細菌介導癌症療法（bacteria-mediated tumor therapy, BMTT）已經有數十年的歷史。然而，由於其可能造成的感染風險抑制了細菌介導癌症療法的臨床應用。為了解決這個問題，本實驗提出了一種與細菌型態相似且具有其多功能性的尖刺狀金屬有機框架（Al-Ru metal-organic frameworks, Al-Ru MOFs），此Al-Ru MOFs不僅不會引起感染，且也能展現細菌介導癌症療法的抗癌效果。這種Al-Ru MOFs是由硫酸鋁、三氯化釕及2-胺基對苯二甲酸透過溶劑熱合成反應（solvothermal reaction）所製備而成。根據所使用的反應溫度，其形態將呈現球狀或是尖刺狀，而無論是球狀或尖刺狀的Al-Ru MOFs均可做為光熱劑（photothermal agents），將所吸收的光能轉換為局部熱量。與球狀Al-Ru MOFs相比較，尖刺狀Al-Ru MOFs表面積較大，因此更容易被巨噬細胞吞噬，以引發更強的免疫反應。此外，在腫瘤內部注射Al-Ru MOFs時，尖刺狀Al-Ru MOFs的滯留時間較球狀Al-Ru MOFs還長，這使其可以重複進行光熱療法以用於癌症治療。在抗腫瘤實驗中，我們於小鼠腫瘤內部施打尖刺狀Al-Ru MOFs後，透過照射近紅外光（near infrared, NIR）所引起的溫和高溫來破壞腫瘤，使整個腫瘤轉化為原位癌症疫苗，引發全身性的免疫反應。並且再配合上免疫檢查點抑制劑PD-1的施打，來進行光熱與免疫之複合治療。上述的實驗結果顯示，此原位癌症疫苗與免疫檢查點抑制劑PD-1具有協同作用，可模擬細菌介導癌症療法的抗腫瘤功能，消除原位腫瘤並抑制腫瘤的復發和轉移，具有臨床應用上的潛力。

The purpose of cancer vaccines is to activate tumor-specific T lymphocytes, subsequently causing tumor regression. Most cancer vaccines under development are associated with defined tumor antigens rather than with all antigens of whole tumor cells, limiting the anti-tumor immune responses that they elicit. This work proposes a multifunctional spiky Al-Ru (III) metal-organic frameworks (Al-Ru MOFs) which can absorb near-infrared light (+NIR) to cause mild hyperthermia which can destroy the tumor. The system prepared herein can convert tumor into an in situ cancer vaccine, thereby inducing systemic immune response. In addition, the combination of Al-Ru MOFs with immune checkpoint inhibitors (+PD-1) cause a powerful synergistic effect, alleviating the tumor immunosuppressive microenvironment, generating a robust long-term anti-tumor immunity. According to the in vivo studies, treatments with Al-Ru MOFs/+NIR can suppress the growth of primary tumors, and induce memory responses against subsequent tumor challenge. The combination of Al-Ru MOFs/+NIR with PD-1 can further increase the rate of tumor rejection. Moreover, mice that had been treated with Al-Ru MOFs/+NIR or Al-Ru MOFs＋PD-1/＋NIR and had rejected the tumors can induce long-lasting tumor-specific immune memory responses. These results suggest that the system that is proposed herein is promising for generating cancer vaccines in situ, by using the tumor itself as the antigen source and combining with PD-1 to generate a long-term anti-tumor immunity for preventing tumor recurrence and metastasis.

內容
摘要 I
Abstract I
目錄 III
圖目錄 V
表目錄 VII
第一章 緒論 1
1.1 癌症治療的發展 1
1.2 細菌介導的癌症療法（BMTT） 2
1.3 癌症的光熱療法（Photothermal therapy） 2
1.4 癌症的免疫療法（Immunotherapy） 4
1.5 PD-1免疫檢查點抑制劑療法之簡介 5
1.6 癌症的光熱與免疫之複合治療 6
1.7 金屬有機框架（Metal-organic frameworks, MOFs）材料之簡介 7
1.8 鋁與釕之簡介 8
1.9 研究目的與實驗設計 9
1.10 實驗流程設計圖 11
第二章 實驗材料與方法 12
2.1 實驗材料 12
2.2 Al-Ru MOFs的製備 12
2.3 Al-Ru MOFs的特性測試 13
2.4 細胞培養及毒性測試 14
2.5 光熱治療對於熱休克蛋白70（HSP70）分泌的影響 14
2.6 體外降解特性實驗 15
2.7 巨噬細胞的吞噬效果分析 15
2.8 巨噬細胞免疫刺激分析 16
2.9 實驗動物 17
2.10 腫瘤內部的滯留實驗 17
2.11 體內光熱能力分析 17
2.12 腫瘤內部細胞的吞噬效果分析 18
2.13 抗腫瘤治療 18
2.14 分泌IFN-γ 之CD8+ T細胞的ELISPOT分析 19
2.15 組織型態學分析 20
2.16 腫瘤微環境之分析 20
2.17 血液生化分析 21
2.18 統計分析 21
第三章 實驗結果與討論 22
3.1 Al-Ru MOFs之特性分析 22
3.2 Al-Ru MOFs之光熱性質分析 24
3.3 Al-Ru MOFs之細胞毒性實驗 25
3.4 Al-Ru MOFs光熱治療對於HSP70分泌的影響 26
3.5 Al-Ru MOFs的體外降解特性 27
3.6 巨噬細胞對Al-Ru MOFs之吞噬效果分析 28
3.7 Al-Ru MOFs對體外巨噬細胞免疫刺激分析 29
3.8 Al-Ru MOFs在腫瘤內部的滯留分析 31
3.9 原位疫苗和免疫檢查點抑制劑的協同作用 34
3.10 全身性的記憶免疫反應 36
3.11 Al-Ru MOFs對腫瘤微環境的調節 38
3.12 Al-Ru MOFs在生物體內的毒性實驗 40
第四章 結論 42
參考文獻 43

圖目錄
圖一、高溫所引發免疫反應示意圖[19] 3
圖二、免疫檢查點的阻斷有助於T細胞活化[31] 6
圖三、MOFs的廣泛應用[39] 8
圖四、實驗樹狀結構圖 11
圖五、Al-Ru MOFs之SEM影像圖 22
圖六、Al-Ru MOFs之STEM及elemental mapping images 23
圖七、Al-Ru MOFs的UV–vis–NIR吸收光譜 24
圖八、不同濃度Al-Ru MOFs經808 nm NIR照射後的升溫曲線圖 25
圖九、不同濃度下Al-Ru MOFs的細胞毒性測試 26
圖十、不同治療方法下CT26細胞內HSP70的表現量 27
圖十一、不同時間點Al-Ru MOFs在PBS溶液中降解之SEM影像圖 28
圖十二、巨噬細胞對Al-Ru MOFs之吞噬效果分析 29
圖十三、巨噬細胞受Al-Ru MOFs刺激後所產生之IL-1β表現量 30
圖十四、巨噬細胞受Al-Ru MOFs刺激後表面CD86及MHC class II之表現量 30
圖十五、Al-Ru MOFs在不同時間點於腫瘤內部滯留清況之活體分子影像圖及總輻射效率曲線圖 32
圖十六、在腫瘤內注射Al-Ru MOFs並暴露於NIR雷射後之不同時間點的腫瘤溫度曲線圖 33
圖十七、腫瘤內部不同種類細胞對Al-Ru MOFs之吞噬效果分析 33
圖十八、CT26腫瘤模型與實驗設計圖 34
圖十九、以不同治療方式治療原位腫瘤之平均生長曲線 35
圖二十、以不同治療方式治療原位腫瘤的個別生長曲線（CR: complete tumor regression） 35
圖二十一、以不同治療方式治療原位腫瘤的生存曲線 36
圖二十二、在第一次腫瘤接種後，經過spiky MOFs+NIR或spiky MOFs+NIR+αPD-1治療而存活的小鼠和對照小鼠之再發癌生長曲線 37
圖二十三、經過spiky MOFs+NIR或spiky MOFs+NIR+αPD-1治療的小鼠脾臟中分離出的CD8+ T細胞所產生IFN-γ+之分析結果圖 38
圖二十四、用anti-CD3, anti-granzyme B, TUNEL或H&E染色之組織學顯微切片圖 39
圖二十五、腫瘤微環境中CD3+ T細胞中CD8+ T細胞的含量及CD8+ T細胞上CD107α（活化標記）的表現量 40
圖二十六、小鼠體重變化圖 41
圖二十七、經過治療過後的血清生化分析結果 41
圖二十八、主要器官組織學染色切片 41

表目錄
表格一、Al及Ru元素在球狀及尖刺狀Al-Ru MOFs中的含量 24

1. Tohme, S., R.L. Simmons, and A. Tsung, Surgery for Cancer: A Trigger for Metastases. Cancer Res, 2017. 77(7): p. 1548-1552.
2. Hannun, Y.A., Apoptosis and the dilemma of cancer chemotherapy. Blood, 1997. 89(6): p. 1845-53.
3. Kaufmann, S.H., Induction of endonucleolytic DNA cleavage in human acute myelogenous leukemia cells by etoposide, camptothecin, and other cytotoxic anticancer drugs: a cautionary note. Cancer Res, 1989. 49(21): p. 5870-8.
4. Vermund, H. and F.F. Gollin, Mechanisms of action of radiotherapy and chemotherapeutic adjuvants. A review. Cancer, 1968. 21(1): p. 58-76.
5. Murphy, J.D., et al., A dosimetric model of duodenal toxicity after stereotactic body radiotherapy for pancreatic cancer. Int J Radiat Oncol Biol Phys, 2010. 78(5): p. 1420-6.
6. Halberg, F.E., et al., Intraoperative radiotherapy with localized radioprotection: diminished duodenal toxicity with intraluminal WR2721. Int J Radiat Oncol Biol Phys, 1991. 21(5): p. 1241-6.
7. Huang, Q., et al., Caspase 3-mediated stimulation of tumor cell repopulation during cancer radiotherapy. Nat Med, 2011. 17(7): p. 860-6.
8. Lauber, K., et al., Apoptosis induction and tumor cell repopulation: the yin and yang of radiotherapy. Radiat Oncol, 2011. 6: p. 176.
9. Joshi, S. and D.L. Durden, Combinatorial Approach to Improve Cancer Immunotherapy: Rational Drug Design Strategy to Simultaneously Hit Multiple Targets to Kill Tumor Cells and to Activate the Immune System. J Oncol, 2019. 2019: p. 5245034.
10. Song, S., M.S. Vuai, and M. Zhong, The role of bacteria in cancer therapy - enemies in the past, but allies at present. Infect Agent Cancer, 2018. 13: p. 9.
11. Karki, R., S.M. Man, and T.D. Kanneganti, Inflammasomes and Cancer. Cancer Immunol Res, 2017. 5(2): p. 94-99.
12. Phan, T.X., et al., Activation of inflammasome by attenuated Salmonella typhimurium in bacteria-mediated cancer therapy. Microbiol Immunol, 2015. 59(11): p. 664-75.
13. Millar, D.G., et al., Hsp70 promotes antigen-presenting cell function and converts T-cell tolerance to autoimmunity in vivo. Nat Med, 2003. 9(12): p. 1469-76.
14. Sedighi, M., et al., Therapeutic bacteria to combat cancer; current advances, challenges, and opportunities. Cancer Med, 2019. 8(6): p. 3167-3181.
15. Forbes, N.S., Engineering the perfect (bacterial) cancer therapy. Nat Rev Cancer, 2010. 10(11): p. 785-94.
16. Kaimala, S., et al., Attenuated Bacteria as Immunotherapeutic Tools for Cancer Treatment. Front Oncol, 2018. 8: p. 136.
17. Stern, C., et al., Induction of CD4(+) and CD8(+) anti-tumor effector T cell responses by bacteria mediated tumor therapy. Int J Cancer, 2015. 137(8): p. 2019-28.
18. Dang, L.H., et al., Combination bacteriolytic therapy for the treatment of experimental tumors. Proc Natl Acad Sci U S A, 2001. 98(26): p. 15155-60.
19. Chu, K.F. and D.E. Dupuy, Thermal ablation of tumours: biological mechanisms and advances in therapy. Nat Rev Cancer, 2014. 14(3): p. 199-208.
20. Aioub, M. and M.A. El-Sayed, A Real-Time Surface Enhanced Raman Spectroscopy Study of Plasmonic Photothermal Cell Death Using Targeted Gold Nanoparticles. J Am Chem Soc, 2016. 138(4): p. 1258-64.
21. Shen, S., et al., Magnetic nanoparticle clusters for photothermal therapy with near-infrared irradiation. Biomaterials, 2015. 39: p. 67-74.
22. Ryu, T.K., et al., Selective Photothermal Tumor Therapy Using Nanodiamond-Based Nanoclusters with Folic Acid. Adv Mater, 2016. 26(23): p. 6428-6436.
23. Lan, G., K. Ni, and W. Lin, Nanoscale Metal-Organic Frameworks for Phototherapy of Cancer. Coord Chem Rev, 2019. 379: p. 65-81.
24. Pardoll, D.M., The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer, 2012. 12(4): p. 252-64.
25. Restifo, N.P., M.E. Dudley, and S.A. Rosenberg, Adoptive immunotherapy for cancer: harnessing the T cell response. Nat Rev Immunol, 2012. 12(4): p. 269-81.
26. Gilboa, E., S.K. Nair, and H.K. Lyerly, Immunotherapy of cancer with dendritic-cell-based vaccines. Cancer Immunol Immunother, 1998. 46(2): p. 82-7.
27. Finn, O.J., Cancer vaccines: between the idea and the reality. Nat Rev Immunol, 2003. 3(8): p. 630-41.
28. Reichert, J.M. and C. Paquette, Therapeutic cancer vaccines on trial. Nat Biotechnol, 2002. 20(7): p. 659-63.
29. Ye, Y., et al., A melanin-mediated cancer immunotherapy patch. Science Immunology, 2017. 2(17): p. eaan5692.
30. Fabregat, I., et al., TGF-beta signaling in cancer treatment. Curr Pharm Des, 2014. 20(17): p. 2934-47.
31. Darvin, P., et al., Immune checkpoint inhibitors: recent progress and potential biomarkers. Exp Mol Med, 2018. 50(12): p. 165.
32. Topalian, S.L., et al., Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med, 2012. 366(26): p. 2443-54.
33. Chen, Q., et al., Photothermal Therapy Promotes Tumor Infiltration and Antitumor Activity of CAR T Cells. Adv Mater, 2019. 31(23): p. e1900192.
34. Chen, Q., et al., Photothermal therapy with immune-adjuvant nanoparticles together with checkpoint blockade for effective cancer immunotherapy. Nat Commun, 2016. 7: p. 13193.
35. Howarth, A.J., et al., Chemical, thermal and mechanical stabilities of metal–organic frameworks. Nature Reviews Materials, 2016. 1(3): p. 15018.
36. Zhou, H.C., J.R. Long, and O.M. Yaghi, Introduction to metal-organic frameworks. Chem Rev, 2012. 112(2): p. 673-4.
37. Wu, M.-X. and Y.-W. Yang, Metal–Organic Framework (MOF)-Based Drug/Cargo Delivery and Cancer Therapy. Advanced Materials, 2017. 29(23): p. 1606134.
38. Park, J., et al., Size-Controlled Synthesis of Porphyrinic Metal-Organic Framework and Functionalization for Targeted Photodynamic Therapy. J Am Chem Soc, 2016. 138(10): p. 3518-25.
39. Chaemchuen, S., et al., Metal-organic frameworks for upgrading biogas via CO2 adsorption to biogas green energy. Chem Soc Rev, 2013. 42(24): p. 9304-32.
40. Sun, B., et al., Engineering an effective immune adjuvant by designed control of shape and crystallinity of aluminum oxyhydroxide nanoparticles. ACS Nano, 2013. 7(12): p. 10834-49.
41. Xu, P., et al., Ultra-small Albumin Templated Gd/Ru Composite Nanodots for In Vivo Dual modal MR/Thermal Imaging Guided Photothermal Therapy. Adv Healthc Mater, 2018. 7(19): p. e1800322.
42. Huang, X., et al., Effective PDT/PTT dual-modal phototherapeutics killing of pathogenic bacteria by using ruthenium nanoparticles. J Mater Chem B, 2016(37): p. 6157-6282.
43. Butterfield, L.H., Cancer vaccines. BMJ, 2015. 350: p. h988.
44. Romero, P., et al., The Human Vaccines Project: A roadmap for cancer vaccine development. Sci Transl Med, 2016. 8(334): p. 334ps9.
45. Keenan, B.P. and E.M. Jaffee, Whole cell vaccines--past progress and future strategies. Semin Oncol, 2012. 39(3): p. 276-86.
46. Melero, I., et al., Therapeutic vaccines for cancer: an overview of clinical trials. Nat Rev Clin Oncol, 2014. 11(9): p. 509-24.
47. Chiang, C.L., F. Benencia, and G. Coukos, Whole tumor antigen vaccines. Semin Immunol, 2010. 22(3): p. 132-43.
48. Kadiyala, P., et al., High-Density Lipoprotein-Mimicking Nanodiscs for Chemo-immunotherapy against Glioblastoma Multiforme. ACS Nano, 2019. 13(2): p. 1365-1384.
49. Min, Y., et al., Antigen-capturing nanoparticles improve the abscopal effect and cancer immunotherapy. Nat Nanotechnol, 2017. 12(9): p. 877-882.
50. Shao, J., et al., Biodegradable black phosphorus-based nanospheres for in vivo photothermal cancer therapy. Nat Commun, 2016. 7: p. 12967.
51. Ahmed, M., et al., Principles of and advances in percutaneous ablation. Radiology, 2011. 258(2): p. 351-69.
52. Rhim, H., et al., Radiofrequency thermal ablation of abdominal tumors: lessons learned from complications. Radiographics, 2004. 24(1): p. 41-52.
53. Schueller, G., et al., Heat shock protein expression induced by percutaneous radiofrequency ablation of hepatocellular carcinoma in vivo. Int J Oncol, 2004. 24(3): p. 609-13.
54. Sgambato, A., et al., Anti PD-1 and PDL-1 Immunotherapy in the Treatment of Advanced Non- Small Cell Lung Cancer (NSCLC): A Review on Toxicity Profile and its Management. Curr Drug Saf, 2016. 11(1): p. 62-8.
55. Ascierto, P.A. and F.M. Marincola, 2015: The Year of Anti-PD-1/PD-L1s Against Melanoma and Beyond. EBioMedicine, 2015. 2(2): p. 92-3.
56. Cheng, X., et al., Size- and morphology-controlled NH2-MIL-53(Al) prepared in DMF-water mixed solvents. Dalton Trans, 2013. 42(37): p. 13698-705.
57. Ahnfeldt, T., et al., Synthesis and modification of a functionalized 3D open-framework structure with MIL-53 topology. Inorg Chem, 2009. 48(7): p. 3057-64.
58. Chen, P.M., et al., Modulation of tumor microenvironment using a TLR-7/8 agonist-loaded nanoparticle system that exerts low-temperature hyperthermia and immunotherapy for in situ cancer vaccination. Biomaterials, 2020. 230: p. 119629.
59. Pan, W.Y., et al., Localized sequence-specific release of a chemopreventive agent and an anticancer drug in a time-controllable manner to enhance therapeutic efficacy. Biomaterials, 2016. 101: p. 241-50.
60. Mazhorova, A., et al., Label-free bacteria detection using evanescent mode of a suspended core terahertz fiber. Opt Express, 2012. 20(5): p. 5344-55.
61. Li, X., et al., New insights into the degradation mechanism of metal-organic frameworks drug carriers. Scientific Reports, 2017. 7(1): p. 13142.
62. Fujiwara, N. and K. Kobayashi, Macrophages in inflammation. Curr Drug Targets Inflamm Allergy, 2005. 4(3): p. 281-6.
63. Kumar, S., et al., Shape and size-dependent immune response to antigen-carrying nanoparticles. J Control Release, 2015. 220(Pt A): p. 141-148.
64. Russell, J.H. and T.J. Ley, Lymphocyte-mediated cytotoxicity. Annu Rev Immunol, 2002. 20: p. 323-70.