Title Page

Contents

List of Abbreviations 10

Abstract 13

CHAPTER 1. INTRODUCTION 15

Part 1. The Regulatory Roles of Mitochondrial Metabolism Dynamics and Mitochondria Calcium Uniporter (MCU) in Bevacizumab Resistance of GBM 15

Part 2. Translational mRNA Profiling Analysis of Pseudopalisading Cells in Bevacizumab Resistance of GBM 18

CHAPTER 2. MATERIALS AND METHODS 23

Part 1. The Regulatory Roles of Mitochondrial Metabolism Dynamics and Mitochondria Calcium Uniporter (MCU) in Bevacizumab Resistance of GBM 23

2.1.1. Cell Culture 23

2.1.2. Lentivirus Production and Infection 23

2.1.3. Cell Proliferation Assays 24

2.1.4. Immunoblot Analysis 25

2.1.5. Immunocytochemical Staining 25

2.1.6. APEX 26

2.1.7. Proteome Digestion and Enrichment of Biotinylated Peptides 27

2.1.8. LC-MS Analysis 29

2.1.9. LC-MS/MS Analysis of Enriched Peptide Samples 32

2.1.10. In Vivo Study 33

2.1.11. Statistical Analysis 34

Part 2. Translational mRNA Profiling Analysis of Pseudopalisading Cells in Bevacizumab Resistance of GBM 35

2.2.1. Cell Culture 35

2.2.2. Lentivirus Production and Infection 35

2.2.3. TRAP 36

2.2.4. Endogenous GFP Imaging 37

2.2.5. LC-MS/MS Analysis and Data Processing 37

2.2.6. Immunoblot Analysis 39

2.2.7. Flow Cytometry 40

2.2.8. Immunohistochemistry 40

2.2.9. RNA-Sequencing Data Processing 41

2.2.10. In Vivo Study 43

2.2.11. Statistics 44

CHAPTER 3. RESULTS AND DISCUSSION 46

Part 1. The Regulatory Roles of Mitochondrial Metabolism Dynamics and Mitochondria Calcium Uniporter (MCU) in Bevacizumab Resistance of GBM 46

3.1.1. Proteomic Analysis of Longitudinal Orthotopic Mouse Brains 46

3.1.2. Investigating Mitochondria-Specific Proteomic Changes in a U87MG Orthotopic Mouse Model Using the Mito-APEX System 52

3.1.3. Mitochondrial Calcium Transporter as a Potential Therapeutic Target 56

3.1.4. The Anti-Cancer Effect of MCU Inhibitor, DS16570511, In vitro and In vivo 62

Part 2. Translational mRNA Profiling Analysis of Pseudopalisading Cells in Bevacizumab Resistance of GBM 68

3.2.1. Enhancement of Hypoxia-Associated Gene Sets in Pseudopalisading Cells by Geographic Tumor Location-Based Classification 68

3.2.2. Establishment of the 5xHRE-TRAP System Under Hypoxic Conditions In Vitro 72

3.2.3. Establishment of the 5xHRE-TRAP System Under Hypoxic Conditions In Vivo 79

3.2.4. Translatomic Analysis in the Brain Tissues of GSC 83NS Transfected with Hypoxic Condition Responsive 5xHRE-TRAP System 86

CHAPTER 4. CONCLUSION 92

Part 1. The Regulatory Roles of Mitochondrial Metabolism Dynamics and Mitochondria Calcium Uniporter (MCU) in Bevacizumab Resistance of GBM 92

Part 2. Translational mRNA Profiling Analysis of Pseudopalisading Cells in Bevacizumab Resistance of GBM 93

References 95

논문요약 105

Figure 1. Proteomic profiling of longitudinal U87MG orthotopic mouse brains resistant to bevacizumab 49

Figure 2. Validation of the mito-APEX system transfected in U87MG GBM cell lines 51

Figure 3. Establishment of an orthotopic mouse model using the mito-APEX system. 55

Figure 4. Proteomic profiling of U87MG brain tumors transfected with the mito-APEX system 59

Figure 5. Proteomic profiling of mito-APEX transfected U87MG brain tumors 61

Figure 6. Evaluation of MCU inhibitor in U87MG cells under normoxic and hypoxic conditions in U87MG cells 66

Figure 7. Evaluation of MCU inhibitor in a U87MG GBM xenograft model 67

Figure 8. Geographic categorization of GBM patient data from IVY GAP based on tumor location 70

Figure 9. Pathway analysis of IVY GAP GBM data according to tumor location-based classification 71

Figure 10. Establishment and validation of the 5 × HRE-TRAP system in vitro 76

Figure 11. Validation of stable GBM cell lines transfected with the 5 ×HRE-TRAP system 78

Figure 12. Establishment and validation of the hypoxia-responsive 5 ×HRE-TRAP system in vivo 83

Figure 13. Establishment and validation of a GBM xenograft mouse model transfected with the 5 × HRE-TRAP system 85

Figure 14. Translatomic analysis in the brain tissues from GSC 83NS transfected with the hypoxia-responsive 5 × HRE-TRAP system 89

Figure 15. Translatomic and proteomic profiling of GSC 83NS cells. 91