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Exosomes: a source of novel disease biomarkers in bladder cancer.

Welton, Joanne Louise 2010. Exosomes: a source of novel disease biomarkers in bladder cancer. PhD Thesis, Cardiff University.

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Abstract

The major aim of this thesis was to perform the first ever proteomics study on bladder cancer exosomes. Initially, exosomes were isolated from urine specimens but hypervariable yields and poor sample quality made proteomics analysis challenging. As an alternative approach, exosomes were isolated from HT1376 bladder cancer cells. Exosomes were purified by ultracentrifugation on a sucrose cushion, and preparations verified as high quality by immunoblotting, flow cytometry and electron microscopy. For global proteomics analysis, the sample was solubilised using SDS and DTT and subjected to LC-MALDI-TOF/TOF MS. We identified 353 proteins with high confidence and 63 of these have not previously been identified in other proteomics studies on human exosomes. Overrepresentation analysis demonstrated that the proteome was consistent with that of other exosomes with significant overlap with exosomes of carcinoma origin. Comparisons with the Gene Ontology database also highlighted strong associations with carcinoma of the bladder and other sites. A GeneGo generated protein interaction network highlighted c-Myc as a major node of protein interaction within this dataset. Several MS-identified proteins were confirmed as genuinely exosomally expressed using a combination of immunoblotting, flotation on continuous sucrose gradients, and flow cytometry. Expression was also verified in exosomes from a variety of sources, including urine. The data will aid our understanding of exosome biogenesis and function and may inform the development of urine exosome-based clinical tools in bladder cancer.

Item Type: Thesis (PhD)
Status: Unpublished
Schools: Medicine
Subjects: R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology (including Cancer)
Date of First Compliant Deposit: 30 March 2016
Last Modified: 04 May 2016 02:54
URI: http://orca-mwe.cf.ac.uk/id/eprint/54114

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