系統識別號 U0026-1707201511104900
論文名稱(中文) 探討活化的巨噬細胞促進大腸癌細胞侵襲的角色
論文名稱(英文) The Role of Activated Macrophages in Enhancing the Invasive Properties of Colon Cancer Cells
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) Department of Biochemistry and Molecular Biology
學年度 103
學期 2
出版年 104
研究生(中文) 張德沛
研究生(英文) Te-Pei Chang
學號 S16021086
學位類別 碩士
語文別 英文
論文頁數 68頁
口試委員 指導教授-吳華林
中文關鍵字 腫瘤相關巨噬細胞  癌細胞爬行  血管新生 
英文關鍵字 Tumor-associated macrophages  Cancer cell migration  Angiogenesis 
中文摘要 腫瘤細胞能夠藉著一系列的細胞因子和趨化因子與在腫瘤微環境的細胞作用,以促進腫瘤細胞發展成更惡性的狀態。有研究指出,腫瘤相關巨噬細胞(TAMs)會誘導大腸癌細胞轉移。然而,關於腫瘤相關巨噬細胞的作用,許多相互矛盾的文獻致無法達成共識。因此本篇研究的目的是探討在大腸癌的腫瘤微環境中腫瘤相關巨噬細胞的功能。我們選用人類大腸癌細胞株 (SW480) 作為研究的體外模式細胞。本研究,所使用的巨噬細胞是從人類單核球細胞 (THP-1) 加入巴豆醇-12-十四烷酸酯-13-乙酸酯 (phorbol 12-myristate 13-acetate) 刺激下成為巨噬細胞 (M0),再藉由γ干擾素的刺激分化成M1型巨噬细胞 (M1),或藉由介白素-4的刺激分化成M2型巨噬细胞 (M2)。由即時聚合酶鏈鎖反應實驗結果顯示大腸癌細胞培養液刺激的M0巨噬細胞會表現高量的M1型巨噬细胞標記基因,代表大腸癌細胞會馴化巨噬細胞成為M1型巨噬细胞。為了探討M0、M1、M2型巨噬细胞培養液對大腸癌細胞爬行能力的影響,藉由細胞穿越實驗(transwell assay) ,我們發現M1型巨噬细胞培養液會吸引大腸癌細胞爬行地最多。為了探討先經由不同的巨噬细胞培養液刺激的大腸癌細胞是否會促進血管新生,我們發現先經由M1型巨噬细胞培養液刺激的大腸癌細胞的培養液會讓人類臍靜脈內皮細胞 (HUVECs) 形成比較多的管柱。此外我們也發現M1型巨噬细胞培養液刺激的大腸癌細胞並不藉由NF-κB的路徑而增加其侵襲和促進血管新生的能力。總括而言,本論文結果顯示人類大腸癌細胞會促使巨噬細胞極化成為M1型巨噬细胞進而增加大腸癌細胞的爬行能力、侵襲能力、和促進血管新生的能力但不藉由活化大腸癌細胞的NF-κB路徑。
英文摘要 Tumor cells are capable of communicating with the cells in the tumor microenvironment via a series of cytokines and chemokines to promote their progression to a more malignant state. In addition, there are studies showing that tumor-associated macrophages (TAMs) induce metastatic behavior of colon cancer cells. However, accumulated conflicting evidences regarding the role of TAMs have not yet reached a consensus. Hence, we aimed to study the functions of TAMs in the tumor microenvironment of colon cancers. We used cell line-SW480 (colorectal adenocarcinoma) in our in vitro model. In the present study, we generated TAMs from THP-1 monocytic cells stimulated with phorbol 12-myristate 13-acetate into macrophages (M0), and M0 were further differentiated into classically activated macrophages (M1) or alternatively activated macrophages (M2) by adding interferon- γ or interleukin-4 respectively. Additionally, we found that SW480 conditioned medium (CM) was able to educate M0 to become M1 that expressed high level of M1 marker genes. To examine the migration ability of SW480 cells under stimuli of M0, M1, and M2 CMs respectively, the transwell assay was performed. SW480 cells migrated more toward the M1 CM than all the other media. To explore the pro-angiogenic ability of SW480 cells with different stimulations of macrophage CM, CM obtained from M1 CM-stimulated SW480 could cause tube formation of human umbilical vein endothelial cells. In addition, we gathered the results from western blotting that stimulation of M1 CM in SW480 cells did not activate their NF-κB pathway. In summary, our data imply that SW480 cells polarize macrophages toward M1 to enhance their migration, invasion and pro-angiogeneic abilities but not by NF-κB pathway.
論文目次 Abstract in Chinese 1
Abstract in English 2
Acknowledgement 3
Content 4
Figure Contents 5
Table Contents 6
Abbreviation 7
Instruments 9
Reagents and Chemicals 11
Introduction 15
Specific Aim 19
Materials and Methods 20
Results 40
Conclusion 43
Discussion 44
References 47
Figures 55
Tables 66
參考文獻 1. Schreuders, E. H., Ruco, A., Rabeneck, L., Schoen, R. E., Sung, J. J., Young, G. P., and Kuipers, E. J. (2015) Colorectal cancer screening: a global overview of existing programmes. Gut
2. Weitz, J., Koch, M., Debus, J., Hohler, T., Galle, P. R., and Buchler, M. W. (2005) Colorectal cancer. Lancet (London, England) 365, 153-165
3. Meira, L. B., Bugni, J. M., Green, S. L., Lee, C. W., Pang, B., Borenshtein, D., Rickman, B. H., Rogers, A. B., Moroski-Erkul, C. A., McFaline, J. L., Schauer, D. B., Dedon, P. C., Fox, J. G., and Samson, L. D. (2008) DNA damage induced by chronic inflammation contributes to colon carcinogenesis in mice. The Journal of clinical investigation 118, 2516-2525
4. Talmadge, J. E., Donkor, M., and Scholar, E. (2007) Inflammatory cell infiltration of tumors: Jekyll or Hyde. Cancer metastasis reviews 26, 373-400
5. Wang, D., Dubois, R. N., and Richmond, A. (2009) The role of chemokines in intestinal inflammation and cancer. Current opinion in pharmacology 9, 688-696
6. Allavena, P., Sica, A., Solinas, G., Porta, C., and Mantovani, A. (2008) The inflammatory micro-environment in tumor progression: the role of tumor-associated macrophages. Critical reviews in oncology/hematology 66, 1-9
7. Mantovani, A., Bottazzi, B., Colotta, F., Sozzani, S., and Ruco, L. (1992) The origin and function of tumor-associated macrophages. Immunology today 13, 265-270
8. Bingle, L., Brown, N. J., and Lewis, C. E. (2002) The role of tumour-associated macrophages in tumour progression: implications for new anticancer therapies. The Journal of pathology 196, 254-265
9. Chanmee, T., Ontong, P., Konno, K., and Itano, N. (2014) Tumor-associated macrophages as major players in the tumor microenvironment. Cancers 6, 1670-1690
10. Gocheva, V., Wang, H. W., Gadea, B. B., Shree, T., Hunter, K. E., Garfall, A. L., Berman, T., and Joyce, J. A. (2010) IL-4 induces cathepsin protease activity in tumor-associated macrophages to promote cancer growth and invasion. Genes & development 24, 241-255
11. Lewis, C. E., and Pollard, J. W. (2006) Distinct role of macrophages in different tumor microenvironments. Cancer research 66, 605-612
12. Wyckoff, J., Wang, W., Lin, E. Y., Wang, Y., Pixley, F., Stanley, E. R., Graf, T., Pollard, J. W., Segall, J., and Condeelis, J. (2004) A paracrine loop between tumor cells and macrophages is required for tumor cell migration in mammary tumors. Cancer research 64, 7022-7029
13. Gil-Bernabe, A. M., Ferjancic, S., Tlalka, M., Zhao, L., Allen, P. D., Im, J. H., Watson, K., Hill, S. A., Amirkhosravi, A., Francis, J. L., Pollard, J. W., Ruf, W., and Muschel, R. J. (2012) Recruitment of monocytes/macrophages by tissue factor-mediated coagulation is essential for metastatic cell survival and premetastatic niche establishment in mice. Blood 119, 3164-3175
14. Ikemoto, S., Yoshida, N., Narita, K., Wada, S., Kishimoto, T., Sugimura, K., and Nakatani, T. (2003) Role of tumor-associated macrophages in renal cell carcinoma. Oncology reports 10, 1843-1849
15. Ueno, T., Toi, M., Saji, H., Muta, M., Bando, H., Kuroi, K., Koike, M., Inadera, H., and Matsushima, K. (2000) Significance of macrophage chemoattractant protein-1 in macrophage recruitment, angiogenesis, and survival in human breast cancer. Clinical cancer research : an official journal of the American Association for Cancer Research 6, 3282-3289
16. Nesbit, M., Schaider, H., Miller, T. H., and Herlyn, M. (2001) Low-level monocyte chemoattractant protein-1 stimulation of monocytes leads to tumor formation in nontumorigenic melanoma cells. Journal of immunology (Baltimore, Md. : 1950) 166, 6483-6490
17. Biswas, S. K., Gangi, L., Paul, S., Schioppa, T., Saccani, A., Sironi, M., Bottazzi, B., Doni, A., Vincenzo, B., Pasqualini, F., Vago, L., Nebuloni, M., Mantovani, A., and Sica, A. (2006) A distinct and unique transcriptional program expressed by tumor-associated macrophages (defective NF-kappaB and enhanced IRF-3/STAT1 activation). Blood 107, 2112-2122
18. Kusmartsev, S., and Gabrilovich, D. I. (2005) STAT1 signaling regulates tumor-associated macrophage-mediated T cell deletion. Journal of immunology (Baltimore, Md. : 1950) 174, 4880-4891
19. Forssell, J., Oberg, A., Henriksson, M. L., Stenling, R., Jung, A., and Palmqvist, R. (2007) High macrophage infiltration along the tumor front correlates with improved survival in colon cancer. Clinical cancer research : an official journal of the American Association for Cancer Research 13, 1472-1479
20. Ohno, S., Inagawa, H., Dhar, D. K., Fujii, T., Ueda, S., Tachibana, M., Suzuki, N., Inoue, M., Soma, G., and Nagasue, N. (2003) The degree of macrophage infiltration into the cancer cell nest is a significant predictor of survival in gastric cancer patients. Anticancer research 23, 5015-5022
21. Khorana, A. A., Ryan, C. K., Cox, C., Eberly, S., and Sahasrabudhe, D. M. (2003) Vascular endothelial growth factor, CD68, and epidermal growth factor receptor expression and survival in patients with Stage II and Stage III colon carcinoma: a role for the host response in prognosis. Cancer 97, 960-968
22. Funada, Y., Noguchi, T., Kikuchi, R., Takeno, S., Uchida, Y., and Gabbert, H. E. (2003) Prognostic significance of CD8+ T cell and macrophage peritumoral infiltration in colorectal cancer. Oncology reports 10, 309-313
23. Barbera-Guillem, E., Nyhus, J. K., Wolford, C. C., Friece, C. R., and Sampsel, J. W. (2002) Vascular endothelial growth factor secretion by tumor-infiltrating macrophages essentially supports tumor angiogenesis, and IgG immune complexes potentiate the process. Cancer research 62, 7042-7049
24. Jedinak, A., Dudhgaonkar, S., and Sliva, D. (2010) Activated macrophages induce metastatic behavior of colon cancer cells. Immunobiology 215, 242-249
25. Herbeuval, J. P., Lelievre, E., Lambert, C., Dy, M., and Genin, C. (2004) Recruitment of STAT3 for production of IL-10 by colon carcinoma cells induced by macrophage-derived IL-6. Journal of immunology (Baltimore, Md. : 1950) 172, 4630-4636
26. Massague, J. (2008) TGFbeta in Cancer. Cell 134, 215-230
27. Peinado, H., Olmeda, D., and Cano, A. (2007) Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype? Nature reviews. Cancer 7, 415-428
28. Illemann, M., Bird, N., Majeed, A., Sehested, M., Laerum, O. D., Lund, L. R., Dano, K., and Nielsen, B. S. (2006) MMP-9 is differentially expressed in primary human colorectal adenocarcinomas and their metastases. Molecular cancer research : MCR 4, 293-302
29. Reinacher-Schick, A., Baldus, S. E., Romdhana, B., Landsberg, S., Zapatka, M., Monig, S. P., Holscher, A. H., Dienes, H. P., Schmiegel, W., and Schwarte-Waldhoff, I. (2004) Loss of Smad4 correlates with loss of the invasion suppressor E-cadherin in advanced colorectal carcinomas. The Journal of pathology 202, 412-420
30. Thuault, S., Valcourt, U., Petersen, M., Manfioletti, G., Heldin, C. H., and Moustakas, A. (2006) Transforming growth factor-beta employs HMGA2 to elicit epithelial-mesenchymal transition. The Journal of cell biology 174, 175-183
31. Dinarello, C. A. (2000) Proinflammatory cytokines. Chest 118, 503-508
32. Mosser, D. M., and Zhang, X. (2008) Interleukin-10: new perspectives on an old cytokine. Immunological reviews 226, 205-218
33. Erreni, M., Mantovani, A., and Allavena, P. (2011) Tumor-associated Macrophages (TAM) and Inflammation in Colorectal Cancer. Cancer microenvironment : official journal of the International Cancer Microenvironment Society 4, 141-154
34. Pancione, M., Forte, N., Sabatino, L., Tomaselli, E., Parente, D., Febbraro, A., and Colantuoni, V. (2009) Reduced beta-catenin and peroxisome proliferator-activated receptor-gamma expression levels are associated with colorectal cancer metastatic progression: correlation with tumor-associated macrophages, cyclooxygenase 2, and patient outcome. Human pathology 40, 714-725
35. Tjiu, J. W., Chen, J. S., Shun, C. T., Lin, S. J., Liao, Y. H., Chu, C. Y., Tsai, T. F., Chiu, H. C., Dai, Y. S., Inoue, H., Yang, P. C., Kuo, M. L., and Jee, S. H. (2009) Tumor-associated macrophage-induced invasion and angiogenesis of human basal cell carcinoma cells by cyclooxygenase-2 induction. The Journal of investigative dermatology 129, 1016-1025
36. Zhang, Y., Sime, W., Juhas, M., and Sjolander, A. (2013) Crosstalk between colon cancer cells and macrophages via inflammatory mediators and CD47 promotes tumour cell migration. European journal of cancer 49, 3320-3334
37. Kobayashi, N., Miyoshi, S., Mikami, T., Koyama, H., Kitazawa, M., Takeoka, M., Sano, K., Amano, J., Isogai, Z., Niida, S., Oguri, K., Okayama, M., McDonald, J. A., Kimata, K., Taniguchi, S., and Itano, N. (2010) Hyaluronan deficiency in tumor stroma impairs macrophage trafficking and tumor neovascularization. Cancer research 70, 7073-7083
38. Kryczek, I., Zou, L., Rodriguez, P., Zhu, G., Wei, S., Mottram, P., Brumlik, M., Cheng, P., Curiel, T., Myers, L., Lackner, A., Alvarez, X., Ochoa, A., Chen, L., and Zou, W. (2006) B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma. The Journal of experimental medicine 203, 871-881
39. Gordon, S., and Mantovani, A. (2011) Diversity and plasticity of mononuclear phagocytes. European journal of immunology 41, 2470-2472
40. Zaynagetdinov, R., Sherrill, T. P., Polosukhin, V. V., Han, W., Ausborn, J. A., McLoed, A. G., McMahon, F. B., Gleaves, L. A., Degryse, A. L., Stathopoulos, G. T., Yull, F. E., and Blackwell, T. S. (2011) A critical role for macrophages in promotion of urethane-induced lung carcinogenesis. Journal of immunology (Baltimore, Md. : 1950) 187, 5703-5711
41. Engstrom, A., Erlandsson, A., Delbro, D., and Wijkander, J. (2014) Conditioned media from macrophages of M1, but not M2 phenotype, inhibit the proliferation of the colon cancer cell lines HT-29 and CACO-2. International journal of oncology 44, 385-392
42. Szlosarek, P., Charles, K. A., and Balkwill, F. R. (2006) Tumour necrosis factor-alpha as a tumour promoter. European journal of cancer 42, 745-750
43. Szlosarek, P. W., Grimshaw, M. J., Kulbe, H., Wilson, J. L., Wilbanks, G. D., Burke, F., and Balkwill, F. R. (2006) Expression and regulation of tumor necrosis factor alpha in normal and malignant ovarian epithelium. Molecular cancer therapeutics 5, 382-390
44. Murakami, A., Kawabata, K., Koshiba, T., Gao, G., Nakamura, Y., Koshimizu, K., and Ohigashi, H. (2000) Nitric oxide synthase is induced in tumor promoter-sensitive, but not tumor promoter-resistant, JB6 mouse epidermal cells cocultured with interferon-gamma-stimulated RAW 264.7 cells: the role of tumor necrosis factor-alpha. Cancer research 60, 6326-6331
45. Rosen, E. M., Goldberg, I. D., Liu, D., Setter, E., Donovan, M. A., Bhargava, M., Reiss, M., and Kacinski, B. M. (1991) Tumor necrosis factor stimulates epithelial tumor cell motility. Cancer research 51, 5315-5321
46. Leibovich, S. J., Polverini, P. J., Shepard, H. M., Wiseman, D. M., Shively, V., and Nuseir, N. (1987) Macrophage-induced angiogenesis is mediated by tumour necrosis factor-alpha. Nature 329, 630-632
47. Hagemann, T., Wilson, J., Kulbe, H., Li, N. F., Leinster, D. A., Charles, K., Klemm, F., Pukrop, T., Binder, C., and Balkwill, F. R. (2005) Macrophages induce invasiveness of epithelial cancer cells via NF-kappa B and JNK. Journal of immunology (Baltimore, Md. : 1950) 175, 1197-1205
48. Coscia, M., Quaglino, E., Iezzi, M., Curcio, C., Pantaleoni, F., Riganti, C., Holen, I., Monkkonen, H., Boccadoro, M., Forni, G., Musiani, P., Bosia, A., Cavallo, F., and Massaia, M. (2010) Zoledronic acid repolarizes tumour-associated macrophages and inhibits mammary carcinogenesis by targeting the mevalonate pathway. Journal of cellular and molecular medicine 14, 2803-2815
49. Zhang, X., Tian, W., Cai, X., Wang, X., Dang, W., Tang, H., Cao, H., Wang, L., and Chen, T. (2013) Hydrazinocurcumin Encapsuled nanoparticles "re-educate" tumor-associated macrophages and exhibit anti-tumor effects on breast cancer following STAT3 suppression. PloS one 8, e65896
50. Gazzaniga, S., Bravo, A. I., Guglielmotti, A., van Rooijen, N., Maschi, F., Vecchi, A., Mantovani, A., Mordoh, J., and Wainstok, R. (2007) Targeting tumor-associated macrophages and inhibition of MCP-1 reduce angiogenesis and tumor growth in a human melanoma xenograft. The Journal of investigative dermatology 127, 2031-2041
51. Dineen, S. P., Lynn, K. D., Holloway, S. E., Miller, A. F., Sullivan, J. P., Shames, D. S., Beck, A. W., Barnett, C. C., Fleming, J. B., and Brekken, R. A. (2008) Vascular endothelial growth factor receptor 2 mediates macrophage infiltration into orthotopic pancreatic tumors in mice. Cancer research 68, 4340-4346
  • 同意授權校內瀏覽/列印電子全文服務,於2020-08-03起公開。

  • 如您有疑問,請聯絡圖書館