癌症幹細胞也被稱為腫瘤起始細胞，具有自我複製和分化的能力，能夠分化成癌細胞且維持腫瘤的生長。然而癌症幹細胞為腫瘤內的小細胞群，且對於癌症治療具有較高耐受性，為了制定有效治療癌症幹細胞的策略，必須要開發出新的技術來區分癌症幹細胞及癌細胞。利用噬菌體展示技術篩選出一組癌症幹細胞標靶胜肽，這些癌症幹細胞標靶胜肽可以識別小鼠的結腸和肝臟癌症幹細胞以及人類胰腺的癌症幹細胞。後續實驗使用醣晶片檢測發現CSC HP-1和CSC HP-hP1主要結合具有Globo四糖根結構的聚醣，如Globo 5和Globo H。而CSC HP-6和HP-9這兩種胜肽則是能結合人類多能幹細胞上的特殊表面多醣SSEA-5。我們利用核磁共振 (Nuclear Magnetic Resonance, NMR) 的滴定實驗研究胜肽與多醣之間的結合及識別機制，首先使用TOCSY (Total Correlation Spectroscopy) 和ROESY (Rotating-frame Effect Overhauser Spectroscopy) 光譜做癌症幹細胞標靶胜肽的光譜訊號標記，再將多醣滴定到胜肽溶液中觀察光譜變化，評估各個胺基酸的化學位移變化或訊號強度變化，確定了標靶胜肽和多醣之間的重要相互作用以及負責結合的關鍵胺基酸。為了進一步研究兩個分子之間的結合模式，我們設計了可生產蛋白質嵌合體的大腸桿菌菌株，將不同的標靶胜肽與泛素 (Ubiquitin) 的C端接合。此菌株可用於製備同位素標記的蛋白質嵌合體，再透過異核核磁共振光譜進行滴定實驗以確認胜肽與多醣的交互作用，然而沒有檢測到蛋白質嵌合體與多醣有直接交互作用，因此我們在此篇研究討論了可能的原因。

Cancer stem cells (CSCs), known as tumor initiation cells, are operationally defined by their exclusive ability to initiate and maintain tumor growth. However, CSCs are general small populations of neoplastic cells within a tumor. To differentiate the CSCs from the normal tumor cells becomes a challenging task and meanwhile, a critical issue for future cancer therapy. A set of cancer stem cell homing peptides (CSC HPs) were screened from a phage displayed random peptide library. These CSC HPs can recognize mouse colon and liver CSCs as well as human pancreatic CSCs. By using glycan microarray chips, two peptides of CSC HP-1 and CSC HP-hP1 were predominantly targeted to the glycans containing Globo tetra-saccharide root structure, such as Globo 5 and Globo H. In addition, two peptides of CSC HP-6 and HP-9 were targeted to SSEA-5, a special surface glycan on human pluripotent stem cells. To confirm the binding and understand the recognition mechanism, we study the interaction through Nuclear Magnetic Resonance (NMR) titration experiments. We firstly used conventional assignment strategy by employing TOCSY (Total Correlation Spectroscopy) and ROESY (Rotating-frame Overhauser Effect Spectroscopy) experiments to finish the assignments of each CSC HP. Glycans were further titrated into the individual peptide solutions to assess the chemical shift or intensity changes. We identified the critical residues responsible for binding. These data revealed the direct evidence of these CSC HPs recognizing the cancer stem cell surface glycans. In addition, we design protein chimeras that the different CSC HPs were fused to the C-terminus of Ubiquitin. The constructs were used to prepare isotope-labeled samples to obtain the detail of the interactions through heteronuclear NMR spectra. However, the direct interaction was not detected in the cases. We discussed the possible reasons here.

ABSTRACT I
中文摘要 II
圖目錄 II
表格目錄 III
英文縮寫 IV
中英文對照 V
第一章、 前言 1
1.1 癌症幹細胞 1
1.2 癌症幹細胞標靶胜肽 3
1.3 癌症幹細胞表面聚醣與標靶胜肽的交互作用 5
1.4 研究目的 6
第二章、 材料與方法 13
2.1 胜肽與多醣的製備 13
2.2 核磁共振1H-1H二維光譜滴定實驗 13
2.3 胜肽接合至泛素C端的大腸桿菌克隆 14
2.4 13C-15N標定之蛋白質嵌合體表現純化 15
2.5 核磁共振15N-1H二維光譜滴定實驗 16
第三章、 結果與討論 24
3.1 胜肽1H-1H二維光譜訊號標定 24
3.2 胜肽與多醣的1H-1H二維光譜滴定實驗 25
3.3 標靶胜肽與多醣之間的解離常數 27
3.4 蛋白質嵌合體15N-1H二維光譜訊號標定 29
3.5 蛋白質嵌合體與多醣的15N-1H二維光譜滴定實驗 29
第四章、 結論 31
參考文獻 65

1. Clarke MF, Dick JE, Dirks PB, Eaves CJ, Jamieson CH, Jones DL, et al. Cancer stem cells--perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res. 2006;66(19):9339-44. Epub 2006/09/23. doi: 10.1158/0008-5472.CAN-06-3126. PubMed PMID: 16990346.
2. Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, Caceres-Cortes J, et al. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature. 1994;367(6464):645-8. Epub 1994/02/17. doi: 10.1038/367645a0. PubMed PMID: 7509044.
3. 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;100(7):3983-8. Epub 2003/03/12. doi: 10.1073/pnas.0530291100. PubMed PMID: 12629218; PubMed Central PMCID: PMCPMC153034.
4. Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J, et al. Identification of a cancer stem cell in human brain tumors. Cancer Res. 2003;63(18):5821-8. Epub 2003/10/03. PubMed PMID: 14522905.
5. Dalerba P, Dylla SJ, Park IK, Liu R, Wang X, Cho RW, et al. Phenotypic characterization of human colorectal cancer stem cells. Proc Natl Acad Sci U S A. 2007;104(24):10158-63. Epub 2007/06/06. doi: 10.1073/pnas.0703478104. PubMed PMID: 17548814; PubMed Central PMCID: PMCPMC1891215.
6. O'Brien CA, Pollett A, Gallinger S, Dick JE. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature. 2007;445(7123):106-10. Epub 2006/11/24. doi: 10.1038/nature05372. PubMed PMID: 17122772.
7. Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peschle C, et al. Identification and expansion of human colon-cancer-initiating cells. Nature. 2007;445(7123):111-5. Epub 2006/11/24. doi: 10.1038/nature05384. PubMed PMID: 17122771.
8. Vinogradov S, Wei X. Cancer stem cells and drug resistance: the potential of nanomedicine. Nanomedicine (Lond). 2012;7(4):597-615. Epub 2012/04/05. doi: 10.2217/nnm.12.22. PubMed PMID: 22471722; PubMed Central PMCID: PMCPMC3376090.
9. Bomken S, Fiser K, Heidenreich O, Vormoor J. Understanding the cancer stem cell. Br J Cancer. 2010;103(4):439-45. Epub 2010/07/29. doi: 10.1038/sj.bjc.6605821. PubMed PMID: 20664590; PubMed Central PMCID: PMCPMC2939794.
10. Greve B, Kelsch R, Spaniol K, Eich HT, Gotte M. Flow cytometry in cancer stem cell analysis and separation. Cytometry A. 2012;81(4):284-93. Epub 2012/02/09. doi: 10.1002/cyto.a.22022. PubMed PMID: 22311742.
11. Vlieghe P, Lisowski V, Martinez J, Khrestchatisky M. Synthetic therapeutic peptides: science and market. Drug Discov Today. 2010;15(1-2):40-56. Epub 2009/11/03. doi: 10.1016/j.drudis.2009.10.009. PubMed PMID: 19879957.
12. Gautam A, Kapoor P, Chaudhary K, Kumar R, Raghava GPS, Consort OSDD. Tumor Homing Peptides as Molecular Probes for Cancer Therapeutics, Diagnostics and Theranostics. Curr Med Chem. 2014;21(21):2367-91. doi: Doi 10.2174/0929867321666140217122100. PubMed PMID: WOS:000337103400005.
13. Smith GP. Filamentous Fusion Phage - Novel Expression Vectors That Display Cloned Antigens on the Virion Surface. Science. 1985;228(4705):1315-7. doi: DOI 10.1126/science.4001944. PubMed PMID: WOS:A1985AJR1500030.
14. Kehoe JW, Kay BK. Filamentous phage display in the new millennium. Chem Rev. 2005;105(11):4056-72. Epub 2005/11/10. doi: 10.1021/cr000261r. PubMed PMID: 16277371.
15. Urbanelli L, Ronchini C, Fontana L, Menard S, Orlandi R, Monaci P. Targeted gene transduction of mammalian cells expressing the HER2/neu receptor by filamentous phage. J Mol Biol. 2001;313(5):965-76. Epub 2001/11/09. doi: 10.1006/jmbi.2001.5111. PubMed PMID: 11700053.
16. Ding H, Prodinger WM, Kopecek J. Identification of CD21-binding peptides with phage display and investigation of binding properties of HPMA copolymer-peptide conjugates. Bioconjug Chem. 2006;17(2):514-23. Epub 2006/03/16. doi: 10.1021/bc0503162. PubMed PMID: 16536485.
17. Spear MA, Breakefield XO, Beltzer J, Schuback D, Weissleder R, Pardo FS, et al. Isolation, characterization, and recovery of small peptide phage display epitopes selected against viable malignant glioma cells. Cancer Gene Ther. 2001;8(7):506-11. Epub 2001/08/11. doi: 10.1038/sj.cgt.7700334. PubMed PMID: 11498772.
18. Jiang YQ, Wang HR, Li HP, Hao HJ, Zheng YL, Gu J. Targeting of hepatoma cell and suppression of tumor growth by a novel 12mer peptide fused to superantigen TSST-1. Mol Med. 2006;12(4-6):81-7. doi: 10.2119/2006-00011.Jiang. PubMed PMID: WOS:000240631200003.
19. Lee TY, Wu HC, Tseng YL, Lin CT. A novel peptide specifically binding to nasopharyngeal carcinoma for targeted drug delivery. Cancer Research. 2004;64(21):8002-8. doi: Doi 10.1158/0008-5472.Can-04-1948. PubMed PMID: WOS:000224790600047.
20. Chang DK, Lin CT, Wu CH, Wu HC. A novel peptide enhances therapeutic efficacy of liposomal anti-cancer drugs in mice models of human lung cancer. PLoS One. 2009;4(1):e4171. Epub 2009/01/13. doi: 10.1371/journal.pone.0004171. PubMed PMID: 19137069; PubMed Central PMCID: PMCPMC2614347.
21. McGuire MJ, Gray BP, Li S, Cupka D, Byers LA, Wu L, et al. Identification and characterization of a suite of tumor targeting peptides for non-small cell lung cancer. Sci Rep. 2014;4:4480. Epub 2014/03/29. doi: 10.1038/srep04480. PubMed PMID: 24670678; PubMed Central PMCID: PMCPMC3967199.
22. Chi YH, Hsiao JK, Lin MH, Chang C, Lan CH, Wu HC. Lung Cancer-Targeting Peptides with Multi-subtype Indication for Combinational Drug Delivery and Molecular Imaging. Theranostics. 2017;7(6):1612-32. doi: 10.7150/thno.17573. PubMed PMID: WOS:000398783200015.
23. Karmali PP, Kotamraju VR, Kastantin M, Black M, Missirlis D, Tirrell M, et al. Targeting of albumin-embedded paclitaxel nanoparticles to tumors. Nanomedicine. 2009;5(1):73-82. Epub 2008/10/03. doi: 10.1016/j.nano.2008.07.007. PubMed PMID: 18829396; PubMed Central PMCID: PMCPMC2824435.
24. Kapoor P, Singh H, Gautam A, Chaudhary K, Kumar R, Raghava GP. TumorHoPe: a database of tumor homing peptides. PLoS One. 2012;7(4):e35187. Epub 2012/04/24. doi: 10.1371/journal.pone.0035187. PubMed PMID: 22523575; PubMed Central PMCID: PMCPMC3327652.
25. Su YH, Lin TY, Liu HJ, Chuang CK. A set of cancer stem cell homing peptides associating with the glycan moieties of glycosphingolipids. Oncotarget. 2018;9(29):20490-507. Epub 2018/05/15. doi: 10.18632/oncotarget.24960. PubMed PMID: 29755667; PubMed Central PMCID: PMCPMC5945507.
26. Merrill AH, Jr. Sphingolipid and glycosphingolipid metabolic pathways in the era of sphingolipidomics. Chem Rev. 2011;111(10):6387-422. Epub 2011/09/29. doi: 10.1021/cr2002917. PubMed PMID: 21942574; PubMed Central PMCID: PMCPMC3191729.
27. Chang WW, Lee CH, Lee P, Lin J, Hsu CW, Hung JT, et al. Expression of Globo H and SSEA3 in breast cancer stem cells and the involvement of fucosyl transferases 1 and 2 in Globo H synthesis. Proc Natl Acad Sci U S A. 2008;105(33):11667-72. Epub 2008/08/08. doi: 10.1073/pnas.0804979105. PubMed PMID: 18685093; PubMed Central PMCID: PMCPMC2575305.
28. Cheung SK, Chuang PK, Huang HW, Hwang-Verslues WW, Cho CH, Yang WB, et al. Stage-specific embryonic antigen-3 (SSEA-3) and beta3GalT5 are cancer specific and significant markers for breast cancer stem cells. Proc Natl Acad Sci U S A. 2016;113(4):960-5. Epub 2015/12/19. doi: 10.1073/pnas.1522602113. PubMed PMID: 26677875; PubMed Central PMCID: PMCPMC4743801.
29. Suzuki Y, Haraguchi N, Takahashi H, Uemura M, Nishimura J, Hata T, et al. SSEA-3 as a novel amplifying cancer cell surface marker in colorectal cancers. Int J Oncol. 2013;42(1):161-7. doi: 10.3892/ijo.2012.1713. PubMed PMID: WOS:000312566900018.
30. Son MJ, Woolard K, Nam DH, Lee J, Fine HA. SSEA-1 Is an Enrichment Marker for Tumor-Initiating Cells in Human Glioblastoma. Cell Stem Cell. 2009;4(5):440-52. doi: 10.1016/j.stem.2009.03.003. PubMed PMID: WOS:000266071100012.
31. Tang C, Lee AS, Volkmer JP, Sahoo D, Nag D, Mosley AR, et al. An antibody against SSEA-5 glycan on human pluripotent stem cells enables removal of teratoma-forming cells. Nat Biotechnol. 2011;29(9):829-34. Epub 2011/08/16. doi: 10.1038/nbt.1947. PubMed PMID: 21841799; PubMed Central PMCID: PMCPMC3537836.
32. Karsten U, Goletz S. What makes cancer stem cell markers different? Springerplus. 2013;2. doi: Artn 301
10.1186/2193-1801-2-301. PubMed PMID: WOS:000209465000099.
33. Bax A, Davis DG. Mlev-17-Based Two-Dimensional Homonuclear Magnetization Transfer Spectroscopy. J Magn Reson. 1985;65(2):355-60. doi: Doi 10.1016/0022-2364(85)90018-6. PubMed PMID: WOS:A1985AVG0100018.
34. Piotto M, Saudek V, Sklenar V. Gradient-Tailored Excitation for Single-Quantum Nmr-Spectroscopy of Aqueous-Solutions. J Biomol Nmr. 1992;2(6):661-5. doi: Doi 10.1007/Bf02192855. PubMed PMID: WOS:A1992KD16100013.
35. Sklenar V, Piotto M, Leppik R, Saudek V. Gradient-Tailored Water Suppression for H-1-N-15 Hsqc Experiments Optimized to Retain Full Sensitivity. J Magn Reson Ser A. 1993;102(2):241-5. doi: DOI 10.1006/jmra.1993.1098. PubMed PMID: WOS:A1993KZ48000018.
36. Bax A, Davis DG. Practical Aspects of Two-Dimensional Transverse Noe Spectroscopy. J Magn Reson. 1985;63(1):207-13. doi: Doi 10.1016/0022-2364(85)90171-4. PubMed PMID: WOS:A1985AKQ4800024.
37. Gross KH, Kalbitzer HR. Distribution of Chemical-Shifts in H-1 Nuclear Magnetic-Resonance Spectra of Proteins. J Magn Reson. 1988;76(1):87-99. doi: Doi 10.1016/0022-2364(88)90203-X. PubMed PMID: WOS:A1988L955200009.
38. Wuthrich K. Sequential individual resonance assignments in the 1H-nmr spectra of polypeptides and proteins. Biopolymers. 1983;22(1):131-8. Epub 1983/01/01. doi: 10.1002/bip.360220121. PubMed PMID: 6673752.
39. Bax A, Ikura M. An efficient 3D NMR technique for correlating the proton and 15N backbone amide resonances with the alpha-carbon of the preceding residue in uniformly 15N/13C enriched proteins. J Biomol Nmr. 1991;1(1):99-104. Epub 1991/05/01. PubMed PMID: 1668719.
40. Palmer AG, 3rd, Fairbrother WJ, Cavanagh J, Wright PE, Rance M. Improved resolution in three-dimensional constant-time triple resonance NMR spectroscopy of proteins. J Biomol Nmr. 1992;2(1):103-8. Epub 1992/01/01. PubMed PMID: 1422144.
41. Kleckner IR, Foster MP. An introduction to NMR-based approaches for measuring protein dynamics. Biochim Biophys Acta. 2011;1814(8):942-68. Epub 2010/11/10. doi: 10.1016/j.bbapap.2010.10.012. PubMed PMID: 21059410; PubMed Central PMCID: PMCPMC3061256.