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研究生： 汪欣儀
Wang, Hsin-I
論文名稱： 腫瘤細胞所表現的Fas ligand對於腫瘤空間上分布及纖維母細胞活化的影響
Effect of tumor Fas ligand on the spatial distribution and activation of fibroblasts
指導教授： 楊倍昌
Yang, Bei-Chang
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： 2016
畢業學年度： 104
語文別： 英文
論文頁數： 57
中文關鍵詞： Fas ligand 、3D腫瘤球 、共培養 、纖維母細胞活化
外文關鍵詞： Fas ligand, 3D tumor spheroid, co-culture, fibroblast activation
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    • 腫瘤浸潤型纖維母細胞是腫瘤基質微環境中的亞細胞群，會促進腫瘤的生長、腫瘤細胞的血管新生成、腫瘤的侵襲能力及轉移。良性腫瘤結節 (tumor nodule)會被纖維母細胞型態的細胞包圍。Fas ligand (FasL) 屬於腫瘤壞死因子家族 (tumor necrosis factor, TNF family)，是二型跨膜蛋白質 (type II transmembrane protein)的一員，並表現於多種癌細胞上。FasL能透過和細胞膜上的Fas受體結合進而誘發細胞凋亡 (apoptosis)，藉此調節免疫系統。FasL在腫瘤細胞上的表現量和癌症的惡性程度及轉移能力有關。在我們實驗室過去的研究中發現使用FasL專一核糖酶 (FasL ribozyme, FasL-RZ) 抑制FasL在人類神經膠質瘤癌細胞上的表現，會讓在裸鼠(Nude mice)中生成的腫瘤結節的邊界較為清晰，且被類纖維母細胞型態之細胞包覆。我們之前以2D共同培養系統比較FasL表現量較低 (FasLlow) 的腫瘤細胞和FasL表現量高 (FasLhigh) 的腫瘤細胞，前者被纖維母細胞所包圍的現象較為明顯，並形成壁壘分明的腫瘤細胞區塊。為了模擬in vivo中癌細胞與纖維母細胞之間的交互作用，本實驗建立了3D (three-dimentional) 腫瘤球共培養系統，以探討腫瘤的FasL與細胞在腫瘤球體上空間分布的關係。在腫瘤細胞與纖維母細胞共培養的腫瘤球中，FasLhigh腫瘤細胞會出現在腫瘤球的表面，顯示FasLhigh腫瘤細胞具有較佳的爬行能力並且能夠穿越纖維母細胞的聚集。當以FasL-RZ抑制腫瘤細胞的FasL表現後，爬行至腫瘤球體表面的腫瘤細胞數量明顯減少。另一方面，當纖維母細胞培養在養過FasL高表現之癌細胞培養液後，纖維母細胞中的TGF-β和PDGFA表現量增加，而且其活化標記蛋白α-smooth muscle actin (α-SMA) 和FAP的表現量也顯著提高。相較之下，在FasL-RZ轉染癌細胞的培養液處理之下，纖維母細胞活化的程度較低。綜合以上結果，我們認為FasL能影響腫瘤球結構以及纖維母細胞的活化。

    Tumor infiltrating fibroblasts are a subpopulation of cells in tumor stroma and may promote tumor growth, angiogenesis, invasion, and metastasis. Benign tumor nodules are frequently encircled by fibroblast-like cells. Fas ligand (FasL), a type II transmembrane protein of the tumor necrosis factor (TNF) family, is the authentic ligand of Fas and widely expressed in a variety of cancers. Fas/FasL interaction induces signal for cell apoptosis and regulates the homeostasis of immune system. The expression of FasL on tumor cells correlates with its malignancy and metastasis. Previously, we have knocked down the expression of FasL in human glioma by FasL ribozymes (FasL-RZ) and found that the boundary of tumor nodules generated by FasL-RZ cells in Nude mice showed less distinctive boundary between tumor and stromal cells. In addition, fibroblasts were able to encircle FasLlow tumor cells to form a capsule-like pattern in a 2D co-culture system but not did FasLhigh tumor cells. To mimic the interaction between cancer cells and fibroblasts in vivo, we applied this study in a three-dimensional (3D) co-culture system to determine the effect of FasL on tumor spheroid architecture. FasLhigh tumor cells appeared at the surface of spheroid indicating that they have capability to migrate through fibroblast cell aggregates. Accordingly, the number of tumor cells on spheroid was reduced when their FasL was suppressed by FasL-RZ transfection. Moreover, when fibroblasts were cultured in the conditioned media of FasLhigh-tumor cells, not only their TGF-β and PDGFA productions but also the activation markers, α-smooth muscle actin (α-SMA) and FAP were significantly elevated. Compared with FasLhigh group, the activation level of fibroblasts was low after treated with the conditioned media of FasL-RZ cells. Taken together, our results reveal a role of FasL in determining the architecture of tumor spheroids and the activation of fibroblasts.

    摘要................................................................................................................... I Abstract............................................................................................................. II Acknowledgement............................................................................................ IV Contents............................................................................................................ VI Figure index...................................................................................................... VIII Introduction...................................................................................................... 1 Tumor metastasis............................................................................................. 1 3D tumor spheroid culture system................................................................... 1 Fibroblasts in cancer........................................................................................ 2 FasL/Fas-induced cell death............................................................................ 3 FasL/Fas interaction for non-apoptotic regulation........................................... 3 FasL in tumor progression and metastasis....................................................... 4 Down regulation FasL in glioma cells results in encapsulated tumor nodule formation.......................................................................................................... 4 Rational in this study....................................................................................... 5 Materials and Methods.................................................................................... 6 Materials.......................................................................................................... 6 Media and Buffer preparation.......................................................................... 11 Methods........................................................................................................... 19 1. Transformation........................................................................................ 19 2. Mini plasmid isolation............................................................................. 19 3. Mixi plasmid isolation............................................................................. 20 4. Cell culture............................................................................................... 21 5. 3D tumor spheroid culture system........................................................... 22 6. Reverse-Transcription Polymerase Chain Reaction (RT-PCR).............. 22 7. Lentivirus production............................................................................... 25 8. Transfection............................................................................................. 25 9. Western-blotting analysis........................................................................ 26 10. Statistical analysis.................................................................................... 27 Results............................................................................................................... 28 Setup the 3D spheroid experimental system.................................................... 28 The spatial distribution of fibroblasts and tumor cells in spheroids................ 29 FasL affects the distribution of tumor cells in spheroids................................. 29 Activation of fibroblasts by tumor cells expression FasL............................... 30 Discussion......................................................................................................... 32 3D co-culture system....................................................................................... 32 The mechanism of FasL-induced fibroblasts activation.................................. 33 The role of FasL in tumor invasion................................................................. 33 References......................................................................................................... 35 Figures and Figure legends............................................................................. 40 Curriculum vitae.............................................................................................. 57   Figure index Figure 1. The 3D tumor spheroid formed conditions in different cell lines and different ways........................................................................... 41 Figure 2. The formation of tumor spheroids with optimal concentration of type I collagen................................................................................. 43 Figure 3. The size and shape of spheroids are more uniform in centrifugation groups...................................................................... 45 Figure 4. The ratio of GFP-tagged fibroblasts and tumor cells in tumor spheroids are better in 1:4............................................................... 46 Figure 5. Kinetics of fibroblasts and tumor cells distribution in tumor spheroids.......................................................................................... 47 Figure 6. FasL affects the migration ability of tumor cells in spheroids........ 49 Figure 7. The mRNA level and Western blot of FasL transfectants.............. 51 Figure 8. The conditioned media of FasL transfectants would affect fibroblasts activation....................................................................... 52 Figure 9. The conditioned media of FasL transfectants would affect fibroblasts activation....................................................................... 54 Figure 10. Summary of the expression of FasL on tumor affects the activation of fibroblasts................................................................... 56

    References
    1. Yokota, J., Tumor progression and metastasis. Carcinogenesis, 2000. 21(3): p. 497-503.
    2. Deryugina, E.I. and J.P. Quigley, Matrix metalloproteinases and tumor metastasis. Cancer Metastasis Rev, 2006. 25(1): p. 9-34.
    3. Kalluri, R. and M. Zeisberg, Fibroblasts in cancer. Nat Rev Cancer, 2006. 6(5): p. 392-401.
    4. Ivascu, A. and M. Kubbies, Rapid generation of single-tumor spheroids for high-throughput cell function and toxicity analysis. J Biomol Screen, 2006. 11(8): p. 922-32.
    5. Weiswald, L.B., D. Bellet, and V. Dangles-Marie, Spherical cancer models in tumor biology. Neoplasia, 2015. 17(1): p. 1-15.
    6. Sutherland, R.M., et al., Oxygenation and differentiation in multicellular spheroids of human colon carcinoma. Cancer Res, 1986. 46(10): p. 5320-9.
    7. Mayer, B., et al., Multicellular gastric cancer spheroids recapitulate growth pattern and differentiation phenotype of human gastric carcinomas. Gastroenterology, 2001. 121(4): p. 839-52.
    8. Grover, A., R.G. Oshima, and E.D. Adamson, Epithelial layer formation in differentiating aggregates of F9 embryonal carcinoma cells. J Cell Biol, 1983. 96(6): p. 1690-6.
    9. Barcellos-Hoff, M.H., et al., Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane. Development, 1989. 105(2): p. 223-35.
    10. Sutherland, R.M., et al., A multi-component radiation survival curve using an in vitro tumour model. Int J Radiat Biol Relat Stud Phys Chem Med, 1970. 18(5): p. 491-5.
    11. Nederman, T., Effects of vinblastine and 5-fluorouracil on human glioma and thyroid cancer cell monolayers and spheroids. Cancer Res, 1984. 44(1): p. 254-8.
    12. Olive, P.L., J.P. Banath, and H.H. Evans, Cell killing and DNA damage by etoposide in Chinese hamster V79 monolayers and spheroids: influence of growth kinetics, growth environment and DNA packaging. Br J Cancer, 1993. 67(3): p. 522-30.
    13. Croft, C.B. and D. Tarin, Ultrastructural studies of wound healing in mouse skin. I. Epithelial behaviour. J Anat, 1970. 106(Pt 1): p. 63-77.
    14. Tarin, D. and C.B. Croft, Ultrastructural studies of wound healing in mouse skin. II. Dermo-epidermal interrelationships. J Anat, 1970. 106(Pt 1): p. 79-91.
    15. Tomasek, J.J., et al., Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol, 2002. 3(5): p. 349-63.
    16. Parsonage, G., et al., A stromal address code defined by fibroblasts. Trends Immunol, 2005. 26(3): p. 150-6.
    17. Mueller, M.M. and N.E. Fusenig, Friends or foes - bipolar effects of the tumour stroma in cancer. Nat Rev Cancer, 2004. 4(11): p. 839-49.
    18. Sappino, A.P., et al., Smooth-muscle differentiation in stromal cells of malignant and non-malignant breast tissues. Int J Cancer, 1988. 41(5): p. 707-12.
    19. Mao, Y., et al., Stromal cells in tumor microenvironment and breast cancer. Cancer Metastasis Rev, 2013. 32(1-2): p. 303-15.
    20. Kopelovich, L., Genetic predisposition to cancer in man: in vitro studies. Int Rev Cytol, 1982. 77: p. 63-88.
    21. Schor, S.L., et al., Occurrence of a fetal fibroblast phenotype in familial breast cancer. Int J Cancer, 1986. 37(6): p. 831-6.
    22. Dimanche-Boitrel, M.T., et al., In vivo and in vitro invasiveness of a rat colon-cancer cell line maintaining E-cadherin expression: an enhancing role of tumor-associated myofibroblasts. Int J Cancer, 1994. 56(4): p. 512-21.
    23. Cornil, I., et al., Fibroblast cell interactions with human melanoma cells affect tumor cell growth as a function of tumor progression. Proc Natl Acad Sci U S A, 1991. 88(14): p. 6028-32.
    24. Olaso, E., et al., Tumor-dependent activation of rodent hepatic stellate cells during experimental melanoma metastasis. Hepatology, 1997. 26(3): p. 634-42.
    25. Waring, P. and A. Mullbacher, Cell death induced by the Fas/Fas ligand pathway and its role in pathology. Immunol Cell Biol, 1999. 77(4): p. 312-7.
    26. Schulze-Osthoff, K., et al., Apoptosis signaling by death receptors. Eur J Biochem, 1998. 254(3): p. 439-59.
    27. Green, D.R. and T.A. Ferguson, The role of Fas ligand in immune privilege. Nat Rev Mol Cell Biol, 2001. 2(12): p. 917-24.
    28. Liu, H.N., et al., The TLR3, PI3K, survivin, FasL, and Fas genes as major risk factors of occurrence and development of cervical cancer disease. Gene, 2014. 550(1): p. 27-32.
    29. Nagata, S. and P. Golstein, The Fas death factor. Science, 1995. 267(5203): p. 1449-56.
    30. Kischkel, F.C., et al., Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor. Embo j, 1995. 14(22): p. 5579-88.
    31. Farley, S.M., et al., Fas ligand elicits a caspase-independent proinflammatory response in human keratinocytes: implications for dermatitis. J Invest Dermatol, 2006. 126(11): p. 2438-51.
    32. Rippo, M.R., et al., Low FasL levels promote proliferation of human bone marrow-derived mesenchymal stem cells, higher levels inhibit their differentiation into adipocytes. Cell Death Dis, 2013. 4: p. e594.
    33. Wisniewski, P., et al., Non-apoptotic Fas signaling regulates invasiveness of glioma cells and modulates MMP-2 activity via NFkappaB-TIMP-2 pathway. Cell Signal, 2010. 22(2): p. 212-20.
    34. Kleber, S., et al., Yes and PI3K bind CD95 to signal invasion of glioblastoma. Cancer Cell, 2008. 13(3): p. 235-48.
    35. Yang, B.C., et al., Mediation of enhanced transcription of the IL-10 gene in T cells, upon contact with human glioma cells, by Fas signaling through a protein kinase A-independent pathway. J Immunol, 2003. 171(8): p. 3947-54.
    36. Su, C.C., et al., Expression of Th17-related genes in PHA/IL-2-activated human T cells by Fas signaling via caspase-1- and Stat3-dependent pathway. Cell Immunol, 2013. 281(2): p. 101-10.
    37. Chun, H.J., et al., Pleiotropic defects in lymphocyte activation caused by caspase-8 mutations lead to human immunodeficiency. Nature, 2002. 419(6905): p. 395-9.
    38. Redondo, P., et al., Fas and Fas ligand: expression and soluble circulating levels in cutaneous malignant melanoma. Br J Dermatol, 2002. 147(1): p. 80-6.
    39. Verbeke, C.S., et al., Fas ligand expression in Hodgkin lymphoma. Am J Surg Pathol, 2001. 25(3): p. 388-94.
    40. Lim, S.C., Expression of Fas ligand and sFas ligand in human gastric adenocarcinomas. Oncol Rep, 2002. 9(1): p. 103-7.
    41. Nozoe, T., et al., Fas ligand expression is correlated with metastasis in colorectal carcinoma. Oncology, 2003. 65(1): p. 83-8.
    42. Muschen, M., et al., CD95 ligand expression as a mechanism of immune escape in breast cancer. Immunology, 2000. 99(1): p. 69-77.
    43. Reimer, T., et al., FasL:Fas ratio--a prognostic factor in breast carcinomas. Cancer Res, 2000. 60(4): p. 822-8.
    44. Shibakita, M., et al., Prognostic significance of Fas and Fas ligand expressions in human esophageal cancer. Clin Cancer Res, 1999. 5(9): p. 2464-9.
    45. Koomagi, R. and M. Volm, Expression of Fas (CD95/APO-1) and Fas ligand in lung cancer, its prognostic and predictive relevance. Int J Cancer, 1999. 84(3): p. 239-43.
    46. Mottolese, M., et al., Prognostic relevance of altered Fas (CD95)-system in human breast cancer. Int J Cancer, 2000. 89(2): p. 127-32.
    47. Chio, C.C., et al., Down-regulation of Fas-L in glioma cells by ribozyme reduces cell apoptosis, tumour-infiltrating cells, and liver damage but accelerates tumour formation in nude mice. Br J Cancer, 2001. 85(8): p. 1185-92.
    48. Hung, J.Y., The role of Fas ligand in the cross-talk of tumor cells and fibroblasts during tumor encapsulation, in Department of Microbiology and Immunology, College of Medicine. 2015, National Cheng Kung University: Tainan. p. 65.
    49. Burleson, K.M., et al., Ovarian carcinoma ascites spheroids adhere to extracellular matrix components and mesothelial cell monolayers. Gynecol Oncol, 2004. 93(1): p. 170-81.
    50. Mueller-Klieser, W., Three-dimensional cell cultures: from molecular mechanisms to clinical applications. Am J Physiol, 1997. 273(4 Pt 1): p. C1109-23.
    51. Liao, C.P., et al., Cancer-associated fibroblasts enhance the gland-forming capability of prostate cancer stem cells. Cancer Res, 2010. 70(18): p. 7294-303.
    52. Brattain, M.G., et al., Enhancement of growth of human colon tumor cell lines by feeder layers of murine fibroblasts. J Natl Cancer Inst, 1982. 69(4): p. 767-71.
    53. Nakashiro, K., et al., Hepatocyte growth factor secreted by prostate-derived stromal cells stimulates growth of androgen-independent human prostatic carcinoma cells. Am J Pathol, 2000. 157(3): p. 795-803.
    54. Kim, S.A., E.K. Lee, and H.J. Kuh, Co-culture of 3D tumor spheroids with fibroblasts as a model for epithelial-mesenchymal transition in vitro. Exp Cell Res, 2015. 335(2): p. 187-96.
    55. Chen, H., et al., TGF-beta induces fibroblast activation protein expression; fibroblast activation protein expression increases the proliferation, adhesion, and migration of HO-8910PM [corrected]. Exp Mol Pathol, 2009. 87(3): p. 189-94.
    56. Wu, I.C., Fas Ligand Expression Alters Tumor Metastasis In B16F10 Cells, in Department of Microbiology and Immunology, College of Medicine. 2005, National Cheng Kung University: Tainan. p. 71.
    57. Hawinkels, L.J., et al., Interaction with colon cancer cells hyperactivates TGF-beta signaling in cancer-associated fibroblasts. Oncogene, 2014. 33(1): p. 97-107.
    58. Kim, R., et al., The role of Fas ligand and transforming growth factor beta in tumor progression: molecular mechanisms of immune privilege via Fas-mediated apoptosis and potential targets for cancer therapy. Cancer, 2004. 100(11): p. 2281-91.
    59. Costea, D.E., et al., Identification of two distinct carcinoma-associated fibroblast subtypes with differential tumor-promoting abilities in oral squamous cell carcinoma. Cancer Res, 2013. 73(13): p. 3888-901.
    60. Thiery, J.P., Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer, 2002. 2(6): p. 442-54.
    61. van Zijl, F., et al., Hepatic tumor-stroma crosstalk guides epithelial to mesenchymal transition at the tumor edge. Oncogene, 2009. 28(45): p. 4022-33.
    62. Lin, H.C., A Novel Signaling Pathway for FasL:FasL Hijacks Met receptor to Enhance Cell Motility and Metastasis, in The Institute of Basic Medical Sciences. 2013, National Cheng Kung University: Tainan. p. 74.
    63. Lin, H.C., et al., Fas ligand enhances malignant behavior of tumor cells through interaction with Met, hepatocyte growth factor receptor, in lipid rafts. J Biol Chem, 2012. 287(24): p. 20664-73.

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