplex measurements, establishing linearity, spike-in recovery, inter- and intra-assay precision, and reproducibility between operators. LuminEV measurements of CD9, CD63, and CD81 in conditioned media from 15 cell lines revealed strong variations between cell types and showed high sensitivity, which enabled EV detection without prior concentration. Using tetraspanin levels as a readout, we noted suppression and induction of EV release from the cultured cells by GW6869 and monensin. Measurement of EV CD9, CD63, and CD81 in blood plasma from 70 disease-free donors showed respective abundance of 72, 16, and 12%. CD63 displayed weak, albeit significant, negative correlation with age and was slightly lower in female samples. The assay was then used to detect cell type-specific EV surface markers, including CD235a (erythrocytes), GAP43 (neurons), and CD68 (macrophages), and to detect differences in tetraspanin profiles between healthy and diseased donors. In summary, LuminEV offers robust and sensitive approach for multiplexed assessment of EV surface proteins, to facilitate the research into EV biology, biomarker, and therapeutic applications.

Proteomic and metabolomic profiles of plasma-derived Extracellular Vesicles differentiate melanoma patients from healthy controls

BackgroundPlasma-derived Extracellular Vesicles (EVs) have been suggested as novel biomarkers in melanoma, due to their ability to reflect the cell of origin and ease of collection. This study aimed to identify novel EV biomarkers that can discriminate between disease stages. This was achieved by characterising the plasma-derived EVs of patients with melanoma, and comparing their proteomic and metabolomic profile to those from healthy controls. MethodsEVs were isolated from the plasma of 36 patients with melanoma and 13 healthy controls using Size Exclusion Chromatography. Proteomic and Metabolomic Analyses were performed, and machine learning algorithms were used to identify potential proteins and metabolites to differentiate the plasma-derived EVs from melanoma patients of different disease stages. ResultsThe concentration and size of the EV population isolated was similar between groups. Proteins (APOC4, PRG4, PLG, TNC, VWF and SERPIND1) and metabolites (lyso PC a C18:2, PC ae C44:3) previously associated with melanoma pathogenesis were identified as relevant in differentiating between disease stages. ConclusionThe results further support the continued investigation of circulating plasma-derived EVs as biomarkers in melanoma. Furthermore, the potential of combined proteo-metabolomic signatures for differentiation between disease stages may provide valuable insights into early detection, prognosis, and personalised treatment strategies.

Rapid Assessment of Biomarkers on Single Extracellular Vesicles Using "Catch and Display" on Ultrathin Nanoporous Silicon Nitride Membranes.

Extracellular vesicles (EVs) are particles released from cells that facilitate intercellular communication and have tremendous diagnostic and therapeutic potential. Bulk assays lack the sensitivity to detect rare EV subsets relevant to disease, and while single EV analysis techniques remedy this, they are often undermined by complicated detection schemes and prohibitive instrumentation. To address these issues, a microfluidic technique for EV characterization called "catch and display for liquid biopsy (CAD-LB)" is proposed. In this method, minimally processed samples are pipette-injected and fluorescently labeled EVs are captured in the nanopores of an ultrathin membrane. This enables the rapid assessment of EV number and biomarker colocalization by light microscopy. Here, nanoparticles are used to define the accuracy and dynamic range for counting and colocalization. The same assessments are then made for purified EVs and for unpurified EVs in plasma. Biomarker detection is validated using CD9 and Western blot analysis to confirm that CAD-LB accurately reports relative protein expression levels. Using unprocessed conditioned media,CAD-LB captures the known increase in EV-associated ICAM-1 following endothelial cell cytokine stimulation. Finally, to demonstrate CAD-LB's clinical potential, EV biomarkers indicative of immunotherapy responsiveness are successfully detected in the plasma of bladder cancer patients treated with immune checkpoint blockade.

Tetraspanins, GLAST and L1CAM Quantification in Single Extracellular Vesicles from Cerebrospinal Fluid and Serum of People with Multiple Sclerosis.

Objective: This study aimed to unravel the single tetraspanin pattern of extracellular vesicles (EVs), L1CAM+ and GLAST+ EV levels as diagnostic biomarkers to stratify people with multiple sclerosis (pwMS), specifically relapsing-remitting (RRMS) and primary progressive (PPMS). Methods: The ExoView platform was used to directly track single EVs using a clinically feasible volume of cerebrospinal fluid (CSF) and serum samples. This technology allowed us to examine the patterns of classical tetraspanin and quantify the levels of L1CAM and GLAST proteins, commonly used to immunoisolate putative neuron- and astrocyte-derived EVs. Results: The tetraspanin EV pattern does not allow us to differentiate RRMS, PPMS and non-MS donors neither in CSF nor serum, but this was associated with the type of biofluid. L1CAM+ and GLAST+ EVs showed a very low presence of tetraspanin proteins. Additionally, a significant decrease in the particle count of L1CAM+ EVs was detected in L1CAM-captured spots, and L1CAM+ and GLAST+ EVs decreased in GLAST-captured spots in the CSF from PPMS subjects compared to RRMS. Interestingly, only GLAST+ EVs exhibited a lower quantity in the CSF from PPMS compared to both MS and non-MS samples. Finally, GLAST+ EVs demonstrated a medium negative and significative correlation with GFAP levels-a biomarker of MS progression, astrocyte damage and neurodegenerative processes. Conclusions: ExoView technology could track neural EV biomarkers and be potentially useful in the diagnostic evaluation and follow-up of pwMS. GLAST+ EVs might provide insights into the etiology of PPMS and could offer small windows to elucidate the molecular mechanisms behind its clinical presentation.

Proteomic Profiling of Serum Extracellular Vesicles Identifies Diagnostic Signatures and Therapeutic Targets in Breast Cancer.

Analysis of extracellular vesicles (EV) is a promising noninvasive liquid biopsy approach for breast cancer detection, prognosis, and therapeutic monitoring. A comprehensive understanding of the characteristics and proteomic composition of breast cancer-specific EVs from human samples is required to realize the potential of this strategy. In this study, we applied a mass spectrometry-based, data-independent acquisition proteomic approach to characterize human serum EVs derived from patients with breast cancer (n = 126) and healthy donors (n = 70) in a discovery cohort and validated the findings in five independent cohorts. Examination of the EV proteomes enabled the construction of specific EV protein classifiers for diagnosing breast cancer and distinguishing patients with metastatic disease. Of note, TALDO1 was found to be an EV biomarker of distant metastasis of breast cancer. In vitro and in vivo analysis confirmed the role of TALDO1 in stimulating breast cancer invasion and metastasis. Finally, high-throughput molecular docking and virtual screening of a library consisting of 271,380 small molecules identified a potent TALDO1 allosteric inhibitor, AO-022, which could inhibit breast cancer migration in vitro and tumor progression in vivo. Together, this work elucidates the proteomic alterations in the serum EVs of breast cancer patients to guide the development of improved diagnosis, monitoring, and treatment strategies. Significance: Characterization of the proteomic composition of circulating extracellar vesicles in breast cancer patients identifies signatures for diagnosing primary and metastatic tumors and reveals tumor-promoting cargo that can be targeted to improve outcomes.

Plasma extracellular vesicle: a novel biomarker for neurodegenerative disease diagnosis.

Extracellular vesicles (EVs) are membrane-bound structures that carry proteins, lipids, RNA, and DNA, playing key roles in cell communication and material transport. Recent research highlights their potential as disease biomarkers due to their stability in bodily fluids. This study explores using tau and TDP-43 proteins in plasma EVs as diagnostic biomarkers for frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Analyzing plasma EVs from clinical cohorts, the study found that the 3R/4R tau ratio and TDP-43 levels effectively differentiate between diagnostic groups with high accuracy. Notably, plasma EV biomarkers demonstrate higher diagnostic accuracy and stability compared to direct plasma testing, providing new insights and approaches for future research and clinical practice. Further research is needed to validate these biomarkers in diverse populations and to establish standardized protocols. Future studies should continue to explore the potential of EV biomarkers in a broader range of neurodegenerative diseases and delve deeper into the mechanisms of EV secretion and sorting to enhance their diagnostic utility.

An aptamer-guided fluorescence polarisation platform for extracellular vesicle liquid biopsy.

The translation of discoveries on extracellular vesicle (EV) based cancer biomarkers to personalised precision oncology requires the development of robust, sensitive and specific assays that are amenable to adoption in the clinical laboratory. Whilst a variety of elegant approaches for EV liquid biopsy have been developed, most of them remain as research prototypes due to the requirement of a high level of microfabrication and/or sophisticated instruments. Hence, this study is set to develop a simple DNA aptamer-enabled and fluorescence polarisation-based homogenous assay that eliminates the need to separate unbound detection ligands from the bound species for EV detection. High specificity is achieved by immobilising EVs with one set of antibodies and subsequently detecting them with a DNA aptamer targeting a distinct EV biomarker. This two-pronged strategy ensures the removal of most, if not all, non-EV substances in the input biofluids, including soluble proteins, protein aggregates or non-vesicular particles, prior to quantifying biomarker-positive EVs. A limit of detection of 5.0 × 106 EVs/mL was achieved with a linear quantification range of 5.0 × 108 to 2.0 × 1010 EVs/mL. Facilitated by a multiple parametric analysis strategy, this aptamer-guided fluorescence polarisation assay was capable of distinguishing EVs from three different types of solid cancer cells based on quantitative differences in the levels of the same sets of biomarkers on EVs. Given the simplicity of the method and its ease of implementation in automated clinical biochemistry analysers, this assay could be exploited for future EV-based continuous and real-time monitoring of the emergence of new macro- or micro-metastasis, cancer progression as well as the response to treatment throughout different stages of cancer management in the clinic.

MiR-182, miR-221 and miR-222 are potential urinary extracellular vesicle biomarkers for canine urothelial carcinoma

Current diagnostic methods for canine urothelial carcinoma (UC) are technically challenging or can lack specificity, hence there is a need for novel biomarkers of UC. To this end, we analysed the microRNA (miRNA) cargo of extracellular vesicles (EVs) from urine samples of dogs with UC to identify candidate miRNA biomarkers. Urine was fractionated using ultrafiltration combined with size-exclusion chromatography and small RNA sequencing analysis was performed on both the EV enriched and (EV free) protein fractions. A greater number of candidate miRNA biomarkers were detected in the EV fraction than the protein fraction, and further validation using droplet digital PCR (ddPCR) was performed on the EV enriched fraction of a second cohort of dogs with UC which indicated that miR-182, miR-221 and miR-222 were significantly overrepresented in dogs with UC when compared with healthy dogs and dogs with urinary tract infections. Pathway analysis confirmed that these three miRNAs are involved in cancer. In addition, their potential downstream gene targets were predicted and PIK3R1, a well-known oncogene is likely to be a shared target between miRNA-182 and miRNA-221/222. In summary, this study highlights the potential of urinary EV-associated miRNAs as a source of biomarkers for the diagnosis of canine UC.

Alzheimer's Disease Related Biomarkers Were Associated with Amnestic Cognitive Impairment in Parkinson's Disease: A Cross-Sectional Cohort Study.

Cognitive impairment is common in patients with Parkinson's disease (PD) and occurs through multiple mechanisms, including Alzheimer's disease (AD) pathology and the involvement of α-synucleinopathies. We aimed to investigate the pathological biomarkers of both PD and AD in plasma and neuronal extracellular vesicles (EVs) and their association with different types of cognitive impairment in PD patients. A total of 122 patients with PD and 30 healthy controls were included in this cross-sectional cohort study between March 2021 and July 2023. Non-dementia PD patients were divided into amnestic and non-amnestic groups according to the memory domain of a neuropsychological assessment. Plasma and neuronal EV biomarkers, including α-synuclein (α-syn), beta-amyloid (Aβ), total tau (T-tau), phosphorylated tau181 (p-tau181), and glial fibrillary acidic protein (GFAP), were measured using a single-molecule array and a chemiluminescence immunoassay, respectively. Neuronal EV but not plasma α-syn levels, were significantly increased in PD as compared to healthy controls, and they were positively associated with UPDRS part III scores and the severity of cognitive impairment. A lower plasma Aβ42 level and higher neuronal EV T-tau level were found in the amnestic PD group compared to the non-amnestic PD group. The results of the current study demonstrate that neuronal EV α-syn levels can be a sensitive biomarker for assisting in the diagnosis and disease severity prediction of PD. Both AD and PD pathologies are important factors in cognitive impairment associated with PD, and AD pathologies are more involved in amnestic memory deficit in PD.

Plasma extracellular vesicle biomarkers for frontotemporal dementia and related disorders.

Plasma extracellular vesicle biomarkers for frontotemporal dementia and related disorders.

Nanoscale flow cytometry-based quantification of blood-based extracellular vesicle biomarkers distinguishes MCI and Alzheimer's disease.

Accurate testing for Alzheimer's disease (AD) represents a crucial step for therapeutic advancement. Currently, tests are expensive and require invasive sampling or radiation exposure. We developed a nanoscale flow cytometry (nFC)-based assay of extracellular vesicles (EVs) to screen biomarkers in plasma from mild cognitive impairment (MCI), AD, or controls. Circulating amyloid beta (Aβ), tau, phosphorylated tau (p-tau)181, p-tau231, p-tau217, p-tauS235, ubiquitin, and lysosomal-associated membrane protein 1-positive EVs distinguished AD samples. p-tau181, p-tau217, p-tauS235, and ubiquitin-positive EVs distinguished MCI samples. The most sensitive marker for AD distinction was p-tau231, with an area under the receiver operating characteristic curve (AUC) of 0.96 (sensitivity 0.95/specificity 1.0) improving to an AUC of 0.989 when combined with p-tauS235. This nFC-based assay accurately distinguishes MCI and AD plasma without EV isolation, offering a rapid approach requiring minute sample volumes. Incorporating nFC-based measurements in larger populations and comparison to "gold standard" biomarkers is an exciting next step for developing AD diagnostic tools. Extracellular vesicles represent promising biomarkers of Alzheimer's disease (AD) that can be measured in the peripheral circulation. This study demonstrates the utility of nanoscale flow cytometry for the measurement of circulating extracellular vesicles (EVs) in AD blood samples. Multiple markers including amyloid beta, tau, phosphorylated tau (p-tau)181, p-tau231, p-tau217, and p-tauS235 accurately distinguished AD samples from healthy controls. Future studies should expand blood and cerebrospinal fluid-based EV biomarker development using nanoflow cytometry approaches.

Differential extracellular vesicle concentration and their biomarker expression of integrin αv/β5, EpCAM, and glypican-1 in pancreatic cancer models

Tumor-derived extracellular vesicles (EVs) show great potential as biomarkers for several diseases, including pancreatic cancer, due to their roles in cancer development and progression. However, the challenge of utilizing EVs as biomarkers lies in their inherent heterogeneity in terms of size and concentration, making accurate quantification difficult, which is highly dependent on the isolation and quantification methods used. In our study, we compared three EV isolation techniques and two EV quantification methods. We observed variations in EV concentration, with approximately 1.5-fold differences depending on the quantification method used. Interestingly, all EV isolation techniques consistently yielded similar EV quantities, overall size distribution, and modal sizes. In contrast, we found a notable increase in total EV amounts in samples from pancreatic cancer cell lines, mouse models, and patient plasma, compared to non-cancerous conditions. Moreover, individual tumor-derived EVs exhibited at least a 3-fold increase in several EV biomarkers. Our data, obtained from EVs isolated using various techniques and quantified through different methods, as well as originating from various pancreatic cancer models, suggests that EV profiling holds promise for the identification of unique and cancer-specific biomarkers in pancreatic cancer.

Characterization of spinal cord tissue-derived extracellular vesicles in neuroinflammation

Extracellular vesicles (EVs) are released by all cells, can cross the blood–brain barrier, and have been shown to play an important role in cellular communication, substance shuttling, and immune modulation. In recent years EVs have shifted into focus in multiple sclerosis (MS) research as potential plasma biomarkers and therapeutic vehicles. Yet little is known about the disease-associated changes in EVs in the central nervous system (CNS). To address this gap, we characterized the physical and proteomic changes of mouse spinal cord-derived EVs before and at 16 and 25 days after the induction of experimental autoimmune encephalomyelitis (EAE), a neuroinflammatory model of MS. Using various bioinformatic tools, we found changes in inflammatory, glial, and synaptic proteins and pathways, as well as a shift in the predicted contribution of immune and glial cell types over time. These results show that EVs provide snapshots of crucial disease processes such as CNS-compartmentalized inflammation, re/de-myelination, and synaptic pathology, and might also mediate these processes. Additionally, inflammatory plasma EV biomarkers previously identified in people with MS were also altered in EAE spinal cord EVs, suggesting commonalities of EV-related pathological processes during EAE and MS and overlap of EV proteomic changes between CNS and circulating EVs.

Extracellular vesicle biomarkers in circulation for colorectal cancer detection: a systematic review and meta-analysis

We provided an overview which evaluated the diagnostic performance of circulation EV biomarkers for CRC from PubMed, Medline, and Web of Science until 21 August 2022.Weidentified 48 studies that involved 7727 participants and evaluated 162 plasma/serum individual EV biomarkers including 117 RNAs and 45 proteins, as well as 45 EV biomarker panels for CRC detection. 12 studies evaluated the diagnostic performance of EV biomarkers for early CRC. The summarized sensitivity, specificity, and AUC value of individual EV RNAs and EV RNA panels were 76%, 75%, 0.87 and 82%, 79% and 0.90, respectively. Meanwhile, those of individual EV proteins and EV protein panels were 85%, 84%, 0.92 and 87%, 83%, 0.92, respectively. These results indicated that EV biomarker panels revealed superior diagnostic performance than the corresponding individual biomarkers. In early CRC, EV biomarkers showed available diagnostic value with the sensitivity, specificity, and AUC value of 80%, 75%, and 0.89.In subgroup analyses, EV miRNAs and LncRNAs held similar diagnostic value with the sensitivity, specificity and AUC value of 75%, 78%, 0.90 and 79%, 72%, 0.83, which was highly consistent with the whole EV RNAs. Significantly, the diagnostic values of EV miRNAs in plasma were marginally higher than those based on serum. In detail, the sensitivity, specificity, and AUC values were 79%, 81%, and 0.92 in plasma, as well as 74%, 77%, and 0.88 in serum, respectively. Therefore, circulation EV biomarkers could be considered as a promising biomarker for the early detection of CRC.

Comparison of cervicovaginal fluid extracellular vesicles isolated from paired cervical brushes and vaginal swabs

AbstractEndometriosis is a common gynaecological condition, with a long diagnostic delay. Surgery is required to confirm a diagnosis, highlighting the need for a non‐invasive biomarker. Extracellular vesicles (EVs) may have a role in endometriosis pathogenesis, yet there is limited EV biomarker literature available. This study aimed to investigate the feasibility of isolating cervico‐vaginal fluid EVs sampled using cervical brushes and vaginal swabs and to compare these methods. After providing informed consent, patients undergoing surgery for suspected endometriosis had cervical brush and vaginal swab samples collected under general anaesthetic. Isolated EVs were characterised through negative stain transmission electron microscopy (TEM), Western blotting (TSG101, CD63, Calnexin, ApoB, Albumin), tunable resistive pulse sensing (TRPS), microBCA assays and RT‐qPCR of miRNAs. PCR was performed on samples prior to EV isolation to assess bacteria present in samples. Cervical brush and vaginal swab EVs were intact vesicles with limited co‐isolated contaminants. Cervical brushes had higher concentrations of particles compared to match vaginal swabs, although both samples had low concentrations. Protein and miRNA yield were similar between matched samples. PCR demonstrated only a small amount DNA within samples was bacterial (>0.5%). Cervico‐vaginal fluids EVs were successfully isolated from cervical brushes and vaginal swabs, demonstrating a new method of sampling reproductive EVs. EV yield from both sample types was low. Similar protein and miRNA levels suggest either sampling method may be suitable for biomarker studies.

Electrochemical detection of extracellular vesicles for early diagnosis: a focus on disease biomarker analysis.

This review article presents a detailed examination of the integral role that electrochemical detection of extracellular vesicles (EVs) plays, particularly focusing on the potential application for early disease diagnostics through EVs biomarker analysis. Through an exploration of the benefits and challenges presented by electrochemical detection vetted for protein, lipid, and nucleic acid biomarker analysis, we underscore the significance of these techniques. Evidence from recent studies renders this detection modality imperative in identifying diverse biomarkers from EVs, leading to early diagnosis of diseases such as cancer and neurodegenerative disorders. Recent advancements that have led to enhanced sensitivity, specificity and point-of-care testing (POCT) potential are elucidated, along with equipment deployed for electrochemical detection. The review concludes with a contemplation of future perspectives, recognizing the potential shifts in disease diagnostics and prognosis, necessary advances for broad adoption, and potential areas of ongoing research. The objective is to propel further investigation into this rapidly burgeoning field, thereby facilitating a potential paradigm shift in disease detection, monitoring, and treatment toward human health management.

Bovine placental extracellular vesicles carry the fusogenic syncytin BERV-K1

Syncytins are endogenous retroviral envelope proteins which induce the fusion of membranes. A human representative of this group, endogenous retrovirus group W member 1 envelope (ERVW-1) or syncytin-1 is present in trophoblast-derived extracellular vesicles and supports the incorporation of these extracellular vesicles into recipient cells. During pregnancy, placenta-derived extracellular vesicles participate in feto-maternal communication. Bovine fetal binucleate trophoblast cells express the syncytin, bovine endogenous retroviral envelope protein K1 (BERV-K1). These cells release extracellular vesicles into the maternal stroma, but it is unclear whether BERV-K1 is included in these extracellular vesicles. Here, extracellular vesicles were isolated from bovine placental tissue using collagenase digestion, ultracentrifugation, and size exclusion chromatography. They were characterized with transmission electron microscopy, nanoparticle tracking analysis, immunoblotting and mass spectrometry. Immunohistochemistry and immunoelectron microscopy were used to localize BERV-K1 within the bovine placental tissue. The isolated extracellular vesicles range between 50 and 300 nm, carrying multiple extracellular vesicle biomarkers. Proteomic analysis and immunoelectron microscopy confirmed BERV-K1 presence on the isolated extracellular vesicles. Further, BERV-K1 was localized on intraluminal vesicles in secretory granules of binucleate trophoblast cells. The presence of BERV-K1 on bovine placental extracellular vesicles suggests their role in feto-maternal communication and potential involvement of BERV-K1 in uptake of extracellular vesicles by target cells.