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Insights Into the Nature and Immune Escape of a Transmissible Cancer in Tasmanian Devils

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Insights Into the Nature and Immune Escape of a Transmissible Cancer in Tasmanian Devils INSIGHTS INTO THE NATURE AND IMMUNE ESCAPE OF A TRANSMISSIBLE CANCER IN TASMANIAN DEVILS César Darío Tovar López, BSc(Hons) GradDipEnvSt(Hons) Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy University of Tasmania July 2012 Declaration of Originality This thesis contains no material which has been accepted for a degree or diploma by the University or any other institution, except by way of background information and duly acknowledged in the thesis, and to the best of the my knowledge and belief no material previously published or written by another person except where due acknowledgement is made in the text of the thesis, nor does the thesis contain any material that infringes copyright. César D. Tovar López i Authority of Access This thesis may be made available for loan and limited copying and communication in accordance with the Copyright Act 1968. César D. Tovar López ii iii iv v Statement of Ethical Conduct The research associated with this thesis abides by the international and Australian codes on human and animal experimentation, the guidelines by the Australian Government's Office of the Gene Technology Regulator and the rulings of the Safety, Ethics and Institutional Biosafety Committees of the University. César D. Tovar López vi Publications Tovar C, Obendorf D, Murchison EP, Papenfuss AT, Kreiss A, Woods GM (2011). Tumor-specific diagnostic marker for transmissible facial tumors of Tasmanian devils: immunohistochemistry studies. Veterinary Pathology 48: 1195-1203 Kreiss A, Tovar C, Obendorf DL, Dun K, Woods GM (2011). A murine xenograft model for a transmissible cancer in Tasmanian devils. Veterinary Pathology 48: 475- 481. Murchison EP, Tovar C, Hsu A, Bender HS, Kheradpour P, Rebbeck CA et al (2010). The Tasmanian devil transcriptome reveals Schwann cell origins of a clonally transmissible cancer. Science 327: 84-87. (Copy of these publications are included in Appendix 1) vii Conference presentations Tovar C., Obendorf D., Kreiss A., and Woods G. 2010. A tumour-specific diagnostic marker for transmissible facial tumours of Tasmanian devils. Oral presentation. Australian Health and Medical Research Congress, Melbourne, Australia. Tovar C., Obendorf D., Kreiss A., and Woods G. 2009. Immunohistochemistry studies of Devil Facial Tumour Disease. Oral presentation. Molecular and Experimental Pathology Society of Australia Conference, Sydney, Australia. Tovar C. 2009. Devil Facial Tumour Disease: Immunohistological evaluation and identification of a biomarker for disease diagnosis. Oral presentation. Wildlife Disease Association International Conference, Blaine, Washington, USA. Tovar C., Obendorf D., Kreiss A., and Woods G. 2009. Periaxin as a diagnostic marker for Devil Facial Tumour Disease (DFTD). Poster presented. Frontiers in Immunology Research Network International Conference, Kona, Hawaii, USA. Tovar C. and Woods G. 2009. Immunohistochemistry of DFTD and identification of a potential marker. Oral presentation. THING Annual Scientific Meeting, Launceston, Australia. Tovar C., Blizzard C., Brown G., and Woods G. 2008. Devil Facial Tumour Disease (DFTD): a diagnosis and vaccination approach. Poster presented. Australian Health and Medical Research Congress, Brisbane, Australia. viii Abstract Devil facial tumour disease (DFTD) is a transmissible cancer that threatens the survival of the Tasmanian devil (Sarcophilus harrisii), the world’s largest living carnivorous marsupial. Despite devils having a competent immune system, there is no evidence of natural immunity to the tumours. Affected animals die within months of tumour appearance. The species could face extinction within 25 to 35 years. Cytogenetic and molecular studies have confirmed that the infectious agent is the cancer cell itself. However, our knowledge about the nature and biology of this tumour is limited. Tumour transcriptome analyses of DFTD tumours and devil tissues revealed that DFTD expresses a set of genes related to the myelination pathway in the peripheral nervous system. This thesis examined the protein expression of peripheral nerve and other neuronal markers in DFTD to further elucidate the nature of the tumour. It also evaluated the utility of neuronal and peripheral nerve proteins as diagnostic markers for the disease. Immunohistochemistry and flow cytometry confirmed that DFTD tumour cells express a number of proteins found throughout the neuronal system. Notably, DFTD tumour cells expressed structural proteins found in myelinating Schwann cells in the peripheral nervous system. These included peripheral myelin protein 22 (PMP22), myelin protein zero (MPZ), myelin basic protein (MBP), and the recently described periaxin (PRX). Nerve growth factor receptor (NGFR), which is involved in the differentiation of Schwann cells, was also detected in DFTD tumour cells. These results coupled with the available genetic data confirm that DFTD cells are of Schwann cell origin. Furthermore, this study showed that periaxin, a specific marker of Schwann cells, is consistently expressed in DFTD primary tumours, DFTD metastases, DFTD cell lines and murine xenografted DFTD tumours. Therefore, this thesis identified periaxin as a sensitive and specific marker for DFTD. This will greatly facilitate the diagnosis of the disease. How DFTD can be transplanted across major histocompatibility (MHC) barriers is an aspect of DFTD that is not understood. Initially this was attributed to the reduced ix diversity at the major histocompatibility complex (MHC) loci. However, recent studies showed that the limited MHC diversity in devils is sufficient to produce measurable mixed lymphocyte reactions and the rejection of skin allografts. Thus, this thesis investigated the expression of MHC to determine if this may explain the lack of immune response to DFTD cells. Analysis of the expression of MHC proteins in devils has been hindered by the lack of cross-reacting reagents. During the course of this study collaborators from the University of Cambridge (UK) developed the first anti-devil MHC class I (MHC-I) antibody. This antibody was used to examine the expression of MHC-I in devil tissues and DFTD. MHC-I protein expression in primary tumours and metastases was lower than normal tissues. Its expression was also variable within a tumour and among different tumours. These results suggest that alteration of MHC-I expression could contribute to the immune escape of DFTD. Immunisation trials with non-viable DFTD cell lines revealed that some Tasmanian devils can produce antibodies against DFTD cells. This thesis utilised an immunoproteomic approach to identify immunogenic proteins in DFTD. Several candidates were identified as DFTD tumour associated antigens. These include heat shock proteins, tubulin, histone H2B, stathmin protein, and proliferating cell nuclear antigen (PCNA). These proteins are excellent targets for the development of early diagnostic tools as well as therapeutic approaches. In summary, this thesis provided strong supporting evidence for a Schwann cell origin of DFTD and generated a specific diagnostic marker for the disease. Additionally, this thesis opened new opportunities for the understanding of the mechanisms that allow immune evasion and the interaction between the tumour cells and the devil’s immune system. These results will have direct implications for the future development of a vaccine approach. x Acknowledgments I primarily wish to express my sincere gratitude to my supervisor Associate Professor Greg Woods for his continued support, guidance, faith and good humour. I am particularly grateful to him for providing the opportunity to undertake this work with his research group at Menzies Research Institute Tasmania. A special thanks to my co-supervisors Dr David Obendorf and Dr Louise Rodman for their friendship, expertise, professional advice and so much appreciated feedback. Special thanks to Alex Kreiss for his friendship and for re-introducing me to the scientific realm and this fascinating research. We have learnt together, shared frustrations and gratifying experiences through our work. Thank you so much. This research would have not been possible without the work and collaboration of friends, colleagues and staff from the Save the Tasmanian devil Program, the Tasmanian Department of Primary Industry, Parks, Water and Environment, Mount Pleasant Laboratory and the University of Tasmania. This research was possible thanks to the sampling material collected and provided by the team. Special thanks to Dr Colette Harmesen, Dr Sarah Peck, Dr Rodrigo Hamede and devil keepers. I appreciate all your support and collaboration. Thank you to the staff at the Department of Pathology of the Royal Hobart Hospital, and especially to Narelle Philips and Steve Weston for all your assistance in the histology lab. Thank you to our collaborators Professor Jim Kaufman and Dr Hannah Siddle for kindly providing precious reagents that facilitate substantial part of this research. Thanks to Associate Professor Katherine Belov and Dr Elizabeth Murchison for their continued support, inspiration and valuable conversations. Thanks to the Dr Eric Guiler Foundation and Philanthropists for their financial assistance that supports our work and effort to save the Tasmanian devil. Working at Menzies Institute Tasmania gave me the opportunity to meet wonderful people. Thanks to my friends in the Immunology Group, Heather, Roslyn, Nick, Gabby, Jess, Terry, Lauren and Takumi. Thank you to Niall, Kathy, Ruth, Stan, Olivier, Jerome and Naseem
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