MESENCHYMAL STEM CELLS AS THERAPY AGAINST HUMAN GLIOBLASTOMA MULTIFORME YULYANA (B.Sc (Hons.), UNSW) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF PHYSIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2013 ACKNOWLEDGEMENTS Research is not a one-man show, and certainly, this thesis would not have been completed without the support of many, to whom I wish to express my gratitudes First and foremost, I am grateful to Assoc Prof Paula Lam for accepting me in the lab, for the opportunity to undertake this M.Sc project and for your guidance, advice, and patience as my supervisor to bring out the best in me To Dr Ivy Ho, for your endless support, guidance and patience; for your encouragement, prayers and positive thinking during trying moments, I am forever grateful For the many lunches, cups of coffee and chats that we shared, they are invaluable to me And your giggles never fail to cast away the gloomy atmosphere To my great lab members, past and present To Dr Sia Kian Chuan, for sharing your journey, for your many advices, a pair of ears, and your pet fishes, I am forever thankful To Jennifer Newman and Toh Xin Yi, I am grateful for your support and dependable assistance To my dearest friends, near and far Your listening ears, comforting words and a pat on my back are more than I could have asked for Thank you for being there when the going got rough Last but not least, to my precious family, without whom, I will not be here Without your unconditional love and support, I would not have been able to weather the storm You are forever my source of strength and motivation iii TABLE OF CONTENTS Declaration Acknowledgements Table of contents Summary List of Tables List of Figures List of Abbreviations ii iii iv vii ix x xii 1 INTRODUCTION 1.1 Glioblastoma Multiforme 1.2 Treatment options for GBMs 1.2.1 Current standard treatment regime for GBMs 1.2.2 Gene therapy for GBMs 4 1.3 Mesenchymal Stem Cell as delivery vector 1.3.1 Mesenchymal Stem Cell (MSCs) 1.3.2 Tumor tracking properties of MSCs 1.3.3 Genetically-engineered MSCs 7 10 1.4 TRAIL as potent apoptosis inducer 1.4.1 TRAIL and its receptors 1.4.2 TRAIL apoptotic pathway 1.4.3 TRAIL resistance and strategies to overcome resistance 10 10 12 14 1.5 Cell communication and adhesion 1.5.1 Gap junctions 1.5.2 Connexin 43 1.5.3 Adhesion-mediated apoptosis resistance 19 20 21 24 1.6 Hypothesis and Study aims 24  2 MATERIALS AND METHODS 26  2.1 Cell culture and reagents 2.1.1 Glioma spheroid culture 2.1.2 MSCs 2.1.3 Primary glioma cell culture 26 27 27 27 2.2 Cloning of pHGCX-TRAIL Herpes Simplex Virus-1 (HSV-1) Amplicon viral vector 2.2.1 Viral packaging and purification 2.2.2 Virus titering 28 29 29 2.3 Cell transfection and transduction 2.3.1 Standard transfection 2.3.2 RNAi transfection 2.3.3 Viral transduction 30 30 30 31 iv 2.4 MSCs characterization 2.4.1 MSCs differentiation 2.4.2 MSCs surface markers analysis 31 31 32 2.5 Enzyme-linked Immunosorbent Assay (ELISA) 33 2.6 Harvesting of CM 33 2.7 In vitro migration assay 34 2.8 Trypan blue dye exclusion assay 34 2.9 Caspase-3 activity assay 35 2.10 FACS analysis 2.10.1 FACS analysis for eGFP 2.10.2 Surface receptor analysis 2.10.3 Cell cycle analysis 35 35 36 36 2.11 Immunofluorescence staining 36 2.12 Dye transfer assay 37 2.13 Intracranial glioma mouse model 37 2.14 Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining 38 2.15 Western blot analysis 39 2.16 RNA isolation 40 2.17 Real Time Reverse Transcriptase Polymerase Chain Reaction 40 2.18 Statistical analysis 41 3 RESULTS 42  3.1 Construction of TRAIL-secreting HSV-1 vector and validation of its functionality 3.1.1 Construction of TRAIL-secreting HSV-1 vector, pHGCX-TRAIL and its functional validation 3.1.2 Functionality of pHGCX-TRAIL in MSCs 3.1.3 Glioma cells response variability to TRAIL 42 42 48 3.2 Physiological effect of CBX 3.2.1 CBX does not affect glioma cells and MSCs viability 3.2.2 CBX blocks GJIC and may affect cell cycle 50 50 54 3.3 CBX enhances TRAIL-induced apoptosis 3.3.1 CBX enhances TRAIL-induced apoptosis in glioma cells 3.3.2 CBX enhances TRAIL-induced apoptosis in patient-derived glioma cultures 3.3.3 Double arm therapy of CBX and MSC-TRAIL prolonged survival of intracranial mouse model 56 56 42 63 64 v 3.4 Mechanisms contributed by CBX in augmenting TRAIL-induced apoptosis 66 3.4.1 CBX downregulates Cx43 66 3.4.2 CBX upregulates DR5 expression 70 4 DISCUSSION 74  4.1 Improvement in therapeutic vector system and its limitation 77 4.2 Cytoplasmic Cx43 may form functional GJ in glioma 83 4.3 GJ-dependent-mediation of cell death 84 4.4 CBX and cellular stress 85 5 FUTURE STUDIES 91 BIBLIOGRAPHY 94 APPENDIX - List of publications 119 vi SUMMARY Glioblastoma multiforme (GBM) is the most common and aggressive brain tumors that to this day are incurable despite the advancement in surgical techniques and standard therapies One contributing factor is the inherent ability of GBMs to disseminate and invade into the normal brain parenchyma, rendering complete removal of tumor cells difficult to achieve The development of anti-glioma gene therapies has become an alternative approach to curb the limitations of standard therapy However, direct administration of gene therapy vectors into brain tumors fails to achieve significant therapeutic efficacy The poor treatment efficacy is attributed to the limited distribution of therapeutic vectors into the brain tumor region, as well as the invasive nature and heterogeneity of GBMs Therefore, improved modalities are needed to effectively circumvent the limitation in the distribution of therapeutics The main objective of this study was to improve the delivery system for GBM treatment by harnessing the tumor-tropic property of human mesenchymal stem cells (MSCs) to deliver therapeutic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) Furthermore, we postulated that the therapeutic efficacy of soluble TRAIL mediated by MSCs could be enhanced when gap junction communication between glioma cells is disrupted To this end, MSCs were transduced with herpes simplex virus-1 amplicon viruses that were engineered to secrete soluble and functional TRAIL (MSC-TRAIL) Carbenoxolone (CBX), a known gap junction inhibitor, was used to interfere with gap junction communication in glioma cells which has been implicated in treatment resistance The therapeutic efficacy of CBX on MSC-TRAILinduced apoptosis was subsequently evaluated in human glioma cell lines, patientderived gliomas, and orthotopic glioma mouse model vii The results in this thesis demonstrated that combination treatment of MSC-TRAIL and CBX significantly enhanced glioma cell death compared to single treatment Enhanced cell death is specific to human gliomas but not normal astrocytes and this included patient-derived isolates that are normally insensitive to TRAIL More importantly, dual arm therapy of MSC-TRAIL and CBX effectively prolonged the survival of orthotopic glioma mice by ~27% when compared with the control mice, indicating that interference of gap junction communication could improve therapeutic efficacy of MSC-TRAIL Molecular evaluation on the mechanisms of enhanced cell death by MSC-TRAIL and CBX showed that it was mediated through an upregulation of C/EBP homologous protein and death receptor expressions Death signals from death receptor were further amplified through the engagement of intrinsic apoptosis pathway and downregulation of anti-apoptotic protein Bcl-2 Furthermore, the results have demonstrated that the downregulation of connexin 43 by CBX further amplified the death signals by preventing these signal molecules to be diluted out and thus sealing the fate of the cells into apoptosis These mechanisms synergistically resulted in the increase in therapeutic efficacy In conclusion, this study has demonstrated that MSC-TRAIL when combined with gap junction inhibitor may serve as an effective therapy against human GBMs It may potentially be applied for clinical use for the following reasons: (1) No obvious physiological or neurological effect was observed in mice administered with CBX; (2) CBX acts synergistically with MSC-TRAIL at multiple levels, which is particularly advantageous as tumor cells employ multiple resistance mechanism to therapeutic agents viii LIST OF TABLES Table 1.1 WHO classification of brain tumors and their features Table 1.2 Compounds used in combinatorial strategies with TRAIL and their mechanism of actions 19 ix LIST OF FIGURES Figure 1.1 Pathological features of malignant gliomas Figure 1.2 Pathways in the development of malignant gliomas Figure 1.3 Glioma tumor tropism of BM-hMSCs Figure 1.4 The TRAIL signaling pathway 13 Figure 1.5 Gap junctions in cell membranes 22 Figure 3.1 Construction and functionality of pHGCX-TRAIL 44 Figure 3.2 Characterization of bone marrow-derived MSCs 45 Figure 3.3 Functionality of pHGCX-TRAIL in MSCs 46 Figure 3.4 Cell surface expression of TRAIL receptors 48 Figure 3.5 Glioma cells response variability to TRAIL 49 Figure 3.6 Effect of CBX on MSCs 52 Figure 3.7 Effect of CBX on glioma cells 53 Figure 3.8 CBX may affect cell cycle progression of glioma cells 55 Figure 3.9 CBX blocks GJIC in glioma cells 56 Figure 3.10 CBX augments MSC-mediated TRAIL-induced apoptosis in human glioma cell lines 57 Figure 3.11 CBX modulates proteins involved in the apoptotic pathway 59 Figure 3.12 CBX augments MSC-mediated TRAIL-induced apoptosis in patient-derived primary glioma cells 62 CBX synergizes with MSC-TRAIL to prolong the survival of glioma-bearing mice 65 Figure 3.14 Connexins expression in glioma cells 67 Figure 3.15 CBX downregulates Cx43 67 Figure 3.16 Downregulation of Cx43 by CBX enhances TRAIL-induced apoptosis in glioma cells 68 Figure 3.13 Figure 3.17 CBX upregulates DR5 expression Figure 3.18 71 Enhanced TRAIL-apoptosis by CBX is partially mediated by x 124 Allensworth, JL, Aird, KM, Aldrich, AJ, Batinic-Haberle, I, and Devi, 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848-58 119 ... blue-stained cells over total cells (blue-stained cells + unstained cells) multiplied by 100 2.9 Caspase activity assay Caspase activity was determined using ApoAlert Caspase Colorimetric Assay Kit... Expression level of caspase also plays a role in determining the sensitivity to TRAIL Increase in caspase degradation, as well as silencing of caspase through methylation, has been reported to... DISC, caspase is activated through dimerization and cleavage Activated caspase triggers the activation of the downstream effector caspase 3, which leads to subsequent cleavage of caspase substrates