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.
Highlights
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
To evaluate the size distribution, exosomes isolated from different methods were subjected to Nanoparticle Tracking Analysis (NTA), which revealed that the size distribution curve of the EE method was broader than those of the UC and EQ methods (Fig. 1a)
Further analysis of the concentration of exosomes by NTA revealed that the EQ method is appropriate for isolation of larger numbers of particles
Summary
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. Small microvesicles of 30–150 nm with endosomal origin, are produced by most cells and are actively released by fusion of the microvesicular bodies with the plasma m embrane[3,4] They carry diverse and complex cargo molecules, such as nucleic acids (DNAs, RNAs), lipids, and proteins, and play an important role in intercellular communication by serving as a carrier for the transfer of these bioactive cargoes between cells. From serum or plasma is especially problematic because of the small volume available, its high viscosity, and its high concentration of proteins and lipoproteins[18] For these reasons, it is very important to find a suitable exosome isolation method and identify its potential for analysis of exosomal cytokine biomarkers
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