Studying the impact of Tspan8 on extracellular vesicles in breast cancer and application of a novel tool for their detection Inaugural-Dissertation zur Erlangung der Doktorwürde der Fakultät für Biologie der Albert-Ludwigs-Universität Freiburg im Breisgau Vorgelegt von Richa Khanduri geboren in Delhi, Indien Freiburg im Breisgau Dezember 2018 Die vorliegende Arbeit entstand in der Arbeitsgruppe Exosomen und Tumorbiologie des Instituts für Infektionsprävention und Krankenhaushygiene am Universitätsklinikum Freiburg unter der Anleitung von PD Dr. Irina Nazarenko. Dekan der Fakultät für Biologie: Prof. Dr. Wolfgang Driever Promotionsvorsitzender: Prof. Dr. Andreas Hiltbrunner Betreuerin der Arbeit: PD Dr. Irina Nazarenko Betreuerin der Fakultät für Biologie: Prof. Dr. Annegret Wilde Referentin: Prof. Dr. Annegret Wilde Koreferent: Prof. Dr. Thomas Reinheckel Drittprüfer: Prof. Dr. Dr. h.c. Christoph Borner Datum der mündlichen Prüfung: 25.02.2019 i DECLARATION I hereby declare, that I am the sole author and composer of my Thesis and that no other sources or learning aids, other than those listed, have been used. Furthermore, I declare that I have acknowledged the work of others by providing detailed references of said work. I hereby also declare, that my Thesis has not been prepared for another examination or assignment, either wholly or excerpts thereof. Freiburg, 28.02.2019 ___________________________________ Place, date Signature Richa Khanduri ii Life consists in penetrating the unknown and fashioning our actions in accord with the new knowledge thus acquired. -Leo Tolstoy iii Table of Contents 1 Summary .......................................................................................................................................................................... 1 2 Introduction .................................................................................................................................................................... 2 2.1 Breast cancer .......................................................................................................................................................... 2 2.2 Extracellular Vesicles .......................................................................................................................................... 3 2.2.1 EV types ........................................................................................................................................................... 4 2.2.2 Biogenesis and secretion of exosomes ................................................................................................ 6 2.2.3 Biogenesis of microvesicles ..................................................................................................................... 9 2.2.4 Molecular composition of exosomes.................................................................................................... 9 2.3 Function of EVs in physiology and pathology ....................................................................................... 12 2.3.1 EVs and tumor microenvironment .................................................................................................... 13 2.3.2 Diagnostic applications of EVs ............................................................................................................ 15 2.3.3 Role of EVs in breast cancer ................................................................................................................. 16 2.4 Tetraspanin 8: characteristic features and its role in cancer .......................................................... 19 2.4.1 Structure of Tetraspanins...................................................................................................................... 20 2.4.2 Tspan8 and its role in cancer ............................................................................................................... 21 2.5 Human Epidermal Growth Factor Receptor 2 ....................................................................................... 23 2.6 WGM lasers .......................................................................................................................................................... 26 3 AIM of the study ......................................................................................................................................................... 28 4 Materials & Methods................................................................................................................................................. 29 4.1 Materials ............................................................................................................................................................... 29 4.1.1 Cell lines ........................................................................................................................................................ 29 4.1.2 Media and supplements ......................................................................................................................... 29 4.1.3 Chemicals and Reagents ......................................................................................................................... 30 4.1.4 Buffers and solutions ............................................................................................................................... 31 4.1.5 Antibodies .................................................................................................................................................... 33 4.1.6 Assay Kits ..................................................................................................................................................... 35 4.1.7 Equipments ................................................................................................................................................. 35 4.1.8 Consumables ............................................................................................................................................... 37 4.1.9 Softwares ...................................................................................................................................................... 38 4.2. Methods ................................................................................................................................................................ 38 4.2.1 Cell culture ................................................................................................................................................... 38 4.2.2 3D cell culture ............................................................................................................................................ 40 4.2.3 Plasmid DNA isolation ............................................................................................................................ 40 iv 4.2.4 Transient transfection ............................................................................................................................ 42 4.2.5 Generation of stable cell lines .............................................................................................................. 43 4.2.6 Flow Cytometry with cells ..................................................................................................................... 43 4.2.7 Fluorescence-activated cell sorting ................................................................................................... 44 4.2.8 Preparation of cell lysate ....................................................................................................................... 44 4.2.9 Western Blotting ....................................................................................................................................... 45 4.2.10 Cell proliferation assays ...................................................................................................................... 45 4.2.11 Adhesion Assay ....................................................................................................................................... 46 4.2.12 Invasion Assay ......................................................................................................................................... 46 4.2.13 EVs isolation from 2D cell culture ................................................................................................... 49 4.2.14 EVs Isolation from 3D cell culture ................................................................................................... 50 4.2.15 EVs isolation from patients’ sera ..................................................................................................... 52 4.2.16 Beads-assisted flow cytometry with EVs ..................................................................................... 53 4.2.17 Electron microscopy ............................................................................................................................. 53 4.2.18 Nanoparticle Tracking Analysis and Zeta Potential ................................................................. 54 4.2.19 DLS ............................................................................................................................................................... 54 4.2.20 Protein quantification .......................................................................................................................... 55 4.2.21 Proteomics (LC-MS/MS).....................................................................................................................