Department of Oncology, Chenggong Hospital Affiliated to Xiamen University, Xiamen University, Xiamen, China.
Medical College, Xiamen University, Xiamen, China.
Neoplasma. 2019 Mar 5;66(2):252-260. doi: 10.4149/neo_2018_180722N513. Epub 2018 Dec 12.
The problems including narrow indications, low drug loading, and difficulty in intervention severely affect the clinical efficacy of anti-tumor embolization. Here, we designed a novel tTF-EG3287 protein consisting of the truncated tissue factor (tTF) fused with the bicyclic polypeptide which was encoded by exons 7 and 8 for accurate localization in the tumor vascular endothelial cells (EG3287). This study aims to explore its anti-cancer effect. Gene sequencing was used to verify the fusion gene and SDS-PAGE gel to confirm the optimal induction time and concentration of tTF-EG3287. Nickel affinity chromatography column was used to purify the fusion protein. Confocal microscopy was used to assess the target activity of tTF-EG3287 on colon cancer cells in vitro. Thrombelastography assay was used to identify the pro-coagulant activity of tTF-EG3287. In in vivo experiments, the specific localization of tTF-EG3287 in tumor tissues and the effect of tTF-EG3287 on tumor thrombosis were further detected by in vivo imaging and HE staining, respectively. The tTF-EG3287 fusion protein was efficiently purified by nickel-affinity chromatography column. Moreover, tTF-EG3287 fusion protein showed strong coagulation a ctivity and specific binding ability to the cell surface of colon cancer. In vivo, tTF-EG3287 stably and persistently accumulated in tumor tissues, and specifically induced mixed thrombus formation in tumor vessels, and then impaired tumor growth (tumor inhibition rate=79.2%, p<0.01). Our data prove that the fusion protein tTF-EG3287 could be used as a novel and promising anti-cancer strategy and has great potential value for clinical applications.
该问题包括狭窄的适应证、低载药量和介入困难，严重影响了抗肿瘤栓塞的临床疗效。在这里，我们设计了一种新型 tTF-EG3287 蛋白，它由融合了双环多肽的截断组织因子 (tTF) 组成，该多肽由外显子 7 和 8 编码，可准确定位在肿瘤血管内皮细胞（EG3287）中。本研究旨在探讨其抗癌作用。基因测序用于验证融合基因，SDS-PAGE 凝胶用于确认 tTF-EG3287 的最佳诱导时间和浓度。镍亲和层析柱用于纯化融合蛋白。共聚焦显微镜用于评估 tTF-EG3287 在体外对结肠癌细胞的靶向活性。血栓弹性图试验用于鉴定 tTF-EG3287 的促凝活性。在体内实验中，通过体内成像和 HE 染色进一步检测 tTF-EG3287 在肿瘤组织中的特异性定位及其对肿瘤血栓形成的影响。tTF-EG3287 融合蛋白通过镍亲和层析柱高效纯化。此外，tTF-EG3287 融合蛋白显示出强烈的凝血活性和对结肠癌细胞表面的特异性结合能力。在体内，tTF-EG3287 稳定且持续地积聚在肿瘤组织中，并特异性诱导肿瘤血管内混合血栓形成，从而损害肿瘤生长（肿瘤抑制率=79.2%，p<0.01）。我们的数据证明，融合蛋白 tTF-EG3287 可用作一种新型有前途的抗癌策略，具有巨大的临床应用潜力。
