Key Laboratory of Molecular Medicine and Biotherapy, Key Laboratory of Medical Molecule Science and Pharmaceutics Engineering, School of Life Science, Advanced Research Institute of Multidisciplinary Science, Beijing Institute of Technology, Beijing 100081, China.
CAMS Key Laboratory of Antiviral Drug Research, Beijing Key Laboratory of Antimicrobial Agents, NHC Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
Molecules. 2023 Feb 9;28(4):1682. doi: 10.3390/molecules28041682.
As a type II transmembrane serine protease, fibroblast activation protein (FAP) is specifically expressed on the surface of fibroblasts associated with a variety of epithelial-derived malignancies such as pancreatic cancer, breast cancer, and colon cancer. It participates in the processes of tumorigenesis, progression, and immunosuppression. FAP constitutes an important target for tumor treatment; however, the current studies on FAP are mainly related to structural characteristics, enzymatic properties, and biological functions, and aptamers of FAP have not been investigated. In this work, by using recombinant human FAP as the target, five candidate aptamers, which are AptFAP-A1, AptFAP-A2, AptFAP-A3, AptFAP-A4, and AptFAP-A5, were selected by capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX), and their secondary structures were predicted to be mainly stem-loop. Moreover, the CE-laser-induced fluorescence (LIF) method was used to determine the equilibrium dissociation constant values between the FAP protein and candidate aptamers, and the value was in the low molar range. Finally, Cy5-labeled aptamers were co-incubated with human pancreatic cancer-associated fibroblasts highly expressing FAP protein, and confocal microscopy imaging showed that aptamer AptFAP-A4 had the highest affinities with the cells. The FAP aptamers screened in this study provide a promising direction for the development of rapid tumor diagnosis and targeted therapy.
作为一种 II 型跨膜丝氨酸蛋白酶，成纤维细胞激活蛋白（FAP）特异性表达于与多种上皮来源的恶性肿瘤相关的成纤维细胞表面，如胰腺癌、乳腺癌和结肠癌。它参与肿瘤发生、进展和免疫抑制的过程。FAP 是肿瘤治疗的重要靶点；然而，目前对 FAP 的研究主要集中在结构特征、酶学特性和生物学功能上，尚未研究 FAP 的适体。在这项工作中，我们以重组人 FAP 为靶标，通过毛细管电泳-指数富集配体系统进化（CE-SELEX）筛选出了五个候选适体，分别为 AptFAP-A1、AptFAP-A2、AptFAP-A3、AptFAP-A4 和 AptFAP-A5，并预测其二级结构主要为茎环结构。此外，我们还使用 CE-激光诱导荧光（LIF）法测定了 FAP 蛋白与候选适体之间的平衡解离常数（Kd 值），Kd 值处于低摩尔范围。最后，我们将 Cy5 标记的适体与高表达 FAP 蛋白的人胰腺癌细胞相关成纤维细胞共孵育，共聚焦显微镜成像显示，适体 AptFAP-A4 与细胞具有最高的亲和力。本研究筛选出的 FAP 适体为快速肿瘤诊断和靶向治疗的发展提供了有前途的方向。
