Department of Nuclear Medicine, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, 430062, China.
The First Affiliated Hospital of Yangtze University, Jingzhou, 434000, China.
Curr Med Sci. 2023 Jun;43(3):623-630. doi: 10.1007/s11596-023-2740-7. Epub 2023 May 24.
Fibroblast activation protein (FAP) has been widely studied and exploited for its clinical applications. One of the difficulties in interpreting reports of FAP-targeted theranostics is due to the lack of accurate controls, making the results less specific and less confirmative. This study aimed to establish a pair of cell lines, in which one highly expresses FAP (HT1080-hFAP) and the other has no detectable FAP (HT1080-vec) as control, to accurately evaluate the specificity of the FAP-targeted theranostics in vitro and in vivo.
The cell lines of the experimental group (HT1080-hFAP) and no-load group (HT1080-vec) were obtained by molecular construction of the recombinant plasmid pIRES-hFAP. The expression of hFAP in HT1080 cells was detected by PCR, Western blotting and flow cytometry. CCK-8, Matrigel transwell invasion assay, scratch test, flow cytometry and immunofluorescence were used to verify the physiological function of FAP. The activities of human dipeptidyl peptidase (DPP) and human endopeptidase (EP) were detected by ELISA in HT1080-hFAP cells. PET imaging was performed in bilateral tumor-bearing nude mice models to evaluate the specificity of FAP.
RT-PCR and Western blotting demonstrated the mRNA and protein expression of hFAP in HT1080-hFAP cells but not in HT1080-vec cells. Flow cytometry confirmed that nearly 95% of the HT1080-hFAP cells were FAP positive. The engineered hFAP on HT1080 cells had its ability to retain enzymatic activities and a variety of biological functions, including internalization, proliferation-, migration-, and invasion-promoting activities. The HT1080-hFAP xenografted tumors in nude mice bound and took up GA-FAPI-04 with superior selectivity. High image contrast and tumor-organ ratio were obtained by PET imaging. The HT1080-hFAP tumor retained the radiotracer for at least 60 min.
This pair of HT1080 cell lines was successfully established, making it feasible for accurate evaluation and visualization of therapeutic and diagnostic agents targeting the hFAP.
成纤维细胞激活蛋白（FAP）已被广泛研究并应用于临床。在解释 FAP 靶向治疗的报告时，其中一个困难是由于缺乏准确的对照，导致结果不够特异和确定。本研究旨在建立一对细胞系，其中一个高度表达 FAP（HT1080-hFAP），另一个作为对照无 FAP 表达（HT1080-vec），以准确评估 FAP 靶向治疗在体外和体内的特异性。
实验组成员（HT1080-hFAP）和空载组（HT1080-vec）细胞系通过重组质粒 pIRES-hFAP 的分子构建获得。PCR、Western blot 和流式细胞术检测 HT1080 细胞中 hFAP 的表达。CCK-8、Matrigel 侵袭实验、划痕实验、流式细胞术和免疫荧光法验证 FAP 的生理功能。ELISA 检测 HT1080-hFAP 细胞中人二肽基肽酶（DPP）和人内肽酶（EP）的活性。在双侧荷瘤裸鼠模型中进行 PET 成像，以评估 FAP 的特异性。
RT-PCR 和 Western blot 证明 HT1080-hFAP 细胞中 hFAP 的 mRNA 和蛋白表达，但 HT1080-vec 细胞中没有。流式细胞术证实近 95%的 HT1080-hFAP 细胞为 FAP 阳性。工程化的 HT1080 细胞上的 hFAP 保留了酶活性和多种生物学功能，包括内化、增殖、迁移和侵袭促进活性。裸鼠 FAP 靶向治疗的 HT1080-hFAP 移植瘤具有良好的选择性结合和摄取 GA-FAPI-04。通过 PET 成像获得高图像对比度和肿瘤-器官比。HT1080-hFAP 肿瘤保留放射性示踪剂至少 60 分钟。
成功建立了这对 HT1080 细胞系，使其能够准确评估和可视化针对 hFAP 的治疗和诊断试剂。
