免疫蛋白质组学筛选肿瘤间质成纤维细胞肿瘤相关抗原及其功能研究

The majority of targeted therapy drugs is directed the tumor cells themselves at present, but the tumor cells are genetically unstable and thus can accumulate adaptive mutations and rapidly acquire drug resistance. Wealth of data suggests that the carcinoma-associated fibroblasts (CAFs) can promote tumorigenesis, progression, and metastasis; in addition, CAFs are presumably genetically stable, which is in contrast to tumor cells. The novel strategies target CAFs to offer new avenues to inhibit the solid tumor growth, and identification of novel molecular marker may provide a potential target for therapeutic intervention. In our study, we used basic fibroblast growth factor (bFGF) to activate normal fibroblasts and these activated fibroblasts have analogous properties with CAFs. We produced polyclonal antibodies by immunizing normal mouse with these activated fibroblasts. The polyclonal antibodies could specifically bind to the activated fibroblasts and induced apoptosis in the activated fibroblasts. Meanwhile, administration of polyclonal antibodies reached significant inhibition against tumor xenografts in BALB/C mice. In the further study, we plan to use immunoproteomic approach to reveal immunogenic antigens of CAFs, which were confirmed by the Protein Bank and clinical data. We choose the target proteins, which are closely related with CAFs, and investigate their role in tumor development by using lentiviral vectors expressing target gene or small interfering RNA. Our study may provide a novel therapy target for solid tumor in clinical application.

目前多数靶向药物是针对肿瘤细胞本身，而肿瘤细胞易通过基因突变等多种途径导致耐药的产生。现研究表明肿瘤间质中的肿瘤相关成纤维细胞（CAFs）与肿瘤的发生、发展及转移密切相关，且基因相对稳定，筛选针对CAFs的分子靶点将为实体瘤的治疗或辅助治疗提供新途径。申请者利用碱性成纤维细胞生长因子激活正常小鼠皮肤来源的成纤维细胞，使其具有CAFs的特性；用激活后的成纤维细胞免疫小鼠，纯化血清获得多克隆抗体。前期实验发现，所得多克隆抗体不仅能与激活后的成纤维细胞特异性结合、抑制其增殖；还能抑制体内肿瘤的生长。我们拟在后续的研究中，用免疫蛋白质组学的方法对多克隆抗体识别的肿瘤相关抗原进行筛选，结合蛋白质功能数据库和临床肿瘤标本检测，确定与肿瘤密切相关的目标蛋白。通过慢病毒介导的基因过表达和 RNA干扰技术，深入研究目标蛋白在肿瘤发生发展中的作用。我们的研究将为实体瘤的分子靶向治疗提供实验基础和新的思路。

肿瘤相关成纤维细胞（CAFs）是肿瘤组织中的主要间质细胞。CAFs在肿瘤的发生、发展、侵袭及转移过程中扮演着重要角色，同时也影响肿瘤细胞对治疗的反应。现研究表明，在临床上单克隆抗体的疗效有限，开发同时针对多个靶点的多克隆抗体是提高其抗肿瘤疗效切实可行的方法。在本研究中，我们用bFGF激活正常小鼠皮肤来源的成纤维细胞，使其具有肿瘤相关成纤维细胞的特性。用激活后的成纤维细胞免疫新西兰大白兔，纯化血清制备多克隆抗体（Poly-Abs）。我们发现抗CAFs多克隆抗体能够在体外和体内抑制肿瘤生长、侵袭和转移。运用免疫共沉淀、免疫蛋白质组学对多克隆抗体识别的CAFs抗原进行筛选，确定14-3-3σ（stratifin）是CAFs相关的肿瘤抗原。14-3-3σ在临床结直肠癌标本中高表达、且与结直肠癌患者的临床病理特征相关。从结直肠癌及其相应的正常组织中分离出成纤维细胞，提取蛋白、western blot分析发现，14-3-3σ在肿瘤相关成纤维细胞中高表达。在本研究中，我们以多克隆抗体抗肿瘤作用为基础，建立了功能导向的免疫蛋白质组学技术，可以更有效高通量筛选肿瘤抗原，发现了肿瘤相关成纤维细胞新的标志性分子14-3-3σ。为靶向CAFs的分子靶向治疗提供新的思路。通过对本项目的开展，我们已发表相关研究论文5篇，其中SCI论文4篇，完成1名硕士研究生的培养。