6 research outputs found

    miRNA profiling of circulating EpCAM(+) extracellular vesicles:promising biomarkers of colorectal cancer

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    Cancer cells secrete small membranous extracellular vesicles (EVs) into their microenvironment and circulation. These contain biomolecules, including proteins and microRNAs (miRNAs). Both circulating EVs and miRNAs have received much attention as biomarker candidates for non-invasive diagnostics. Here we describe a sensitive analytical method for isolation and subsequent miRNA profiling of epithelial-derived EVs from blood samples of patients with colorectal cancer (CRC). The epithelial-derived EVs were isolated by immunoaffinity-capture using the epithelial cell adhesion molecule (EpCAM) as marker. This approach mitigates some of the specificity issues observed in earlier studies of circulating miRNAs, in particular the negative influence of miRNAs released by erythrocytes, platelets and non-epithelial cells. By applying this method to 2 small-scale patient cohorts, we showed that blood plasma isolated from CRC patients prior to surgery contained elevated levels of 13 EpCAM+-EV miRNAs compared with healthy individuals. Upon surgical tumour removal, the plasma levels of 8 of these were reduced (miR-16-5p, miR-23a-3p, miR-23b-3p, miR-27a-3p, miR-27b-3p, miR-30b-5p, miR-30c-5p and miR-222-3p). These findings indicate that the miRNAs are of tumour origin and may have potential as non-invasive biomarkers for detection of CRC. This work describes a non-invasive blood-based method for sensitive detection of cancer with potential for clinical use in relation to diagnosis and screening. We used the method to study CRC; however, it is not restricted to this disease. It may in principle be used to study any cancer that release epithelial-derived EVs into circulation

    Tumour exosomes display differential mechanical and complement activation properties dependent on malignant state:implications in endothelial leakiness

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    Background: Exosomes have been implicated in tumour progression and metastatic spread. Little is known of the effect of mechanical and innate immune interactions of malignant cell-derived exosomes on endothelial integrity, which may relate to increased extravasation of circulating tumour cells and, therefore, increased metastatic spread. Methods: Exosomes isolated from non-malignant immortalized HCV-29 and isogenic malignant non-metastatic T24 and malignant metastatic FL3 bladder cells were characterized by nanoparticle tracking analysis and quantitative nanomechanical mapping atomic force microscopy (QNM AFM) to determine size and nanomechanical properties. Effect of HCV-29, T24 and FL3 exosomes on human umbilical vein endothelial cell (HUVEC) monolayer integrity was determined by transendothelial electrical resistance (TEER) measurements and transport was determined by flow cytometry. Complement activation studies in human serum of malignant and non-malignant cell-derived exosomes were performed. Results: FL3, T24 and HCV-29 cells produced exosomes at similar concentration per cell (6.64, 6.61 and 6.46×104 exosomes per cell for FL3, T24 and HCV-29 cells, respectively) and of similar size (120.2 nm for FL3, 127.6 nm for T24 and 117.9 nm for HCV-29, respectively). T24 and FL3 cell-derived exosomes exhibited a markedly reduced stiffness, 95 MPa and 280 MPa, respectively, compared with 1,527 MPa with non-malignant HCV-29 cell-derived exosomes determined by QNM AFM. FL3 and T24 exosomes induced endothelial disruption as measured by a decrease in TEER in HUVEC monolayers, whereas no effect was observed for HCV-29 derived exosomes. FL3 and T24 exosomes traffic more readily (11.6 and 21.4% of applied exosomes, respectively) across HUVEC monolayers than HCV-29 derived exosomes (7.2% of applied exosomes). Malignant cell-derived exosomes activated complement through calcium-sensitive pathways in a concentration-dependent manner. Conclusions: Malignant (metastatic and non-metastatic) cell line exosomes display a markedly reduced stiffness and adhesion but an increased complement activation compared to non-malignant cell line exosomes, which may explain the observed increased endothelial monolayer disruption and transendothelial transport of these vesicles
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