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The Interaction of the Epha2 Receptor and Microglia in Glioblastoma

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The Interaction of the Epha2 Receptor and Microglia in Glioblastoma The Interaction of The EphA2 Receptor and Microglia in Glioblastoma Dr Manasi Jiwrajka B.A Neuroscience (Hons.)., MBBS 0000-0003-4040-124X A thesis submitted for the degree of Master of Philosophy at The University of Queensland in 2020 Medicine Faculty ABSTRACT Introduction Glioblastoma (GBM) is the most malignant primary tumour of the central nervous system (CNS) in adults. The median survival for patients despite maximal surgical resection and chemoradiation therapy is approximately 15 months. Resident macrophages of the brain, microglia, have been implicated in promoting glioblastoma despite their immune function. Eph receptors and their corresponding ephrin ligands are tyrosine kinase receptors that promote cell invasion and proliferation in the context of tumours. However, the role of Eph receptors on microglia have not been investigated to-date. Aim The aim of this study is to investigate the role of microglia and the EphA2 receptor in the context of GBM by focusing on three main components: (i) Eph and ephrin expression on microglia, (ii) the functional role of EphA2 in a human microglia cell line and in co-culture with primary GBM cell line, and (iii) the expression and co-localization of EphA2 and microglia markers in patient GBM samples as they correlate to clinical demographic and outcomes. Methods The study utilises a variety of techniques to investigate the above-mentioned aims, especially cell culture, flow cytometry, quantitative polymerase chain reaction (qPCR) and immunohistochemistry. Microglia cytokine release, phagocytosis, migration, and proliferation were assessed using new and unique models involving microscopy. Additionally, fluorescent labelling of microglia and primary tumour cells (from intraoperative tumour specimens) allowed for novel methods of 2D and 3D coculture to be conducted. A 3D human GBM organoid model was created for the first time to observe the behaviour of both the tumour and microglia cells. Tissue microarrays were created for tumour immunohistochemistry. Iba1 was used as a microglia marker, and EphA2 antibody (both in-house and commercial) in conjunction with its high-affinity ligand, EphrinA1 was utilised for receptor activation assays. The Queensland Oncology Online (QOOL) database was used in conjunction with patient files to obtain clinical data. i Jiwrajka 2020 Results The primary findings of this study show that a number of Eph and ephrin family members are expressed on microglia, and in particular EphA2 is highly expressed and functional on human microglia cells. EphA2 activation induced microglia cells to undergo a phenotypic change with increased proliferation and decreased migration. The opposite effect was achieved when EphA2 was down regulated using shRNA. EphA2 knockdown induced increased phagocytosis while decreased microglia proliferation. In co-culture, with primary human GBM cells, microglia promoted GBM cell growth while the proliferation rate of microglia was reduced under these conditions. To better assess the interaction of microglia with primary GBM cells a 3D organoid approach combined with optical barcoding was utilised. Results under organoid conditions highlighted a contact repulsion mechanism, where microglia rapidly segregated and encircled GBM cells. In patient GBM samples, EphA2 and the microglia marker Iba1 were co- localised, but not as frequently as expected from our in vitro studies. Interestingly, patient samples with a high level of co-localisation of EphA2 on microglia had a worse survival prognosis compared to those who have a lower co-localization level. Additionally, as predicted and validated by The Cancer Genome Atlas (TCGA) datasets, high grade GBM have a higher expression of EphA2 and Iba1 compared to low grade glioma. Patients who are IDH mutant (a positive prognostic marker), have a lower EphA2 expression. Interestingly paired patients with ‘normal’ and tumour cores demonstrated an increase in Iba1 expression in tumour samples. Paired patients who developed recurrent GBM displayed lower Iba1 expression. Conclusion In summary, this study has identified novel findings regarding the expression of Eph and ephrin family members on microglia and further identifies a functional role of EphA2 in mediating the proliferation and migration capabilities of microglia. This is significant in the context of GBM as microglia actively migrate to the tumour site and promote tumour growth. This study is a first of its kind and paves the way for future opportunities to target EphA2 present on microglia which may result in significant GBM anti-tumour responses. ii Jiwrajka 2020 DECLARATION BY THE AUTHOR This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co- authors for any jointly authored works included in the thesis. iii Jiwrajka 2020 PUBLICATIONS INCLUDED IN THE THESIS No publications included SUBMITTED MANUSCRIPTS INCLUDED IN THIS THESIS No manuscripts submitted for Publication OTHER PUBLICATIONS DURING CANDIDATURE Pratap, K., Jiwrajka, M. Weber, L., Richardson, A 2019 “Severe Symptomatic Hyponatraemia Secondary to Bowel Preparation" BMJ Case Reports Jiwrajka, M., Ranasinghe, S., Yaxley, W., Perera M., and Yaxley, J. 2018 “Drugs for benign prostatic hypertrophy” Australian Prescriber Jiwrajka, M., Yaxley, W., Perera, M., Roberts, M., Dunglison, N., Yaxley, J., Esler, R. 2018 “Review and Update of Benign Prostatic Hyperplasia (BPH) in General Practice” Australian Journal of General Practice Jiwrajka, M. Pratap, K. Yaxley, W. Dunglison, N. 2017 “Result of Health Illiteracy and Cultural Stigma: Fournier’s Gangrene, A Urological Emergency” BMJ Case Reports Jiwrajka, M. Mahmoud, A., Uppal, M. 2017 “A Rohingya Refugee’s Journey in Australia And The Barriers To Accessing Healthcare” BMJ Case Reports Jiwrajka, M., Biswas, S., 2016 “Why Should Students Write a Global Health Case Report?” Australian Medical Student Journal Jiwrajka, M., Phillips, A., Butler, M., Rossi, M., Pocock, JM. 2016, “The Plant-Derived Chalcone 2,2′,5′-Trihydroxychalcone Provides Neuroprotection against Toll-Like Receptor 4 Triggered Inflammation in microglia,” Oxidative Medicine and Cellular Longevity, vol. 2016, Article ID 6301712, 10 pages. http://dx.doi.org/10.1155/2016/6301712 iv Jiwrajka 2020 CONTRIBUTION BY OTHERS TO THE THESIS The cell lines HMC3 and BV2 were donated by Prof Glen Boyle and Prof Peter Parsons. The PLVX virus used to transfect microglia cells with synthetic proteins was designed by Dr Seckin Akgul. Tissue Microarrays were created by Ms Crystal Chang from QIMR Histology department. Immunohistochemistry staining was performed with the assistance of Ms Crystal Chang and Mr Clay Winterford. from the QIMR Histology department. All microscopy was with the assistance of Dr Tam Hong Nguyen from the QIMR Flow Cytometry and Imaging Department. The organoid matrix was created by Dr Ulrich Baumgartner. FACS sorting was performed by Dr Amanda Stanley from the QIMR Flow Cytometry and Imaging Department. Statistical analysis on the tumour bank was performed with the assistance of Ms. Anita Pelecanos from the QIMR Statistics Department. v Jiwrajka 2020 STATEMENT OF PARTS OF THE THESIS SUBMITTED TO QUALIFY FOR THE AWARD OF ANOTHER DEGREE None vi Jiwrajka 2020 RESEARCH INVOLVING HUMAN OR ANIMAL SUBJECTS HREC/17/QRBW/577 Novel Therapies for Brain Cancer approved by the RBWH HREC vii Jiwrajka 2020 ACKNOWLEDGEMENTS Firstly, I would like to sincerely thank Prof Bryan Day for agreeing to supervise this project, and for his support throughout. My sincere thanks to my review panel who provided feedback at each milestone: Prof Glen Boyle, Dr Adrian Wiegmans and Dr Hamish Alexander. Dr Tam Nguyen, Ms Crystal Chang, Mr Clay Winterford and Ms Anita Pelecanos, thank you for your considerable assistance, time, and teaching. I would also like to acknowledge the wonderful scientists of the Sid Faithfull Cancer Laboratory who helped me with every Western Blot that went wrong and cheered me on when an experiment worked. In particular, I would like to thank Dr Rochelle
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