Identification of exosomal proteins in primary human bronchial tracheal epithelial cell HBTE and the H358, THP1 and MCF7 cell lines MD Faruk Abdullah Saurav (BPharm, MSc) A thesis submitted in partial fulfilment of the requirements of the University of Greenwich for the Degree of Doctor of Philosophy. February 2018 Declaration I certify that the work contained in this thesis, or any part of it, has not been accepted in substance for any previous degree awarded to me, and is not concurrently being submitted for any degree other than that of Doctor of Philosophy being studied at the University of Greenwich. I also declare that, this work is the result of my own investigations, except where otherwise identified by references and that the contents are not the outcome of any form of research misconduct. Candidate: MD Faruk A Saurav Supervisor: Dr Azizur Rahman i Acknowledgement First of all I would like to thank almighty Allah for providing me to chase my dream. I would like to thank my Supervisor Dr Azizur Rahman for supporting me in every step even if when I was down for providing me enough opportunity to expand my project beyond my initial plan and allowing me to learn new techniques. I would also like to thank my second supervisor Dr. Lauren Pecorino for showing me the right way. I would also like to thank my colleagues who supported me by helping me throughout my project. It would also like to express gratitude to the entire technician team in the link lab, especially John and Cliff for supporting me all the time sometime even out of their way. The best support is the support from family. I want to thank my family members, each one of them. My father, my first hero, who supported me when I fell down, encourages me to get up again. My mother, who prays for me all the time; my brother and sister and as well as my in- laws who always pray for my success. Lastly, I want to thank my lovely wife, Tanzena Akter, without whom my study would not been finished. Her tiring effort to keep in my foot, continuous praying for my wellbeing and the support she gave me was the essential element of my path from beginning to the end. MD Faruk Abdullah Saurav ii Abstract Background: Early detection of cancer is of paramount importance for successful treatment. Unfortunately, this is currently complex and time consuming. New sources of biomarkers are needed to improve. Exosomes are nano sized extracellular vesicles released by almost all cells have gained much interest as a cancer biomarker source due to their ability to transfer genetic materials, stability and ease of availability. Aims: The aim of this study is to investigate the potentials of exosomes as a source of biomarkers of cancer in general and lung cancer in particular. To achieve this, exosomes from three difference cancer cell lines (lung cancer H358, leukaemia THP1 and breast cancer MCF7) and a primary lung cell HBTE were isolated and their protein contents were analysed to establish wheather cancer specific proteins are present. Methods and Materials: Exosomes were isolated and characterized by scanning (SEM) and transmission electron microscopy (TEM), dynamic light scattering (DLS) and Western Blot analysis. The exosome number and protein profile were analysed at different cellular growth stages using Exo-Elisa based on exosomal marker CD63 and mass spectrometry (MS) respectively. LC-MS based proteomic approach has been used to compare the proteomic profile of exosomes from three cancer cells. Finally, comparative proteomic study and gene expression analysis were carried out between exosomes from lung cancer cell (H358) and its counterpart normal cell (HBTE) Results and Discussions: Successful isolation of exosomes from H358, THP1 and MCF7 was confirmed based on their size distribution (ranging from 96.54±28.53nm to 128.06±17.74nm) and by the presence of exosomal markers (CD63, CD81, CD9 and Hsp70). The number of exosomes released/cell was shown to increase with time ranging from 14500 on day 1 to 18000 at day 15. Interestingly, MS analysis revealed that, alpha-2-macroglobulin (A2M) and pregnancy zone protein was present only in stationary phase indicative of the oxidative stress. Comparative proteomic study by LC-MS identified a total of 613 proteins commonly found across three cell lines. A large proportion of membranous proteins were identified including integrins, catenins, cadherins, and cathepsins. Most of which are involved in molecular signalling, cellular growth and transport, supporting the role of exosomes in cellular communication. Several adhesion molecules such as integrins, laminlins, catenins as well as cadherins and cathepsins were also identified as differentially expressed between different types of cancer derived exosomes. Proteomic profiling of HBTE cell derived exosomes revealed 1011 proteins which only partially overlapped with those identified in H358 exosomes. A total of 205 proteins were specific for the cancerous lung cell line derived exosomes. Of particular interest was the identification of CTNNB1, an adhesion molecule known to be present in several other lung cancers, making it an ideal candidate for biomarker for non-small cell lung cancer, bronchioalveloar carcinoma. Gene expression analysis revealed that this protein is expressed at cellular level in both cancerous and normal lung cells, albeit found in a significantly higher level in H358 (p≤0.05). Conclusion: This is the first report to show successful isolation and characterization of exosomes from H358 and HBTE. Comparative proteomic analysis of the newly isolated exosomes not only revealed that exosomes are a good source of lung cancer biomarkers but identified a potential candidate. CTNNB1 was selectively found in lung cancer cell derived exosomes but not in the counterpart normal cell (HBTE) and was selected for further investigation. iii Table of Contents Declaration i Acknowledgements ii Abstract iii Table of Contents iv List of Tables Xii List of Figures xiii List of Abbreviations xvi Chapter 1: ............................................................................................................ 1 Introduction: ........................................................................................................ 1 1.1 Overview of the project: .............................................................................................. 1 1.2 Cancer: ........................................................................................................................ 1 1.3 Cancer Discussed in this study: ................................................................................... 2 1.3.1 Lung Cancer: ........................................................................................................ 3 1.3.2 Breast Cancer: ...................................................................................................... 4 1.3.3 Leukaemia: ........................................................................................................... 5 1.4 Mechanism of Metastasis: ........................................................................................... 7 1.5 Exosome: ................................................................................................................... 10 1.5.1 Role of exosomes in metastasis: ........................................................................ 11 1.6 Composition of Exosome: ......................................................................................... 14 1.6.1 Lipid Composition: ............................................................................................ 14 1.6.2 Exosomal Protein Composition: ........................................................................ 15 1.6.3 Exosomal RNA composition: ............................................................................ 18 1.6.4 Biogenesis of Exosomes: ................................................................................... 19 iv 1.7 Isolation of Exosomes: .............................................................................................. 21 1.7.1 Ultracentrifugation: ............................................................................................ 21 1.7.2 Density Gradient Ultracentrifugation: ............................................................... 23 1.7.3 Size exclusion chromatography: ........................................................................ 23 1.7.4 Ultrafiltration: .................................................................................................... 24 1.7.5 Magnetic Beads:................................................................................................. 24 1.7.6 Precipitation methods: ....................................................................................... 25 1.7.7 Isolation of exosomes by sieving: ...................................................................... 25 1.8 Detection and characterisation of Exosomes: ........................................................... 26 1.8.1 Electron Microscopy: ......................................................................................... 26 1.8.2 Western Blot: ..................................................................................................... 26 1.8.3 Flow Cytometry: ................................................................................................ 27 1.8.4 Atomic force microscopy:.................................................................................. 27 1.8.5 Nano Particle Tracking Analysis (NTA): .........................................................