Year: 2016
Type: dissertation
Abstract: Exosomes are membrane-enclosed vesicles, 30 to 150 nm in diameter, formed into the lumen of cellular structures called multivesicular bodies.They are released into the extracellular environment upon fusion of multivesicular bodies with the plasma membrane.Exosomes carry proteins, lipids and nucleic acids.Mounting evidence shows that exosomes function as intercellular signaling organelles, which can transfer their cargo to target cells and induce phenotypic changes in the recipients.Exosomes can be isolated from several, easily accessible body fluids such as serum, plasma and urine.In cancer patients, they represent a potential source of non-invasive biomarkers, as the cancer-derived exosomes carry proteins or other biomolecules originating from the tumor.Several exosome-isolation methods are currently available, but there is no consensus on the best protocol to use.The most preferred method is differential centrifugation, consisting of a series of centrifugations using increasing force of gravity to deplete the liquid of cell debris and larger vesicles, before saving the last pallet with the lightest vesicles.In this study, we investigated the effect of force reduction on the purity and the yield of isolated exosomal material.We found that reduction in the applied forces in step two of the protocol is directly proportional to purity and inversely proportional to yield.We also showed that the exosome markers CD63 and Flotillin-1 are not specific to exosomes and that CD9 expression in exosomes is cell type specific.We further performed protein analysis of cancer exosomes derived from cell culture media of metastatic melanoma and colorectal cancer cell lines, as well as of exosomes isolated from plasma of patients with rectal cancer.We found that several proteins were selectively enriched in exosomes from these cell lines.We further found evidence of different protein modifications in cells and exosomes.No specific protein signatures were identified in the small cohort of the patient extracted exosomes we analyzed.Finally, we demonstrated that melanoma-derived exosomes are internalized by melanoma cells and fibroblasts.Fibroblasts that have been co-cultured with melanoma exosomes did not show changes in metabolic activity.
Author: Galina Dimitrova Pavlova
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2586322393