Specimen-specific drift of densities defines distinct subclasses of extracellular vesicles from human whole saliva.
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ABSTRACT:
SUBMITTER: Yamamoto S
PROVIDER: S-EPMC8032098 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Extracellular vesicles (EVs) are cell-derived membrane vesicles that are released into the extracellular space. EVs encapsulate key proteins and mediate intercellular signalling pathways. Recently, primary cilia have been shown to release EVs under fluid-shear flow, but many proteins encapsulated in these vesicles have never been identified. Primary cilia are ubiquitous mechanosensory organelles that protrude from the apical surface of almost all human cells. Primary cilia also serve as compartments for signalling pathways, and their defects have been associated with a wide range of human genetic diseases called ciliopathies. To better understand the mechanism of ciliopathies, it is imperative to know the distinctive protein profiles of the differently sourced EVs (cilia vs cytosol). Here, we isolated EVs from ciliated wild-type (WT) and non-ciliated IFT88 knockout (KO) mouse endothelial cells using fluid-shear flow followed by a conventional method of EV isolation. EVs isolated from WT and KO exhibited distinctive sizes. Differences in EV protein contents were studied using liquid chromatography with tandem mass spectrometry (LC-MS-MS) and proteomic comparative analysis, which allowed us to classify proteins between ciliary EVs and cytosolic EVs derived from WT and KO, respectively. A total of 79 proteins were exclusively expressed in WT EVs, 145 solely in KO EVs, and 524 in both EVs. Our bioinformatics analyses revealed 29% distinct protein classes and 75% distinct signalling pathways between WT and KO EVs. Based on our statistical analyses and in vitro studies, we identified NADPH-cytochrome P450 reductase (POR), and CD166 antigen (CD166) as potential biomarkers for ciliary and cytosolic EVs, respectively. Our protein-protein interaction network analysis revealed that POR, but not CD166, interacted with either established or strong ciliopathy gene candidates. This report shows the unique differences between EVs secreted from cilia and the cytosol. These results will be important in advancing our understanding of human genetic diseases.
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Project description:Circulating and interstitial small membrane-bound extracellular vesicles (EVs) represent promising targets for the development of novel diagnostic or prognostic biomarker assays, and likely serve as important players in the progression of a vast spectrum of diseases. Current research is focused on the characterization of vesicles secreted from multiple cell and tissue types in order to better understand the role of EVs in the pathogenesis of conditions including neurodegeneration, inflammation, and cancer. However, globally consistent and reproducible techniques to isolate and purify vesicles remain in progress. Moreover, methods for extraction of EVs from solid tissue ex vivo are scarcely described. Here, we provide a detailed protocol for extracting small EVs of interest from whole fresh or frozen tissues, including brain and tumor specimens, for further characterization. We demonstrate the adaptability of this method for multiple downstream analyses, including electron microscopy and immunophenotypic characterization of vesicles, as well as quantitative mass spectrometry of EV proteins.
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Project description:AimTo confirm the presence of extracellular vesicles (EVs) in cell-free saliva (CFS) of children with asthma and describe the isolated EV population.MethodsA pooled sample of CFS EVs isolated from 180 participants using ExoQuick-TC was examined in downstream analyses. Transmission electron microscopy (TEM) was used to confirm the presence of EVs. Nanoparticle tracking analysis (NTA) and single particle interferometric reflectance imaging sensing (SP-IRIS) with fluorescence were used for sizing, counting, and phenotyping of EVs. Capillary immunoassays were used for protein quantitation.ResultsTEM confirmed the presence of EVs of diverse sizes, indicating the prep contained a heterogeneous population of EVs. Capillary immunoassays confirmed the presence of EV-associated proteins (CD9, CD63, CD81, ICAM-1, and ANXA5) and indicated limited cellular contamination. As others have also reported, there were discrepancies in the EV sizing and enumeration across platforms. Fluorescent NTA detected particles with a mode diameter of ~90 nm, whereas SP-IRIS reported sizes of ~55-60 nm that more closely approximated the TEM results. Consistent with protein immunoassay results, SP-IRIS with fluorescence showed that the majority of these EVs were CD9- and CD63-positive, with little expression of CD81.ConclusionEVs from CFS can be isolated using a high-throughput method that can be scaled to large epidemiological studies. To our knowledge, we are the first to characterize CFS EVs from patients with asthma. The use of CFS EVs as potential novel biomarkers in asthma warrants further investigation and opens a new avenue of research for future studies.
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Project description:In-depth knowledge of bodily fluid phosphoproteomes, such as whole saliva, is limited. To better understand the whole saliva phosphoproteome, we generated a large-scale catalog of phosphorylated proteins. To circumvent the wide dynamic range of phosphoprotein abundance in whole saliva, we combined dynamic range compression using hexapeptide beads, strong cation exchange HPLC peptide fractionation, and immobilized metal affinity chromatography prior to mass spectrometry. In total, 217 unique phosphopeptides sites were identified representing 85 distinct phosphoproteins at 2.3% global FDR. From these peptides, 129 distinct phosphorylation sites were identified of which 57 were previously known, but only 11 of which had been previously identified in whole saliva. Cellular localization analysis revealed salivary phosphoproteins had a distribution similar to all known salivary proteins, but with less relative representation in "extracellular" and "plasma membrane" categories compared to salivary glycoproteins. Sequence alignment showed that phosphorylation occurred at acidic-directed kinase, proline-directed, and basophilic motifs. This differs from plasma phosphoproteins, which predominantly occur at Golgi casein kinase recognized sequences. Collectively, these results suggest diverse functions for salivary phosphoproteins and multiple kinases involved in their processing and secretion. In all, this study should lay groundwork for future elucidation of the functions of salivary protein phosphorylation.
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Project description:Small extracellular vesicles from saliva (SEVs) have high potential as biomarkers in Head and Neck cancer (HNC). However, there is no common consensus on the ideal method for their isolation. This study compared different ultracentrifugation (UC) methods (durations and + /- additional purification) with size exclusion chromatography (SEC) and investigated the potential of SEVs as diagnostic biomarkers and their biological activity on NK and CD8+ T cells. SEVs from 19 HNC patients and 8 healthy donors (HDs) were thoroughly characterized. Transmission electron microscopy confirmed the isolation of vesicles by all methods. The average size determined via nanoparticle-tracking analysis was smaller for SEVs isolated by SEC than UC. The highest particle-to-protein yield was achieved by UC (3 h + 3 h) (UCopt) and SEC. However, SEC yielded considerably fewer SEVs. Comparing the surface marker cargo, SEVs isolated by UCopt from HNC patients carried more PD-L1, FasL, and TGF-β than SEVs from HDs. These levels correlated with tumor stage and HPV status. SEVs downregulated NKG2D expression on primary NK cells. HNC SEVs accelerated CD8+ T cell death compared to HD SEVs. This study suggests that UCopt is preferable when isolation of a high particle-to-protein load is required. Especially PD-L1 and FasL on SEVs hold substantial potential as diagnostic biomarkers.
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Project description:Extracellular vesicles (EVs) are ideal for liquid biopsy, but distinguishing cancer cell-derived EVs and subpopulations of biomarker-containing EVs in body fluids has been challenging. Here, we identified that the glycoproteins CD147 and CD98 define subpopulations of EVs that are distinct from classical tetraspanin+ EVs in their biogenesis. Notably, we identified that CD147+ EVs have substantially higher microRNA (miRNA) content than tetraspanin+ EVs and are selectively enriched in miRNA through the interaction of CD147 with heterogeneous nuclear ribonucleoprotein A2/B1. Studies using mouse xenograft models showed that CD147+ EVs predominantly derive from cancer cells, whereas the majority of tetraspanin+ EVs are not of cancer cell origin. Circulating CD147+ EVs, but not tetraspanin+ EVs, were significantly increased in prevalence in patients with ovarian and renal cancers as compared to healthy individuals and patients with benign conditions. Furthermore, we found that isolating miRNAs from body fluids by CD147 immunocapture increases the sensitivity of detecting cancer cell-specific miRNAs, and that circulating miRNAs isolated by CD147 immunocapture more closely reflect the tumor miRNA signature than circulating miRNAs isolated by conventional methods. Collectively, our findings reveal that CD147 defines miRNA-enriched, cancer cell-derived EVs, and that CD147 immunocapture could be an effective approach to isolate cancer-derived miRNAs for liquid biopsy.
| S-EPMC10042814 | biostudies-literature
Project description:Extracellular vesicles (EVs) mediate intercellular signaling by transferring their cargo to recipient cells, but the functional consequences of signaling are not fully appreciated. RBC-derived EVs are abundant in circulation and have been implicated in regulating immune responses. Here, we use a transgenic mouse model for fluorescence-based mapping of RBC-EV recipient cells to assess the role of this intercellular signaling mechanism in heart disease. Using fluorescent-based mapping, we detected an increase in RBC-EV-targeted cardiomyocytes in a murine model of ischemic heart failure. Single cell nuclear RNA sequencing of the heart revealed a complex landscape of cardiac cells targeted by RBC-EVs, with enrichment of genes implicated in cell proliferation and stress signaling pathways compared with non-targeted cells. Correspondingly, cardiomyocytes targeted by RBC-EVs more frequently express cellular markers of DNA synthesis, suggesting the functional significance of EV-mediated signaling. In conclusion, our mouse model for mapping of EV-recipient cells reveals a complex cellular network of RBC-EV-mediated intercellular communication in ischemic heart failure and suggests a functional role for this mode of intercellular signaling.
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Project description:BACKGROUND:Activated microglia play an essential role in inflammatory responses elicited in the central nervous system (CNS). Microglia-derived extracellular vesicles (EVs) are suggested to be involved in propagation of inflammatory signals and in the modulation of cell-to-cell communication. However, there is a lack of knowledge on the regulation of EVs and how this in turn facilitates the communication between cells in the brain. Here, we characterized microglial EVs under inflammatory conditions and investigated the effects of inflammation on the EV size, quantity, and protein content. METHODS:We have utilized western blot, nanoparticle tracking analysis (NTA), and mass spectrometry to characterize EVs and examine the alterations of secreted EVs from a microglial cell line (BV2) following lipopolysaccharide (LPS) and tumor necrosis factor (TNF) inhibitor (etanercept) treatments, or either alone. The inflammatory responses were measured with multiplex cytokine ELISA and western blot. We also subjected TNF knockout mice to experimental stroke (permanent middle cerebral artery occlusion) and validated the effect of TNF inhibition on EV release. RESULTS:Our analysis of EVs originating from activated BV2 microglia revealed a significant increase in the intravesicular levels of TNF and interleukin (IL)-6. We also observed that the number of EVs released was reduced both in vitro and in vivo when inflammation was inhibited via the TNF pathway. Finally, via mass spectrometry, we identified 49 unique proteins in EVs released from LPS-activated microglia compared to control EVs (58 proteins in EVs released from LPS-activated microglia and 37 from control EVs). According to Gene Ontology (GO) analysis, we found a large increase of proteins related to translation and transcription in EVs from LPS. Importantly, we showed a distinct profile of proteins found in EVs released from LPS treated cells compared to control. CONCLUSIONS:We demonstrate altered EV production in BV2 microglial cells and altered cytokine levels and protein composition carried by EVs in response to LPS challenge. Our findings provide new insights into the potential roles of EVs that could be related to the pathogenesis in neuroinflammatory diseases.
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Project description:Most cancer-related deaths are caused by distant metastases, which are tumour cells that have escaped from a primary tumour and passed into the bloodstream to colonize a new organ. In this context, communication between tumour and stromal cells is essential. Indeed, tumor cells interact with cells in the tumor microenvironment and are able to modify them to their advantage. Both extracellular vesicles (EVs) and exosomes are heterogeneous populations of small vesicles present in the tumor microenvironment and in body fluids that have recently emerged as powerful mediators involved in this communication and their transport in fluids. Tumor cells release large quantities of exosomes containing tumor markers, which can then spread to distant locations.
The exosomes are of endosomal origin. They are composed of proteins, lipids, RNA and DNA, and they circulate in the bloodstream. They can be internalized by specific distant cells and thus deliver a functional message. It has recently been shown that tumor exosomes containing pro-metastatic factors form pre-metastatic niches, before the tumor cells actually arrive, while determining the metastatic organotropism of tumors. These properties are now opening up new avenues of research in tumor biomarkers. In recent years, several studies have highlighted different markers contained specifically in exosomes derived from cancer cells. Consequently, exosomes are considered as potential reservoirs of tumor biomarkers that could be clinically useful for the non-invasive diagnosis of cancer, with the advantage of being performed by liquid biopsy. The study of microRNA (miRNA) is of particular interest. Indeed, miRNAs are small non-coding RNAs (between 21 and 25 nucleotides) involved in the regulation of gene expression and which are frequently deregulated in cancer. Several studies underline that the variation of free miRNAs in the blood is correlated with the progression of the disease, particularly in colon cancer. However, the stability of free miRNAs is controversial. Therefore, exosomes represent a very advantageous means of transporting miRNAs in the blood, as they are able to protect miRNAs from degradation by RNAase.
The hypothesis of the project is that circulating exosomes derived from tumours contain markers including specific miRNAs that could be used as biomarkers of early prognosis (survival and progression), easily measured in blood samples from patients with colon cancer. But other molecules contained in exosomes could also be of interest.
| 2349603 | ecrin-mdr-crc
Project description:The development and progression of oral cavity squamous cell carcinoma (OSCC) involves complex cellular mechanisms that contribute to the low five-year survival rate of approximately 20% among diagnosed patients. However, the biological processes essential to tumor progression are not completely understood. Therefore, detecting alterations in the salivary proteome may assist in elucidating the cellular mechanisms modulated in OSCC and improve the clinical prognosis of the disease. The proteome of whole saliva and salivary extracellular vesicles (EVs) from patients with OSCC and healthy individuals were analyzed by LC-MS/MS and label-free protein quantification. Proteome data analysis was performed using statistical, machine learning and feature selection methods with additional functional annotation. Biological processes related to immune responses, peptidase inhibitor activity, iron coordination and protease binding were overrepresented in the group of differentially expressed proteins. Proteins related to the inflammatory system, transport of metals and cellular growth and proliferation were identified in the proteome of salivary EVs. The proteomics data were robust and could classify OSCC with 90% accuracy. The saliva proteome analysis revealed that immune processes are related to the presence of OSCC and indicate that proteomics data can contribute to determining OSCC prognosis.
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