Extracellular Vesicles Proteome Research Articles

Extracellular Vesicles heterogeneity through the lens of multiomics.

Extracellular vesicles (EVs) are heterogenous in size, biogenesis, cargo and function. Beside small EVs, aggressive tumor cells release a population of particularly large EVs, namely large oncosomes (LO). This study provides the first resource of label-free quantitative proteomics of LO and small EVs obtained from distinct cancer cell types (prostate, breast, and glioma). This dataset was integrated with a SWATH Proteomic assay on LO, rigorously isolated from the plasma of patients with metastatic prostate cancer (PC). Proteins enriched in LO, which were identified also at the RNA level, and found in plasma LO significantly correlated with PC progression. Single EV RNA-Seq of the PC cell-derived LO confirmed some of the main findings from the bulk RNA-Seq, providing first evidence that single cell technologies can be successfully applied to EVs. This multiomics resource of cancer EVs can be leveraged for developing a multi-analyte approach for liquid biopsy.

Proteomics of plasma-derived extracellular vesicles reveals S100A8 as a novel biomarker for Alzheimer's disease: A preliminary study

Extracellular vesicles (EVs) act as mediators for intercellular transfer of Aβ and tau proteins, promoting the propagation of these pathological misfolded proteins throughout the brain in Alzheimer's disease (AD). Levels of blood exosomal Aβ42, total Tau (t-Tau) and phosphorylated Tau (p-Tau) had a high correlation with their concentrations in cerebrospinal fluid (CSF), demonstrating that exosomal biomarkers have equal contribution as those in CSF for the diagnosis of AD. We aimed to comprehensively characterize the proteome of plasma-derived EVs to identify differentially expressed proteins (DEPs) and pathways in AD. Tandem mass tag (TMT) labeled quantitative proteomics was applied to analyze plasma-derived EV proteins in 9 AD patients and 9 healthy controls. 335 proteins were quantified, and 12 DEPs were identified including seven upregulated proteins and five down-regulated proteins. Oligomerized Aβ1–42 induced SH-SY5Y cell damage model was built to mimic the pathological changes of AD, and small interfering RNA (siRNA) against S100A8 was used to knock down S100A8 expression. Results displayed S100A8 was down regulated in plasma-derived EVs from AD patients, while enriched in EVs derived from Aβ1–42-induced SH-SY5Y cells. Furthermore, Aβ1–42-induced SH-SY5Y cells treated with S100A8 siRNA showed decreased Aβ levels in cell lysate and EVs, especially in EVs. SignificanceThe investigation aimed to comprehensively characterize the proteome of plasma-derived EVs to identify DEPs and potential biomarker of AD. S100A8 was found down regulated in plasma-derived EVs from AD patients using TMT labeled quantitative proteomics. The diagnostic value of S100A8 was also confirmed using receiver operating characteristic curve (ROC) analysis. Furthermore, Aβ1–42-induced SH-SY5Y cells treated with S100A8 siRNA showed decreased Aβ levels in cell lysate and EVs, especially in EVs. The preliminary findings suggest that suppression of S100A8 expression inhibits Aβ aggregation both in cell lysate and EVs from Aβ1–42-induced SH-SY5Y cells, and S100A8 more likely regulates Aβ aggregation via EVs. Therefore, plasma-derived EV S100A8 might be a potential biomarker of AD. Manipulation of S100A8 expression may be a novel therapeutic strategy in the treatment of AD.

Enhancing outcome prediction of concurrent chemoradiation treatment in patients with locally advanced cervical cancer through plasma extracellular vesicle proteomics

Most patients with locally advanced cervical cancer (LACC) are primarily treated using concurrent chemoradiation (CCRT); however, LACC lacks reliable predictive biomarkers. Extracellular vesicles (EVs) could define the dynamic biological response to CCRT. However, the relationship between EVs and the therapeutic response to LACC is unestablished. Thus, we aimed to determine the relationship of plasma EVs pre- and post-CCRT in 62 patients with LACC. For proteomic analyses, EVs were isolated using ultracentrifugation (UC) with size exclusion chromatography or UC alone. We found that plasma particle concentration was significantly increased post-treatment in non-responders. After CCRT, there was a decrease in proteins related to serine protease and fibrinogen, which contribute to tumor microenvironment alteration. This reduction also extended to proteins involved in innate immune and viral immune responses, correlating with reduced tumor burden. Sparse partial least squares discriminant analysis revealed 8, 13, and 19 proteins at diagnosis, one month, and three months, respectively, influencing the CCRT response. Among these, FIBG, TFR1, HBA, and FINC are prognostic markers according to The Cancer Genome Atlas tissue gene expression database. Our discriminant model demonstrated excellent specificity and negative predictive value, underscoring the model's reliability in determining responsiveness to CCRT and highlighting the potential clinical applicability of EVs in improving outcomes in LACC.

Proteomic profiling of human plasma extracellular vesicles identifies PF4 and C1R as novel biomarker in sarcopenia.

Sarcopenia, the gradual and generalized loss of muscle mass and function with ageing, is one of the major health problems in older adults, given its high prevalence and substantial socioeconomic implications. Despite the extensive efforts to reach consensus on definition and diagnostic tests and cut-offs for sarcopenia, there is an urgent and unmet need for non-invasive, specific and sensitive biomarkers for the disease. Extracellular vesicles (EVs) are present in different biofluids including plasma, whose cargo reflects cellular physiology. This work analysed EV proteome in sarcopenia and robust patients in the search for differentially contained proteins that can be used to diagnose the disease. Plasma small EVs (sEVs) from a total of 29 robust controls (aged 73.4±5.6years; 11 men and 18 women) and 49 sarcopenic patients (aged 82.3±5.4years; 15 men and 34 women) aged 65years and older were isolated and their cargo was analysed by proteomics. Proteins whose concentration in sEVs was different between sarcopenic and robust patients were further validated using ELISA. The concentration of these candidates was correlated to the EWGSOP2 sarcopenia tests for low muscle strength and low physical performance, and receiver operating characteristic (ROC) curve analyses were carried out to evaluate their diagnostic power, sensitivity and specificity. Proteomic analysis identified 157 sEVs proteins in both sarcopenic and robust samples. Among them, 48 proteins had never been reported in the ExoCarta nor Vesiclepedia databases. Statistical analysis revealed eight proteins whose concentration was significantly different between groups: PF4 (log2 FC=4.806), OIT3 (log2 FC=-1.161), MMRN1 (log2 FC=-1.982), MASP1 (log2 FC=-0.627), C1R (log2 FC=1.830), SVEP1 (log2 FC=1.295), VCAN (FC=0.937) and SPTB (log2 FC=1.243). Among them, platelet factor 4 (PF4) showed the lowest concentration while Complement C1r subcomponent (C1R) increased the most in sarcopenic patients, being these results confirmed by ELISA (P=1.07E-09 and P=0.001287, respectively). The concentrations of candidate proteins significantly correlated with EWGSOP2 tests currently used. ROC curve analysis showed an area under the curve of 0.8921 and 0.7476 for PF4 and C1R, respectively. Choosing the optimal for PF4, 80% sensitivity and 85.71% specificity was reached while the optimal cut-off value of C1R would allow sarcopenia diagnosis with 75% sensitivity and 66.67% specificity. Our results support the determination of EV PF4 and C1R as plasma diagnostic biomarkers in sarcopenia and open the door to investigate the role of the content of these vesicles in the pathogeny of the disease.

Acute neuroinflammation promotes a metabolic shift that alters extracellular vesicle cargo in the mouse brain cortex.

Neuroinflammation is initiated through microglial activation and cytokine release which can be induced through lipopolysaccharide treatment (LPS) leading to a transcriptional cascade culminating in the differential expression of target proteins. These differentially expressed proteins can then be packaged into extracellular vesicles (EVs), a form of cellular communication, further propagating the neuroinflammatory response over long distances. Despite this, the EV proteome in the brain, following LPS treatment, has not been investigated. Brain tissue and brain derived EVs (BDEVs) isolated from the cortex of LPS-treated mice underwent thorough characterisation to meet the minimal information for studies of extracellular vesicles guidelines before undergoing mass spectrometry analysis to identify the differentially expressed proteins. Fourteen differentially expressed proteins were identified in the LPS brain tissue samples compared to the controls and 57 were identified in the BDEVs isolated from the LPS treated mice compared to the controls. This included proteins associated with the initiation of the inflammatory response, epigenetic regulation, and metabolism. These results allude to a potential link between small EV cargo and early inflammatory signalling.

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

Analysis of extracellular vesicles (EVs) is a promising noninvasive liquid biopsy approach for breast cancer (BC) detection, prognosis, and therapeutic monitoring. A comprehensive understanding of the characteristics and proteomic composition of BC-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 (DIA) proteomic approach to characterize human serum EVs derived from patients with BC (n = 126) and healthy donors (HDs, n = 70) in a discovery cohort and validated the findings in five independent cohorts. Examination of the EV proteomes enabled construction of specific EV protein classifiers for diagnosing BC and distinguishing patients with metastatic disease. Of note, TALDO1 was found to be an EV biomarker of distant metastasis of BC. In vitro and in vivo analysis confirmed the role of TALDO1 in stimulating BC 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 BC migration in vitro and tumor progression in vivo. Together, this work elucidates the proteomic alterations in the serum EVs of BC patients to guide development of improved diagnosis, monitoring, and treatment strategies.

Prostate cancer reshapes the secreted and extracellular vesicle urinary proteomes

Urine is a complex biofluid that reflects both overall physiologic state and the state of the genitourinary tissues through which it passes. It contains both secreted proteins and proteins encapsulated in tissue-derived extracellular vesicles (EVs). To understand the population variability and clinical utility of urine, we quantified the secreted and EV proteomes from 190 men, including a subset with prostate cancer. We demonstrate that a simple protocol enriches prostatic proteins in urine. Secreted and EV proteins arise from different subcellular compartments. Urinary EVs are faithful surrogates of tissue proteomes, but secreted proteins in urine or cell line EVs are not. The urinary proteome is longitudinally stable over several years. It can accurately and non-invasively distinguish malignant from benign prostatic lesions and can risk-stratify prostate tumors. This resource quantifies the complexity of the urinary proteome and reveals the synergistic value of secreted and EV proteomes for translational and biomarker studies.

Non-targeted analysis of the plasma proteome between early and late rugby playing career

Plasma proteomics can offer objective biological insight into the physiological responses of athletes. Rugby players may be repeatedly exposed to high-impact collisions that could predispose them to a higher risk of neurodegenerative conditions. The potential mechanisms underlying the heightened risk are unclear and discovery proteomics could provide unbiased insight into the pathways involved. Plasma extracellular vesicles (EV) are a novel system of intercellular communication which have been implicated in the propagation of misfolded proteins in the brain and could carry putative diagnostic biomarkers. This project investigates whether differences in plasma EV proteome exist between early and late career rugby players. Twenty-four male participants were recruited, including 8 academy players (18 ± 1 years), 8 rugby players (33 ± 5 years) with 10-year professional rugby career and 8 CrossFit athletes (32 ± 5 years) with no history of collision-related sports injuries. Participants gave their informed consent to the ethically approved procedures. Venous blood samples were collected in EDTA blood sample tubes, according to standard operating procedures. Hyper-porous strong-anion exchange magnetic microparticles (MagReSyn® SAX) were used to capture membrane-bound particles (i.e. EV) from plasma. EV-enriched samples were digested with trypsin and analysed using nano-flow liquid chromatography and high-resolution tandem mass spectrometry (Thermo, U3000 and Q-Exactive mass spectrometer). Label free quantitation was performed using Progenesis QI-P (Waters Corp) and proteins were identified by Mascot (Matrix Sci) searches of the UniProt Knowledge base. Statistical differences were investigated by one-way analysis of variance (with correction for multiple testing) in R (v4.3.2). Functional enrichment analysis was performed using ShinyGO (0.80). In total, 449 proteins were confidently identified (false discovery rate; FDR <1%) and gene ontology profiling confirmed 414 of these proteins were of EV origin (Jensen COMPARTMENTS). One-way ANOVA highlighted 192 significantly (P < 0.05, q < 0.02) different proteins across the 3 participant groups, of which 24 proteins were specific to professional rugby players. Functional enrichment analysis revealed key pathways related to Vitamin digestion and absorption, complement and coagulation cascade, inflammatory response, neuron death and negative regulation of cell death (FDR=5%).In conclusion, proteomic profiling is able to detect robust differences between the plasma EV proteome of long-serving professional rugby players compared to younger rugby players or age-matched athletes that did not have a history of collision-related sports injuries. Our findings shed new light on processes affected by a professional rugby playing career, further exploitation of this type of analysis could be used to discover sport-specific biomarkers.

Effect of hypercholesterolemia on circulating and cardiomyocyte-derived extracellular vesicles

Hypercholesterolemia (HC) induces, propagates and exacerbates cardiovascular diseases via various mechanisms that are yet not properly understood. Extracellular vesicles (EVs) are involved in the pathomechanism of these diseases. To understand how circulating or cardiac-derived EVs could affect myocardial functions, we analyzed the metabolomic profile of circulating EVs, and we performed an in-depth analysis of cardiomyocyte (CM)-derived EVs in HC. Circulating EVs were isolated with Vezics technology from male Wistar rats fed with high-cholesterol or control chow. AC16 human CMs were treated with Remembrane HC supplement and EVs were isolated from cell culture supernatant. The biophysical properties and the protein composition of CM EVs were analyzed. THP1-ASC-GFP cells were treated with CM EVs, and monocyte activation was measured. HC diet reduced the amount of certain phosphatidylcholines in circulating EVs, independently of their plasma level. HC treatment significantly increased EV secretion of CMs and greatly modified CM EV proteome, enriching several proteins involved in tissue remodeling. Regardless of the treatment, CM EVs did not induce the activation of THP1 monocytes. In conclusion, HC strongly affects the metabolome of circulating EVs and dysregulates CM EVs, which might contribute to HC-induced cardiac derangements.

Comparative extracellular vesicles proteomics unravels host-pathogen interactions: New insights in bacterial and fungal endophthalmitis in murine models

PurposeEndophthalmitis, an intraocular infection, often results in devastating outcomes due to frequent misdiagnosis and delayed treatment initiation. Extracellular Vesicles (EVs) have long been lauded for its potential as a biomarker in several infections. Previously, we have profiled EVs in bacterial and fungal infected murine model of endophthalmitis. In this study, we reported a comparative analysis of bacterial and fungal endophthalmitis to understand the disease pathobiology and identify a potential signature molecule for diagnosis of infectious endophthalmitis. MethodsEndophthalmitis was induced by intravitreal injection of bacteria (S. aureus and P. aeruginosa) and fungus (A. flavus and C. albicans) in C57BL/6 mice followed by enucleation at 24 h. EVs were isolated by ultracentrifugation and total proteins were subjected to LC-MS/MS. A comparative analysis of the EV protein cargo during bacterial and fungal endophthalmitis was performed in this study along with the pathway assessment by Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology. ResultsProteome analysis of EVs revealed the presence of over 1500 proteins in each of the infected and control groups. Comparative (bacteria versus fungal) study revealed that the protein cargo of the EVs was very distinct among the infected sets with a very few common proteins. Crucial differentially expressed proteins (DEPs) common to both infections were Complement Cascade 8-alpha (C8α), chymase, sorting nexin-29 and glutathione-S-transferase and common pathways were metabolic pathways, Staphylococcus aureus infection, PPAR signalling pathway, cAMP signalling pathway, TGFβ signalling pathway and HIF-1 signalling pathway. ConclusionThis study comprehensively investigates the global proteome of EVs derived from mice model of bacterial and fungal endophthalmitis. Proteomic analysis reveals the disparate EV protein cargo underlining the differences in the pathogenesis of bacterial and fungal endophthalmitis with C8α, chymase, sorting nexin 29 and glutathione-S-transferase common to both infections.

Extracellular vesicles secreted by 3D tumor organoids are enriched for immune regulatory signaling biomolecules compared to conventional 2D glioblastoma cell systems.

Newer 3D culturing approaches are a promising way to better mimic the in vivo tumor microenvironment and to study the interactions between the heterogeneous cell populations of glioblastoma multiforme. Like many other tumors, glioblastoma uses extracellular vesicles as an intercellular communication system to prepare surrounding tissue for invasive tumor growth. However, little is known about the effects of 3D culture on extracellular vesicles. The aim of this study was to comprehensively characterize extracellular vesicles in 3D organoid models and compare them to conventional 2D cell culture systems. Primary glioblastoma cells were cultured as 2D and 3D organoid models. Extracellular vesicles were obtained by precipitation and immunoaffinity, with the latter allowing targeted isolation of the CD9/CD63/CD81 vesicle subpopulation. Comprehensive vesicle characterization was performed and miRNA expression profiles were generated by smallRNA-sequencing. In silico analysis of differentially regulated miRNAs was performed to identify mRNA targets and corresponding signaling pathways. The tumor cell media and extracellular vesicle proteome were analyzed by high-resolution mass spectrometry. We observed an increased concentration of extracellular vesicles in 3D organoid cultures. Differential gene expression analysis further revealed the regulation of twelve miRNAs in 3D tumor organoid cultures (with nine miRNAs down and three miRNAs upregulated). MiR-23a-3p, known to be involved in glioblastoma invasion, was significantly increased in 3D. MiR-7-5p, which counteracts glioblastoma malignancy, was significantly decreased. Moreover, we identified four miRNAs (miR-323a-3p, miR-382-5p, miR-370-3p, miR-134-5p) located within the DLK1-DIO3 domain, a cancer-associated genomic region, suggesting a possible importance of this region in glioblastoma progression. Overrepresentation analysis identified alterations of extracellular vesicle cargo in 3D organoids, including representation of several miRNA targets and proteins primarily implicated in the immune response. Our results show that 3D glioblastoma organoid models secrete extracellular vesicles with an altered cargo compared to corresponding conventional 2D cultures. Extracellular vesicles from 3D cultures were found to contain signaling molecules associated with the immune regulatory signaling pathways and as such could potentially change the surrounding microenvironment towards tumor progression and immunosuppressive conditions. These findings suggest the use of 3D glioblastoma models for further clinical biomarker studies as well as investigation of new therapeutic options.

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).

Changes Induced by P2X7 Receptor Stimulation of Human Glioblastoma Stem Cells in the Proteome of Extracellular Vesicles Isolated from Their Secretome.

Extracellular vesicles (EVs) are secreted from many tumors, including glioblastoma multiforme (GBM), the most common and lethal brain tumor in adults, which shows high resistance to current therapies and poor patient prognosis. Given the high relevance of the information provided by cancer cell secretome, we performed a proteomic analysis of microvesicles (MVs) and exosomes (EXOs) released from GBM-derived stem cells (GSCs). The latter, obtained from the brain of GBM patients, expressed P2X7 receptors (P2X7Rs), which positively correlate with GBM growth and invasiveness. P2X7R stimulation of GSCs caused significant changes in the EV content, mostly ex novo inducing or upregulating the expression of proteins related to cytoskeleton reorganization, cell motility/spreading, energy supply, protection against oxidative stress, chromatin remodeling, and transcriptional regulation. Most of the induced/upregulated proteins have already been identified as GBM diagnostic/prognostic factors, while others have only been reported in peripheral tumors. Our findings indicate that P2X7R stimulation enhances the transport and, therefore, possible intercellular exchange of GBM aggressiveness-increasing proteins by GSC-derived EVs. Thus, P2X7Rs could be considered a new druggable target of human GBM, although these data need to be confirmed in larger experimental sets.

Abstract 6965: Characterizing the proteome landscape of extracellular vesicles from prostate cancer cell lines by mass spectrometry

Abstract Multiple subtypes of prostate cancer can emerge in advanced stages of disease, such as tumors that maintain androgen receptor features (AR+), those without AR but with neuroendocrine features (AR-/NE+), and those without AR or NE features (AR-/NE-). These latter subtypes are particularly aggressive and incredibly lethal. Extracellular vesicles (EVs) are small particles that are mediators of cell-cell communication and have been nominated as potential biomarkers of disease state. We hypothesized that the proteome of EVs contains information about their originating cell that can distinguish between prostate cancer subtypes. To address this, we isolated EVs from cell lines of differing subtypes – AR+ (LNCaP, LNCaP95, C4-2, 22RV1), AR-/NE+(NCI-H660, EF1, LASCPC-01), and AR-/NE- (PC3, DU145) – and prepared the EV proteome for quantitative, label-free mass spectrometry analysis. This identified 2,000+ proteins in the EVs for each cell line where EVs from similar subtypes clustered together. Interestingly, we also found that EVs contained enough protein information to utilize developed gene signatures for AR or NE activity. EVs isolated from AR-/NE+ cells contained known NE signature proteins such as SYP, CHGA, and UCHL1. EVs from the AR+ cell lines displayed >50% AR activity driven by PSA and STEAP1 proteins in addition to other significant distinguishing protein markers. This supports the potential for using the proteome of tumor-derived EVs to identify novel signature molecular markers besides those already known to the field for developing liquid-based biomarker assays. In addition, our work characterizes the EV surfaceome which is critical for enriching signals from tumor-derived EVs feasible for clinical diagnostics assay development. While PSMA has been a reliable marker for cells reliant on AR signaling, less is known about protein markers in the AR- subset. Our work highlights TROP2 as especially enriched in the EVs from AR-/NE- cell lines, supporting its relevance as a target in patients without treatment options. To evaluate disease outcomes, further clinical studies are needed to develop specific and sensitive tests based on the EV proteome derived from advanced prostate cancer patients. Citation Format: Megan L. Ludwig, Abderrahman Day, Hannah E. Bergom, Zoi Sychev, Alec Horrmann, Justin Hwang, Justin M. Drake. Characterizing the proteome landscape of extracellular vesicles from prostate cancer cell lines by mass spectrometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6965.

Extracellular vesicles from virulent P. brasiliensis induce TLR4 and dectin-1 expression in innate cells and promote enhanced Th1/Th17 response

ABSTRACT Extracellular vesicles (EVs) are membrane-enclosed nanoparticles that transport several biomolecules and are involved in important mechanisms and functions related to the pathophysiology of fungal diseases. EVs from Paracoccidioides brasiliensis, the main causative agent of Paracoccidioidomycosis (PCM), modulate the immune response of macrophages. In this study, we assessed the EVs proteome from a virulent P. brasiliensis isolated from granulomatous lesions and compared their immunomodulatory ability with EVs isolated from the fungus before the animal passage (control EVs) when challenging macrophages and dendritic cells (DCs). Proteome showed that virulent EVs have a higher abundance of virulence factors such as GP43, protein 14-3-3, GAPDH, as well as virulence factors never described in PCM, such as aspartyl aminopeptidase and a SidJ analogue compared with control EVs. Virulent extracellular vesicles induced higher expression of TLR4 and Dectin-1 than control EVs in macrophages and dendritic cells (DCs). In opposition, a lower TLR2 expression was induced by virulent EVs. Additionally, virulent EVs induced lower expression of CD80, CD86 and TNF-α, but promoted a higher expression of IL-6 and IL-10, suggesting that EVs isolated from virulent P. brasiliensis-yeast promote a milder DCs and macrophage maturation. Herein, we showed that EVs from virulent fungi stimulated a higher frequency of Th1/Tc1, Th17, and Treg cells, which gives new insights into fungal extracellular vesicles. Taken together, our results suggest that P. brasiliensis utilizes its EVs as virulence bags that manipulate the immune system in its favour, creating a milder immune response and helping with fungal evasion from the immune system.

Proteomic characterization of extracellular vesicles in programmed cell death.

Programmed cell death (PCD) is a fundamental biological process that plays a critical role in cell development, differentiation, and homeostasis. The secretion and uptake of extracellular vesicles (EVs) is one of the important regulatory mechanisms for PCD. EVs are natural membrane structures secreted by cells that contain a variety of proteins, lipids, nucleic acids, and other bioactive molecules. Due to their important roles in intercellular communication and disease progression, there is great interest in studying EVs and their cargo. Different protein components are sorted and packaged in EVs, allowing EVs to perform their functions. The study of EV proteomics helps us understand the role of PCD in the development of diseases. Meanwhile, proteomics is a powerful tool for studying the composition and function of EVs, which assists in the identification, quantification, and profiling of protein components of EVs, and provides insight into the molecular mechanisms involved in PCD and related diseases. In this review, we summarize the characteristics of EV proteomics in different types of PCD, compare different proteomic profiling strategies for EVs, and discuss the impact of EV proteomics on cell function and regulation during PCD, to understand its role in the pathogenesis of related diseases.

Polycarbonate Ultracentrifuge Tube Re-use in Proteomic Analyses of Extracellular Vesicles.

Single-use laboratory plastics exacerbate the pollution crisis and contribute to consumable costs. In extracellular vesicle (EV) isolation, polycarbonate ultracentrifuge (UC) tubes are used to endure the associated high centrifugal forces. EV proteomics is an advancing field and validated re-use protocols for these tubes are lacking. Re-using consumables for low-yield protein isolation protocols and downstream proteomics requires reagent compatibility with mass spectroscopy acquisitions, such as the absence of centrifuge tube-derived synthetic polymer contamination, and sufficient removal of residual proteins. This protocol describes and validates a method for cleaning polycarbonate UC tubes for re-use in EV proteomics experiments. The cleaning process involves immediate submersion of UC tubes in H2O to prevent protein drying, washing in 0.1% sodium dodecyl sulfate (SDS) detergent, rinsing in hot tap water, demineralized water, and 70% ethanol. To validate the UC tube re-use protocol for downstream EV proteomics, used tubes were obtained following an experiment isolating EVs from cardiovascular tissue using differential UC and density gradient separation. Tubes were cleaned and the experimental process was repeated without EV samples comparing blank never-used UC tubes to cleaned UC tubes. The pseudo-EV pellets obtained from the isolation procedures were lysed and prepared for liquid chromatography-tandem mass spectrometry using a commercial protein sample preparation kit with modifications for low-abundance protein samples. Following cleaning, the number of identified proteins was reduced by 98% in the pseudo-pellet versus the previous EV isolation sample from the same tube. Comparing a cleaned tube against a blank tube, both samples contained a very small number of proteins (≤20) with 86% similarity. The absence of polymer peaks in the chromatograms of the cleaned tubes was confirmed. Ultimately, the validation of a UC tube cleaning protocol suitable for the enrichment of EVs will reduce the waste produced by EV laboratories and lower the experimental costs.

Multiple Myeloma Derived Extracellular Vesicle Uptake by Monocyte Cells Stimulates IL-6 and MMP-9 Secretion and Promotes Cancer Cell Migration and Proliferation.

Multiple Myeloma (MM) is an incurable haematological malignancy caused by uncontrolled growth of plasma cells. MM pathogenesis is attributed to crosstalk between plasma cells and the bone marrow microenvironment, where extracellular vesicles (EVs) play a role. In this study, EVs secreted from a panel of MM cell lines were isolated from conditioned media by ultracentrifugation and fluorescently stained EVs were co-cultured with THP-1 monocyte cells. MM EVs from three cell lines displayed a differential yet dose-dependent uptake by THP-1 cells, with H929 EVs displaying the greatest EV uptake compared to MM.1s and U266 EVs suggesting that uptake efficiency is dependent on the cell line of origin. Furthermore, MM EVs increased the secretion of MMP-9 and IL-6 from monocytes, with H929 EVs inducing the greatest effect, consistent with the greatest uptake efficiency. Moreover, monocyte-conditioned media collected following H929 EV uptake significantly increased the migration and proliferation of MM cells. Finally, EV proteome analysis revealed differential cargo enrichment that correlates with disease progression including a significant enrichment of spliceosome-related proteins in H929 EVs compared to the U266 and MM.1s EVs. Overall, this study demonstrates that MM-derived EVs modulate monocyte function to promote tumour growth and metastasis and reveals possible molecular mechanisms involved.

The Proteome of Extracellular Vesicles Released from Pulmonary Microvascular Endothelium Reveals Impact of Oxygen Conditions on Biotrauma.

The lung can experience different oxygen concentrations, low as in hypoxia, high as under supplemental oxygen therapy, or oscillating during intermittent hypoxia as in obstructive sleep apnea or intermittent hypoxia/hyperoxia due to cyclic atelectasis in the ventilated patient. This study aimed to characterize the oxygen-condition-specific protein composition of extracellular vesicles (EVs) released from human pulmonary microvascular endothelial cells in vitro to decipher their potential role in biotrauma using quantitative proteomics with bioinformatic evaluation, transmission electron microscopy, flow cytometry, and non-activated thromboelastometry (NATEM). The release of vesicles enriched in markers CD9/CD63/CD81 was enhanced under intermittent hypoxia, strong hyperoxia and intermittent hypoxia/hyperoxia. Particles with exposed phosphatidylserine were increased under intermittent hypoxia. A small portion of vesicles were tissue factor-positive, which was enhanced under intermittent hypoxia and intermittent hypoxia/hyperoxia. EVs from treatment with intermittent hypoxia induced a significant reduction of Clotting Time in NATEM analysis compared to EVs isolated after normoxic exposure, while after intermittent hypoxia/hyperoxia, tissue factor in EVs seems to be inactive. Gene set enrichment analysis of differentially expressed genes revealed that EVs from individual oxygen conditions potentially induce different biological processes such as an inflammatory response under strong hyperoxia and intermittent hypoxia/hyperoxia and enhancement of tumor invasiveness under intermittent hypoxia.

Size-exclusion chromatography combined with DIA-MS enables deep proteome profiling of extracellular vesicles from melanoma plasma and serum

Extracellular vesicles (EVs) are important players in melanoma progression, but their use as clinical biomarkers has been limited by the difficulty of profiling blood-derived EV proteins with high depth of coverage, the requirement for large input amounts, and complex protocols. Here, we provide a streamlined and reproducible experimental workflow to identify plasma- and serum- derived EV proteins of healthy donors and melanoma patients using minimal amounts of sample input. SEC–DIA-MS couples size-exclusion chromatography to EV concentration and deep-proteomic profiling using data-independent acquisition. From as little as 200 µL of plasma per patient in a cohort of three healthy donors and six melanoma patients, we identified and quantified 2896 EV-associated proteins, achieving a 3.5-fold increase in depth compared to previously published melanoma studies. To compare the EV-proteome to unenriched blood, we employed an automated workflow to deplete the 14 most abundant proteins from plasma and serum and thereby approximately doubled protein group identifications versus native blood. The EV proteome diverged from corresponding unenriched plasma and serum, and unlike the latter, separated healthy donor and melanoma patient samples. Furthermore, known melanoma markers, such as MCAM, TNC, and TGFBI, were upregulated in melanoma EVs but not in depleted melanoma plasma, highlighting the specific information contained in EVs. Overall, EVs were significantly enriched in intact membrane proteins and proteins related to SNARE protein interactions and T-cell biology. Taken together, we demonstrated the increased sensitivity of an EV-based proteomic workflow that can be easily applied to larger melanoma cohorts and other indications.