脑胶质瘤新型靶点- - TEM8受体高选择性双模态多肽分子探针的筛选构建及活体成像研究

The upregulation of tumor endothelial marker 8 (TEM8) on the endothelial cells of blood vessels has been proved highly related to tumor angiogenesis, making this receptor a particularly superior new candidate for future tumor angiogenesis targeting studies. Several antiangiogenesis studies have been conducted based on the interaction between the ligand (such as peptides, fusion protein) and the TEM8 receptor. The applicant had reported a molecular tracer of 18F-FP-QQM that was the first time that TEM8 expression has been visualized with PET using a peptidic probe. Glioma showed highly infiltrative properties in the central nervous system tumors. There is an urgent clinical need for its early diagnosis and treatment. Our preliminary data showed a high expression of TEM8 on C6 glioblastoma cell line. Therefore, we are going to perform parallel in vitro and in vivo selection using phage display to identify peptides that accumulate within tumors in vivo and target to the TEM8 receptor on glioma. By comparing to structure of QQM, we aim to develop a newly, highly selective targeting peptide sequence, that easily go across the brain blood barrier. This binding peptide has both organ-specific and molecular-specific properties. Furthermore, we would develop a dual-modality tumor objective targeting nanoparticles with both MR and optical imaging features. This probe is following targeting TEM8 receptor of the orthotropic U87 tumor model in vivo by tail vein injection. The biological characteristics of TEM8 expression and tumor angiogenesis would be visualized and integrated by the data obtained from MR and optical imaging. Overall, the most important, we would be able to achieve non-invasive, dynamic, quantitative analysis of TEM8 expression associated micro-vessels density on glioma. This new peptide-based dual-modality tumor targeting nanoparticles provide the possibility of dynamic monitoring of the progression of the disease, and improve the early diagnosis, treatment and assessment of the therapeutic efficacy.

TEM8受体与肿瘤新生血管密切相关，是一种新型优越的评估肿瘤血管化程度的标记，逐渐成为国际肿瘤分子影像学研究的希望。将TEM8做为靶标研发多肽、融合蛋白等进行抗肿瘤血管化治疗在国际上有一定报道。申请人也在国际上首次合成18F-FP-QQM多肽分子探针可视化肿瘤TEM8蛋白表达水平。脑胶质瘤在中枢神经系统肿瘤中侵袭性最高，其早期诊断及治疗是临床研究难点，我们前期实验结果显示C6胶质母细胞瘤细胞高表达TEM8蛋白。 因此，本项目拟以QQM多肽为基础展开双重噬菌体展示，筛选易于透过血脑屏障的靶向脑胶质瘤TEM8受体的更高特异性多肽序列，兼具器官及分子特异性，并构建多肽靶向的双模态成像分子探针，通过MR及光学成像获得原位接种U87模型TEM8受体生物学特征的可视化数据，整合分析，实现无创，动态，定量分析TEM8相关的脑胶质瘤发展，为早期诊断，动态监控病情提供新方法，为合理治疗并评估疗效提供新思路。

通过本项目的资助，按照预期目标，课题组通过western-blot检测脑胶质瘤C6细胞内TEM8受体高表达。应用ATPS包被的纳米氧化铁颗粒在体外成功标记C6细胞，并对细胞活性及增殖力无明显抑制。成功构建了QQM多肽介导的MR及光学成像探针，并在脑胶质瘤原位接种模型中长期示踪肿瘤的生长演变。另一方面应用课题责任人理性推理获得与TEM8受体高亲和力的靶向多肽QQM，成功构建靶向TEM8的特异性MRI 及光学成像双功能分子探针Z-IO-Q，以QQM多肽介导，利用该分子探针可进行4T1乳腺癌细胞体外摄取及双功能成像，再进一步实现了裸鼠皮下移植瘤模型的MR及近红外光学成像，达到在分子水平双模态可视化脑胶质瘤及乳腺癌TEM8新型受体的表达。同时进行的体内阻断成像，证实了QQM多肽对TEM8受体的靶向性。体外重要器官的探针分布的研究进一步说明QQM在特异性靶向TEM8受体的同时较少被非靶向器官摄取。本项目并为建立以TEM8为靶点的新型肿瘤血管生成相关标记的诊断、治疗及疗效评估的研究体系提供了可能性。 课题责任人及研究团队成员共同发表受本项目资助的SCI收录论文1篇。课题组获2014年上海市科学技术奖二等奖。课题责任人受2013欧洲放射学年会及2015北美放射学年会邀请大会发言。课题责任人协助培养2名博士研究生完成学位论文，获得博士学位。目前在培博士生1人。另有标明受本项目助的3篇SCI论文待发表。 综上，本项目在科研内容及人才培养方面均达到预期目标。