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系統識別號 U0026-0808201121455600
論文名稱(中文) 口腔癌表面抗原轉化與癌症幹細胞之分析
論文名稱(英文) Transition Effects of Stem Cell Markers in Oral Cancer Cell
校院名稱 成功大學
系所名稱(中) 口腔醫學研究所
系所名稱(英) Institute of Oral Medicine
學年度 99
學期 2
出版年 100
研究生(中文) 李宗儒
研究生(英文) Tsung-Ju Li
學號 T46984011
學位類別 碩士
語文別 英文
論文頁數 67頁
口試委員 指導教授-謝達斌
口試委員-陳玉玲
口試委員-袁國
中文關鍵字 口腔癌  癌症幹細胞  表面抗原轉化 
英文關鍵字 OEC-M1  CD133  CD44  CSCs  Transition  Oral cancer 
學科別分類
中文摘要 幹細胞是具有分裂增殖自己與分化成多種特定功能體細胞的兩種特性。有研究證實癌症幹細胞是從正常的幹細胞轉變而來。而近期研究發現口腔癌幹細胞的存在與癌症的復發性有關。故本研究的方向是找出口腔癌症幹細胞的候選人與其表面上特定抗原的關係。我們實驗室選擇兩種幹細胞的表面抗原CD133和CD44來篩選癌症幹細胞的候選人。在研究的過程中發現兩者抗原在台灣口腔癌的細胞株OEC-M1其實有上下游相關特性。
在Wildtype的OEC-M1中其細胞族群為CD133-/CD44+和CD133-/CD44-。利用流式細胞分選儀我們篩選出CD133+/CD44-細胞群後發現在培養過程中會分化出CD133+/CD44+的細胞群,最後分化成與Wildtype的OEC-M1類似的CD133-/CD44+和CD133-/CD44-抗原表現。在細胞的增生率實驗結果,帶有CD133抗原的細胞群明顯與沒有CD133抗原細胞群來的低。進一步的利用抗癌藥物的測試,我們也發現帶有CD133抗原表現的細胞群明顯對藥物的耐受度也比較高。在動物實驗的驗證下,我們成功的將1000顆帶有CD133抗原的細胞群在非肥胖糖尿病/重症联合免疫缺陷小鼠上長出新的腫瘤。先前有文獻證明癌症幹細胞比其它分化後的癌細胞容易產生瘤球(Tumorsphere),我們的實驗利用無血清的3D 培養方式,發現帶有CD133抗原表現的細胞群也較容易生成瘤球,間接證明帶有CD133抗原表現的細胞群是處於在上游。最後利用雷射掃描共軛焦顯微鏡來拍攝這些瘤球與從瘤球分化出來的細胞群,我們發現帶有癌症幹細胞特性的瘤球依然留著CD133幹細胞的標示,然而分化出來的細胞群缺乏CD133的標示,證實CD133抗原表現的細胞群確實是處於在上游。
以上的研究觀察到CD133與CD44幹細胞抗原間之轉換特性,也證實CD133是處於在上游的幹細胞抗原。希望這個發現在後續之後的口腔癌中能找出治療癌症的新思維,並有效降低癌症的復發。
英文摘要 Accumulation of evidence shows that only a small minority of cancer cells have stem cell properties – known as cancer stem cell (CSC) (Visvader and Lindeman 2008). Whether CSC truly exists within a solid tumor has always been a critically debated topic. Nevertheless, cancer subpopulations that have the capability for self-renewing and exhibit differentiation similar to stem cells were reported by many research groups using cell-line models and surgical specimens. In this study, we aimed to identify CSC-like candidates using a matrix of stem cell markers in an oral cancer cell line and characterize the dynamics of these markers in subsequent culture and in vivo growth.
In the primary oral cell lines studied, OEC-M1, we noticed that most wild-type OEC-M1 cells are CD133-/CD44+ (>50%) and CD133-/CD44-. We used FACS ARIA to purify the CD133+/CD44- subpopulation. It was discovered that cultivation of CD133+/CD44- for 9 days render a significant enrichment of the CD133+/CD44+ subpopulation. Upon culturing CD133+/CD44+ cells for 9 days, a significant decrease in the CD133+/CD44+ subpopulation (>80%) and increase in the CD133-/CD44+ (>80%) subpopulation was observed. In proliferation study, we noticed subpopulations with CD133 antigen showed a lower proliferation rate than non-CD133 subpopulations. As for cell toxicity test, subpopulations with CD133 markers showed higher tolerance to Cisplatin toxicity. Animal studies further showed that as few as 1,000 CD133+ cancer cells can generate tumor pulps in NOD/SCID mice. Previous studies show cancer stem cells have the capability of forming tumorspheres than differentiated cancer cells. We cultured different subpopulations in serum-free medium and obtained the same result published by other groups - CD133 populations were able to form tumorsphere easier than non-CD133 subpopulation. Using confocal microscopy, we further verified these tumorspheres could maintain CD133 marker while the differentiated cells that grow out from the sphere did not retain CD133 marker.
This possible transition between CD133 and CD44 stem cell maker can be study further to show a better understanding of cancer stem cell. In combination with animal study, we hope for a new clinical treatment by targeting this pathway and stop recurrence within patient.
論文目次 English abstract 1
Chinese abstract 3
Acknowledgement 5
Contents 6
Table contents 8
Figure contents 9
1. Introduction 11
1.1 Oral cancer 11
1.2 Cancer Heterogeneity Model 12
1.3 Cancer Stem Cell: History and Background 13
1.4 Cancer Stem Cell: Definition 14
1.5 Cancer and role of stem cell molecular marker 14
1.6 Rationale 15
2. Material and Methods 16
2.1 Cell culture and stem marker subpopulation maintenance 16
2.2 Proliferation and chemosensitivity assay 16
2.3 Tumor sphere formation assay 17
2.4 Flow cytometric analysis and cell sorting 17
2.5 Real-time reverse transcription-PCR 18
2.6 In vivo tumorigenic assay 19
2.7 Confocal study of tumorsphere stem cell marker 19
3. Results 21
3.1 CD133 marker subpopulation shows cancer stem cell phenotype in vitro 21
3.2 Drug resistance comparison between subpopulation 21
3.3 CD133 marker subpopulation favors tumor formation in vivo 22
3.4 CD133+/CD44- subpopulation present as upstream cancer stem cells 22
3.5 Transition of stem cell marker present different level of Oct-4, Bmi-1 expression 23
3.6 Upstream subpopulations have high level of EMT gene expression 23
3.7 Upstream subpopulations have high potential of forming tumorsphere 24
3.8 Tumorsphere shows ability to produce downstream subpopulation 24
4. Discussion 26
5. Summary 33
6. Reference 34
7. Figures and Legends 42
8. Background 66
參考文獻 Al-Hajj, M., M. S. Wicha, et al. "Prospective identification of tumorigenic breast cancer cells." Proceedings of the National Academy of Sciences 100(7): 3983-3988 (2003).

Bao, S., Q. Wu, et al. "Glioma stem cells promote radioresistance by preferential activation of the DNA damage response." Nature 444(7120): 756-760 (2006).

Bauer, N., A. V. Fonseca, et al. "New Insights into the Cell Biology of Hematopoietic Progenitors by Studying Prominin-1 (CD133)." Cells Tissues Organs 188(1-2): 127-138 (2008).

Bertolini, G., L. Roz, et al. "Highly tumorigenic lung cancer CD133+ cells display stem-like features and are spared by cisplatin treatment." Proceedings of the National Academy of Sciences 106(38): 16281-16286 (2009).

Charafe-Jauffret, E., F. Monville, et al. "Cancer Stem Cells in Breast: Current Opinion and Future Challenges." Pathobiology 75(2): 75-84 (2008).

Chen, Y.-C., H.-S. Hsu, et al. "Oct-4 Expression Maintained Cancer Stem-Like Properties in Lung Cancer-Derived CD133-Positive Cells." PLoS One 3(7): e2637 (2008).

Chiou, S.-H., C.-C. Yu, et al. "Positive Correlations of Oct-4 and Nanog in Oral Cancer Stem-Like Cells and High-Grade Oral Squamous Cell Carcinoma." Clinical Cancer Research 14(13): 4085-4095 (2008).

Chute, J. P., G. G. Muramoto, et al. "Inhibition of aldehyde dehydrogenase and retinoid signaling induces the expansion of human hematopoietic stem cells." Proceedings of the National Academy of Sciences 103(31): 11707-11712 (2006).

Clarke, M. F., J. E. Dick, et al. "Cancer Stem Cells—Perspectives on Current Status and Future Directions: AACR Workshop on Cancer Stem Cells." Cancer Research 66(19): 9339-9344 (2006).

Dembinski, J. and S. Krauss "Characterization and functional analysis of a slow cycling stem cell-like subpopulation in pancreas adenocarcinoma." Clinical and Experimental Metastasis 26(7): 611-623 (2009).

Dick, J. E. "Stem cell concepts renew cancer research." Blood 112(13): 4793-4807 (2008).

Dick, J. E. "Looking ahead in cancer stem cell research." Nat Biotech 27(1): 44-46 (2009).

Dontu, G., W. M. Abdallah, et al. "In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells." Genes & Development 17(10): 1253-1270 (2003).

Farrar, W. L. Cancer Stem Cells, Cambridge University Press. (2009).

Ganguly, A., H. Yang, et al. "Paclitaxel-Dependent Cell Lines Reveal a Novel Drug Activity." Molecular Cancer Therapeutics 9(11): 2914-2923 (2010).

Gao, M. Q., Y. P. Choi, et al. "CD24+ cells from hierarchically organized ovarian cancer are enriched in cancer stem cells." Oncogene 29(18): 2672-2680 (2010).

Haraguchi, N., M. Ohkuma, et al. "CD133+CD44+ population efficiently enriches colon cancer initiating cells." Ann Surg Oncol 15(10): 2927-2933 (2008).

Hill, R. P. and R. Perris. ""Destemming" cancer stem cells." J Natl Cancer Inst 99(19): 1435-1440 (2007).

Ho, M. M., A. V. Ng, et al. "Side Population in Human Lung Cancer Cell Lines and Tumors Is Enriched with Stem-like Cancer Cells." Cancer Research 67(10): 4827-4833 (2007).

Hollier, B., K. Evans, et al. The Epithelial-to-Mesenchymal Transition and Cancer Stem Cells: A Coalition Against Cancer Therapies." Journal of Mammary Gland Biology and Neoplasia 14(1): 29-43-43 (2009). "

Huang, S.-D., Y. Yuan, et al. "Self-renewal and chemotherapy resistance of p75NTR positive cells in esophageal squamous cell carcinomas." BMC Cancer 9(1): 9 (2009).

Hurt, E. M., K. Chan, et al. "Identification of Vitronectin as an Extrinsic Inducer of Cancer Stem Cell Differentiation and Tumor Formation." Stem Cells 28(3): 390-398 (2010).

Hurt, E. M., B. T. Kawasaki, et al. "CD44+CD24- prostate cells are early cancer progenitor/stem cells that provide a model for patients with poor prognosis." Br J Cancer 98(4): 756-765 (2008).

Jemal, A., T. Murray, et al. "Cancer Statistics, 2005." CA Cancer J Clin 55(1): 10-30 (2005).

Joshua, B., M. J. Kaplan, et al. "Frequency of cells expressing CD44, a Head and Neck cancer stem cell marker: Correlation with tumor aggressiveness." Head & Neck: n/a-n/a. (2011).

Kemper, K., M. R. Sprick, et al. "The AC133 Epitope, but not the CD133 Protein, Is Lost upon Cancer Stem Cell Differentiation." Cancer Research 70(2): 719-729 (2010).

Ko, Y. C., Y. L. Huang, et al. "Betel quid chewing, cigarette smoking and alcohol consumption related to oral cancer in Taiwan." J Oral Pathol Med 24(10): 450-453 (1995).

Kong-Chao, C., S. Ih-Jen, et al. "Pathological Features of Betel Quid-Related Oral Epithelial Lesions in Taiwan with Special Emphasis on the Tumor Progression and Human Papillomavirus Association." Oncology 63(4): 362-369 (2002).

Lapidot, T., C. Sirard, et al. "A cell initiating human acute myeloid leukaemia after transplantation into SCID mice." Nature 367(6464): 645-648 (1994).

Leemans, C. R., B. J. M. Braakhuis, et al. "The molecular biology of head and neck cancer." Nat Rev Cancer 11(1): 9-22 (2011).

Lin, W. J., R. S. Jiang, et al. "Smoking, alcohol, and betel quid and oral cancer: a prospective cohort study." J Oncol 2011: 525976 (2011).

Lo, W. L., S. Y. Kao, et al. "Outcomes of oral squamous cell carcinoma in Taiwan after surgical therapy: factors affecting survival." J Oral Maxillofac Surg 61(7): 751-758 (2003).

Lobo, N. A., Y. Shimono, et al. "The Biology of Cancer Stem Cells." Annual Review of Cell and Developmental Biology 23(1): 675-699 (2007).

Mack, B. and O. Gires (2008). "CD44s and CD44v6 Expression in Head and Neck Epithelia." PLoS One 3(10): e3360.

Mani, S. A., W. Guo, et al. "The Epithelial-Mesenchymal Transition Generates Cells with Properties of Stem Cells." Cell 133(4): 704-715 (2008).

Monroe, M. M., E. C. Anderson, et al. "Cancer stem cells in head and neck squamous cell carcinoma." J Oncol 2011: 762780 (2011).

Nagano, O. and H. Saya "Mechanism and biological significance of CD44 cleavage." Cancer Science 95(12): 930-935 (2004).

Niwa, H., J. Miyazaki, et al. "Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells." Nat Genet 24(4): 372-376 (2000).

Okamoto, A., K. Chikamatsu, et al. "Expansion and characterization of cancer stem-like cells in squamous cell carcinoma of the head and neck." Oral oncology 45(7): 633-639 (2009).

Park, I. K., S. J. Morrison, et al. "Bmi1, stem cells, and senescence regulation." J Clin Invest 113(2): 175-179 (2004).

Patrawala, L., T. Calhoun, et al. "Highly purified CD44+ prostate cancer cells from xenograft human tumors are enriched in tumorigenic and metastatic progenitor cells." Oncogene 25(12): 1696-1708 (2006).

Perez-Losada, J. and A. Balmain "Stem-cell hierarchy in skin cancer." Nat Rev Cancer 3(6): 434-443 (2003).

Pietersen, A. M., B. Evers, et al. "Bmi1 Regulates Stem Cells and Proliferation and Differentiation of Committed Cells in Mammary Epithelium." Current biology : CB 18(14): 1094-1099 (2008).

Prince, M. E., R. Sivanandan, et al. "Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma." Proceedings of the National Academy of Sciences 104(3): 973-978 (2007).

Reya, T., S. J. Morrison, et al. "Stem cells, cancer, and cancer stem cells." Nature 414(6859): 105-111 (2001).

Roesch, A., M. Fukunaga-Kalabis, et al. "A Temporarily Distinct Subpopulation of Slow-Cycling Melanoma Cells Is Required for Continuous Tumor Growth." Cell 141(4): 583-594 (2010).

Saigusa, S., K. Tanaka, et al. "Correlation of CD133, OCT4, and SOX2 in Rectal Cancer and Their Association with Distant Recurrence After Chemoradiotherapy." Annals of Surgical Oncology 16(12): 3488-3498-3498 (2009).

Saini, V. and R. H. Shoemaker "Potential for therapeutic targeting of tumor stem cells." Cancer Science 101(1): 16-21 (2010).

Sayed, S. I., R. C. Dwivedi, et al. "Implications of understanding cancer stem cell (CSC) biology in head and neck squamous cell cancer." Oral Oncol 47(4): 237-243 (2011).

Sell, S. "Stem cell origin of cancer and differentiation therapy." Critical Reviews in Oncology/Hematology 51(1): 1-28 (2004).

Shackleton, M., E. Quintana, et al. "Heterogeneity in Cancer: Cancer Stem Cells versus Clonal Evolution." Cell 138(5): 822-829 (2009).

Shmelkov, S. V., R. St.Clair, et al. "AC133/CD133/Prominin-1." The International Journal of Biochemistry & Cell Biology 37(4): 715-719 (2005).

Singh, A. and J. Settleman "EMT, cancer stem cells and drug resistance: an emerging axis of evil in the war on cancer." Oncogene 29(34): 4741-4751 (2010).

Singh, S. K., I. D. Clarke, et al. "Identification of a Cancer Stem Cell in Human Brain Tumors." Cancer Research 63(18): 5821-5828 (2003).

Singh, S. K., C. Hawkins, et al. "Identification of human brain tumour initiating cells." Nature 432(7015): 396-401 (2004).

Song, L. B., J. Li, et al. "The polycomb group protein Bmi-1 represses the tumor suppressor PTEN and induces epithelial-mesenchymal transition in human nasopharyngeal epithelial cells." J Clin Invest 119(12): 3626-3636 (2009).

Song, Z., W. Yue, et al. "Sonic Hedgehog Pathway Is Essential for Maintenance of Cancer Stem-Like Cells in Human Gastric Cancer." PLoS One 6(3): e17687 (2011).

Southam, C. M. and A. Brunschwig "Quantitative studies of autotransplantation of human cancer. Preliminary report." Cancer 14(5): 971-978 (1961).

Stordal, B. and M. Davey "Understanding cisplatin resistance using cellular models." IUBMB Life 59(11): 696-699 (2007).

Takaishi, S., T. Okumura, et al. "Identification of Gastric Cancer Stem Cells Using the Cell Surface Marker CD44." Stem Cells 27(5): 1006-1020 (2009).

Tsantoulis, P. K., N. G. Kastrinakis, et al. "Advances in the biology of oral cancer." Oral oncology 43(6): 523-534 (2007).

Visvader, J. E. and G. J. Lindeman "Cancer stem cells in solid tumours: accumulating evidence and unresolved questions." Nat Rev Cancer 8(10): 755-768 (2008).

Wang, R., K. Chadalavada, et al. "Glioblastoma stem-like cells give rise to tumour endothelium." Nature 468(7325): 829-833 (2010).

Xu, J., S. Lamouille, et al. "TGF-[beta]-induced epithelial to mesenchymal transition." Cell Res 19(2): 156-172 (2009).

Yang, Z.-J. and R. J. Wechsler-Reya "Hit 'Em Where They Live: Targeting the Cancer Stem Cell Niche." Cancer Cell 11(1): 3-5 (2007).

Yi, L., Z.-h. Zhou, et al. "Isolation and characterization of stem cell-like precursor cells from primary human anaplastic oligoastrocytoma." Mod Pathol 20(10): 1061-1068 (2007).

Yin, A. H., S. Miraglia, et al. "AC133, a Novel Marker for Human Hematopoietic Stem and Progenitor Cells." Blood 90(12): 5002-5012 (1997).

Zhang, Q., S. Shi, et al. "A subpopulation of CD133+ cancer stem-like cells characterized in human oral squamous cell carcinoma confer resistance to chemotherapy." Cancer letters 289(2): 151-160 (2010).

Zhou, L., X. Wei, et al. "CD133, One of the Markers of Cancer Stem Cells in Hep-2 Cell Line." The Laryngoscope 117(3): 455-460 (2007).

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