新型EpCAM分子导向多肽与干细胞微环境抑制多肽对结直肠癌干细胞三位一体的靶向治疗研究

Many efforts have been made to design therapies that specifically target the colorectal cancer stem cells (CSCs) population, since this was the crucial population for metastasis and relapse. However, therapies that specifically target the colorectal CSC have to eliminate CSC, normal colorectal cancer cells and inhibit CSC niche which made of Cancer-associated fibroblasts(CAF) and TGF-β because of the CSC dynamics. EpCAM is a good target to eliminate CSC and normal cancer cells simultaneously as it is overepressed on CSC and normal cancer cells. AcSDKP is a small peptide which has the potential to inhibit CAF and the production of TGF-β in CSC niche. In previous work, we got a EpCAM specific binding peptide SNF through phages library screening and we also synthesised an analogue of AcSDKP (AcSDDKDP) which exhibits longer half-life in mice. In present study, we will prove SNF specific binding ability to colorectal CSC and normal colorectal cancer cells, use SNF as a guiding peptide to synthesis EpCAM targeted pH sensitive Rapacymin nano particles for eliminating CSC and colorectal cancer cells simultaneously. We also want to prove the ability of AcSDDKDP to inhibit colorectal CSC niche, to synthesis a pH sensitive nano particels to improve the pharmacokinetics characteristics of AcSDDKDP. At the end, we will use SNF-rapamycin nano particle and AcSDDKDP nano particle to realize a threefold therapeutic regimen for colorectal cancer stem cell therapy. It means we will eliminate CSC, normal cancer cells and inhibte CSC niche at same time. The project will give more thoughts for CSC targeted therapies and give more experience for development of peptied and protein medicines.

结直肠癌的肿瘤干细胞(CSC)靶向治疗必须依照“动态肿瘤干细胞模型”，消灭CSC、消灭普通结直肠癌细胞、抑制肿瘤成纤维细胞CAF和转化生长因子TGF-β形成的结直肠CSC微环境。针对上皮细胞黏附分子EpCAM的治疗可同时打击CSC和普通结直肠癌细胞。AcSDKP是具有抑制结直肠CSC微环境多种成分潜能的小肽。前期我们筛选到EpCAM特异性结合多肽SNF，还构建了AcSDKP长效异构体(AcSDDKDP)。本课题将证明SNF能够靶向结直肠CSC；构建SNF导向雷帕霉素酸敏纳米粒实现CSC、普通结直肠癌细胞的共同杀伤；证明AcSDDKDP能够抑制CAF和TGF-β形成的CSC微环境；构建AcSDDKDP酸敏纳米粒改善其药代特性；最终联合这两种纳米粒在CSC裸鼠移植瘤模型、结直肠癌转移模型和自发结直肠癌的CPC;APC基因敲除小鼠实现三位一体的结直肠CSC靶向治疗，为CSC靶向治疗提供新思路。

早期诊断是提高结直肠癌五年生存率的唯一方法。然而，普通结肠镜的结直肠癌漏检率高。显微内镜分子成像依赖激光共聚焦显微内镜观察和分子探针对肿瘤标志物的特异性结合客观的实现肿瘤早期诊断，是未来内镜的发展方向。多肽因质量小、免疫原性低、易于穿过病变黏膜、亲和力高、特异性高、易于大量合成、造价低廉等优点成为构建探针的首选。前期工作中，申请人利用噬菌体随机呈现肽库筛选得到一条能够特异性与食管腺癌组织结合的七肽（SNFYMPL）。以该七肽构建的荧光探针在能够特异性显现食管组织中微小的恶性病变，为食管腺癌的早期荧光内窥镜诊断提供可能。目前该七肽已获得国际专利授权（US2010/0310459A1）。相关文章发表在Gastroenterology 杂志（IF 13.90）。可是，该分子在细胞表面结合的靶分子不明。本课题拟在筛选到靶分子EpCAM的基础上，扩展该小肽的结肠癌诊断和治疗中的应用。本课题经实验获得的实验结果如下：免疫荧光和小鼠移植瘤的在体检测证实，SNF*-FITC能够与EpCAM高表达的结肠癌，胃癌细胞及组织特异性结合；SNF*作用后，不促进肿瘤细胞的增殖，不激活EpCAM下游信号通路；在小鼠荧光内窥镜平台下，Cy5.5偶联的SNF*小肽可以明显结合APC条件基因敲除小鼠的自发结肠肿瘤；SNF*偶联后，明显改善SNF*-PHIS-DSPE-PTX纳米胶束与结肠和胃腺癌细胞的靶向结合能力，增强药物利用率，提高抗肿瘤活性，减少PTX化疗体内毒副作用。本课题的完成将为我国结直肠癌的基础和临床提供大量有意义的资料。