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研究生：	吳宜臻 Wu, Yi-Zhen
論文名稱：	蕾莎瓦在肝癌小鼠中會誘發T細胞免疫檢查受體的表現 Sorafenib induces expression of immune checkpoint receptors on T cells in hepatoma-bearing mice
指導教授：	張志鵬 Chang, Chih-Peng
學位類別：	碩士 Master
系所名稱：	醫學院 - 微生物及免疫學研究所 Department of Microbiology & Immunology
論文出版年：	2018
畢業學年度：	106
語文別：	英文
論文頁數：	52
中文關鍵詞：	蕾莎瓦、肝癌、程序化死亡分子-1 、細胞毒性T淋巴細胞相關抗原4 、信號轉導及轉錄激活蛋白3
外文關鍵詞：	Sorafenib, Hepatocellular carcinoma, Programmed death-1, Cytotoxic T-lymphocyte-associated protein 4, Signal transducer and activator of transcription 3
相關次數：	點閱：147 下載：4
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肝細胞癌(以下簡稱肝癌)是現居全球前五名的常見癌症，亦是高居前三名的高致死率癌症。而在台灣，更是在十大致死癌症中的第二名。為了治療肝癌末期病患，一個多激酶抑制藥劑稱為蕾莎瓦，是唯一通過核准的臨床用藥。然而，近十年來許多研究發現，在長期服用蕾莎瓦的肝癌病患中，對蕾莎瓦會出現抗藥性的情形。在包含肝癌的許多癌症中顯示，免疫細胞的高度浸潤與對化療藥物的低反應和預後有關。然而，化學藥物如何去調控免疫細胞仍需要更進一步的探討。在本篇研究中，我們發現以蕾莎瓦治療肝癌小鼠時，不論在周邊血液或是腫瘤內部都可以發現在CD4+和CD8+T細胞表面，抑制受體PD-1(程序化死亡分子1)和CTLA-4(細胞毒性T 淋巴細胞相關抗原4)的表現量明顯增加。我們也進一步證實，蕾莎瓦不但會增加抑制受體的表現也會抑制T細胞的增生。STAT3 (信號轉導及轉錄激活蛋白3)是腫瘤增生的重要的調節分子，已知它會促進PD-1和CTLA-4的表現。因此，我們利用STAT3的抑制劑stattic來研究STAT3在蕾莎瓦誘導的PD-1表現中的角色。我們發現抑制STAT3的活性能夠減少PD-1在T細胞上的表現，並改善蕾莎瓦在肝癌模式小鼠中的療效。從上述結果，我們認為蕾莎瓦所誘導的STAT3在T細胞去活化和對蕾莎瓦治療的反應中扮演一個非常重要的角色。以STAT3為標的來改善蕾莎瓦在肝癌病患上的效果可能是未來可以嘗試的治療方式。

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the third leading cause of cancer-related mortality in the world. In Taiwan, HCC is the second among the top ten leading causes of death in cancer. To treat advanced HCC, the multiple-kinase inhibitor, sorafenib, is the only standard clinical drug. However, in recent decades, many studies have shown that HCC patients exhibited resistance towards sorafenib during long-term treatment. In many cancers, including HCC, high infiltration of immune cells is associated with poor response to chemodrugs and prognosis. Nevertheless, how chemodrugs regulate these immune cells is yet to be determined. In this study, we found that the HCC-bearing mice treated with sorafenib showed an increased expression of inhibitory receptor PD-1 (Programmed death-1) and CTLA-4 (Cytotoxic T-lymphocyte-associated protein 4) on both peripheral and intra-tumor CD4+ and CD8+ T cells. We also provided evidence to show that sorafenib not only increased inhibitory receptor expression but also inhibited T cell proliferation. STAT3 (Signal transducer and activator of transcription 3), the key mediator of tumor progression is known to induce PD-1 and CTLA-4. Hence, we used STAT3 inhibitor stattic to examine its role in sorafenib-induced PD-1 expression. We found that inactivation of STAT3 was able to decrease PD-1 expression on T cells and improve sorafenib efficacy in HCC-bearing mice. According to these results, we suggest that sorafenib-induced STAT3 plays a critical role on T cell inactivation and limited responses to sorefenib treatment. Targeting STAT3 may serve a promising therapeutic intervention to improve sorafenib responses in HCC patients.

中文摘要	I
Abstract	II
Acknowledgement	III
Table of contents	IV
Abbreviations	VII
I. Introduction	1
1.	Etiology of hepatocellular carcinoma	1
2.	Sorafenib	3
3.	Immune checkpoint	6
4.	STAT3	8
II. Objective and Specific Aims	11
III. Materials and methods	12
1.	  Materials	12
1.1	Animal	12
1.2	Cells	12
1.3	Antibody	13
1.4	Reagents	14
2.	  Methods	18
2.1	Mice in situ hepatoma model	18
2.2	Flow cytometry	18
2.3	Immunofluorescence assay	19
2.4	ELISA	19
2.5	Primary splenocyte isolation	20
2.6    Preparation of hepatoma cell condition medium	20
2.7    T cell proliferation assay	21
2.8    Intrahepatic lymphocytes (IHLs) isolation	21
2.9	Statistical analysis	21
IV. Results	22
IV-1. Dose-related responses and liver damage in sorafenib-treated HCC-bearing mice	22
IV-2. Sorafenib treatment increases expression of CTLA-4 and PD-1 on peripheral T cells of HCC-bearing mice.	22
IV-3. 	Sorafenib treatment increases infiltration of CTLA-4 and PD-1 expressed T cells to HCC tissues..	23
IV-4. T cells from sorafenib-treated HCC-bearing mice reduces proliferation activity in responses to Con A..	24
IV-5. 5.	Sorafenib treatment increases expression of CTLA-4 and PD-1 on splenocytes in vitro	24
IV-6. Inhibition of STAT3 by inhibitor stattic reduces sorafenib-induced PD-1 expression on T cells.	25
IV-7. Stattic improves sorafenib efficacy in treating HCC-bearing mice.	25
V. Conclusion	26
VI. Discussion	27
VII. References	30
VIII. Figures	36
Figure 1. Dose-related responses and liver damage by treatment of sorafenib in HCC-bearing mice	36
Figure 2. Sorafenib treatment increases expression of CTLA-4 and PD-1 on both CD4+ and CD8+ peripheral T cells of HCC-bearing mice.	38
Figure 3. Sorafenib treatment downregulates the serum levels of IL-6, IL-12 and IL-33 in HCC-bearing mice.	40
Figure 4. Sorafenib treatment increases infiltration of PD-1+ CD4+ and CTLA-4+CD4+ T cells in hepatoma tissues.	41
Figure 5. Sorafenib treatment increases infiltration of PD-1+ CD8+ and CTLA-4+CD8+ T cells in hepatoma tissues.	43
Figure 6. T cells from sorafenib-treated HCC-bearing mice shows low proliferation responses to Con A.	45
Figure 7. Co-treatment of MCM and sorafenib increases expression of PD-1 and CTLA4 on splenocytes.	46
Figure 8. Stattic reduces sorafenib-induced PD-1 expression on T cells .	48
Figure 9. Stattic improves sorafenib efficacy in HCC-bearing mice..	49
Figure 10. Sorafenib treatment increases suppressive receptors PD-1 and CTLA-4 expression on T cells may be via STAT3 related pathway in hepatoma-bearing mice.	51
IX. Appendix	52
Appendix 1. Sorafenib treatment increases infiltration of regulatory T cells in hepatoma tissues.	52

                                    

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