Title Page
Abstract
Contents
ABBREVIATIONS 19
LITERATURE REVIEW 22
1. Current immunotherapy for breast cancer in human and veterinary medicine 22
2. To overcome the immune evasion by immunotherapy targeting tumor microenvironment 25
3. Therapeutic cancer vaccine in breast cancer 29
4. TSG-6 in cancer 31
CHAPTER 1. Tumor secreted TSG-6 promotes cell proliferation, migration, and invasion effects, regulates immune checkpoint proteins and immunomodulation in vitro 35
1. Introduction 35
2. Materials and Methods 37
2.1. Cell culture 37
2.2. siRNA transfection on breast cancer cells 37
2.3. Isolation of canine peripheral blood mononuclear cells (PBMCs) and non-adherent cells 38
2.4. Cell proliferation assay 38
2.5. Cell count assay 39
2.6. Wound healing assay 39
2.7. Invasion assay 39
2.8. Cell cycle assay 40
2.9. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) 40
2.10. Direct and indirect co-culture experiments 41
2.11. Antibodies 41
2.12. Western blot analysis 42
2.13. Immunofluorescence 43
2.14. Flow cytometry 43
2.15. Statistical analysis 44
3. Results 44
3.1. TSG-6 down-regulation on breast cancer cell lines using siRNA transfection 44
3.2. TSG-6 down-regulation induce impaired cancer cell proliferation, migration, and invasion ability 45
3.3. TSG-6 is related to the expression of stem cell signaling pathway 45
3.4. TSG-6 down-regulation suppresses the expression of immune checkpoint protein PD-L1 in tumor cells by regulating CD44 expression 46
3.5. TSG-6 down-regulation activates macrophages in breast cancer microenvironment 47
3.6. TSG-6 down-regulation activates CD3+/CD8α+ T cells in breast cancer microenvironment 48
4. Discussion 48
5. Figures and Tables 52
CHAPTER II. Tumor secreted TSG-6 promotes tumor growth, metastasis, regulates immune checkpoint proteins, and immunomodulation in vivo 74
1. Introduction 74
2. Materials and Methods 76
2.1. Cell culture 76
2.2. siRNA transfection on breast cancer cells 76
2.3. Animals 76
2.4. Single guide RNA (sgRNA) design for TSG-6 knock-out 77
2.5. Single cell culture and isolation of TSG-6 knock-out cell line 77
2.6. Breast cancer mouse model 78
2.7. Histological examination 78
2.8. Cell proliferation assay 79
2.9. Cell count assay 79
2.10. Wound healing assay 79
2.11. Invasion assay 80
2.12. Cell cycle assay 80
2.13. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) 81
2.14. Antibodies 82
2.15. Western blot analysis 82
2.16. Immunofluorescence (IF) 83
2.17. Flow cytometry 83
2.18. Statistical analysis 84
3. Results 84
3.1. TSG-6 down-regulation on breast cancer cell lines using siRNA transfection 84
3.2. TSG-6 down-regulation induce impaired cancer cell proliferation, migration, and invasion ability 85
3.3. TSG-6 down-regulation inhibited NF-κB, STAT3, SOX2 pathways, and CD-44 and PD-L1 expression 86
3.4. Establishment of TSG-6 knock-out breast cancer cell line for in vivo experiments 86
3.5. Tumor volume was reduced in the TSG-6 knock out breast cancer mouse model 88
3.6. Lung and spleen metastasis were reduced in the TSG-6 knock out breast cancer mouse model 88
3.7. Immune checkpoint protein expression was regulated in the TME of TSG-6 knock out breast cancer mouse model 89
3.8. Antitumor immunity was increased in the TME of TSG-6 knock out breast cancer mouse model 89
3.9. Systemic immunity was activated in the TSG-6 knock out breast cancer mouse model 90
4. Discussion 90
5. Figures and Tables 95
CHAPTER III. TSG-6 knock-out autologous whole cell vaccine improves therapeutic efficacy against triple-negative mouse breast cancer model 121
1. Introduction 121
2. Materials and Methods 123
2.1. Cell culture 123
2.2. Animals 123
2.3. Preparation of whole-cell vaccine 124
2.4. Breast cancer mouse model and therapeutic immunotherapy 124
2.5. Adoptive transfer in vivo 124
2.6. CCK assay 125
2.7. In vitro cytotoxicity assay 125
2.8. Annexin V/PI staining 126
2.9. Histological examination 126
2.10. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) 127
2.11. Antibodies 127
2.12. Western blot analysis 128
2.13. Immunofluorescence (IF) 129
2.14. Flow cytometry 129
2.15. Statistical analysis 129
3. Results 130
3.1. Development of TSG-6 knock out whole-cell vaccine 130
3.2. TSG-6 knock-out increased tumor immunogenicity in in vitro cytotoxicity assay 131
3.3. Adoptive cell transfer using serum and splenic immune cells of TSG-6 knock out cell immunized mice showed increased tumor suppression 131
3.4. Adoptive cell transfer did not affect tumor metastasis 132
3.5. Adoptive cell transfer increased infiltration of immune cells into TME, but did not induce sufficient systemic immune response 132
3.6. TSG-6 knock out whole-cell vaccine therapy prolonged survival in breast cancer mouse model 133
3.7. TSG-6 knock out whole cell vaccine therapy inhibited tumor growth and prevented metastasis to the lungs and spleen 134
3.8. TSG-6 knockout whole-cell vaccine therapy induced systemic immune activation 134
4. Discussion 135
5. Figures and Tables 140
GENERAL CONCLUSION 158
REFERENCES 161
국문초록 179
Table 1. Mechanisms orchestrated by the tumor that contribute to its escape from the host immune system 34
Table 2. Lists of primers used for RT-qPCR in Chapter 1 72
Table 3. Primer design for genomic DNA detection 118
Table 4. RT-qPCR primer used in Chapter 2 119
Table 5. Table of RT-qPCR primer sequences in Chapter 3 157
Figure 1. Summary of actions attributed to TSG-6, secreted by MSC and contained also in MSC-EV upon TNFα stimulation. 33
Figure 2. CIPp, CIPm, and BT-20 cell lines were transfected with TSG-6 specific siRNA (siTSG-6). 52
Figure 3. Cell proliferation was reduced in siTSG-6 group. 53
Figure 4. Cell cycle assay performed in siTSG-6 group. 55
Figure 5. Regulation of cyclin D1 in siTSG-6 group. 56
Figure 6. Wound healing assay performed in siTSG-6 group. 57
Figure 7. Invasion assay performed in siTSG-6 group. 58
Figure 8. Expression level of CD44, PD-L1, and STAT3, Nf-κB, and Sox2 pathways were analyzed in siTSG-6 group. 60
Figure 9. CD44 and PD-L1 expression in siTSG-6 group were analyzed with immunofluorescence. 62
Figure 10. Schematic illustration of study design of indirect and direct co-culture with canine macrophage cell line (DH82) or canine peripheral blood... 63
Figure 11. Indirect co-culture with DH82 cells and siTSG-6 group showed macrophage phenotypic conversion from M2 to M1 type. 65
Figure 12. Indirect co-culture with DH82 cells and siTSG-6 group increased PD-1 expression on macrophages. 66
Figure 13. Indirect and direct co-cultures with DH82 cells and siTSG-6 groups showed the same tendency for macrophage phenotypic conversion. 68
Figure 14. Indirect and direct co-cultures with cPBMCs increase the population of cytotoxic T cells. 69
Figure 15. Anti-tumor immunomodulatory effect of TSG-6 on co-cultured cPBMCs. 70
Figure 16. Relative mRNA expression of TSG-6 in cancer cells co-cultured indirectly with DH82 cells. 71
Figure 17. 4T1 cells were transfected with TSG-6 specific siRNA (siTSG-6), and cell proliferation was reduced in siTSG-6 group. 95
Figure 18. Cell cycle assay on siTSG-6 transfected 4T1 cell line. 96
Figure 19. Wound healing assay, invasion assay, and cadherin expression analysis were performed in siTSG-6 group. 98
Figure 20. Expression level of CD44, PD-L1, and STAT3, Nf-κB, and Sox2 pathways were analyzed in siTSG-6 group. 100
Figure 21. Selection of CRISPR targets for murine TSG-6. 101
Figure 22. murine TSG-6 targeting sgRNA was synthesized in abundance. 102
Figure 23. T7 endonuclease I (T7E1) assay was conducted on sgTSG6 transfected pooled cells to identify fragmented target regions. 103
Figure 24. T7E1 assay results on single cell cultured colonies. 105
Figure 25. Verification of isolated TSG-6 knock out cells (KO-4T1). 106
Figure 26. TSG-6, CD-44, PD-L1 and B7-H3 expressions in KO-4T1. 108
Figure 27. In vivo experiment to confirm the role of TSG-6 in tumor microenvironment. 110
Figure 28. Analysis of tumor metastasis to the spleen and lung in TSG-6 KO breast cancer mouse models. 112
Figure 29. Expression of immune checkpoint proteins in TSG-6 KO tumor microenvironment. 113
Figure 30. Populations of tumor-infiltrated lymphocytes in TSG-6 KO breast cancer mouse models. 114
Figure 31. PD-1 expressions on tumor-infiltrated lymphocytes in TSG-6 KO breast cancer mouse models. 115
Figure 32. PD-L1 expressions on tumor cells in TSG-6 KO breast cancer mouse models. 116
Figure 33. Systemic immune cell populations and cytokine levels in TSG-6 KO breast cancer mouse models. 117
Figure 34. Development of TSG-6 KO whole-cell vaccine. 141
Figure 35. In vitro cytotoxicity assay on 4T1 breast cancer cells. 142
Figure 36. Tumor growth after adoptive cell transfer using TSG-6 KO cell-immunized mice. 144
Figure 37. Tumor metastasis after adoptive cell transfer using TSG-6 KO cell-immunized mice. 146
Figure 38. Population of tumor-infiltrated lymphocytes after adoptive cell transfer using TSG-6 KO cell-immunized mice. 147
Figure 39. Systemic immune response after adoptive cell transfer using TSG-6 KO cell-immunized mice. 148
Figure 40. Survival of breast cancer mouse model treated with TSG-6 KO whole-cell vaccine. 149
Figure 41. Tumor growth and metastasis in breast cancer mouse model treated with TSG-6 KO whole-cell vaccine. 151
Figure 42. TSG-6 KO whole-cell vaccine therapy induce activation of cytotoxic T cells. 153
Figure 43. TSG-6 KO whole-cell vaccine therapy induce systemic immune activation. 155
Figure 44. Mechanism of increasing immunogenicity of TSG-6 KO tumor cell vaccine. 156