胰管腺癌占胰臟癌總病例之90%，其五年存活率低於5%。由於腫瘤微環境之免疫反應被抑制，以及被分泌之細胞外基質形成纖維化結構，此屏障大為限制化療或標靶藥物之療效。因此，新型療法被廣泛地開發以滿足治療胰臟癌之醫療需求。其中因溶瘤病毒可選擇性的瞄準癌細胞，並能夠同時引起免疫反應，故本研究欲透過此方式結合標靶與免疫療法，希望可以克服治療胰臟癌之困境。使用溶瘤腺病毒血清型C5與F41 (OAdV-C5與-F41)，利用其具專一性、安全性、易操作性與複製能力之特性，於細胞與動物模式中探討OAdV-C5與-F41治療胰管腺癌之療效。首先，建立標準之病毒生產期純化流程，並運用反轉錄聚合酶連鎖反應技術與免疫螢光染色法分析OAdV-C5與-F41。進一步探討OAdV-C5與-F41對於癌細胞是否具選擇性，結果發現此二病毒較傾向感染胰管腺癌細胞並可擊殺之。接著，建立原位胰管腺癌裸鼠模型以了解OAdV治療PDAC之有效性。利用腫瘤內注射法給予OAdV-C5與-F41，經兩週動物活體影像分析與存活期觀察，發現OAdV-C5與-F41有效抑制腫瘤生長且具劑量相關性，整體存活率亦延長1.5倍。實驗結果亦發現OAdV-C5與-F41可增加裸鼠體內之單核球、自然殺手細胞與B細胞且具時間相關性，顯示OAdV療法可顯著提升免疫反應。此外，於OAdV-C5與-F41治療初期，腫瘤生長受抑制且PD-1表現亦下降；然而，治療後期PD-1表現上升，腫瘤大小亦增大，顯示腫瘤內PD-1表現與治療效果可能相關。高表現量之PD-1可能造成免疫抑制反應，減弱免疫細胞活性，而降低其攻擊腫瘤之能力。上述結果指出結合免疫檢查點抑制劑延長抑制腫瘤生長效果之潛力，期望可用以開發為胰管腺癌之新療法。

Pancreatic ductal adenocarcinoma (PDAC) with an extremely low 5-year survival rate is one of the most disastrous diseases. Some advanced therapies have been developed to meet the unmet medical need. However, owing to the immunosuppressive microenvironment and chemoresistant fibrous barriers, the efficacies of treatments are limited. Because oncolytic viruses (OVs) can specifically lyse the tumor cells and trigger anti tumor immune responses, they may provide alternative strategy as the cancer-targeted and immune therapy. In this study, oncolytic adenoviruses serotype C5 and F41 (OAdV-C5 and -F41), which are specific, replicative, relatively safe and easily manipulated, were used to treat PDAC in vitro and in vivo. OAdV-C5 and -F41 were isolated and characterized using RT-qPCR and immunofluorescence assay. As infected by OAdV-C5 or -F41, the tested cancer cells, especially in pancreatic malignancy, were more prone to be killed than normal cells. Therefore, the orthotopic PDAC nude mouse model was established and used to evaluate the efficacy of intratumor injection of OAdV-C5 or -F41. It was found that the tumor size was reduced in a dose dependence, and overall survival rates of the mice were accordingly prolonged 1.5-fold. Moreover, the infiltration of monocytes, NK cells and B cells in the PDAC microenvironment was increased in a time-dependent manner by analyzing the gene expression of Mcp1 (monocytes), Ncr1 (NK cells) and Cxcl13 (B cells). It was found that the expression of PD-1 was down-regulated at 2-wk post OAdV treatment. Afterwards PD-1 level increased at 1-3 months post treatment, and it could lead to the immunosuppression and attenuate the tumor-eliminating capability of immune cells. Taken together, the OAdV has the therapeutic potential, and its combination with immune checkpoint inhibitors may be promising for PDAC treatment.

中文摘要 I
Abstract II
圖目錄 VII
表目錄 VIII
縮寫 IX
一、研究背景介紹 1
1.1 胰臟癌 1
1.1.1 全球盛行率 1
1.1.2 種類與分期 1
1.1.3整體存活率 2
1.1.4症狀與診斷 3
1.1.5治療方式 4
1.1.6 胰臟癌腫瘤微環境 (Tumor microenvironment, TME) 5
1.1.7 胰臟癌術後與困境 6
1.2 溶瘤病毒 (Oncolytic virus, OV) 6
1.2.1腫瘤毒殺機制 6
1.2.1.1標靶治療特性 6
1.2.1.2免疫療法特性 7
1.2.2 癌症治療之臨床現況 7
1.2.3 胰臟癌治療之臨床現況 8
1.2.4 腺病毒 9
1.2.5 本研究使用之腺病毒 10
1.2.6 腺病毒與免疫反應 10
1.3 腺病毒結合免疫檢查點抑制劑 10
1.3.1 免疫檢查點PD-1與PD-L1 10
二、研究動機與目的 12
三、材料與方法 13
3.1 實驗流程設計 13
3.1.1 細胞實驗流程圖 13
3.1.2 動物實驗流程圖 13
3.2 細胞來源與培養 13
3.3 病毒來源與生產 15
3.4 細胞病變作用 (Cytopathic effect, CPE)觀察 15
3.5 半細胞感染劑量 (50% cell culture infective dose, CCID50)與半細胞致死劑量 (50% lethal concentration, LC50)測定 16
3.6 細胞存活觀察 16
3.7 實驗動物培養 16
3.8 原位胰臟癌動物模型建立 17
3.9 溶瘤腺病毒治療 17
3.10 反轉錄聚合酶連鎖反應 (Reverse transcription polymerase chain reaction, RT-PCR) 17
3.11 西方點墨法 (Western blotting analysis) 19
3.12組織切片 20
3.13免疫螢光染色 20
四、實驗結果 21
4.1 OAdV-C5與F41定性與定量 21
4.2 OAdV-C5與F41感染與毒殺胰管腺癌細胞之效力高於正常細胞 22
4.3 高劑量之OAdV-C5與F41抑制胰臟腫瘤生長並延長整體存活率 23
4.4 OAdV-C5與F41感染腫瘤細胞並持續存在於腫瘤內 24
4.5 OAdV-C5與F41吸引免疫細胞至腫瘤微環境並活化免疫反應 25
4.6 OAdV-C5與F41增加腫瘤微環境內免疫檢查點PD-1之表現 26
五、討論 28
六、結論 31
七、參考文獻 32

Adamska, A., et al. (2017). Pancreatic ductal adenocarcinoma: current and evolving therapies. Int J Mol Sci, 18(7). doi:10.3390/ijms18071338
Altinoz, M. A., et al. (2017). Rabies virus vaccine as an immune adjuvant against cancers and glioblastoma: new studies may resurrect a neglected potential. Clin Transl Oncol, 19(7), 785-792. doi:10.1007/s12094-017-1613-6
Ballehaninna, U. K., et al. (2012). The clinical utility of serum CA 19-9 in the diagnosis, prognosis and management of pancreatic adenocarcinoma: An evidence based appraisal. J Gastrointest Oncol, 3(2), 105-119. doi:10.3978/j.issn.2078-6891.2011.021
Burris, H. A., 3rd, et al. (1997). Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial. J Clin Oncol, 15(6), 2403-2413. doi:10.1200/JCO.1997.15.6.2403
Capasso, C., et al. (2014). The evolution of adenoviral vectors through genetic and chemical surface modifications. Viruses, 6(2), 832-855. doi:10.3390/v6020832
Chen, C. Y., et al. (2018). Oncolytic virus and PD-1/PD-L1 blockade combination therapy. Oncolytic Virother, 7, 65-77. doi:10.2147/OV.S145532
Chu, L. C., et al. (2017). Diagnosis and detection of pancreatic cancer. Cancer J, 23(6), 333-342. doi:10.1097/PPO.0000000000000290
Cicenas, J., et al. (2017). KRAS, TP53, CDKN2A, SMAD4, BRCA1, and BRCA2 mutations in pancreatic cancer. Cancers (Basel), 9(5). doi:10.3390/cancers9050042
Conroy, T., et al. (2011). FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer. N Engl J Med, 364(19), 1817-1825. doi:10.1056/NEJMoa1011923
Ebrahimi, S., et al. (2017). Interferon-mediated tumor resistance to oncolytic virotherapy. J Cell Biochem, 118(8), 1994-1999. doi:10.1002/jcb.25917
Eissa, I. R., et al. (2018). The current tatus and future prospects of oncolytic viruses in clinical trials against melanoma, glioma, pancreatic, and breast cancers. Cancers (Basel), 10(10). doi:10.3390/cancers10100356
Eriksson, E., et al. (2017). Shaping the tumor stroma and sparking immune activation by CD40 and 4-1BB signaling induced by an armed oncolytic virus. Clin Cancer Res, 23(19), 5846-5857. doi:10.1158/1078-0432.CCR-17-0285
Fajardo, C. A., et al. (2017). Oncolytic adenoviral delivery of an EGFR-targeting T-cell engager improves antitumor efficacy. Cancer Res, 77(8), 2052-2063. doi:10.1158/0008-5472.CAN-16-1708
Feng, M., et al. (2017). PD-1/PD-L1 and immunotherapy for pancreatic cancer. Cancer Lett, 407, 57-65. doi:10.1016/j.canlet.2017.08.006
Fukuhara, H., et al. (2016). Oncolytic virus therapy: a new era of cancer treatment at dawn. Cancer Sci, 107(10), 1373-1379. doi:10.1111/cas.13027
Gong, J., et al. (2016). Clinical development of reovirus for cancer therapy: An oncolytic virus with immune-mediated antitumor activity. World J Methodol, 6(1), 25-42. doi:10.5662/wjm.v6.i1.25
Gupta, R., et al. (2017). Current and future therapies for advanced pancreatic cancer. J Surg Oncol, 116(1), 25-34. doi:10.1002/jso.24623
Hendrickx, R., et al. (2014). Innate immunity to adenovirus. Hum Gene Ther, 25(4), 265-284. doi:10.1089/hum.2014.001
Heo, J., et al. (2013). Randomized dose-finding clinical trial of oncolytic immunotherapeutic vaccinia JX-594 in liver cancer. Nat Med, 19(3), 329-336. doi:10.1038/nm.3089
Idachaba, S., et al. (2019). A review of pancreatic cancer: epidemiology, genetics, screening, and management. Open Access Maced J Med Sci, 7(4), 663-671. doi:10.3889/oamjms.2019.104
Johnson, B. A., 3rd, et al. (2017). Strategies for increasing pancreatic tumor immunogenicity. Clin Cancer Res, 23(7), 1656-1669. doi:10.1158/1078-0432.CCR-16-2318
Jost, S., et al. (2013). Control of human viral infections by natural killer cells. Annu Rev Immunol, 31, 163-194. doi:10.1146/annurev-immunol-032712-100001
Jung, B. K., et al. (2017). A hydrogel matrix prolongs persistence and promotes specific localization of an oncolytic adenovirus in a tumor by restricting nonspecific shedding and an antiviral immune response. Biomaterials, 147, 26-38. doi:10.1016/j.biomaterials.2017.09.009
Karakas, Y., et al. (2018). Recent advances in the management of pancreatic adenocarcinoma. Expert Rev Anticancer Ther, 18(1), 51-62. doi:10.1080/14737140.2018.1403319
Kaufman, H. L., et al. (2015). Oncolytic viruses: a new class of immunotherapy drugs. Nat Rev Drug Discov, 14(9), 642-662. doi:10.1038/nrd4663
Khanal, S., et al. (2018). The repertoire of adenovirus in human disease: the innocuous to the deadly. Biomedicines, 6(1). doi:10.3390/biomedicines6010030
Klasse, P. J. (2015). Molecular determinants of the ratio of inert to infectious virus particles. Prog Mol Biol Transl Sci, 129, 285-326. doi:10.1016/bs.pmbts.2014.10.012
Lafaro, K. J., et al. (2019). The paradoxical web of pancreatic cancer tumor microenvironment. Am J Pathol, 189(1), 44-57. doi:10.1016/j.ajpath.2018.09.009
Lee, J. C., et al. (2018). Management of recurrent pancreatic cancer after surgical resection: a protocol for systematic review, evidence mapping and meta-analysis. BMJ Open, 8(4), e017249. doi:10.1136/bmjopen-2017-017249
Mulvihill, S., et al. (2001). Safety and feasibility of injection with an E1B-55 kDa gene-deleted, replication-selective adenovirus (ONYX-015) into primary carcinomas of the pancreas: a phase I trial. Gene Ther, 8(4), 308-315. doi:10.1038/sj.gt.3301398
Murakami, T., et al. (2019). Role of the tumor microenvironment in pancreatic cancer. Ann Gastroenterol Surg, 3(2), 130-137. doi:10.1002/ags3.12225
Muruve, D. A. (2004). The innate immune response to adenovirus vectors. Hum Gene Ther, 15(12), 1157-1166. doi:10.1089/hum.2004.15.1157
Nakao, A., et al. (2011). A phase I dose-escalation clinical trial of intraoperative direct intratumoral injection of HF10 oncolytic virus in non-resectable patients with advanced pancreatic cancer. Cancer Gene Ther, 18(3), 167-175. doi:10.1038/cgt.2010.65
Packiam, V. T., et al. (2018). An open label, single-arm, phase II multicenter study of the safety and efficacy of CG0070 oncolytic vector regimen in patients with BCG-unresponsive non-muscle-invasive bladder cancer: Interim results. Urol Oncol, 36(10), 440-447. doi:10.1016/j.urolonc.2017.07.005
Pishvaian, M. J., et al. (2017). Therapeutic implications of molecular subtyping for pancreatic cancer. Oncology (Williston Park), 31(3), 159-166, 168.
Poruk, K. E., et al. (2013). Screening for pancreatic cancer: why, how, and who? Ann Surg, 257(1), 17-26. doi:10.1097/SLA.0b013e31825ffbfb
Rahal, A., et al. (2017). Oncolytic viral therapy for pancreatic cancer. J Surg Oncol, 116(1), 94-103. doi:10.1002/jso.24626
Schnurr, M., et al. (2015). Strategies to relieve immunosuppression in pancreatic cancer. Immunotherapy, 7(4), 363-376. doi:10.2217/imt.15.9
Seth, R. B., et al. (2006). Antiviral innate immunity pathways. Cell Res, 16(2), 141-147. doi:10.1038/sj.cr.7310019
Sharma, P., et al. (2015). The future of immune checkpoint therapy. Science, 348(6230), 56-61. doi:10.1126/science.aaa8172
Siegel, R. L., et al. (2018). Cancer statistics, 2018. CA Cancer J Clin, 68(1), 7-30. doi:10.3322/caac.21442
Simon, S., et al. (2017). PD-1 expression on tumor-specific T cells: friend or foe for immunotherapy? Oncoimmunology, 7(1), e1364828. doi:10.1080/2162402X.2017.1364828
Takeuchi, O., et al. (2010). Pattern recognition receptors and inflammation. Cell, 140(6), 805-820. doi:10.1016/j.cell.2010.01.022
Ueno, H., et al. (2013). Randomized phase III study of gemcitabine plus S-1, S-1 alone, or gemcitabine alone in patients with locally advanced and metastatic pancreatic cancer in Japan and Taiwan: GEST study. J Clin Oncol, 31(13), 1640-1648. doi:10.1200/JCO.2012.43.3680
Wang-Gillam, A., et al. (2019). NAPOLI-1 phase 3 study of liposomal irinotecan in metastatic pancreatic cancer: Final overall survival analysis and characteristics of long-term survivors. Eur J Cancer Care (Engl), 108, 78-87. doi:10.1016/j.ejca.2018.12.007
Woller, N., et al. (2015). Viral infection of tumors overcomes resistance to PD-1-immunotherapy by broadening neoantigenome-directed T-cell responses. Mol Ther, 23(10), 1630-1640. doi:10.1038/mt.2015.115
Wu, H., et al. (2019). An oncolytic adenovirus 11p vector expressing adenovirus death protein in the E1 region showed significant apoptosis and tumour-killing ability in metastatic prostate cells. Oncotarget, 10(20), 1957-1974. doi:10.18632/oncotarget.26754