Evaluating and Controlling Glioblastoma Infiltration by Joseph F. Georges A Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Approved March 2014 by the Graduate Supervisory Committee: Burt Feuerstein, Co-Chair Brian Smith, Co-Chair Kendall Van Keuren-Jensen Pierre Deviche Kevin Bennett ARIZONA STATE UNIVERSITY May 2014 ABSTRACT Glioblastoma (GBM) is the most common primary brain tumor with an incidence of approximately 11,000 Americans. Despite decades of research, average survival for GBM patients is a modest 15 months. Increasing the extent of GBM resection increases patient survival. However, extending neurosurgical margins also threatens the removal of eloquent brain. For this reason, the infiltrative nature of GBM is an obstacle to its complete resection. The central hypothesis of this dissertation is that targeting genes and proteins that regulate GBM motility, and developing techniques that safely enhance extent of surgical resection, will improve GBM patient survival by decreasing infiltration into eloquent brain regions and enhancing tumor cytoreduction during surgery. Chapter 2 of this dissertation describes a gene and protein; aquaporin-1 (aqp1) that enhances infiltration of GBM. In chapter 3, a method is developed for enhancing the diagnostic yield of GBM patient biopsies which will assist in identifying future molecular targets for GBM therapies. In chapter 4, an intraoperative optical imaging technique is developed for improving identification of GBM and its infiltrative margins during surgical resection. This dissertation aims to target glioblastoma infiltration from molecular and cellular biology and neurosurgical disciplines. In the introduction; 1. A background of GBM and current therapies is provided. 2. A protein that decreases GBM survival is discussed. 3. An imaging modality utilized for improving the quality of accrued GBM patient samples is described. 4. An overview of intraoperative contrast agents available for neurosurgical resection of GBM, and discussion of a new agent for intraoperative visualization of GBM is provided. i ACKNOWLEDGMENTS I would like to express the greatest appreciation to my advisor, Burt Feuerstein MD/PhD. Burt selflessly prioritized my training as a graduate student, and spent substantial time mentoring me in experiment design, data analysis, scientific writing, and grantsmanship. He taught me the value of collaborative research, and helped me acquire training at highly regarded research institutes. Burt always had a plan for my continued growth as a physician-scientist. This was evident in the numerous educational opportunities he provided for me in clinical neuroscience. Without Burt’s mentoring and support, this dissertation would not have been possible. I would also like to thank my committee for supporting my development as a scientist, and creating opportunities for my continued learning. I thank Kendall Van Keuren-Jensen for the countless times she allowed me to interrupt her work to casually chat about neuroscience and experiment design. I also thank Kevin Bennett for providing me opportunities to learn MRI, and for the great memories of working with him and his students. I am grateful to Pierre Deviche for serving as my undergraduate research mentor beginning in 2003, providing me opportunities to work on neuroendocrine projects, and making graduate school a possibility for me. I would also like to thank Brian Smith for his part in creating the interdisciplinary neuroscience program between Arizona State University and Barrow Neurological Institute (ASU-BNI), and for his personal support during graduate school. Additionally, I thank Trent Anderson and Peter Nakaji for their friendship and mentoring during graduate school. Trent helped me troubleshoot many experiments and always provided great advice towards improving manuscripts. I enjoyed working with ii Peter Nakaji at BNI. Dr Nakaji strongly mentored me in clinical neurosurgery research, and he provided me endless opportunities to design and implement molecular imaging projects. My training as a physician-scientist would not have been possible without the infrastructure and research opportunities created by faculty at ASU-BNI. I would like to thank the researchers who have made this program a great educational experience for myself and fellow classmates. I would also like to thank the labmates I have enjoyed working with in graduate school. I am grateful for your support during my training and the great times we shared working together. Also, I would like thank my wife Jackie and my family who supported and encouraged me throughout medical and graduate school. iii TABLE OF CONTENTS Page LIST OF TABLES .................................................................................................................... v LIST OF FIGURES ................................................................................................................. vi CHAPTER 1 INTRODUCTION .......................................................................................................1 2A AQUAPORIN 1 PREDICTS SURVIVAL OF MALIGNANT GLIOMA PATIENTS AND INCREASES GLIOMA CELL INVASIVENESS ..................................................... 14 Introduction .............................................................................................. 15 Materials and Methods ............................................................................. 17 Results ...................................................................................................... 25 Discussion ................................................................................................ 38 Conclusion ................................................................................................ 41 References ................................................................................................ 44 2B AQUAPORIN-1 OVEREXPRESSION ENHANCES GLIOMA CELL MOTILITY INDEPENDENTLY OF WATER TRANSPORT ............................................................. 47 Introduction .............................................................................................. 47 Materials and Methods ............................................................................. 49 Results ...................................................................................................... 51 Discussion ................................................................................................ 59 References ................................................................................................ 61 iv CHAPTER Page 3 CONTRAST-FREE MICROSCOPIC ASSESSMENT OF GLIOBLASTOMA BIOSPECIMENS PRIOR TO BIOBANKING ………………………………….…………63 Introduction .............................................................................................. 64 Materials and Methods ............................................................................. 68 Results ...................................................................................................... 71 Discussion ................................................................................................ 77 Conclusion ................................................................................................ 79 References ................................................................................................ 80 4 RAPID AND SPECIFIC DIAGNOSIS OF HUMAN ASTROCYTIC BRAIN TUMORS BY IMMEDIATE EX VIVO IMAGING WITH SULFORHODAMINE 101 82 Introduction .............................................................................................. 83 Materials and Methods ............................................................................. 85 Results ...................................................................................................... 90 Discussion .............................................................................................. 101 Conclusion .............................................................................................. 103 References .............................................................................................. 107 5 DISCUSSION ................... .............................................................................................. 110 REFERENCES....... ................................................................................................................. 117 APPENDIX A: Cirriculum Vitae ........................................................................................... 123 APPENDIX B: AQP1 and Glioblastoma Infiltration .............................................................. 129 APPENDIX C: Sulforhodamine 101 Specifically Labels Human Astrocytoma .................... 131 APPENDIX D: Reflectance Microscopy Screening of Glioblastoma Biospecimens ............. 133 v LIST OF TABLES Table Page 1. Characteristics of patients examined for aqp1 expression .................................. 42 2. Aqp1 expression is an independent predictor of survival ................................... 43 3. Distribution of human biopsies stained with SR101 and final diagnosis .......... 105 4. Stereology counts per region of interest ............................................................ 106 vi LIST OF FIGURES Figure Page 1. Aquaporin-1 expression is prognostic in GBM ............................................ 27 2. AQP1 overexpression enhances U251 motility ............................................ 29 3. AQP1 enhances matrigel invasion. ..............................................................