Clonogenicity and Stem Cells Charlotte Mary Beaver Submitted for the degree of Doctor of Philosophy to University College London Division of Surgery and Interventional Science, UCL March 2013 1 I, Charlotte Beaver, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Abstract Primary keratinocytes form 3 types of colony with different morphologies termed holoclones, meroclones and paraclones, thought to be derived from stem, early and late stage precursor cells respectively (Barrandon and Green, 1987b, Rochat et al., 1994). Cancer cell lines produce colonies with morphologies analogous to those of holoclones, meroclones and paraclones, and consequently holoclone morphology is used as a surrogate marker for stem cell colonies. The aim of this study was to elucidate the relationship between clonogenicity, colony morphology and stem cells. Colonies formed by primary prostate epithelial cells and prostate cancer cell lines (DU145, PC3, LNCaP) were characterised. The proportions of colonies were not altered significantly by modification of culture conditions. In contrast to cancer cells, primary prostate epithelial cells form only two types of colony, termed types 1 and 2, which are analogous to holoclones and paraclones. Only type 1 colonies were highly proliferative, able to self-renew and express putative stem cell markers. Paradoxically, cells from DU145 meroclones formed holoclones and had self- renewal capacity (by serial cloning and xenografting). It is concluded that the major difference between holoclone and meroclone colonies from the cancer cell line DU145 is the proportion of stem cells within each colony, not the presence or absence of stem cells. Phage display was used to look for targets on the surface of cells in Type 1 colonies. Various experimental protocols were tested, but no targets were identified. 3 Acknowledgements To my supervisors Professor John Masters and Dr Aamir Ahmed for their guidance and support. To Dr Stephen Hart for his help and knowledge of phage display. To Ben Challacombe at Guy’s hospital for providing tissue, even when he was on holiday on the other side of the world. To those at the Prostate Cancer Research Centre and the Cecil Pilkington Charitable Trust who generously funded this work (2009-2013). To all my friends PCRC. Thanks for all the help, support and friendship. It’s been a great few years. To my family and especially my parents who have supported by ambitions. I wouldn’t be here without you. To Mark who has supported me all these years and made me lots of cups of tea. 4 Table of Contents Abstract .......................................................................................................... 3 Acknowledgements ........................................................................................ 4 List of Figures .............................................................................................. 13 List of Tables................................................................................................ 17 Abbreviations ............................................................................................... 19 1 Introduction ........................................................................................... 24 1.1 The Prostate and it’s Diseases ....................................................... 24 1.1.1 Prostate Anatomy ..................................................................... 24 1.1.1 Prostate Histology .................................................................... 26 1.1.2 Benign Prostatic Hyperplasia ................................................... 27 1.1.3 Prostate Cancer ....................................................................... 28 1.2 Adult Stem Cells ............................................................................. 30 1.2.1 The Adult Stem Cell Hierarchy ................................................. 30 1.2.2 Stem Cells in the Haematopoietic System ............................... 32 1.2.3 Adult Stem Cell Identification ................................................... 34 1.3 Prostate Stem Cells ........................................................................ 37 1.3.1 Prostate Stem Cell Identification .............................................. 37 1.4 Cancer Stem Cells .......................................................................... 39 1.4.1 Models of Tumour Heterogeneity ............................................. 39 1.4.2 Cancer Stem Cell Definition ..................................................... 43 1.4.3 Identification of Cancer Stem Cells .......................................... 43 5 1.4.4 Identification of CSCs: In Vivo Tumourigenicity ........................ 45 1.5 Prostate Cancer Stem Cells ............................................................ 50 1.5.1 Cellular Origin of Prostate Cancer ............................................ 50 1.5.2 Prostate Cancer Stem Cell Identification .................................. 51 1.6 Clonogenicity .................................................................................. 52 1.6.1 The Clonogenic Assay ............................................................. 52 1.6.2 The Human Tumour Stem Cell Assay (HTSCA)....................... 54 1.6.3 Non-adherent Colonies ............................................................ 56 1.6.4 Barrandon and Green: Holoclones, Meroclones and Paraclones 57 1.6.5 Cancer Colony Morphology ...................................................... 59 1.7 Drug Discovery by Phage Display .................................................. 62 1.7.1 Bacteriophages ........................................................................ 62 1.7.2 Bacteriophage Infection of an E.coli Host ................................. 63 1.7.3 Peptide phage display .............................................................. 64 1.7.4 Targeting Tumours by Phage Display ...................................... 66 1.8 Experimental Aims .......................................................................... 68 1.8.1 Hypothesis ............................................................................... 68 1.8.2 Objectives ................................................................................ 69 2 Materials and Methods ......................................................................... 70 2.1 Prostate Cancer Cell Lines ............................................................. 70 2.2 Primary Human Prostate Epithelial Cells ........................................ 71 6 2.2.1 Tissue Collection ...................................................................... 71 2.2.2 Tissue Digestion ....................................................................... 71 2.2.3 Single Cell Suspension ............................................................ 72 2.3 Clonogenic Assays ......................................................................... 72 2.3.1 Cell Lines ................................................................................. 72 2.3.2 Primary Prostate Epithelial Cells .............................................. 73 2.3.3 Analysis .................................................................................... 74 2.4 Effect of Culture Conditions on Clonogenicity ................................. 76 2.4.1 Seeding Density ....................................................................... 76 2.4.2 FBS Concentration ................................................................... 76 2.5 Secondary Cloning ......................................................................... 80 2.5.1 Cell Lines ................................................................................. 80 2.5.2 Primary Prostate Epithelial Cells .............................................. 81 2.6 Serial Cloning ................................................................................. 81 2.7 Serial Passage ................................................................................ 82 2.7.1 Cell Lines ................................................................................. 82 2.7.2 Prostate Epithelial Cells ........................................................... 83 2.8 Sphere Formation ........................................................................... 83 2.8.1 Cell Lines ................................................................................. 84 2.8.2 Analysis .................................................................................... 85 2.9 Tumourigenicity .............................................................................. 85 2.9.1 Primary Tumourigenicity ........................................................... 87 7 2.9.2 Clonogenicity of Xenografts ..................................................... 88 2.9.3 Secondary Tumourigenicity ...................................................... 89 2.10 Immunocytochemistry .................................................................. 90 2.10.1 Sample Preparation .............................................................. 90 2.10.2 Staining ................................................................................. 91 2.10.3 Two Colour Staining .............................................................. 92 2.10.4 Image analysis ...................................................................... 93 2.11 Incucyte Analysis of Clonal Growth ............................................. 94 2.12 Peptide Phage Display