斑馬魚異種腫瘤移植模型在腫瘤誘發血管新生研究上的應用

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研究生：	童凱澤 Tung, Kai-Che
論文名稱：	斑馬魚異種腫瘤移植模型在腫瘤誘發血管新生研究上的應用 The Application of Zebrafish Tumor Xenograft Model in Tumor-Induced Angiogenesis Research
指導教授：	莊永仁 Chuang, Yung-Jen
口試委員:
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2010
畢業學年度：	98
語文別：	英文
論文頁數：	39
中文關鍵詞：	血管新生、巨噬細胞、人類組織蛋白酶S 、血管新生因子、斑馬魚異種腫瘤移植模型、斑馬魚、卵巢癌
外文關鍵詞：	Angiogenesis, Macrophages, Cathepsin S, Vascular endothelial growth factor A, Zebrafish Tumor Xenograft Model, Zebrafish, Ovarian cancer
相關次數：	點閱：2 下載：0
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近年來斑馬魚生物模型被廣泛地應用於癌症研究。其中，斑馬魚異種腫瘤移植模型具有多項獨特優點，包含：斑馬魚飼養維護設備價格便宜、容易操作、可降低實驗藥物的使用量、在基因體學/蛋白質體學研究上有多種工具與方法可以搭配應用，如斑馬魚胚胎原位雜合技術以及免疫螢光染色技術等。不同於其他常見的模式生物，斑馬魚胚胎具有高度透明性，此優點對於在活體上觀測癌症發展以及癌症誘發之血管新生研究有莫大的助益。因此我們利用此模型來研究癌症誘發之血管新生。
卵巢癌近年來為婦科癌症死亡率較高的癌症之一。其惡性程度與腫瘤誘發之血管新生有密切的關係。近年來有報導指出，惡性腫瘤以及其誘發之血管新生與其周遭之M2表現型之腫瘤相關巨噬細胞有高度相關性，然而對於腫瘤以及腫瘤相關巨噬細胞之交互作用機制目前仍待釐清。
本篇研究中，我建立了斑馬魚腫瘤異種移植模型，並結合活體外實驗，釐清在腫瘤微環境中惡性腫瘤誘發血管新生的分子機制。在活體外實驗中，我們發現卵巢癌細胞 (SKOV3 cells) 之血管新生因子 (VEGFA) 以及人類組織蛋白酶S (cathepsin S) 之表現量會在其與M2極化之巨噬細胞共同培養後大量上升。而在斑馬魚異種腫瘤移植模型上，我們發現當M2極化之巨噬細胞與卵巢癌細胞被共同移植時，腫瘤誘發之血管新生現象會顯著提升。我們的結果顯示腫瘤相關巨噬細胞主要是藉由提升卵巢癌細胞之血管新生相關基因表現，促進腫瘤誘發之血管新生。

Zebrafish model have become a powerful tool in cancer research in recent years. The advantages of the zebrafish tumor xenograft model include: low cost, easy experimentation, reduced dosage for drug test, feasibility of various genetics/proteomics approaches such as whole mount in situ hybridization and whole mount immunocytochemistry. Different from other vertebrate organisms, the transparency of zebrafish embryo allowed us to monitor tumor progression and the tumor-induced angiogenesis in live embryos. In addition, zebrafish tumor xenograft model is much more rapid and cheaper than the current mouse model. Thus, here I aimed to use this model in tumor-induced angiogenesis research.
Ovarian carcinoma is considered as one of the leading gynecologic cancers with high mortality rate. The tumor malignancy is highly associated with tumor-induced angiogenesis. The tumor-associated macrophages (TAMs) with M2-like phenotype have been reported with tumor malignancy by promoting tumor-induced angiogenesis; however, the mechanisms of the interaction between cancer cells and macrophages are incompletely understood.
In this study, I established the zebrafish tumor xenograft model to accompany the in vitro cell-based assays to elucidate the molecular mechanism of tumor-induced angiogenesis in the cancer microenvironment. I have identified that the VEGFA and cathepsin S are induced in SKOV3 cells after co-cultured with M2-polarized macrophages. Furthermore, the zebrafish tumor xenograft model indicated when co-injected with M2-polarized macrophages, the tumor-induced angiogenesis was significantly increased. In conclusion, my results revealed that the tumor-associated macrophages could trigger the ovarian cancer cells to up-regulate angiogenesis-related genes in promoting tumor-induced angiogenesis.

List of contents
中文摘要    I
Abstract        II
誌謝        III
Abbreviations    VII
1. Introduction    1
1.1 Angiogenesis and current assay models    1
1.2 The advantages of zebrafish model    2
1.3 Ovarian cancer and tumor-associated macrophages    2
1.4 The angiogenesis-related factors in tumor microenvironment    3
1.5 The objective of this study    4
2.1 Zebrafish tumor xenograft model.    5
2.2 Cell culture and cell preparation    6
2.3 Construction of nls-mCherry expression vector    6
2.4 CL1-5 cell transfection    7
2.5 Cryosection and histochemistry staining    7
2.6 M1 and M2-polarized macrophage preparation    7
2.7 M2 macrophages and ovarian cancer cell co-culture procedure    7
2.8 Real-time PCR analysis    8
2.9 Western blot    8
2.10 Cathepsin S enzyme kinetic assay    9
2.11 Co-injection of SKOV3 cells and M2-polarized macrophages for tumor-induced neovascularization assay    9
3. Results    10
3.1 The control microspheres stayed immobilized at the injection site and did not induce angiogenesis in zebrafish embryos    10
3.2 Cancer cells could survive and proliferate in zebrafish embryo    10
3.3 Tumor-induced angiogenesis in zebrafish embryos    11
3.4 THP-1 cells were induced to M2-polarized macrophages    12
3.5 The VEGFA and cathepsin S gene expression level of ovarian cancer cells were increased after co-culture with M2 cells    13
3.6 M2-polarized macrophages promote SKOV3 tumor-induced angiogenesis  in vivo    14
4. Discussion    15
4.1 Zebrafish tumor xenograft model    15
4.1.1 Micropipettes preparation    15
4.1.2 The host vs. graft rejection of tumor xenograft does not occur in zebrafish xenograft model    16
4.1.3 The choice of xenograft injection site    17
4.1.4 The limitations of this model    18
4.2 Tumor-associated macrophages promote ovarian cancer cells induced-angiogenesis    19



List of Tables
Table 1. The sequence of primers used for real-time quantitative PCR    25



List of Figures
Figure 1. Zebrafish tumor xenograft model    26
Figure 2. The control microspheres did not migrate or induce angiogenesis in zebrafish    27
Figure 3. CL1-5 with endogenous nuclear mCherry indicated human cancer cells could survive and migrate in zebrafish    28
Figure 4. Cancer cell mitosis in zebrafish    29
Figure 5. Cancer cell mass was formed at the perivitelline space    30
Figure 6. No cancer-host immune cell interaction was observed around the tumor xenograft    31
Figure 7. The dynamic process of tumor-induced angiogenesis was monitored for up to 80 hours    32
Figure 8. THP-1 cells were polarized into the M2 macrophages    33
Figure 9. Expression level of CTSS was increased in SKOV3 cells co-cultured with polarized M2 macrophages    34
Figure 10. Polarized M2 macrophages effectively promoted SKOV3 cells to induce angiogenesis    35
Figure 11. Proposed model for the role of tumor-associated macrophages in tumor-induced angiogenesis    36



List of Supplemental Figures
Figure S1. The tip of micropipettes was created with blunt end    37
Figure S2. Time-course analysis of tumor cell dissemination throughout the perivitelline space    38
Figure S3. In vivo cell migration assay    39

                                

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