Exosomes are cell-derived vesicles and are abundant in biological fluids; they contain RNA molecules which may serve as potential diagnostic biomarkers in 'precision medicine'. To promote the clinical application of exosomal RNA (exoRNA), many isolation methods must be compared and validated. Exosomes in cell culture medium (CCM) and serum may be isolated using ultracentrifugation (UC), ExoQuick or Total Exosome Isolation Reagent (TEI), and exoRNA may be extracted using TRIzol-LS, SeraMir, Total Exosome RNA Isolation (TER), HiPure Liquid RNA/miRNA kit (HLR), miRNeasy or exoRNeasy. ExoRNA was assessed using NanoDrop, Bioanalyzer 2100, quantitative polymerase chain reaction and high-throughput sequencing. UC showed the lowest recovery of particles, but the highest protein purity for exosome isolation. For isolation of exoRNA, we found that combinations of the TEI and TER methods resulted in high extraction efficiency and purity of small RNA obtained using CCM. High yield and a narrow size distribution pattern of small RNA were shown in exoRNA isolated by exoRNeasy from serum. In RNA profile analysis, the small RNA constituent ratio, miRNA content and amount varied as a result of methodological differences. This study showed that different methods may introduce variations in the concentration, purity and size of exosomes and exoRNA. Herein we discuss the advantages and disadvantages of each method and their application to different materials, therefore providing a reference according to research design.

Exosomes, membrane vesicles of 40–100 nm in diameter, are derived from endosomes in various cells. The bioactive molecules specifically packed into exosomes can be horizontally transferred into recipient cells changing their biological properties, by which tumour cells continuously modify their surrounding microenvironment and distant target cells favouring cancer metastasis. It has been suspected for a long time that exosomes participate in the whole process of tumour metastasis. Although there is much unknown and many controversies in the role of cancer exosome, the major contribution of tumour-associated exosomes to different steps of cancer metastasis are demonstrated in this review. Mainly because these exosomes are easily accessible and capable of representing their parental cells, exosomes draw much attention as a promising biomarker for tumour screening, diagnosis and prognosis. Currently, researchers have found numerous biomarkers in exosomes with great potential to be utilized in personalized medicine. In this article, we summarize the roles of biomarkers, which are validated by clinical samples. Even though many conundrums remain, such as exosome extraction, large multicentre validation of biomarkers and data interpretation, exosomes are certain to be used in clinical practice in the near future as the field rapidly expands.

HFD is appropriate for UEVs isolation and miRNA analysis when compared with conventional UC. miR-145 in UEVs is upregulated from PCa patients compared BPH patients and healthy controls. We suggest the potential use of UEVs miR-145 as a biomarker of PCa.

BackgroundEpithelial–mesenchymal transition (EMT) is regarded as a critical event during tumor metastasis. Recent studies have revealed changes and the contributions of proteins in/on exosomes during EMT. Besides proteins, microRNA (miRNA) is another important functional component of exosomes. We hypothesized that the miRNA profile of exosomes may change following EMT and these exosomal miRNAs may in return promote EMT, migration and invasion of cancer cells.ResultsThe small RNA profile of exosomes was altered following EMT. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the specific miRNAs of M-exosomes have the potential to drive signal transduction networks in EMT and cancer progression. Co-culture experiments confirmed that M-exosomes can enter epithelial cells and promote migration, invasion and expression of mesenchymal markers in the recipient cells.ConclusionOur results reveal changes in the function and miRNA profile of exosomes upon EMT. M-exosomes can promote transfer of the malignant (mesenchymal) phenotype to epithelial recipient cells. Further, the miRNAs specifically expressed in M-exosomes are associated with EMT and metastasis, and may serve as new biomarkers for EMT-like processes in lung cancer.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-5143-6) contains supplementary material, which is available to authorized users.

Extracellular vesicles (EVs) mediate intercellular communication via transferring proteins and other biological molecules and have been recentlyinvestigated as biomarkers of disease. Sensitive and specific biomarkers are required for lung cancer diagnosis and prognosis. The present study screens for abnormal EV proteins in non-small cell lung cancer (NSCLC) using a quantitative proteomics strategy involving LC-MS/MS to identify ideal biomarkers for NSCLC diagnosis. EVs are enriched from the sera of early and advanced NSCLC patients and healthy controls and from cell culture supernatants of lung adenocarcinoma and bronchial epithelial cell lines. In the sera and supernatants, 279 and 632 differentially expressed proteins, respectively, are associated with signaling pathways including extracellular membrane-receptor interaction, focal adhesion, and regulation of the actin cytoskeleton. Thirty-two EV proteins are identified at the intersection of differentially expressed proteins between the NSCLC groups and cell lines. Based on bioinformatics analysis, in silico immunohistochemical, and PRM verification, fibronectin is selected for following in vitro studies and validation with an independent cohort. Fibronectin on EVs is estimated to perform well in the diagnosis of NSCLC patients based on AUC, showing great potential for clinical use and demonstrating the efficacy of this method for EV-associated biomarker screening.

Background: Circulating tumor cells (CTCs) has been demonstrated as a promising liquid biopsy marker for breast cancer (BC). However, the intra-patient heterogeneity of CTCs remains a challenge to clinical application. We aim at profiling aggressive CTCs subpopulation in BC utilizing the distinctive metabolic reprogramming which is a hallmark of metastatic tumor cells. Methods: Oncomine, TCGA and Kaplan-Meier plotter databases were utilized to analyze expression and survival relevance of the previously screened metastasis-promoting metabolic markers (PGK1/G6PD) in BC patients. CTCs detection and metabolic classification were performed through micro-filtration and multiple RNA in situ hybridization using CD45 and PGK1/G6PD probes. Blood samples were collected from 64 BC patients before treatment for CTCs analysis. Patient characteristics were recorded to evaluate clinical applications of CTCs metabolic subtypes, as well as morphological EMT subtypes classified by epithelial (EpCAM/CKs) and mesenchymal (Vimentin/Twist) markers. Results: PGK1 and G6PD expressions were up-regulated in invasive BC tissues compared with normal mammary tissues. Increased tissue expressions of PGK1 or G6PD indicated shortened overall and relapse-free survival of BC patients (P < 0.001). Blood GM + CTCs (DAPI + CD45 − PGK1/G6PD +) was detectable (range 0-54 cells/5 mL) in 61.8% of tCTCs > 0 patients. Increased GM + CTCs number and positive rate were correlated with tumor metastasis and progression (P < 0.05). The GM + CTCs ≥ 2/5 mL level presented superior AUC of ROC at 0.854 (95% CI 0.741-0.968) in the diagnosis of BC metastasis (sensitivity/specificity: 66.7%/91.3%), compared with that of tCTCs (0.779) and CTCs-EMT subtypes (E-CTCs 0.645, H-CTCs 0.727 and M-CTCs 0.697). Moreover, GM + CTCs + group had inferior survival with decreased 2 years-PFS proportion (18.5%) than GM + CTCs − group (87.9%; P = 0.001). Conclusions: This work establishes a PGK1/G6PD-based method for CTCs metabolic classification to identify the aggressive CTCs subpopulation. Metabolically active GM + CTCs subtype is suggested a favorable biomarker of distant metastasis and prognosis in BC patients.

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