Dataset Information

Cytokine profiling in serum-derived exosomes isolated by different methods.

ABSTRACT: Exosomes in blood play an important role in cell-to-cell signaling and are a novel source of biomarkers for the diagnosis and prognosis of diseases. Recently, evidence has accumulated that cytokines are released from encapsulated exosomes and are capable of eliciting biological effects upon contact with sensitive cells. However, there is currently limited information on exosome isolation methods for cytokine research. In this study, we evaluated three exosome isolation methods for their usability, yield, purity, and effectiveness in subsequent cytokine profiling. We found that ultracentrifugation (UC) and Exoquick (EQ), but not exoEasy, yielded appropriate exosome sizes, and EQ had higher exosome extraction efficiency than the other two methods. Although UC generated markedly fewer particles than EQ, it yielded a relatively high purity. Next, we performed a multiplex assay with the ProcartaPlex Immune Monitoring 65-Plex Panel to determine the feasibility of these methods for cytokine profiling. The results indicated significant differences among isolation methods when analyzing exosomal cytokine profiles. We further investigated the changes of exosomal cytokines according to breast cancer progression in triple-negative breast cancer. We found significantly decreased concentrations of MIP-3 alpha, IL-23, M-CSF, Eotaxin-3, BLC, SDF-1 alpha, IL-2R, MDC, FGF-2, IL-22, and IL-31 in exosomes from metastatic breast cancer (MBC) patients.

SUBMITTER: Jung HH

PROVIDER: S-EPMC7442638 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Cytokine profiling in serum-derived exosomes isolated by different methods.

Jung Hae Hyun HH Kim Ji-Yeon JY Lim Ji Eun JE Im Young-Hyuck YH

Scientific reports 20200821 1

Exosomes in blood play an important role in cell-to-cell signaling and are a novel source of biomarkers for the diagnosis and prognosis of diseases. Recently, evidence has accumulated that cytokines are released from encapsulated exosomes and are capable of eliciting biological effects upon contact with sensitive cells. However, there is currently limited information on exosome isolation methods for cytokine research. In this study, we evaluated three exosome isolation methods for their usabilit ...[more]

PMID: 32826923

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Project description:Cytokines and chemokines circulate in plasma and may be transferred to distant sites, via exosomes. HIV infection is associated with dysregulation of cytokines and chemokines, which subsequently contribute to the pathogenesis of HIV. Alcohol and tobacco exposure, which are prevalent in HIV-infected individuals, may induce changes in the expression of cytokines and chemokines. Therefore, our aim in this study was to quantify plasma exosomal cytokines and chemokines that we expect to exacerbate toxicity or disease progression in HIV-positive drug abusers. We measured the levels of cytokines and chemokines in the plasma and plasma exosomes of 39 patients comprising six groups: HIV-negative and HIV-positive non drug abusers, HIV-negative and HIV-positive alcohol users, and HIV-negative and HIV positive tobacco smokers. We measured six cytokines (TNF-?, IL-1?, IL-8, IL-6, IL-1ra, IL-10) and two chemokines (MCP-1 and RANTES). All were present in exosomes of healthy subjects, but their levels varied between different study groups. HIV-positive alcohol drinkers had higher levels of plasma IL-8 compared to those of HIV-positive non-drinkers. The IL-1ra level was significantly higher in exosomes of non-HIV-infected alcohol drinkers compared to those of HIV-positive alcohol drinkers. Interestingly, the IL-10 level was higher in exosomes compared with their respective plasma levels in all study groups except HIV-positive non-alcohol drinkers. IL-10 was completely packaged in exosomes of HIV-positive smokers. HIV-positive smokers had significantly higher levels of plasma IL-8 compared with HIV-positive non-smokers and significantly higher exosomal IL-6 levels compared with HIV-negative subjects. HIV-positive smokers had significantly increased plasma levels of IL-1ra compared to HIV-positive non-smokers. The MCP-1 levels in the plasma of HIV-positive smokers was significantly higher than in either HIV-positive non-drug abusers or HIV-negative smokers. Overall, the findings suggest that plasma cytokines and chemokines are packaged in exosomes at varying degrees in different study groups. Exosomal cytokines and chemokines are likely to have a significant biological role at distant sites including cells in the brain.

Project description:Background: Clinical management of metastatic gastric cancer (mGC) remains a major challenge due to a lack of specific biomarkers and effective therapeutic targets. Recently, accumulating evidence has suggested that exosomes play an essential role in cancer metastasis and can be an excellent reservoir of novel biomarkers and candidate therapeutic targets for cancer. Therefore, in this study, we aimed to reveal the proteomic profile of mGC-derived exosomes. Methods: Exosomes were isolated from pooled serum samples of 20 mGC patients and 40 healthy controls (HC) by ultracentrifugation. Next, quantitative proteomic analyses were applied to analyze the protein profiles of the exosomes, and bioinformatic analyses were conducted on the proteomic data. Finally, the expression of exosomal protein candidates was selectively validated in individual subjects by western blot analysis. Results: We isolated exosomes from serum samples. The size of the serum derived exosomes ranged from 30 to 150 nm in diameter. The exosomal markers CD9 and CD81 were observed in the serum exosomes. However, the exosomal negative marker calnexin, an endoplasmic reticulum protein, was not detected in exosomes. Overall, 443 exosomal proteins, including 110 differentially expressed proteins (DEPs) were identified by quantitative proteomics analyses. The bioinformatics analyses indicated that the upregulated proteins were enriched in the process of protein metabolic, whereas the downregulated proteins were largely involved in cell-cell adhesion organization. Surprisingly, 10 highly vital proteins (UBA52, PSMA1, PSMA5, PSMB6, PSMA7, PSMA4, PSMA3, PSMB1, PSMA6, and FGA) were filtered from DEPs, most of which are proteasome subunits. Moreover, the validation data confirmed that PSMA3 and PSMA6 were explicitly enriched in the serum derived exosomes from patients with mGC. Conclusion: The present study provided a comprehensive description of the serum exosome proteome of mGC patients, which could be an excellent resource for further studies of mGC.

Dataset Information

Cytokine profiling in serum-derived exosomes isolated by different methods.

Publications

Cytokine profiling in serum-derived exosomes isolated by different methods.

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