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Validation and Identification of Tumour Endothelial Markers and Their Uses in Cancer Vaccine

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Validation and Identification of Tumour Endothelial Markers and Their Uses in Cancer Vaccine Validation and identification of tumour endothelial markers and their uses in cancer vaccine _______________________________________________________________________________ by Xiaodong Zhuang A thesis submitted to The University of Birmingham for the degree of DOCTOR OF PHILOSOPHY Molecular Angiogenesis Group Department of Immunity and Infection College of Medical and Dental Sciences The University of Birmingham September 2012 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Abstract The abnormal tumour microenvironment, which is typically hypoxic, acidic and with poor blood flow, induces the endothelial expression of genes not found on normal microvessels. By selectively targeting these tumour endothelial markers (TEMs) it is possible to induce tumour regression, presenting a potential strategy for therapeutic intervention. Potential TEMs were predicted by bioinformatics data mining. Validation of these TEM candidates identified a novel TEM CLEC14A. Functional characterization suggests a regulatory role of CLEC14A in endothelial cell migration. Inhibition of endothelial migration by CLEC14A antisera or monoclonal antibody holds therapeutic promise for the treatment of cancer. Differential gene expression analysis of freshly isolated lung tumour endothelium by 2nd generation sequencing identified 13 putative TEMs. Subsequent validation work confirmed six of which to be expressed on lung tumour vasculature. Finally, a pre-validated marker, Robo4, was investigated as a cancer vaccine. A strong antibody response was induced by delivery of pure mouse Robo4 protein or a Robo4 conjugate. The in vivo sponge assay in Robo4 vaccinated mice showed a significant reduction in vessel invasion. Tumour implantation experiments in vaccinated mice showed a marked delay in tumour growth. Acknowledgements My deepest thanks go to Professor Roy Bicknell for being an inspirational mentor and for sharing his scientific insights. I sincerely thank him for helping me through many difficulties in the past six years. I would like to thank my co-supervisor, Dr. Zsuzsa Nagy for her guidance, advice, support and encouragement during the course of my PhD study. I thank Gordon Ryan, Jane Steele, James Beesley, Rajeeb Swain and Victoria Heath for teaching me essential lab techniques and skills. I express my gratitude to John Herbert for being a great friend and adventuring the TEM hunting with me. I extremely appreciate his excellent bioinformatics work that enabled us to generate interesting data. I also express my thanks to Cancer Research UK and KTP for their funding. My heartfelt thanks go to my wonderful wife Xue Lin for her endless support, constant encouragement and friendship. I am equally indebted to my grandmother and my parents for their continuous support throughout these years. List of contents Introduction ..................................................................................................................................... 1 1.1 Endothelial cells .................................................................................................................. 2 1.2 Angiogenesis ....................................................................................................................... 3 1.3 Tumour angiogenesis .......................................................................................................... 6 1.4 Anti-angiogenic therapy ...................................................................................................... 8 1.5 VEGF as a target ................................................................................................................. 9 1.6 Tumour vascular targeting ................................................................................................. 14 1.7 Tumour endothelial markers (TEMs) ................................................................................ 16 1.8 Identification of novel TEMs in lung cancer ..................................................................... 23 1.9 Targeting tumour vasculature by vaccination .................................................................... 27 1.10 Aims and objectives ........................................................................................................ 32 Materials and methods ................................................................................................................. 34 2.1 Materials ........................................................................................................................... 35 2.2 Molecular biology methods............................................................................................... 44 2.3 Mammalian cell isolation and culture ............................................................................... 50 2.4 Transfection and transduction of mammalian cells ........................................................... 54 2.5 Protein analysis methods ................................................................................................... 57 2.6 Protein purification and Fc fragment removal .................................................................. 59 2.7 Methods used in expression analysis ................................................................................ 60 2.8 Gene expression profiling methods ................................................................................... 62 2.9 In vitro angiogenesis assays .............................................................................................. 64 2.10 In vivo angiogenesis assay .............................................................................................. 65 2.11 Generation of CLEC14A Knockout mice........................................................................ 66 2.12 Immunogenicity assay ..................................................................................................... 67 2.13 Mouse immunization ....................................................................................................... 68 2.14 Bioinformatics websites and programs ........................................................................... 70 i 2.15 Statistical methods .......................................................................................................... 71 Validation of potential TEMs ....................................................................................................... 72 3.1 Introduction ....................................................................................................................... 73 3.2 CLEC14A is predominantly expressed in endothelial cells .............................................. 74 3.3 CLEC14A siRNA knockdown in HUVEC........................................................................ 76 3.4 CLEC14A is expressed on vessels in tumours but not those in healthy tissue .................. 77 3.5 Confirmation of CLEC14A as a tumour endothelial marker by immunohistochemistry .. 85 3.6 GBP4 is highly expressed on tumour vessels and on vessels in some adjacent tissue ...... 88 3.7 GBP4 is absent from vessels in healthy tissue .................................................................. 93 3.8 Validation of an IKBKE antibody by siRNA knockdown ................................................. 96 3.9 Immunofluorescence of IKBKE on tissue samples ........................................................... 97 3.10 Conclusions ................................................................................................................... 102 Functional chareacterization of CLEC14A .............................................................................. 103 4.1 Introduction ..................................................................................................................... 104 4.2 CLEC14A knockdown inhibits endothelial cell migration ............................................. 105 4.3 Polyclonal antisera to CLEC14A inhibits endothelial cell migration ............................. 107 4.4 Production of human and mouse CLEC14A protein using lentivirus ............................. 109 4.5 Generation of monoclonal antibodies to both human and mouse CLEC14A ................. 111 4.6 Papain cleavage of CLEC14A-Fc protein and Fc fragment depletion ............................ 112 4.7 Evaluation of the CLEC14A monoclonal antibodies ...................................................... 114 4.8 Monoclonal antibody to CLEC14A inhibits endothelial cell migration .......................... 116 4.9 Overexpression of CLEC14A in HUVEC using a lentiviral system ............................... 118 4.10 Overexpression of CLEC14A inhibits HUVEC migration ........................................... 120 4.11 Generation of germline transmission mice carrying a CLEC14A knockout allele ........ 122 4.12 Generation of CLEC14A knockout mice ...................................................................... 123 4.13 Confirmation of CLEC14A knockout in mice at the protein
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