Title Page
국문 초록
Abstract
Contents
Ⅰ. Introduction 14
1. The important of ICD in overcoming the limitations of cancer immunotherapy 14
2. Mechanisms by which ICDs promote the immunogenicity of tumors 17
3. Advanced HTS system needed for ICD inducer screening 20
4. Image-based AI analysis: the key to develop HTS for ICD inducer 20
Ⅱ. Materials and Methods 24
1. Cell lines and culture 24
2. Reagents and antibodies 24
2.1. Reagents 24
2.2. Antibodies and fluorescent labelling probes 25
3. Crystal violet assay 26
4. Caspase 3/7 activity live imaging 26
5. Western blotting 27
6. LDH assay 28
7. Silver staining 28
8. Calreticulin IF staining 29
9. Extracellular ATP assay 30
10. Bone marrow-derived immune cells culture 30
11. Phagocytosis 31
12. Statistical analysis 31
Ⅲ. Results 32
1. Development of the AI ICD-HTS system through pseudo-labeling 32
2. Blinded testing of unknown compounds using real-time AI imaging analysis 35
3. Exclusion of apoptosis inducers from candidates 41
4. Confirmation of membrane rupture and release of internal materials by the selected candidates 45
5. Verification of DAMPs release by the selected candidates 47
6. Validation of antigen-presenting cell activation by the selected candidates 49
7. Cross-checking reported information about candidates with experimental results 51
Ⅳ. Discussion 54
Ⅴ. References 58
Table 1. Cross-checking reported information about candidates with experimental results. 53
Figure 1. Development of the AI ICD-HTS system through pseudo-labeling. 34
Figure 2A. Blinded testing of unknown compounds using real-time AI imaging analysis. 37
Figure 2B. Blinded testing of unknown compounds using real-time AI imaging analysis. 38
Figure 2C. Blinded testing of unknown compounds using real-time AI imaging analysis. 39
Figure 2D. Blinded testing of unknown compounds using real-time AI imaging analysis. 40
Figure 3A. Exclusion of apoptosis inducers from candidates. 43
Figure 3B. Exclusion of apoptosis inducers from candidates. 44
Figure 4. Confirmation of membrane rupture and release of internal materials by the selected candidates. 46
Figure 5. Verification of DAMPs release by the selected candidates. 48
Figure 6. Validation of antigen-presenting cell activation by the selected candidates. 50
Scheme 1. A schematic diagram of the molecular mechanisms of ICDs. 16
Scheme 2. A schematic diagram of the roles of DAMPs in ICD. 19
Scheme 3. A schematic figure of the overall AI based ICD-HTS system. 23