Development of an organotypic 3D in vitro model of normal human breast tissue: a tool for cancer initiation studies Claire Elizabeth Nash BSc (Hons) Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds School of Medicine March 2014 i The candidate confirms that the work submitted is her own, except where work which has formed part of jointly authored publications has been included. The contribution of the candidate and the other authors to this work has been explicitly indicated below. The candidate confirms that appropriate credit has been given within the thesis where reference has been made to the work of others. The work in Chapter 1 and introduction of Chapter 3 of the thesis has appeared in publication as follows: Nash, C. and V. Speirs, Pre-Clinical Modelling of Breast Cancer: Which Model to Choose?, in Breast Cancer Metastasis and Drug Resistance. Progress and Prospects, A. Ahmad, Editor 2013, Springer. I was first author of the chapter. The other author, Prof Valerie Speirs, contributed through reviewing and editing the work prior to final submission to the book editor. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. © 2014 The University of Leeds and Claire Elizabeth Nash ii Candidate Achievements Publications Nash, C. and V. Speirs, Pre-Clinical Modelling of Breast Cancer: Which Model to Choose?, in Breast Cancer Metastasis and Drug Resistance. Progress and Prospects, A. Ahmad, Editor 2013, Springer. D. Holliday, M. Moss, S. Pollock, S. Lane, A. Shabaan, R. Millican-Slater, C. Nash, A. Hanby & V. Speirs. The practicalities of using tissue slices as pre-clinical organotypic breast cancer models. J Clin Pathol, 2013, 66(3), p. 253-5. Verghese, E., Drury, R., Green, C.A., Holliday, D.L., Lu, X., Nash, C., Speirs, V., Thorne, J.L., Thygesen, H.H., Zougman, A., Hull, M.A., Hanby, A.M. and Hughes, T.A. Role of miR-26b in carcinoma-associated fibroblasts and effect on migration and invasion of breast cancer epithelial cells. The Lancet, 2014. 383: p. S103. Millican-Slater, R., Good, R., Nash, C., Heads, J.A., Pollock, S., Gomm, J., Jones, J.L., Sundara- Rajan, S., Horgan, K., Hanby, A.M., Speirs, V. (2014) Pathobiology of breast tissue and primary cell cultures obtained from a female-to-male transgender patient. Cell and Tissue Banking. (submitted) Poster and Oral Presentations Nash, C. A 3D in vitro model of normal breast tissue: A tool for breast cancer initiation studies. [Poster] National Cancer Research Institute, Liverpool, UK, November 2012. Nash, C. A 3D tri-culture model of normal mammary gland: A tool for breast cancer initiation studies. [Poster] CTRC-AACR San Antonio Breast Cancer Symposium, San Antonio, USA, December 2012. iii Nash, C. Developing and Characterising a Novel 3D in vitro Model of Normal Breast for Cancer Initiation Studies. The Pathological Society and British Division of the IAP national meeting, Edinburgh, UK, June 2013. Nash, C. Developing and Characterising a Novel 3D in vitro Model of Normal Breast for Cancer Initiation Studies. Leeds Institute of Molecular Medicine Postgraduate Research Symposium, Leeds, UK, April 2013. Nash, C. Developing and Characterising a Novel 3D in vitro Model of Normal Breast for Cancer Initiation Studies. The University of Leeds Faculty of Medicine and Health Postgraduate Conference, Leeds, UK, July 2013. Awards Awarded 1st prize for best plenary oral presentation at The Pathological Society and British Division of the IAP national meeting, Edinburgh, UK, June 2013. Awarded 2nd prize oral presentation at Leeds Institute of Molecular Medicine Postgraduate Research Symposium, Leeds, UK, April 2013. Awarded 2nd prize oral presentation at The University of Leeds Faculty of Medicine and Health Postgraduate Conference, Leeds, UK, July 2013. Awarded joint 3rd Postgraduate Researcher of the Year at the Showcase University of Leeds Postgraduate Researcher conference, Leeds, UK, December 2014 Awarded BACR/ CRUK Travel Bursary to attend CTRC-AACR San Antonio Breast Cancer Symposium in USA, December 2012 iv Acknowledgements I would like to thank my primary supervisor, Prof Valerie Speirs, for her guidance, dedication, encouragement, passion, and most of all patience throughout the last few years. Without her this PhD would not have been possible. I would also like to thank my co-supervisors: Prof Andrew Hanby for all his help with pathology and for making me laugh throughout these years, Dr Georgia Mavria and Dr Darren Tomlinson for their extensive knowledge and guidance throughout and Dr Debbie Holliday for training me and for her friendship giving me a solid foundation to build my thesis upon. I’d also like to thank our collaborators Dr Fedor Berditchevski and Prof Louise Jones (and her group) for their help throughout. I’d like to thank the Leeds Institute of Molecular Medicine for funding me and all the patients who donated to the Leeds Breast Tissue Bank for making this work possible. I big thank you goes out to all of the people I have shared the laboratory and department with who have not just been a massive help but like a family to me over the last few years. Dr Stanley Ko’s generosity, knowledge and support was greatly appreciated and Dr Euan Baxter and Dr Laura Smith have been especially helpful with their help and guidance throughout. A special thank you goes out to Emily Smart for being a fantastic friend and keeping me sane throughout my PhD. I’d like to thank my family for believing in me, getting me through my education and encouraging me to pursue my dreams. Their support has been crucial in allowing me to build a career in science and will never go unappreciated. Finally I’d like to give a huge thank you to my fiancé Michael Baron. He has supported me both financially and emotionally throughout this project and without him I would have certainly crumbled under the pressure. His continued enthusiasm for life and positive attitude has kept me balanced throughout the entirety of this PhD. v Abstract The mechanisms involved in breast cancer initiation are not well understood. This may in part be due to a lack of an in vitro model that faithfully recapitulates the morphology, phenotype and in vivo architecture of the normal human mammary gland. Most in vitro models of normal breast have relied on the use of reconstituted basement membrane gels to induce luminal epithelial cell polarity and have neglected the role of myoepithelial cells and fibroblasts in this process. The aim of this thesis was to develop a three dimensional in vitro culture system of normal breast which included three of the major functional cell types of breast embedded in a more physiologically relevant collagen I matrix. It was then sought to use the system to investigate the mechanisms behind breast cancer initiation via genetic manipulation of well- known oncogenes and tumour suppressors involved in breast cancer progression. To achieve this, myoepithelial cells (Myo1089, originally isolated from breast reduction mammoplasty sample, gift of Dr Mike O’Hare) and fibroblasts (isolated and immortalised from breast reduction mammoplasty samples collected with ethical approval in house) were characterised by immunofluorescence to assess their suitability. Following characterisation, these were virally transfected with Turbo Green Fluorescent Protein (tGFP) and dsRed protein respectively to enable tracking. Three-dimensional tri-cultures were established in collagen I and included the non-tumorigenic luminal epithelial cell line HB2 with GFP Myo1089 cells and dsRed fibroblasts. Cells were cultured for three weeks in Transwell™ cell culture inserts. Following fixation these were analysed by haematoxylin and eosin staining, confocal microscopy and immunohistochemistry. Morphology and immunostaining profiles were compared to sections of a normal human in vivo breast tissue specimen. Immunohistochemical characterisation using the following antibodies: E-cadherin, epithelial membrane antigen, vimentin, laminin 5, collagen IV plus luminal and basal cytokeratins, demonstrated polarised epithelial structures with lumen formation and basement membrane vi production with a similar immunostaining profile to normal breast tissue. The importance of including myoepithelial cells and fibroblasts in maintaining these structures was demonstrated. We established this model was amenable to genetic engineering by overexpressing HER2 and HER3 in HB2 cells, and knocking out ERβ1 in Myo1089 cells and DOCK4 in fibroblast cell lines using siRNA/shRNA techniques respectively. These were included in separate models with morphological and phenotypic effects determined by haematoxylin and eosin staining, immunohistochemistry and quantification of HB2 structures formed. We further investigated the intracellular signalling cascades stimulated by heregulin in order to validate our findings upon overexpression of HER2 and HER3 and to investigate the cancer initiation potential of heregulin in the breast. In summary, an in vitro model of normal breast tissue that includes three of the major functional breast cell types cultured in a physiologically relevant three dimensional matrix has been developed. The morphology and protein expression profile of the model was validated against a human breast tissue specimen and confirmed that it is a suitable model of normal breast. The model proved to be reproducible, suitable for experimentation using genetic engineering