癌症疫苗可以提供腫瘤相關抗原(tumor-associated antigens)給抗原呈現細胞，佐劑的刺激更可使之活化，進而引發強烈的後天性免疫反應。近年來的研究指出透過免疫刺激因子活化抗原呈現細胞，例如樹突細胞可以引起樹突細胞活化並引發後續專一性免疫反應。於此，我們致力開發一種可注射型癌症疫苗，利用蠶絲蛋白水膠作為載體，搭載由奈米粒子遞送的樹突細胞免疫刺激因子以及免疫原性細胞死亡(immunogenic cell death, ICD)誘導劑阿黴素(doxorubicin, Dox)促使原位的腫瘤相關抗原釋放。藉由蠶絲蛋白水膠緩慢釋放的特性，樹突細胞免疫刺激因子以及阿黴素可以持續的存在腫瘤微環境吸引免疫細胞的滲透，並活化免疫細胞誘發強烈並持久的免疫反應以抑制腫瘤生長。實驗結果顯示，我們的癌症疫苗在小鼠原位乳癌模型中能抑制腫瘤生長和癌症的肺部轉移，並且分析經癌症疫苗注射後小鼠的腫瘤、淋巴結和脾臟，發現樹突細胞的大量活化以及胞殺型T細胞的增生分化。因此，我們希望透過此癌症疫苗的研發可以為未來的癌症治療提供新的免疫療法。

Cancer vaccine has been shown to provoke strong adaptive immune response mediated by the antigen-presenting cells (APCs) that acquired antigens and were further activated with the assistant of adjuvant. Recently, delivery of immunostimulants for APCs activation such as dendritic cells (DCs) have been reported to elicit a protected anti-tumor effect. Here, we perform an injectable cancer vaccine comprises silk hydrogel as an injectable implant and immunostimulants-loaded nanoparticles (NPs) as a potential adjuvant cooperated with an immunogenic cell death (ICD) inducer, doxorubicin (Dox) to in situ generate tumor-associated antigens. We hypothesize that due to the controlled releasing properties of silk hydrogel, Dox and immunostimulants-NPs could be sustain released from the hydrogel, resulting in the recruitment and activation of DCs which in turn induce a long-lasting anti-tumor immune response. The result indicated that our Dox and immunostimulants-NPs co-loaded silk hydrogel vaccine inhibit the tumor progression and lung metastasis against murine orthotopic breast cancer model. Besides, DCs activation and CD8+ T cell expansion were found in the tumor and the draining lymph node (LN) of tumor-bearing mice after treated with our injectable immunostimulants-loaded vaccine. These findings demonstrated the therapeutic efficacy of cancer vaccine and revealed the potential application of in situ vaccination for advanced cancer immunotherapy.

Table of content
中文摘要 i
Abstract ii
致謝 iii
Table of Charts vi
Abbreviation viii
Chapter 1 Motivation and Aims 1
1.1 Motivation 2
1.2 Aims 3
Chapter 2 Literature Review 5
2.1 Breast cancer 6
2.2 Cancer immunity 7
2.2.1 Dendritic cells 7
2.2.2 T cells 8
2.2.3 Macrophages 9
2.3 Immunotherapy for cancer treatment 10
2.3.1 Immune checkpoint blockade 10
2.3.2 Adoptive T cell therapy (ATC therapy) 12
2.3.3 DC-based cancer vaccine 13
2.3.4 DC-based cancer vaccine – Immunogenic cell death (ICD) 14
2.3.5 DC-based cancer vaccine – STING-based therapy 14
2.4 Advantages of hydrogel as an injectable implant to load of immunostimulants for cancer treatment 17
2.5 Advantages of utilizing NPs for cancer immunotherapy 18
2.5.1 NPs for immunomodulation of TME 19
2.5.2 Nanoscale cancer vaccine 20
Chapter 3 Materials and Methods 22
3.1 Materials 23
3.2 Animals 23
3.3 Cell lines 23
3.4 Isolation of bone marrow-derived dendritic cells 24
3.5 Preparation of LPD-cGAMP nanoparticle 24
3.6 Characterization of nanoparticle 25
3.7 Preparation and characterization of silk hydrogel 25
3.8 Cumulative release of Dox and cGAMP 26
3.9 Reverse transcription-quantitative real-time PCR 26
3.10 Western blot analysis 27
3.11 In vitro DCs activation through LPD-cGAMP NPs treatment 28
3.12 Flow cytometry analysis of anti-tumor study using orthotopic breast cancer model. 28
3.13 Statistics 29
Chapter 4 Results 30
4.1 Characterization of LPD-cGAMP NPs delivered in injectable implant 31
4.2 In vitro efficacy of LPD-cGAMP NPs induced STING activation in BMDCs and Dox-induced ICD signal in 4T1 cells. 33
4.3 In vivo therapeutic efficacy of injectable hydrogel loaded with LPD-cGAMP and Dox in murine orthotopic breast cancer model 35
Chapter 5 Conclusion 48
Chapter 6 Discussion and Prospect 51
Chapter 7 Refercences 55

Table of Charts
Figure 2.3.1. T cell inhibition through PD1/PDL1 immune checkpoint. 12
Figure 2.3.5. STING activation pathway. 16
Figure 2.4. Solution to Gel transition of silk hydrogel. 18
Figure 1. Physical properties and encapsulation efficiency of LPD-cGAMP. 38
Figure 2. The gelation process of silk solution. 38
Figure 3. Scanning electron microscopy image of silk hydrogel and LPD-cGAMP
NPs-loaded silk hydrogel. 39
Figure 4. Cumulative in vitro release of Dox and LPD-cGAMP from silk hydrogel. 39
Figure 5. STING-related genes expression in LPD-cGAMP NPs-treated BMDCs. 40
Figure 6. STING activation and DC activation in LPD-cGAMP NPs-treated BMDCs. 41
Figure 7. Dox-induced danger signal calrecticulin (CRT) expression of gene and
protein in 4T1 cells. 42
Figure 8. Treatment schedule of LPD-cGAMP NPs and Dox co-loaded hydrogel
vaccine in the orthotopic breast cancer model. 43
Figure 9. LPD-cGAMP NPs and Dox co-loaded hydrogel vaccine inhibited tumor
growth and lung metastasis. 44
Figure 10. LPD-cGAMP NPs and Dox co-loaded hydrogel vaccine induced DC
activation and CD8+ T cell expansion in tumor. 45
Figure 11. LPD-cGAMP NPs and Dox co-loaded hydrogel vaccine induced DC
activation and CD8+ T cell expansion in LN. 46
Figure 12. LPD-cGAMP NPs and Dox co-loaded hydrogel vaccine induced CD8+ T
cell expansion in spleen. 47
Figure 13. Schematics of LPD-cGAMP NPs and Dox co-loaded hydrogel vaccine for
cancer immunotherapy of 4T1 breast cancer model. 50

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