成功大學博碩士論文系統

簡易檢索 / 詳目顯示

回結果列表

研究生：	郭佩芳 Kuo, Pei-Fang
論文名稱：	鑑定食道鱗狀上皮細胞癌癌症幹細胞之特性與其對於合併化放療之反應 Identification and characterization of esophageal squamous cell carcinoma cancer stem cells and their chemoradiation therapy
指導教授：	呂佩融 Lu, Pei-Jung
學位類別：	碩士 Master
系所名稱：	醫學院 - 臨床醫學研究所 Institute of Clinical Medicine
論文出版年：	2013
畢業學年度：	101
語文別：	英文
論文頁數：	94
中文關鍵詞：	癌幹細胞、食道癌、合併化放療抗性
外文關鍵詞：	CSC, ESCC, CCRT resistance
相關次數：	點閱：137 下載：0
分享至:	分享至facebook 分享至twitter

查詢本校圖書館目錄查詢臺灣博碩士論文知識加值系統勘誤回報

食道癌可區分成鱗狀細胞癌和腺癌，只有少數的食道癌病患能利用手術治療，其餘病患則需依靠放射線治療以及化學治療。但即使經過切除手術以及合併化學和放射線治療，病患5年的存活率仍低於25％。癌症幹細胞被認為具有和正常幹細胞一樣的細胞特性和分子特徵。而在許多癌症中，已有數個和癌症幹細胞相關的生物標誌被證實與病人的癒後較差有關，但食道癌幹細胞相關的生物標記仍未被發現。在此研究中，我們的研究目的是探討食道鱗狀上皮細胞癌的癌症幹細胞對於合併化放療的抗性，並探討新穎的生物標誌以應用於食道鳞狀上皮細胞癌病人的合併化放療反應及癒後情形。為了此研究目標我們建立了一個能增加癌症幹細胞比例的腫瘤細胞球團培養平台。功能性分析方面，證實腫瘤細胞球團具有自我更新，腫瘤發生，分化以及順鉑抗藥性。基因表現維陣列分析以及即時定量聚合酶連鎖反應顯示腫瘤細胞團中與幹細胞特性及抗藥性相關的基因有表現量上升。相反的，腫瘤細胞團中與分化及上皮間質轉化的基因表現則受到抑制，同時顯示食道鳞狀上皮細胞株所形成的腫瘤球團具有上皮細胞屬性。我們也發現CLDN4，CLDN7 和 ABCB1具有作為鱗狀上皮細胞癌幹細胞生物標誌的潛力，利用這些基因的mRNA表現，公開的資料庫也發現，與其他癌症病人有較差的癒後相關。另一方面腫瘤細胞球團在細胞及動物模式中對合併化放療都具有更佳的抗性，證實腫瘤細胞團同時具有幹細胞特性及合併化放療抗性。我們也發現合併化放療性的細胞株，並不具有幹細胞特性。綜合以上實驗結果，我們證明腫瘤細胞球團具有幹細胞特性及合併化放療抗性，並且CLDN4，CLDN7 和 ABCB1具有作為食道鱗狀上皮細胞癌幹細胞的生物標誌潛力，在未來可能利用來預測病人的合併化放療的預後狀況。

Squamous cell cancer and adenocarcinoma is the major cancer type in esophageal cancers. Even after surgery and/or concurrent chemoradiation therapy (CCRT), 5-year survival of patients is rarely >25%. Cancer stem cells (CSCs) were responsible for tumor initiation and progression in different cancers. CSCs were believed exhibiting the cellular and molecular characteristics of normal stem cells. Several CSC markers have been shown to associate with poor prognosis of many cancers. However, the correlation between CSC markers and tumor progression of ESCC remains unclear. In this study, we aimed to investigate the role CSCs of esophageal squamous cell carcinoma (ESCC) in CCRT resistance, and to explore the novel biomarkers that were associated with CCRT response and prognosis of ESCC. To accomplish this goal, we developed an in vitro tumor sphere culture, which enriched CSCs in the tumor spheres. Functional assays demonstrated that the tumor spheres had the properties of self-renewal, tumor initiating, differentiation, and cisplatin resistance. Gene expression microarray and quantitative real-time PCR confirmed that the stemness- and drug resistant-associated genes were up-regulated in the tumor spheres. In contrast, the genes involved in differentiation and epithelial-to mesenchymal transition were down-regulated in the tumor spheres, and the tumor spheres of ESCC cell lines belonged to the epithelial type. In addition, CLDN4, CLDN7 and ABCB1 were identified as the potential biomarkers of ESCC CSCs, and their mRNA expression levels were correlated with poor prognosis of the patients in different cancer types from the public available database. On the other hand, in vitro and in vivo experiments demonstrated that the tumor-sphere cultures and tumor sphere-generated tumors in nude mice were more resistant to CCRT, indicating that the tumor spheres had both stemness and CCRT-resistant properties. However, we also found that the CCRT-resistance cell lines did not had stemness properties. Together, we demonstrated that the tumor spheres, which were enriched of CSCs, had stemness and CCRT-resistant properties, and CLDN4, CLDN7 and ABCB1 were identified as the potential biomarkers of ESCC CSCs, which could be used to predict the prognosis and CCRT response of ESCC patients in the future.

Abstract-----------------------------------------------	I
中文摘要-------------------------------------------------	III
誌謝----------------------------------------------------	V
Abbreviations------------------------------------------	1
Introduction-------------------------------------------	2
Materials and Methods----------------------------------	10
Results------------------------------------------------	20
Conclusions and Discussion-----------------------------	39
Figure and Table---------------------------------------	44
Figure 1. Identification of tumor sphere culture harbored self-renewal property in ESCC cell lines.--------------	44
Figure 2. Compared with parental cells, tumor spheres have better tumor initiation capacity.----------------------	45
Figure  3. Tumor spheres exhibited differentiation property.	--------------------------------------------------------48
Figure 4. Tumor sphere cells were resistant to cisplatin than parental cells.-----------------------------------	49
Figure 5. Compare with parental cell, the up-regulated  biological events and genes of tumor sphere  from Gene Ontology (GO) analysis.--------------------------------	50
Figure 6. Compare with parental cell, the down-regulated  biological events and genes of tumor sphere  from Gene Ontology (GO) analysis.--------------------------------	51
Figure 7. The gene expression profiles of stemness, drug-resistant, EMT  and Epithelial differentiation association genes--------------------------------------------------	53
Figure 8. The stemness medium culture did not change stemness- associated genes expression and proteins level.	--------------------------------------------------------55
Figure 9. The tumor spheres harbored stemness-associated genes expression properties.---------------------------	56
Figure 10. The protein expression level of stemness and drug-resistant associated genes were up-regulated in tumor spheres of ESCC cell lines.----------------------------	57
Figure 11. ESCC CSC-like cells exhibited epithelial-like phenotype.---------------------------------------------	58
Figure 12. TGF-β can induce EMT and decrease p53 level in CE48T cell line.---------------------------------------	59
Figure 13. Quantitative RT-PCR analysis of DSG3, GJB5, CLDN4 and CLDN7 were up-regulation in tumor spheres of ESCC cell lines.-------------------------------------------------	60
Figure 14. Surface protein expression of DSG3, CLDN4, CLDN7 and ABCB1 were up-regulation in tumor spheres of ESCC cell lines.-------------------------------------------------	61
Figure 15. The association of DSG3, ABCB1, CLDN4 and CLDN7 with poor cancer prognosis-----------------------------	63
Figure 16. CSCs can not charicterized from CD44/CD24 phenotype in ESCC cell lines.--------------------------	65
Figure 17. Tumor sphere cells showed more CCRT-resistant ability than parental cells.---------------------------	66
Figure 18. Tumor sphere-drivered tumors were more resistant to CCRT in vivo----------------------------------------	68
Figure 19. CE48T CCRT-resistant cells were more radio- resistant and  cisplatin -resistant than parental CE48T cells.-------------------------------------------------	69
Figure 20. KYSE70 CCRT-resistant cells were more CCRT-resistant than parental KYSE70 cells.------------------	70
Figure 21. The CE48T CCRT-resistant cells and parental cells showed similar sphere formation ability and stemness associated gene expression profile.--------------------	71
Figure 22. The KYSE70 CCRT-resistant cells did not distinguish with parental cells.-----------------------	72
Table 1. Primer of quantitative reverse transcription PCR	--------------------------------------------------------73
Table 2. Antibodies of immunoblot and flow cytometry---	75
References---------------------------------------------	76
Appendix-----------------------------------------------	84
Appendix 1.--------------------------------------------	84
Appendix 2.--------------------------------------------	85
Appendix 3.--------------------------------------------	86
Appendix 4.--------------------------------------------	87
Appendix 5.--------------------------------------------	88
Appendix 6.--------------------------------------------	90
Appendix 7.--------------------------------------------	91
Appendix 8.--------------------------------------------	92
Appendix 9.--------------------------------------------	93
Appendix 10.-------------------------------------------	94
                                    

1. Ke L. Mortality and incidence trends from esophagus cancer in selected geographic areas of China circa 1970-90.Int J Cancer. 2002 Nov 20;102(3):271-4.
2. Sitas F, Egger S, Urban MI, Taylor PR, Abnet CC, Boffetta P, O'Connell DL, Whiteman DC, Brennan P, Malekzadeh R, Pawlita M, Dawsey SM, Waterboer T; InterSCOPE Collaboration. InterSCOPE Study: Associations Between Esophageal Squamous Cell Carcinoma and Human Papillomavirus Serological Markers. J Natl Cancer Inst. 2012 Jan 18;104(2):147-58.
3. 行政院衛生署. 國人100年度衛生統計資料 http://www.doh.gov.tw/CHT2006/DM/DM2_2_p02.aspx?class_no=440&now_fod_list_no=11468&level_no=1&doc_no=77184. 2012.
4. Cameron AJ, Romero Y. Symptomatic gastro-oesophageal reflux as a risk factor for oesophageal adenocarcinoma. Gut. 2000 Jun;46(6):754-5.
5. American cancer society - information and resources for cancer http://www.cancer.org/acs/groups/cid/documents/webcontent/003098-pdf.pdf
6. Toh Y, Oki E, Ohgaki K, Sakamoto Y, Ito S, Egashira A, Saeki H, Kakeji Y, Morita M, Sakaguchi Y, Okamura T, Maehara Y. Alcohol drinking, cigarette smoking, and the development of squamous cell carcinoma of the esophagus: molecular mechanisms of carcinogenesis. Int J Clin Oncol. 2010 Apr;15(2):135-44.
7. Hsu PK, Wang BY, Huang CS, Wu YC, Hsu WH. Prognostic factors for post-recurrence survival in esophageal squamous cell carcinoma patients with recurrence after resection. J Gastrointest Surg. 2011 Apr;15(4):558-65.
8. Kato H, Fukuchi M, Miyazaki T, Nakajima M, Kimura H, Faried A, Sohda M, Fukai Y, Masuda N, Manda R, Ojima H, Tsukada K, Kuwano H. Classification of Recurrent Esophageal Cancer after Radical Esophagectomy with Two- or Three-field Lymphadenectomy. Anticancer Res. 2005 Sep-Oct;25(5):3461-7.
9. The National Comprehensive Cancer Network. http://www.nccn.org/index.asp. 2012
10. Scarpa M, Valente S, Alfieri R, Cagol M, Diamantis G, Ancona E, Castoro C. Systematic review of health-related quality of life after esophagectomy for esophageal cancer. World J Gastroenterol. 2011 Nov 14;17(42):4660-74.
11. Lv J, Cao XF, Zhu B, Ji L, Tao L, Wang DD. Effect of neoadjuvant chemoradiotherapy on prognosis and surgery for esophageal carcinoma. World J Gastroenterol. 2009 Oct 21;15(39):4962-8.
12. Comprehensive Cancer Information - National Cancer Institute http://seer.cancer.gov/csr/1975_2009_pops09/index.html. 2012.
13. Vaiopoulos AG, Kostakis ID, Koutsilieris M, Papavassiliou AG. Colorectal cancer stem cells. Stem Cells. 2012 Mar;30(3):363-71.
14. Anderson EC, Hessman C, Levin TG, Monroe MM, Wong MH. The Role of Colorectal Cancer Stem Cells in Metastatic Disease and Therapeutic Response. Cancers (Basel). 2011 Jan 1;3(1):319-339.
15. SEER Stat Fact Sheets: Esophagus. http://seer.cancer.gov/statfacts/html/esoph.html
16. Siegel R, Ward E, Brawley O, Jemal A. Cancer statistics, 2011: the impact of eliminating socioeconomic and racial disparities on premature cancer deaths. CA Cancer J Clin. 2011 Jul-Aug;61(4):212-36.
17. Clevers H. The cancer stem cell: premises, promises and challenges. Nat Med. 2011 Mar;17(3):313-9.
18. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med. 1997 Jul;3(7):730-7.
19. Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, Henkelman RM, Cusimano MD, Dirks PB. Identification of human brain tumour initiating cells. Nature. 2004 Nov 18;432(7015):396-401.
20. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A. 2003 Apr 1;100(7):3983-8. Epub 2003 Mar 10.
21. Alvarez M. A question of supply and demand. Nature. 2007 Jan 4;445(7123):124.
22. Li C, Heidt DG, Dalerba P, Burant CF, Zhang L, Adsay V, Wicha M, Clarke MF, Simeone DM. Identification of pancreatic cancer stem cells. Cancer Res. 2007 Feb 1;67(3):1030-7.
23. Zhang S, Balch C, Chan MW, Lai HC, Matei D, Schilder JM, Yan PS, Huang TH, Nephew KP. Identification and characterization of ovarian cancer-initiating cells from primary human tumors. Cancer Res. 2008 Jun 1;68(11):4311-20.
24. Maitland NJ, Collins AT. Prostate cancer stem cells: a new target for therapy. J Clin Oncol. 2008 Jun 10;26(17):2862-70. doi: 10.1200/JCO.2007.15.1472.
25. Lawson DA, Witte ON. Stem cells in prostate cancer initiation and progression.J Clin Invest. 2007;117(8):2044–2050
26. Seufferlein T, Ahn J, Krndija D, Lother U, Adler G, von Wichert G. Tumor biology and cancer therapy - an evolving relationship. Cell Commun Signal. 2009 Aug 13;7:19
27. Cheng TS, Chang LK, Howng SL, Lu PJ, Lee CI, Hong YR. SUMO-1 modification ofcentrosomal protein hNinein promotes hNinein nuclear localization. Life Sci. 2006 Feb 2;78(10):1114-20
28. Schepers AG, Snippert HJ, Stange DE, van den Born M, van Es JH, van de Wetering M, Clevers H. Lineage Tracing Reveals Lgr5+Stem Cell Activity in Mouse Intestinal Adenomas. Science. 2012 Aug 10;337(6095):730-5.
29. Driessens G, Beck B, Caauwe A, Simons BD, Blanpain C. Defining the mode of tumour growth by clonal analysis. Nature. 2012 Aug 23;488(7412):527-30.
30. Chen J, Li Y, Yu TS, McKay RM, Burns DK, Kernie SG, Parada LF. A restricted cell population propagates glioblastoma growth after chemotherapy. Nature. 2012 Aug 23;488(7412):522-6.
31. Gilbertson RJ, Graham TA. Cancer: Resolving the stem-cell debate.Nature. 2012 Aug 1.
32. Hu Y, Fu L. Targeting cancer stem cells: a new therapy to cure cancer patients. Am J Cancer Res 2012;2(3):340-356
33. Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, Brown M, Jacquemier J, Viens P, Kleer CG, Liu S, Schott A, Hayes D, Birnbaum D, Wicha MS, Dontu G. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell. 2007 Nov;1(5):555-67.
34. Huang SD, Yuan Y, Liu XH, Gong DJ, Bai CG, Wang F, Luo JH, Xu ZY. Self-renewal and chemotherapy resistance of p75NTR positive cells in esophageal squamous cell carcinomas. BMC Cancer. 2009 Jan 10;9:9.
35. Zhao JS, Li WJ, Ge D, Zhang PJ, Li JJ, Lu CL, Ji XD, Guan DX, Gao H, Xu LY, Li EM, Soukiasian H, Koeffler HP, Wang XF, Xie D. Tumor initiating cells in esophageal squamous cell carcinomas express high levels of CD44. PLoS One. 2011;6(6):e21419.
36. Soda Y, Marumoto T, Friedmann-Morvinski D, Soda M, Liu F, Michiue H, Pastorino S, Yang M, Hoffman RM, Kesari S, Verma IM. Transdifferentiation of glioblastoma cells into vascular endothelial cells. Proc Natl Acad Sci U S A. 2011 Mar 15;108(11):4274-80
37. Schepers AG, Snippert HJ, Stange DE, van den Born M, van Es JH, van de Wetering M, Clevers H. Lineage Tracing Reveals Lgr5+Stem Cell Activity in Mouse Intestinal Adenomas. Science. 2012 Aug 10;337(6095):730-5.
38. Mizuno H, Kitada K, Nakai K, Sarai A. PrognoScan: a new database for meta-analysis of the prognostic value of genes. BMC Med Genomics. 2009 Apr 24;2:18.
39. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A. 2003 Apr 1;100(7):3983-8.
40. Yi H, Cho HJ, Cho SM, Jo K, Park JA, Lee SH, Chang BJ, Kim JS, Shin HC. Effect of 5-FU and MTX on the Expression of Drug-resistance Related Cancer Stem Cell Markers in Non-small Cell Lung Cancer Cells. Korean J Physiol Pharmacol. 2012 Feb;16(1):11-6.
41. Lane DP. Cancer. p53, guardian of the genome. Nature. 1992 Jul 2;358(6381):15-6.
42. Levine AJ, Oren M. The first 30 years of p53: growing ever more complex. Nat Rev Cancer. 2009 Oct;9(10):749-58.
43. Zhao Z, Zuber J, Diaz-Flores E, Lintault L, Kogan SC, Shannon K, Lowe SW. p53 loss promotes acute myeloid leukemia by enabling aberrant self-renewal. Genes Dev. 2010 Jul 1;24(13):1389-402.
44. Mizuno H, Spike BT, Wahl GM, Levine AJ. Inactivation of p53 in breast cancers correlates with stem cell transcriptional signatures. Proc Natl Acad Sci U S A. 2010 Dec 28;107(52):22745-50.
45. S Singh A, Settleman J. EMT, cancer stem cells and drug resistance: an emerging axis of evil in the war on cancer. Oncogene. 2010 Aug 26;29(34):4741-51
46. May CD, Sphyris N, Evans KW, Werden SJ, Guo W, Mani SA. Epithelial-mesenchymal transition and cancer stem cells: a dangerously dynamic duo in breast cancer progression. Breast Cancer Res. 2011 Feb 8;13(1):202.
47. Chen C, Wei Y, Hummel M, Hoffmann TK, Gross M, Kaufmann AM, Albers AE. Evidence for Epithelial-Mesenchymal Transition in Cancer Stem Cells of Head and Neck Squamous Cell Carcinoma. PLoS One. 2011 Jan 27;6(1):e16466.
48. Tsai JH, Donaher JL, Murphy DA, Chau S, Yang J. Spatiotemporal regulation of epithelial-mesenchymal transition is essential for squamous cell carcinoma metastasis. Cancer Cell. 2012 Dec 11;22(6):725-36.
49. Rassouli FB, Matin MM, Bahrami AR, Ghaffarzadegan K, Cheshomi H, Lari S, Memar B, Kan MS. Evaluating stem and cancerous biomarkers in CD15+CD44 + KYSE30 cells. Tumour Biol. 2013 Jun 25.
50. Tang KH, Dai YD, Tong M, Chan YP, Kwan PS, Fu L, Qin YR, Tsao SW, Lung HL, Lung ML, Tong DK, Law S, Chan KW, Ma S, Guan XY.A CD90(+) tumor-initiating cell population with an aggressive signature and metastatic capacity in esophageal cancer. Cancer Res. 2013 Apr 1;73(7):2322-32.

校內：2023-12-31公開
校外：不公開電子論文尚未授權公開，紙本請查館藏目錄

簡易檢索 / 詳目顯示

相關論文