Bead-Based Extracellular Vesicle Analysis Using Flow Cytometry.
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ABSTRACT:
SUBMITTER: Yang KS
PROVIDER: S-EPMC7718389 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Background: Extracellular vesicles (EVs) are secreted by cells from their membrane within circulation and body fluids. Knowledge of the involvement of EVs in pathogenesis of lung diseases is increasing. The present study aimed to evaluate the expression of exosomal surface epitopes in a cohort of idiopathic pulmonary fibrosis (IPF) patients followed in two Italian Referral Centres for Interstitial Lung Diseases, comparing them with a group of healthy volunteers. Materials and Methods: Ninety IPF patients (median age and interquartile range (IQR) 71 (66-75) years; 69 males) were selected retrospectively. Blood samples were obtained from patients before starting antifibrotic therapy. A MACSPlex Exosome Kit, human, (Miltenyi Biotec, Bergisch-Gladbach, Germany), to detect 37 exosomal surface epitopes, was used. Results: CD19, CD69, CD8, and CD86 were significantly higher in IPF patients than in controls (p = 0.0023, p = 0.0471, p = 0.0082, and p = 0.0143, respectively). CD42a was lower in IPF subjects than in controls (p = 0.0153), while CD209, Cd133/1, MCSP, and ROR1 were higher in IPF patients than in controls (p = 0.0007, p = 0.0050, p = 0.0139, and p = 0.0335, respectively). Kaplan-Meier survival analysis for IPF patients: for median values and a cut-off of 0.48 for CD25, the two subgroups showed a significant difference in survival rate (p = 0.0243, hazard ratio: 0.52 (95%CI 0.29-0.92); the same was true for CD8 (cut-off 1.53, p = 0.0309, hazard ratio: 1.39 (95%CI 0.75-2.53). Conclusion: Our multicenter study showed for the first time the expression of surface epitopes on EVs from IPF patients, providing interesting data on the communication signatures/exosomal profile in serum from IPF patients and new insights into the pathogenesis of the disease and a promising reliability in predicting mid-term survival of IPF patients.
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Project description:Extracellular vesicles (EVs) can be harvested from cell culture supernatants and from all body fluids. EVs can be conceptually classified based on their size and biogenesis as exosomes and microvesicles. Nowadays, it is however commonly accepted in the field that there is a much higher degree of heterogeneity within these two subgroups than previously thought. For instance, the surface marker profile of EVs is likely dependent on the cell source, the cell's activation status, and multiple other parameters. Within recent years, several new methods and assays to study EV heterogeneity in terms of surface markers have been described; most of them are being based on flow cytometry. Unfortunately, such methods generally require dedicated instrumentation, are time-consuming and demand extensive operator expertise for sample preparation, acquisition, and data analysis. In this study, we have systematically evaluated and explored the use of a multiplex bead-based flow cytometric assay which is compatible with most standard flow cytometers and facilitates a robust semi-quantitative detection of 37 different potential EV surface markers in one sample simultaneously. First, assay variability, sample stability over time, and dynamic range were assessed together with the limitations of this assay in terms of EV input quantity required for detection of differently abundant surface markers. Next, the potential effects of EV origin, sample preparation, and quality of the EV sample on the assay were evaluated. The findings indicate that this multiplex bead-based assay is generally suitable to detect, quantify, and compare EV surface signatures in various sample types, including unprocessed cell culture supernatants, cell culture-derived EVs isolated by different methods, and biological fluids. Furthermore, the use and limitations of this assay to assess heterogeneities in EV surface signatures was explored by combining different sets of detection antibodies in EV samples derived from different cell lines and subsets of rare cells. Taken together, this validated multiplex bead-based flow cytometric assay allows robust, sensitive, and reproducible detection of EV surface marker expression in various sample types in a semi-quantitative way and will be highly valuable for many researchers in the EV field in different experimental contexts.
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