肝癌转移干细胞在肝癌转移中的作用和分子基础及其抗体靶向治疗的研究

Emerging evidence suggests that cancer stem cells (CSCs) are heterogeneous populations, and the roles of metastatic cancer stem cell (MCSCs) has become a major focus in cancer research. Elucidating the roles of MCSCs in metastasis might contribute to understand the mechanism of cancer metastasis and provide an important experimental basis for targeted MCSC treatment. Previously, we had successfully established the functional monoclonal antibody library against liver cancer stem cell by using the immuno-proteomic technology. From it, we preliminarily screened and identified numbers of mAbs targeting CSC. We also found that 4 mAbs might target MCSCs. In this studies, we would focus on identifying MCSC by these Mabs and CSC markers. Moreover, cancer stem cell antigens that recognized by the functional monoclonal antibodies were obtained by immunoprecipitation and then identified by mass spectrometry analysis. Then our study would focus on elucidating the molecular mechanisms of antigen function and clarifying the clinical significance of MCSCs. It would provide potential molecule markers and candidate targets for predicting the liver cancer metastasis, recurrence and prognosis. Furthermore, two monoclonal antibodies were also selected for evaluating the efficacy of treating liver cancer metastasis in human liver cancer xenograph model.

最新研究发现肿瘤干细胞（CSC）具有明显的异质性，存在着多种生物学特性的亚群，其中肿瘤转移干细胞(MCSC)亚群成为最新的研究热点。对MCSC研究不仅能进一步解密肿瘤转移机制，也将为靶向肿瘤干细胞治疗肿瘤转移提供重要的实验依据。申请者在前期研究中成功制备了抗肝癌干细胞单抗库，已筛选获得多株识别肝癌干细胞的功能性单抗，并初步证明其中4株可能是识别MCSC亚群的单抗。本研究拟应用上述单抗和肿瘤干细胞标志物从人肝癌细胞系和肝癌组织中分离鉴定肝癌转移干细胞，应用免疫质谱技术分离获得与MCSC特异反应的单抗所识别的功能性抗原基因，进行临床相关性及分子机制研究，阐明MCSC在肝癌转移、复发及预后中的作用，为临床检测MCSC用于预示肿瘤转移及预后提供有应用潜力的分子标志物和候选靶标。同时完成1-2株靶向MCSC治疗性单抗在肝癌移植瘤模型中对肝癌转移和复发的药效学和安全性研究，评价其临床应用价值。

本项目筛选获得4株抗肝癌转移干细胞单抗，命名为Hab-1，28C10(Hab-2)，7B2(Hab-3)，11B7(Hab-4)。体外鉴定发现4株单抗能够识别肿瘤干细胞不同亚群，单抗Hab-1能够识别MHHCC97L细胞系中的ESA+、CD90+的肝癌干细胞；单抗28C10能够识别CD90+的肿瘤干细胞；另外两株单抗特异识别的7B2+、11B7+MHCC97L细胞成瘤性较阴性细胞高100倍，证明该两株单抗靶向肝癌干细胞。体外功能研究发现4株单抗均能显著抑制肝癌干细胞自我更新、耐药和侵袭；体内抑制移植瘤的生长和转移。单抗28C10体内药效实验结果显示28C10低剂量(2.5mg/kg)和高剂量(40mg/kg)对于肝癌移植瘤生长的抑制率分别为24.0%和67.7%，单顺铂治疗组和顺铂联合单抗28C10高剂量抗体组抑制率分别为35.9%和70.1%，表明该单抗治疗肝癌极具临床应用价值。.同时研究了所获肝癌干细胞靶标基因CPA4的表达功能和分子机制。免疫组化结果显示，CPA4在肝癌组织中表达的阳性率为48％，正常肝组织低表达；且CPA4和CD90在肝癌组织中的表达呈显著正相关。采用Real-Time PCR方法检测显示肝癌干细胞中CPA4与干性基因Sox-2和Oct-4呈正相关。体外功能研究显示CPA4可以促进肝癌细胞MHCC97L的迁移和侵袭能力。分子机制研究发现，CPA4基因参与激活STAT3信号通路，维持肿瘤干细胞干性。.为了研究所获抗肝癌干细胞单抗与抗PD-1或PD-L1单抗联合治疗肝癌提供实验证据。我们采用免疫组织化学的方法检测和分析PD-L1在肝细胞肝癌和胆管细胞癌中的表达和临床意义。结果显示PD-L1在肝癌组织中表达的阳性率为69.1%，且与肝细胞肝癌组织类型、浸润深度、TNM分期显著相关。PD-L1膜阳性表达与肝细胞肝癌病理分级显著相关。另外，在该项目支持下，我们通过多组学数据分析得出CEP170基因与肝癌预后不良呈正相关；免疫组化结果及数据库分析证明肝癌组织中CEP170表达量高于正常组织；敲降CEP170，肝癌细胞发生G0/G1细胞周期阻滞，显著抑制肝癌细胞增殖及克隆形成能力。.综上，本项目支持下筛选获得4株抗肝癌干细胞单抗和2个功能基因，有望为靶向肝癌转移干细胞治疗肝癌提供候选单抗药物。