Exosomes: A source of novel disease biomarkers in bladder cancer Joanne Louise Welton BSc (Hons), MPhil Thesis presented for the degree of Philosophiae Doctor July 2010 Section of Oncology and Palliative Medicine School of Medicine Velindre Cancer Centre Whitchurch Cardiff CF15 2TL UMI Number: U514248 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U514248 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 DECLARATION This work has not previously been accepted in substance for any degree and is not concurrently submitted in candidature for any degree. Signed ... .................................................................. (candidate) Date STATEMENT 1 This thesis is being submitted in partial fulfillment of the requirements for the degree of PhD Signed ...................................................... (candidate) Date .!2%/05?\/2,QV.O STATEMENT 2 This thesis is the result of my own independent work/investigation, except where otherwise stated. Other sources are acknowledged by explicit references. Signed ....................................................... (candidate) Date STATEMENT 3 I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter- library loan, and for the title and summary to be made available to outside organisations. Signed ............................................. r^TTT................................ (candidate) Date STATEMENT 4 - BAR ON ACCESS APPROVED I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter- library loans after expiry of a bar on access approved bv the Graduate Development Committee. Signed ........................................................................................(candidate) Date........................................ Dedication This thesis is dedicated to my family, especially my fiance Sean and parents who have believed in me and supported me wholeheartedly throughout all my studies. Acknowledgements I am indebted too my supervisors, Dr Aled Clayton, Dr Ian Brewis, and Professor Malcolm D. Mason, for their support, encouragement and guidance throughout my PhD. I would also like to thank Dr John Staffurth for his advice and encouragement. I would like to thank Mrs Lyn Court at Velindre Cancer Centre for teaching me immunoblotting. I am also grateful for to all the people at the Central Biotechnology Services (CBS) Proteomics facility, Henry Wellcome Building, University Hospital of Wales, particularly Dr Sanjay Khanna and Mrs Swee Nixon for their technical expertise. I would like to thank Dr Peter Giles for his expert help with the bioinformatics part of my project. I also owe Dr Paul Brennan a debt of gratitude in allowing me to pick his brain with respect to the proteomics workflow. Thanks also go to Dr Anthony Hann of the biosciences electron microscopy unit for his help with exosome imaging. I would also like to thank all my donors for their kind specimen donations. Finally I would like to thank my friends and colleagues at the Cancer Research Wales Laboratories and the Henry Wellcome building, UHW, especially Dr Paul Mitchell, Dr Jason Webber, Dr Lisa Spary and Miss Sophie Betteridge as it has been your unwavering support, mentally and physically, throughout which has made my PhD possible. Summary Exosomes are nanometre-sized vesicles secreted by most cells into the extracellular milieu. They have been proposed as a good source of disease-related markers as they are available non-invasively (for example from urine) and express a repertoire of proteins enriched in cancer antigens and stress proteins. 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 been previously 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. In conclusion we report the first proteomics dataset on exosomes derived from bladder cancer cells. We identified 353 exosomal proteins with high quality MS data and highly pure exosomes. 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. Publications and presentations Publications Welton JL. Khanna S, Giles PJ, Brennan P, Brewis IA, Staffurth J, Mason MD, Clayton A. (2010) Proteomic analysis of bladder cancer exosomes. Molecular and Cellular Proteomics. 9(6): 1324-38 Mitchell PJ*, Welton J*. Staffurth J, Court J, Mason MD, Tabi Z, Clayton A. (2009). Can urinary exosomes act as treatment response markers in prostate cancer?J Transl Med 7:4 (*equal contribution to manuscript) Presentations and conferences attended Welton JL. Brewis 1A, Brennan P, Staffurth J, Mason MD, Clayton A (2009). Bladder cancer exosome proteomics for biomarker discovery (Oral Presentation). 24 th Annual Postgraduate Research Day, Graduate School of Biomedical and Life Sciences. School of Medicine, Cardiff University, Cardiff, UK. Welton JL. Brewis IA, Brennan P, Staffurth J, Mason MD, Clayton A (2009). Proteomic analysis of bladder cancer exosomes (Oral Presentation). Velindre NHS Trust R&D conference. Village Hotel, Cardiff. UK Welton JL. Brewis LA, Brennan P, Staffurth J, Mason MD, Clayton A (2009). Proteomic analysis of bladder cancer exosomes (Poster Presentation). EMBO conference “Cancer Proteomics 2009: Mechanistic Insights, Technological Advances, and Molecular Medicine”. University College Dublin, Ireland Welton JL. Brewis IA, Staffurth J, Clayton A (2008). Differential proteomic analysis of exosomes in association with cancer: method development (Poster Presentation). 5th Joint BSPRJEBI Proteomics Meeting. Hinxton, Cambridge, UK Welton JL. Brewis IA, Tabi Z, Staffurth J, Mason MD, Clayton A (2007). Differential proteomic analysis of exosomes isolated from human urine (Poster Presentation) 22nd Annual Postgraduate Research Day, Graduate School of Biomedical and Life Sciences . School of Medicine, Cardiff University, Cardiff, UK. Welton JL, Brewis IA, Tabi Z, Staffurth J, Mason MD, Clayton A (2007). Developing methods for differential protein analysis in cancer (Poster Presentation). Velindre NHS Trust R&D conference. Village Hotel, Cardiff, UK. Contents Table of contents 1 Chapter 1: Introduction ..................................................................................................1 1.1 Bladder cancer.......................................................................................................2 1.1.1 Risk factors and aetiology ............................................................................. 4 1.1.2 Diagnosis, treatment and monitoring ........................................................... 4 1.1.3 Molecular alterations in bladder cancer .......................................................9 1.1.4 Bladder cancer markers.................................................................................. 9 1.1.5 The future for bladder cancer markers ..................................................... 21 1.2 Exosome biology and purification .................................................................... 24 1.2.1 Exosome form ation ........................................................................................ 24 1.2.2 What is the composition of exosomes? ......................................................31 1.2.3 Comparison of exosomes with other secreted vesicles ..........................37 1.2.4 Defining exosomes ..........................................................................................38 1.2.5 What functions do exosomes have? .......................................................... 40 1.2.6 Exosome purification strategies .................................................................. 45 1.3 Proteomics analysis of exosomes ....................................................................49