癌旁激活的肝星状细胞促进肝癌介入后残癌存活的分子机制及其靶向干预

Residue tumor locating at the periphery of lesions suggests that signals from peri-tumoral tissues promote the survival of tumor cells to resist the attacks of chemotherapy, ischemia and hypoxia. Hepatic stellate cells(HSC) play the important roles during the progresses of hepatitis, cirrhosis and HCC. In our previous work, we found that activated HSC secreted FGF-9 to promote the resistance of HCC cells to apoptosis induced by chemotherapeutic agents and hypoxia.Activation of hedgehog signal pathway were involved in these processes. We hypothesis that survival signals from HSC promote the formation of residue tumors after interventional therapy, and interrupting the interaction between HSC cells and cancer cells would improve the therapeutic effects of interventional treatment. We will study the influences of activated HSC on the apoptosis of HCC cells under the conditions of chemotherapeutic agents and hypoxia, by using the in vitro two-dimensional, three-dimensional cell co-culture system. We will also explore the roles of FGF-9/FGF receptor 2/Hedgehog/Gli-2 signal transduction,and especially pay attention to the roles of PKA and SMO. Moreover, we will testify the effects of combining interventional therapy with Hedgehog signaling inhibitor targeting SMO in the HCC animal modle. Our study will provide a new mechanism of suvival of residue tumor, and a novel experimental basis of Hedgehog targeted therapy for residue tumor after interventional therpy.

介入后残癌多发生在病灶的周边，提示癌-癌旁组织相互作用为癌细胞抵抗化疗、缺血缺氧"打击"提供了生存信号。肝星状细胞(HSC)在"肝炎-肝硬化-肝癌"发展中起重要作用。前期研究发现HSC分泌成纤维细胞生长因子9(FGF-9）诱导肝癌细胞抵抗化疗、缺氧引起凋亡作用；且存在Hedgehog通路激活。本课题提出"HSC提供生存信号促进残癌存活；阻断HSC-肝癌细胞之间关键分子，抑制残癌"的设想。利用体外二维、三维细胞共培养装置，研究在化疗、缺氧条件下，HSC对肝癌细胞凋亡的影响，重点探讨FGF-9/FGFR-2/ Hedgehog/GLI-2通路在两者之间的作用；明确PKA分子在介导FGF-9/FGFR-2信号、Hedgehog通路激活之间"桥梁"作用；以及SMO分子细胞膜募集、激活对Hedgehog通路关键作用；肝癌动物模型中，验证介入联合针对SMO的Hedgehog抑制剂，抑制残癌发生的效果。

介入（化疗栓塞）治疗肝癌面临的最大问题是残癌，导致治疗失败。除了癌细胞本身因素之外，癌周的微环境为癌细胞存活发展提供重要支撑。激活的肝星状细胞(HSC)是肝癌微环境中重要间质细胞，具有多样功能。本研究使用化疗、缺氧条件模拟临床介入，利用激活HSC上清液或者HSC共培养残癌细胞，研究激活的HSC促进肝癌存活机制，寻找促进残癌存活的关键分子及其靶点；并用动物模型及其临床标本进行验证。通过研究我们得出以下结论：（1）激活的肝星状细胞（HSC）通过分泌成纤维细胞生长因子9 (FGF-9) 促进化疗药物、缺氧处理后残癌细胞的存活、增殖和侵袭能力，此过程与Hedgehog通路激活、上皮间质转化（EMT）相关分子改变有关。Hedgehog通路中PKA分子激活、SMO移位及细胞膜募集、GLI-2在其中起重要作用。（2）建立肝癌介入的实验动物模型模拟临床介入化疗栓塞治疗；介入化疗栓塞联合Hedgehog抑制剂能够明显抑制肝癌的生长，延长生存。（3）介入后残癌切除标本中，FGF-9表达与GIL-2表达相关，FGF-9水平和介入治疗反应、患者预后明显相关。这个研究从肿瘤间质角度对肝癌介入治疗抵抗进行了新的解释，即激活的星状细胞分泌FGF-9促进残癌细胞对介入治疗的抵抗，依赖于Hedgehog通路激活，提示了介入联合Hedgehog通路抑制剂可以提高疗效。此外，还对化疗损伤后HSC分泌的生长分化因子15(GDF15)、CCL26促进肝癌治疗抵抗进行了研究；高硬度基质促进癌细胞对化疗的耐药；以及对介入治疗后残癌的控制策略进行研究。