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研究生： 王秋樺
Wang, Chiu-Hua
論文名稱： 開發癌症診斷的CDCP1適體
Development of aptamers targeting CUB Domain-Containing Protein 1 for cancer diagnosis
指導教授： 黃暉升
Huang, Huei-Sheng
學位類別： 碩士
Master
系所名稱： 醫學院 - 醫學檢驗生物技術學系
Department of Medical Laboratory Science and Biotechnology
論文出版年： 2025
畢業學年度： 113
語文別： 英文
論文頁數： 73
中文關鍵詞： CDCP1 、適體 、細胞-SELEX 、癌症診斷
外文關鍵詞： CDCP1, Aptamer, Cell-SELEX, Cancer diagnosis
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    • 癌症是全球主要死因之一，因此早期診斷對提升治療成效與病患存活率至關重要。標靶性生物標誌物的發掘與應用已成為癌症診斷與治療領域的重點。其中，CUB domain-containing protein 1（CDCP1）是一種穿膜蛋白，已被證實在多種癌症中高度表達，並與腫瘤進展及預後不良密切相關。CDCP1在細胞表面以全長（flCDCP1）及切割變體（clCDCP1）存在，CDCP1被切割後會釋放出65 kDa的氨基端片段（ATF）。這些不同形式的CDCP1在癌症中具有促進腫瘤幹細胞特性及增壓藥物抗性等多重功能。近期研究已在癌症患者的臨床體液中檢測到ATF，顯示其作為癌症檢測生物標誌物的潛力。適體（aptamer）是一種單鏈DNA或RNA分子，因具備高穩定性、低生產成本及低免疫原性等優勢，逐漸成為癌症診斷與治療的新興工具，可用於標靶檢測或治療。本研究旨在開發針對CDCP1不同形式（flCDCP1、clCDCP1及ATF）的高特異性適體，以輔助癌症診斷。研究中利用細胞-SELEX技術，篩選CDCP1過度表達或剔除的T24細胞，並結合次世代定序分析，成功獲得五個候選適體。經點墨法及流式細胞術驗證，A2與A4適體對clCDCP1相對具高結合力， A5與A6適體能有效檢測條件培養基中的ATF，且其結合效力優於現有ATF特異性抗體。綜上所述，本研究篩選出的適體對CDCP1各形式均具高親和力與特異性，展現其於癌症液體活檢等臨床診斷應用的潛力。

    Cancer ranks as the top cause of death worldwide, making early diagnosis critical for improving treatment efficacy and patient survival rates. The discovery and application of targeted biomarkers have become important in cancer diagnosis and therapy. CUB domain-containing protein 1 (CDCP1) is highly expressed in various cancers and is closely associated with tumor progression and poor prognosis. CDCP1 is present on the cell surface in two forms: full-length (flCDCP1) and cleaved (clCDCP1), with the latter releasing an amino-terminal fragment (ATF). These different CDCP1 forms exhibit multiple functions in cancer, including promoting cancer stemness and enhancing drug resistance. Recent studies have detected ATF in the clinical fluids of cancer patients, highlighting its potential as a biomarker for cancer detection. Aptamers, which are single-stranded DNA or RNA, offer advantages such as high stability, low production costs, and low immunogenicity, making them valuable tools for targeted cancer diagnosis and therapy. This study aimed to develop specific aptamers targeting different CDCP1 isoforms to advance cancer diagnostics. Using cell-SELEX, we identified specific aptamers by screening T24 cells engineered for CDCP1 overexpression or knockout, and subsequently analyzed them via next-generation sequencing. Five candidate aptamers were identified and validated using dot blot and flow cytometry. Aptamers A2 and A4 had relatively high binding affinity toward clCDCP1, while aptamers A5 and A6 effectively recognized the ATF fragment in culture supernatants, demonstrating superior affinity and detection sensitivity compared to commercially available ATF-specific antibodies. The selected aptamers exhibit high affinity and specificity toward various CDCP1 isoforms, highlighting their strong potential for clinical diagnostic applications such as cancer liquid biopsy.

    Abstract I 致謝 III Contents IV List of Abbreviations VI Introduction 1 Materials and methods 7 Results 20 Establishing a cell-SELEX platform for selecting CDCP1 aptamers. 20 Assessing the DNA pools from the cell-SELEX. 21 Determining the binding affinity of CDCP1 aptamers. 23 The aptamers can bind to CDCP1 through the conformation of the aptamers. 25 Using aptamers to detect ATF 27 Aptamers do not affect the cleavage of CDCP1 and its function. 29 Discussion 30 Figures 34 Figure 1. Establishing a CDCP1 cell-SELEX platform for selecting aptamers 36 Figure 2. Assessing the DNA pools from the cell-SELEX 38 Figure 3. Analyzing the CDCP1 aptamers using NGS sequencing and confirming the target of aptamer binding. 41 Figure 4. Determining the binding affinity of CDCP1 aptamers 45 Figure 5. The aptamers can bind to CDCP1 through the conformation of the aptamers 50 Figure 6. Using aptamers to detect ATF 52 Figure 7. Aptamers do not affect the cleavage of CDCP1 and its function. 54 Figure 8. CDCP1 structure predicted by AlphaFold 55 References 56 Appendix 60 Appendix 1. List of Experimental Materials 61 Appendix 2. List of cell types in research. 62 Appendix 3. The structure and pathway of CUB domain-containing protein 1. 63 Appendix 4. The aptamer is folded into a 3D structure that enables target recognition. 64 Appendix 5. The number of cells used in Cell SELEX, the amount of input DNA, and the incubation time with the cells. 65

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