Title Page
Contents
Abstract 17
Chapter I. Introduction 21
References 24
Chapter II. Optimal Voltage and Electrical Pulse Conditions for Irreversible Electroporation (IRE) to Induce Immunogenic Cell Death (ICD) 27
2.1. Introduction 28
2.2. Materials and Methods 32
A. Finite Element Method (FEM) Simulation 32
B. In Vitro 2 Dimension (2D) and 3 Dimension (3D) Cell Culture 33
C. IRE Treatment Conditions 34
D. In Vitro Cell Viability Assay 35
E. Immunofluorescence (IF) Assay for Calreticulin (CRT) 35
F. Measurement of High Mobility Group Box 1 (HMGB1) Released in Response to IRE 36
G. Measurement of Adenosine Triphosphate (ATP) Released in Response to IRE 37
H. Animal Testing 37
I. Statistical Analysis 38
2.3. Results 39
A. FEM Simulation 39
B. In Vitro Cytotoxicity 41
C. Expression of Damage-associated Molecular Patterns (DAMPs) in Response to IRE 45
D. In Vivo Tumor Growth Suppression by IRE 49
2.4. Conclusions 55
2.5. References 56
Chapter III. Combination of Irreversible Electroporation (IRE) and Stimulator of Interferon Genes (STING) Agonist for Effective Cancer Immunotherapy 65
3.1. Introduction 66
3.2. Materials and Methods 71
A. Finite Element Method (FEM) Simulation 71
B. In Vitro Cell culture 72
C. In Vitro 3 Dimension (3D) Tumor Model 72
D. In Vitro IRE Treatment 73
E. In Vitro Cell Viability Analysis 73
F. In Vitro Analysis of the Expression of Damage-associated Molecular Patterns (DAMPs) 74
G. In Vivo Tumor Treatment with a Combination of IRE and STING Agonist 75
H. Tumor-Infiltrating Immune Cell Analysis Using Flow Cytometry 76
I. Statistical Analysis 78
3.3. Results 79
A. In Vitro Cellular Experiments to Establish Electrical Conditions for IRE 79
B. In Vitro Immunogenic Cell Death (ICD) Induced by IRE 83
C. In Vivo Tumor Growth Suppression by Combinational Treatment with IRE and STING Agonist in an LLC Tumor Model 90
D. Induction of Anti-Cancer Immunity in Tumor Microenvironment (TME) by a Combination of IRE and STING Agonist 94
3.4. Conclusions 99
3.5. References 100
Chapter IV. Triple Combination of Irreversible Electroporation (IRE), Fusogenic Liposome Loaded with STING (cFLs-STING) and Anti-programmed Cell Death Protein-1 (aPD-1) Antibody for Effective Cancer Immunotherapy 111
4.1. Introduction 112
4.2. Materials and Methods 119
A. Lipid 119
B. Preparation of Liposomes 119
C. Characterization of Liposome 120
D. Size Exclusion Chromatography 121
E. Liposome Encapsulation Efficiency of STING 121
F. In Vitro Cell Culture 122
G. In Vitro Cell Viability Assay 122
H. Reporter Assay of Type I interferon (IFN) Activity 123
I. Confocal Microscopy 123
J. Flow Cytometry Analysis 124
K. IRE Treatment Condition 124
L. Animal Testing 124
4.3. Result 128
A. Characterization of Liposome and STING Encapsulation Efficiency 128
B. In Vitro Cellular Uptake and Cytotoxicity of Liposome 132
C. Immune Activation of Liposome Encapsulating STING (Lipo-STING) in Various Cells 134
D. Optical Imaging of Tumor Accumulation according to the Injection Method of Anionic Non-fusogenic Liposomes (aNFLs) and Cationic Fusogenic Liposomes (cFLs) 137
E. In vivo Tumor Treatment Effect Combined with IRE, cFLs-STING and aPD-1 142
4.4. Conclusions 146
4.5. References 148
Chapter V. General Conclusion 158
ABSTRACT IN KOREAN 162
Table 1. The critical electrical field (Ec) values for the boundary... 33
Figure 1-1. Ablation zone areas predicted for irreversible... 40
Figure 1-2. Viability of LLC cells grown in 2 dimension (2D) and... 44
Figure 1-3. Expression of damage associate molecular patterns... 48
Figure 1-4. Tumor growth suppression by irreversible... 51
Figure 1-5. Tumor growth suppression by irreversible... 54
Figure 2-1. In vitro cellular experiments to establish electrical... 82
Figure 2-2. In vitro immunogenic cell death (ICD) was induced by... 88
Figure 2-3. In vitro immunogenic cell death (ICD) was induced by... 89
Figure 2-4. In vivo tumor growth suppression by combinational... 93
Figure 2-5. Combination treatment with irreversible electroporation... 98
Figure 3-1. Composition and properties of anionic non-fusogenic... 131
Figure 3-2. In vitro cellular uptake and cytotoxicity of liposomes... 133
Figure 3-3. Activation of the immune response by stimulator of... 136
Figure 3-4. Biodistribution images of anionic non-fusogenic... 141
Figure 3-5. Tumor growth suppression of triple combinational... 145
Scheme 1. Schematic illustration of the optimal conditions for... 31
Scheme 2. Schematic illustration of a combination of irreversible... 70
Scheme 3. Schematic illustration of a combination of irreversible... 118