MR/NIRF双模态分子影像指导下靶向肽-siRNA复合体在乳腺癌脑转移中的应用研究

A key factor to determine the tumor organ-specific metastasis is the adhesion and growth of tumor cells in the target organ, which is associated with the organ-specific homing receptors and the corresponding ligands expressed on the surface of the cells. Therefore, it will be of great theoretical and practical importance in the diagnosis and treatment of brain metastases in breast cancer, to monitor real-timely the brain-seeking breast carcinoma cells, and to silence the brain-specific homing receptor on their surface before metastasis. We have successfully screened and obtained a novel phage display-derived peptide, denoted BrBP1, which can specifically bind to the human brain-seeking breast carcinoma cells. In this project, we are going to find the receptor of BrBP1 by mass spectrometry, GST-pull down and so on, then develop siRNA to silence it. On the basis of BrBP1, we will construct and characterize a multi-purpose and dual-modality molecular probe, which can deliver the BrBP1-receptor-silencing siRNA to the human brain-seeking breast carcinoma cells through the interaction of BrBP1 and its receptor. The specificity and functions of the molecular probe will be verified in vitro. We will further study the specificity, the biodistribution, the efficiency of gene silencing and inhibition of brain metastases by MR / NIRF imaging in vivo, ex vivo imaging and histological examination. We believe that our work will lay the foundation for early diagnosis, treatment and real-time monitoring of brain metastases in breast cancer. Meanwhile, it may provide a novel approach to diagnose and treat other metastatic disease.

决定肿瘤器官特异性转移的关键因素是器官特异性归巢受体及相应配体。实时监测具有脑转移潜能的乳腺癌细胞群并在转移之前沉默该群细胞特异性归巢受体对乳腺癌脑转移的诊治具有重要价值。我们前期已成功获得了乳腺癌脑转移细胞靶向性多肽BrBP1。本课题拟寻找与BrBP1特异性结合的受体分子并设计高效沉默该受体的siRNA。在此基础上，构建和表征乳腺癌脑转移靶向性、荷载siRNA的多功能多模态分子探针siRNA-BrBP1-PEI/SPIONs/Cy5.5，体外鉴定该探针的靶向性和功能，通过MR/NIRF双模态活体成像、离体组织器官成像及组织学分析研究该探针体内肿瘤靶向性、分布、沉默基因效率及对脑转移的抑制作用。本项目研究结果将为乳腺癌脑转移早期诊断、治疗及疗效实时监测提供重要的实验基础，也可为临床诊治其他具备合适靶点的肿瘤远处转移开辟一条新的途径。

乳腺癌脑转移（BCBM）是乳腺癌最严重的并发症，目前缺乏脑转移性乳腺癌分子成像探针及有效抑制BCBM的分子靶向药物，开发靶向脑转移性乳腺癌的分子成像探针对BCBM早期诊断和高灵敏度探测具有重要意义。.本项目通过噬菌体展示技术筛选乳腺癌脑转移细胞靶向性多肽，成功获得与乳腺癌脑转移细胞系特异性及高亲和力结合的多肽BRBP1，并获得发明专利授权。通过临床样本分析显示BRBP1肽可与人乳腺浸润性导管癌组织标本特异性结合且倾向结合高转移潜能的肿瘤细胞。基于该靶向多肽BRBP1构建的脂质体纳米探针，能够在活体水平上检测乳腺癌皮下瘤及脑转移病灶。目前基于该靶向分子及TWF1 siRNA联合化疗药物PTX的诊断-治疗一体化脂质体探针研究正在进行中。成功构建了靶向乳腺癌脑转移的MR/NIRF双模态Fe3O4/DiR探针，该探针能够特异性富集到乳腺癌皮下瘤病灶，并且能穿透血脑屏障在脑转移病灶聚集，提高T2磁共振成像敏感性，提示该纳米探针可检测乳腺癌脑转移微小病灶，为乳腺癌脑转移的早期诊断提供实验基础。将BRBP1肽与细胞渗透肽TAT及促凋亡肽KLA连接，成功构建及优化BRBP1-TAT-KLA复合肽，该复合肽可显著抑制脑转移性乳腺癌的生长及乳腺癌脑转移灶的形成。通过IP、质谱分析等寻找及鉴定与乳腺癌脑转移细胞靶向性多肽BRBP1结合的细胞表面分子或受体，已经获得多个备选分子，相关研究正在进行中。发现TCF21基因作为抑癌基因在人乳腺癌细胞及大多数乳腺癌组织中低表达，并且TCF21基因低表达与乳腺癌淋巴结转移、肿瘤体积增大及不良预后有关。体外实验证实TCF21基因过表达能够抑制乳腺癌MDA-MB-231细胞的增殖并促进其凋亡。TCF21基因3'UTR 中的rs12190287位点多态性与中国汉族女性乳腺癌发病风险有关。与携带有rs12190287 C等位基因的个体相比，携带有G等位基因的个体患乳腺癌的风险显著降低。rs12190287 G等位基因能够干扰hsa-miR-224对TCF21基因表达的调控。因此该多态性位点可能作为中国汉族女性乳腺癌发病风险的生物标记物。.本项目完成了预定的研究内容，研究结果为乳腺癌脑转移早期诊断、治疗及疗效实时监测提供了重要的实验基础。