癌周热激活的肝星状细胞促进肝癌热消融后残癌生长、侵袭转移分子机制及其靶向干预

Tumor microenvironment influences the tumor response to various anti-cancer treatments. Residue tumor after thermal ablation (eg. Radio-frequency ablation, microwave) is a major barrier to improve the therapeutic effect. Our previous study has found that heat-activated hepatic stellate cells (HSC) secrete an amount of collagen I. However, it is unclear whether collagen I can modulate the tumor response to thermal ablation in hepatocellular carcinoma(HCC). In this project, we propose the hypothesis that heat-activated HSC cells secrete collagen I to promote the formation of residue HCC, and to enhance the invasion and metastasis. Firstly, we will testify whether HSC cells can prevent the apoptosis of HCC cells under heat stress by co-culturing HCC cells and HSC in vitro and in vivo. If blocking the secretion of collagen I, whether can it reverse the protective effects of HSC on HCC cells? Secondly, we will study the effects of collagen I on the residue HCC cells after thermal attack. Particularly, how does the collagen I modulate the pseudopodial formation, migration of residue HCC cells and the activated signal transduction of integrinβ1/RhoA/ROCK1/MLC/FAK. Thirdly, using the drug FK506 targeting FKBP65 (molecular chaperon of collagen I) reduces the secretion of collagen I in HSC cells. Whether can FK506 combined with thermal ablation reduce the formation of residue tumor in HCC xenograft model? Lastly, the relationships among HSC, collagen I and residue tumor will be investigated in clinical residue HCC specimens after thermal ablation.

癌周微环境影响癌对各种治疗的反应。残癌是肝癌热消融治疗（射频、微波）疗效难以提高主要因素，多发生在癌周。前期发现热激活肝星状细胞（HSC）分泌I型胶原增加，但它在肝癌对热消融反应中作用尚不清楚。本课题提出"热激活HSC通过分泌I型胶原促进热消融后残癌发生，促进残癌细胞生长、侵袭转移"假说。通过体外二维、三维及动物体内共培养肝癌细胞和HSC，在热应激作用下，HSC能否减少肝癌细胞凋亡，促进存活；抑制HSC分泌I型胶原能否逆转其 "保护"作用。进一步，研究I型胶原对热应激后残癌生物学行为影响，重点是I型胶原对残癌细胞伪足形成、运动影响及整合素β1/RhoA/ROCK1/MLC/FAK通路改变；应用FK506靶向结合I型胶原分子伴侣FKBP65，能否抑制I型胶原分泌；FK506联合热消融治疗肝癌动物移植模型,能否减少残癌发生；应用射频后残癌临床切除标本，检验癌周HSC、I型胶原表达与残癌的关系。

热消融是不能手术早期肝癌一线治疗；由于其微创性，已被应用在中期肝癌、大肝癌消融治疗。热消融后残癌是导致治疗后肝癌复发，甚至是肿瘤快速进展主要原因，也是阻碍消融疗效提高主要因素。本课题聚焦热消融后微环境如何影响残癌细胞存活、进展分子机制，尤其是肝星状细胞（HSC）在其中作用，以及潜在干预措施，获得一系列原创性成果：（1）Collagen I对热消融后残癌的影响。热处理后HSC分泌Collagen I增加；肝癌不完全消融动物模型中，消融周边Collagen I沉积增加；Collagen I能明显促进热处理后残癌细胞增殖、EMT、干细胞样表型，与ERK磷酸化激活有关；索拉非尼逆转Collagen I促消融后残癌进展的效应；Collagen I激活整合素β1/RhoA/ROCK1/MLC/FAK通路，促进残癌细胞伪足形成；FK506抑制HSC分泌Collagen I，与FKBP65有关；肝癌不完全消融动物模型，FK506抑制后残癌体内生长；消融后残癌临床切除标本，癌周HSC、Collagen I表达增加，其表达增加与消融残癌切除术后的复发、患者预后有关。（2）Collagen I增加会导致细胞外基质硬度增加。将热处理后残癌细胞培养在不同基质硬度3D凝胶上，模拟消融后物理微环境。基质硬度增加促进残癌细胞增殖，这与硬度依赖性ERK磷酸化有关；维生素K1抑制ERK磷酸化，抑制残癌细胞增殖。维生素K1与索拉非尼联用明显抑制残癌体内生长。（3）肝癌细胞如何在亚致死性热应激中生存并迅速生长的机制。激活HSC分泌HGF，增强残癌细胞24h内自噬性存活。HGF通过c-Met/ERK及其下游ATG5/Beclin1或cyclinD1，调控热损伤后残癌细胞自噬存活和增殖的状态。肝癌不完全消融动物模型中，抑制自噬结合c-Met抑制剂通过抑制自噬、减少增殖和增加凋亡，明显抑制残癌进展。（4）HSC分泌骨膜蛋白（POSTN）促进热消融后残癌进展。POSTN能明显增强热处理后残癌细胞增殖、运动、侵袭、EMT活化和抗凋亡、干性特征。POSTN通过整合素β1诱导相关的通路促进增殖或干性分子表达。二甲双胍或维生素D类似物抑制POSTN分泌，明显抑制残癌的生长。本课题从不同层面阐述了热消融后微环境促进残癌存活、进展分子机制及潜在干预措施，对提高消融疗效，具有潜在临床应用价值。