噬菌體展示技術乃是利用噬菌體外套膜上隨機胜肽鏈與目標分子鍵結力的不同，分離出對不同目標分子(或不同細胞)具有專一性、高親和力的噬菌體株，透過定序而得到其引導胜肽序列。此引導胜肽可以結合於病毒或非病毒載體，促進治療載體的專一性。也可以經由引導胜肽本身與腫瘤的某一特定分子結合，誘發或阻斷特定的訊息傳遞路徑，甚或導致腫瘤細胞的凋亡，來達到治療的目的。噬菌體展示技術在癌症的研究上已受到廣泛的注意並且蓬勃發展，已應用於部分腫瘤抗癌胜肽的篩選，如淋巴癌、乳癌、前列腺癌等，應用的範圍也越來越廣。目前口腔癌佔臺灣十大男性癌症的第四位，且其發生率有逐年增加的趨勢。口腔癌主要的治療方式為根除性手術或合併術後放射線治療及化學治療。然而近年來口腔癌患者的存活率並未顯著上升，其治療對臨床醫師仍深具挑戰性，也迫切需要新的治療方式。本研究主要在利用噬菌體展示技術，來篩選對口腔癌細胞具專一性的引導胜肽，作為未來發展口腔癌治療載體的基礎，並進一步研究其臨床運用的可行性。在本研究中，我們運用了這項技術，篩選出數十個對人類口腔癌細胞株(舌癌細胞株，HSC-3)具有高親和力的噬菌體株並加以定序。定序的結果並利用BLAST生物資訊工具與公開資料庫進行相似性序列比對，顯示某些噬菌體株所攜帶的引導胜肽，與許多重要的生物蛋白質有極為相似的胜肽序列。我們並利用各種免疫組織化學的方法證實了其中的一個噬菌體株(噬菌體株-29)，能夠藉由其所攜帶的引導胜肽和舌癌細胞表面的αvβ6結合而快速進入舌癌細胞中。此外，我們也證實了這個引導胜肽不只在人類的口腔癌，甚至在其他常見的頭頸部癌症都可能有極佳的臨床應用。我們目前正針對其餘具有臨床潛力的噬菌體株，持續進行定性的研究當中。

Phage display is a molecular technique accomplished by incorporation of the nucleotide sequence encoding the protein to be displayed into a phage or phagemid genome as a fusion to a gene encoding a phage coat protein. After several rounds of selection and amplification, high affinity phage clones, and thus high affinity “homing peptides” can be obtained. Cell-binding homing peptides selected in this manner could be linked by physical or genetic manipulation to gene therapy vectors that mediate their own entry (viral or non-viral vectors) to facilitate targeting. Homing peptides that target specific cellular receptors can also be used as a treatment modality to induce various signal transduction pathways or even apoptotic signals of cancer cells. Oral squamous cell carcinoma (OSCC) is one of the most common cancers in the world. It has become the fourth cancer death reason of males in Taiwan. Radical surgery combined with postoperative chemotherapy and/or radiotherapy is still the major modality for treatment of OSCC. The 5-year survival rate of OSCC is still discouraged in recent years. Patients with OSCC present numerous challenges to treating physicians. In this study, we aimed to isolate and identify homing phage clones specific to oral cancer cells by panning with a random phage peptide library. The homing phage clones will be used as a basis to improve targeting specificity of gene therapy vectors. A NCBI BLAST search was performed and close similarities were found to several important molecules biologically with the homing peptides carried by phage clones. Characterization of the selected phage-29 was then studied by immunohistochemical methods. Internalization of this phage-29 is sequence-specific and mediated by integrin αvβ6 in HSC-3 cells rapidly. We also confirmed that the integrin αvβ6-targeting homing peptide is universally useful in all major kinds of head and neck cancer. We will further study the possible biological functions of the other homing peptides to see whether these peptides could have potential applications for oral cancer treatment.

中文摘要 ----------------------------------------------------------i
ABSTRACT ------------------------------------------------------ii
ABBREVIATIONS -----------------------------------------------iii
INTRODUCTION -----------------------------------------------1
MATERIALS AND METHODS -------------------------------4
Materials --------------------------------------------------------4
Cells -----------------------------------------------------------4
Labwares and reagents----------------------------------4
Primary antibodies and isotype control --------------4
Secondary antibodies -------------------------------------5
Peptides ------------------------------------------------------5
Specimens of various head and neck squamous
cell carcinoma -------------------------------------------5
Methods --------------------------------------------------------6
Panning ------------------------------------------------------6
Phage amplification --------------------------------------7
Phage titering ----------------------------------------------7
Plaque amplification and DNA sequencing -------8
NCBI BLAST search -------------------------------------8
Phage titration ELISA-------------------------------------8
Phage immunocytochemistry--------------------------9
Internalization assay -------------------------------------9
Competition assay ---------------------------------------9
Immunofluorescence confocal microscopy ------10
Western blot -----------------------------------------------11
Phage immunohistochemistry------------------------11
Immunopathological study of integrin αvβ6--------11
RESULTS -------------------------------------------------------13
Results of Phage Clone Selection-----------------------13
Characterization of the Selected Phage clones-------13
Phage titration ELSIA --------------------------------------13
Selected phage clones attached to and internalized
by HSC-3 cells--------------------------------------------14
Phage-29 is rapidly internalized into HSC-3 cells by
active cellular transportation --------------------------14
Internalization of phage-29 is sequence-specific -15
Disulfide-constrained, cyclic-RGDLASL peptide
efficiently inhibits the internalization of phage-29
as linear RGDLASL peptide---------------------------15
Internalization of phage-29 is mediated by integrin
αvβ6 ---------------------------------------------------------16
HSC-3 cells express more integrin β6 than NOK
cells at fully confluency ---------------------------------16
αvβ6-targeting phage interacts with oral cancer
cells at invasion front, stroma invasion and
extracapsular spread in lymph node metastasis
-----------------------------------------------------------------17
Integrin αvβ6 is universally expressed in all major
kinds of HNSCC------------------------------------------17
DISCUSSION --------------------------------------------------19
CONCLUSION -------------------------------------------------25
REFERENCES -------------------------------------------------26
TABLES -----------------------------------------------------------33
FIGURES ---------------------------------------------------------36

Albert JM, Cao C, Geng L, Leavitt L, Hallahan DE, Lu B. Integrin αvβ3 antagonist Cilengitide enhances efficacy of radiotherapy in endothelial cell and non-small-cell lung cancer models. Int J Radiat Oncol Biol Phys 2006, 65(5):1536-43.
Allman R, Cowburn P, Mason M. In vitro and in vivo effects of a cyclic peptide with affinity for the αvβ3 integrin in human melanoma cells. Eur J Cancer 2000, 36(3):410-22.
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997, 25(17):3389-402.
Azzazy HM, Highsmith WE Jr. Phage display technology: Clinical applications and recent innovations. Clin Biochem 2002, 35(6):425-45.
Barry MA, Dowers WJ, Johnston SA. Toward cell-targeting gene therapy vectors: selection of cell-binding peptides from random peptide-presenting phage libraries. Nat Med 1996, 2(3):299-305.
Berryman S, Clark S, Monaghan P, Jackson T. Early events in integrin αvβ6-mediated cell entry of foot-and-mouth disease virus. J Virol 2005, 79(13):8519-34.
Breuss JM, Gallo J, DeLisser HM, Klimanskaya IV, Folkesson HG, Pittet JF, Nishimura SL, Aldape K, Landers DV, Carpenter W, et al. Expression of the β6 integrin subunit in development, neoplasia and tissue repair suggests a role in epithelial remodeling. J Cell Sci 1995, 108 (Pt 6):2241-51.
Burman A, Clark S, Abrescia NGA, Fry EE, Stuart DI, Jackson T. Specificity of the VP1 GH loop of foot-and-mouth disease virus for αv integrins. J Virol 2006, 80(19):9798-810.
Busk M, Pytela R, Sheppard D. Characterization of the integrin αvβ6 as a fibronectin-binding protein. J Biol Chem 1992, 267(9):5970-6.
Carreiras F, Lehmann M, Sichel F, Marvaldi J, Gauduchon P, Le Talaer JY. Implication of the αvβ3 integrin in the adhesion of the ovarian-adenocarcinoma cell line IGROV1. Int J Cancer 1995, 63(4):530-6.
Chang MC, Chiang CP, Lin CL, Lee JJ, Hahn LJ, Jeng JH. Cell-mediated immunity and head and neck cancer: With special emphasis on betel quid chewing habit. Oral Oncol 2005, 41(8):757-75.
Chen YK, Huang HC, Lin LM, Lin CC. Primary oral squamous cell carcinoma: an analysis of 703 cases in southern Taiwan. Oral Oncol 1999, 35(2):173-9.
Clark RA, Ashcroft GS, Spencer MJ, Larjava H, Ferguson MW. Re-epithelialization of normal human excisional wounds is associated with a switch from αvβ5 to αvβ6 integrins. Br J Dermatol 1996, 135(1):46-51.
Davies L, Welch HG. Epidemiology of head and neck cancer in the United States. Otolaryngol Head Neck Surg 2006, 135(3):451-7.
DOH Report, Department of Health, Taiwan, Republic of China, Cancer Registration Report, 2003.
Fanucchi M, Khuri FR, Shin D, Johnstone PAS, Chen A. Update in the management of head and neck cancer. Update cancer ther 2006, 1(2):211-9.
Gurrath M, Muller G, Keseller H, Aumailly M, Timpl R. Conformation/activity studies of rationally designed potent anti-adhesive RGD peptides. Eur J Biochem 1992, 210(3):911-21.
Hamidi S, Salo T, Kainulainen T, Epstein J, Lerner K, Larjava H. Expression of αvβ6 integrin in oral leukoplakia. Br J Cancer 2000, 82(8):1433-40.
Hasenburg A, Fischer DC, Tong XW, Rojas-Martinez A, Kaufman RH, Ramzy I, Kohlberger P, Orlowska-Volk M, Aguilar-Cordova E, Kieback DG. Adenovirus-mediated thymidine kinase gene therapy for recurrent ovarian cancer: expression of coxsackie-adenovirus receptor and integrins αvβ3 and αvβ5. J Soc Gynecol Investig 2002, 9(3):174-80.
Healy JM, Murayama O, Maeda T, Yoshino K, Sekiguchi K, Kikuchi M. Peptide ligands for integrin αvβ3 selected from random phage display libraries. Biochemistry 1995, 34(12):3948-55.
Hosotani R, Kawaguchi M, Masui T, Koshiba T, Ida J, Fujimoto K, Wada M, Doi R, Imamura M. Expression of integrin αvβ3 in pancreatic carcinoma: relation to MMP-2 activation and lymph node metastasis. Pancreas 2002, 25(2):e30-5.
Hyland S, Beerli RR, Barbas CF, Hynes NE, Wels W. Generation and functional characterization of intracellular antibodies interacting with the kinase domain of human EGF receptor. Oncogene 2003, 22(10):1557-67.
Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell 2002, 110(6):673-87.
Impola U, Uitto VJ, Hietanen J, Hakkinen L, Zhang L, Larjava H, Isaka K, Saarialho-Kere U. Differential expression of matrilysin-1 (MMP-7), 92 kD gelatinase (MMP-9), and metalloelastase (MMP-12) in oral verrucous and squamous cell cancer. J Pathol 2004, 202(1):14-22.
Jackson T, Clark S, Berryman S, Burman A, Cambier S, Mu D, Nishimura S, King AM. Integrin αvβ8 functions as a receptor for foot-and-mouth disease virus: role of the beta-chain cytodomain in integrin-mediated infection. J Virol 2004, 78(9):4533-40.
Jackson T, Mould AP, Sheppard D, King AM. Integrin αvβ1 is a receptor for foot-and-mouth disease virus. J Virol 2002, 76(3):935-41.
Jackson T, Sheppard D, Denyer M, Blakemore W, King AM. The epithelial integrin αvβ6 is a receptor for foot-and-mouth disease virus. J Virol 2000, 74(11):4949-56.
Janes SM, Watt FM. Switch from αvβ5 to αvβ6 integrin expression protects squamous cell carcinomas from anoikis. J Cell Biol 2004, 166(3):419-31.
Jensen KB, Jensen ON, Ravn P, Clark BF, Kristensen P. Identification of keratinocyte-specific markers using phage display and mass spectrometry. Mol Cell Proteomics 2003, 2(2):61-9.
Jones J, Watt FM, Speight PM. Changes in the expression of αv integrins in oral squamous cell carcinomas. J Oral Pathol Med 1997, 26(2):63-8.
Karlin S, Altschul SF. Applications and statistics for multiple high-scoring segments in molecular sequences. Proc Natl Acad Sci USA 1993, 90(12):5873-7.
Karlin S, Altschul SF. Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes. Proc Natl Acad Sci USA 1990, 87(6):2264-8.
Koch J, Breitling F, and Dübel S. Rapid titration of filamentous bacteriophage (M13) on nitrocellulose membranes. BioTechniques 2000, 29(6): 1196-1202.
Kraft S, Diefenbach B, Mehta R, Jonczyk A, Luckenbach GA, Goodman SL. Definition of an unexpected ligand recognition motif for αvβ6 integrin. J Biol Chem 1999, 274(4):1979-85.
Koistinen P, Heino J. The selective regulation of αvβ1 integrin expression is based on the hierarchical formation of αv-containing heterodimers. J Biol Chem 2002, 277(27):24835-41.
Koivisto L, Grenman R, Heino J, Larjava H. Integrins α5β1, αvβ1, and αvβ6 collaborate in squamous carcinoma cell spreading and migration on fibronectin. Exp Cell Res 2000, 255(1):10-7.
Koivunen E, Wang B, Ruoslahti E. Phage libraries displaying cyclic peptides with different ring sizes: ligand specificities of the RGD-directed integrins, Biotechnology (NY) 1995, 13(3):265-70.
Kotz JD, Bond CJ, Cochran AG. Phage-display as a tool for quantifying protein stability determinants. Eur J Biochem 2004, 271(9):1623-9.
Lafrenie RM, Lee SF, Hewlett IK, Yamada KM, Dhawan S. Involvement of integrin αvβ3 in the pathogenesis of human immunodeficiency virus type 1 infection in monocytes. Virology 2002, 297(1):31-8.
Lessey BA, Young SL. Integrins and other cell adhesion molecules in endometrium and endometriosis. Semin Reprod Endocrinol 1997, 15(3):291-9.
Li J, Tan H, Dong X, Xu Z, Shi C, Han X, Jiang H, Krissansen GW, Sun X. Antisense integrin αvβ3 gene therapy suppresses subcutaneously implanted hepatocellular carcinomas. Dig Liver Dis 2007, 39(6):557-65.
Li X, Yang Y, Hu Y, Dang D, Regezi J, Schmidt BL, Atakilit A, Ellis D, Ramos DM. αvβ6-Fyn signaling promotes oral cancer progression. J Biol Chem 2003, 278(43):41646-53.
Lim M, Guccione S, Haddix T, Sims L, Cheshier S, Chu P, Vogel H, Harsh G. Integrin αvβ3 in central nervous system tumors. Hum Pathol 2005, 36(6):665-9.
Lo WL, Kao SY, Chi LY, Wong YK, Chang CS. Outcomes of oral squamous cell carcinoma in Taiwan after surgical therapy: factors affecting survival. J Oral Maxillofac Surg 2003, 61:751-8.
Lupold SE, Rodriguez R. Disulfide-constrained peptides that bind to the extracellular portion of the prostate-specific membrane antigen. Mol Cancer Ther 2004, 3(5):597-603.
Maragou P, Bazopoulou-Kyrkanidou E, Panotopoulou E, Kakarantza-Angelopoulou E, Sklavounou-Andrikopoulou A, Kotaridis S. Alteration of integrin expression in oral squamous cell carcinomas. Oral Dis 1999, 5(1):20-6.
Maruta F, Parker AL, Fisher KD, Hallissey MT, Ismail T, Rowlands DC, Chandler LA, Kerr DJ, Seymour LW. Identification of FGF receptor-binding peptides for cancer gene therapy. Cancer Gene Ther 2002, 9(6):543-52.
Mateu MG, Valero ML, Andreu D, Domingo E. Systematic replacement of amino acid residues within an Arg-Gly-Asp-containing loop of foot-and-mouth disease virus and effect on cell recognition. J Biol Chem 1996, 271(22):12814-9.
Matsui T, Ota T, Ueda Y, Tanino M, Odashima S. Isolation of a highly metastatic cell line to lymph node in human oral squamous cell carcinoma by orthotopic implantation in nude mice. Oral Oncol 1998, 34(4):253-6.
Maubant S, Cruet-Hennequart S, Dutoit S, Denoux Y, Crouet H, Henry-Amar M, Gauduchon P. Expression of αV-associated integrin β subunits in epithelial ovarian cancer and its relation to prognosis in patients treated with platinum-based regimens. J Mol Histol 2005, 36(1-2):119-29.
Menendez JA, Vellon L, Mehmi I, Teng PK, Griggs DW, Lupu R. A novel CYR61-triggered 'CYR61-αvβ3 integrin loop' regulates breast cancer cell survival and chemosensitivity through activation of ERK1/ERK2 MAPK signaling pathway. Oncogene 2005, 24(5):761-79.
Momose F, Araida T, Negishi A, Ichijo H, Shioda S, Sasaki S. Variant sublines with different metastatic potentials selected in nude mice from human oral squamous cell carcinomas. J Oral Pathol Med 1989, 18(7): 391-5.
Moyle M, Napier MA, McLean JW. Cloning and expression of a divergent integrin subunit β8. J Biol Chem 1991, 266(29):19650-8.
Nilsson F, Tarli L, Viti F, Neri D. The use of phage display for the development of tumour targeting agents. Adv Drug Deliv Rev 2000, 43(2-3):165-96.
Niu J, Ahmed N, Andrews S, Turton J, Rates R, Agrez M. The αvβ6 integrin regulates its own expression with cell crowding: implications for tumour progression. Int J Cancer 2001, 92(1):40-8.
O'Donnell V, LaRocco M, Duque H, Baxt B. Analysis of foot-and-mouth disease virus internalization events in cultured cells. J Virol 2005, 79(13):8506-18.
Pasqualini R, Koivunen E, Ruoslahti E. αv integrins as receptors for tumor targeting by circulating ligands. Nat Biotechno. 1997, 15(6):542-6.
Pinholt EM, Rndum J, Pindborg JJ. Oral cancers: a retrospective study of 100 Danish cases. Br J Oral Maxillofac Surg 1997, 35(2):77-80.
Puri SK, Fan CY, Hanna E. Significance of extracapsular lymph node metastases in patients with head and neck squamous cell carcinoma. Curr Opin Otolaryngol Head Neck Surg 2003, 11(2):119-23.
Puy LA, Pang C, Librach CL. Immunohistochemical analysis of αvβ5 and αvβ6 integrins in the endometrium and endometriosis. Int J Gynecol Pathol 2002, 21(2):167-77.
Rasmussen UB, Schreiber V, Schultz H, Mischler F, Schughart K. Tumor cell-targeting by phage-displayed peptides. Cancer Gene Ther 2002, 9(7):606-12.
Regezi JA, Ramos DM, Pytela R, Dekker NP, Jordan RC. Tenascin and β6 integrin are overexpressed in floor of mouth in situ carcinomas and invasive squamous cell carcinomas. Oral Oncol 2002, 38(4):332-6.
Renschler MF, Bhatt RR, Dower WJ, Levy R. Synthetic peptide ligands of the antigen binding receptor induce programmed cell death in a human B-cell lymphoma. Proc Natl Acad Sci USA 1994, 91(9):3623-7.
Sadeghi MM, Bender JR. Activated αvβ3 integrin targeting in injury-induced vascular remodeling. Trends Cardiovasc Med 2007, 17(1):5-10.
Sakuma T, Sari I, Goodman CN, Lindner JR, Klibanov AL, Kaul S. Simultaneous integrin αvβ3 and glycoprotein IIb/IIIa inhibition causes reduction in infarct size in a model of acute coronary thrombosis and primary angioplasty. Cardiovasc Res 2005, 66(3):552-61.
Sankaranarayanan R. Health care auxiliaries in the detection and prevention of oral cancer. Eur J Cancer B Oral Oncol 1997, 33(3):149-54.
Sharma A, Rao Z, Fry E, Booth T, Jones EY, Rowlands DJ, Simmons DL, Stuart DI. Specific interactions between human integrin αvβ3 and chimeric hepatitis B virus core particles bearing the receptor-binding epitope of foot-and-mouth disease virus. Virology 1997, 239(1):150-7.
Silverman SJ, Gorsky M, Lozada F. Oral leukoplakia and malignant transformation. A follow-up study of 257 patients. Cancer 1984, 53(3):563-8.
Sipos B, Hahn D, Carceller A, Piulats J, Hedderich J, Kalthoff H, Goodman SL, Kosmahl M, Kloppel G. Immunohistochemical screening for β6-integrin subunit expression in adenocarcinomas using a novel monoclonal antibody reveals strong up-regulation in pancreatic ductal adenocarcinomas in vivo and in vitro. Histopathology 2004, 45(3):226–36.
Smith GP. Filamentous fusion phage: Novel expression vectors that display cloned antigens on the virion surface. Science 1985; 228(4705):1315-7.
Sun YX, Fang M, Wang J, Cooper CR, Pienta KJ, Taichman RS. Expression and activation of αvβ3 integrins by SDF-1/CXC12 increases the aggressiveness of prostate cancer cells. Prostate 2007, 67(1):61-73.
Thomas GJ, Jones J, Speight PM. Integrins and oral cancer. Oral Oncol 1997, 33(6):381-8.
Thomas GJ, Nystrom ML, Marshall JF. αvβ6 integrin in wound healing and cancer of the oral cavity. J Oral Pathol Med 2006, 35(1):1–10.
Thomas GJ, Poomsawat S, Lewis MP, Hart IR, Speight PM, Marshall JF. αvβ6 integrin upregulates matrix metalloproteinase 9 and promotes migration of normal oral keratinocytes. J Invest Dermatol 2001, 116(6):898-904.
Westernoff TH, Jordan RCK, Regezi JA, Ramos DM, Schmidt BL. β6 Integrin, tenascin-C, and MMP-1 expression in salivary gland neoplasms. Oral Oncol 2005, 41(2):170-4.
Xue H, Atakilit A, Zhu W, Li X, Ramos DM, Pytela R. Role of the αvβ6 integrin in human oral squamous cell carcinoma growth in vivo and in vitro. Biochem Biophys Res Commun 2001, 288 (3):610–18.
Zhang Y, Lu Hai, Dazin P, Kapila Y. Squamous cell carcinoma cell aggregates escape suspension-induced, p53-mediated anoikis. J Biol Chem 2004, 279(46):48342-9.