LOX介导基质硬度“土壤”改造参与肝癌预转移龛形成的实验基础研究

Pre-metastatic niche formation, a rate-limiting step in the process of tumor metastasis, accelerates and favors the emergence of metastasis in the target organ. In recent years, the related studies on premetastatic niche has just started, and of particular note,to date whether matrix stiffness “soil” microenvironment involves in the formation of pre-metastatic niche and further modulates the survival and growth of HCC cells in the target organ is still largely unknown. Our previous studies suggest that increasing matrix stiffness drives and regulates the initiation stage of HCC metastasis, and high expression of LOX is stiffness-dependent, therefore, we speculate that LOX mediated-matrix stiffness alteration may involve in the formation of pre-metastatic niche of HCC and further influence the colonization and growth of metastatic tumor cells in the target organ. This project is mainly to elucidate the effects of LOX-mediated matrix stiffness “soil” amelioration on pre-metastatic niche formation and its related mechanism based on the established novel experimental platforms in vivo and in vitro simulating pre-metastatic niche stiffness micronenvironment of HCC, and provide more evidences to validate the roles of stiffness-dependant tumor derived soluable factor LOX during pre-metastatic niche formation and stiffness “soil” amelioration in the target organ including BMDCs recruitment, mutual interaction between BMDCs and pre-resided fibroblast cells, matrix stiffness “soil” environment remodeling, pre-metastatic niche formation, growth and survival of HCC cells in niche. Undoubtedly, these results will build up the solid theoretical and experimental basis for intervention of pre-metastatic niche of HCC.

预转移龛形成可加速、促成转移在靶器官的最终实现，是肿瘤转移中具有“限速”作用的关键节点，目前肝癌预转移龛研究尚处起步阶段，从基质硬度“土壤”微环境角度，解析其调控肝癌靶器官预转移龛形成，癌细胞转移定植生长均未见相关报道。项目组前期研究显示，基质硬度增加可参与肝癌侵袭转移始动阶段调控，且LOX上调表达呈硬度依赖性，其介导基质硬度微环境改变是否同样参与肝癌预转移龛形成，影响转移在靶器官实现成为课题新的增长点。本项目通过构建具有创新意义的肝癌预转移龛实验平台，对目标LOX作为硬度依赖可溶性因子“特使”，诱导募集BMDCs到达远端特异靶器官，并与定植此处的基质细胞相互作用，协同重塑基质硬度土壤微环境，参与肝癌预转移龛构建，形成循环游离癌细胞粘附、定植生长的适宜“土壤”等相关过程进行解析，以期获得基质硬度土壤参与预转移龛形成及其调控肝癌细胞转移特征的系列证据，为针对预转移龛干预提供理论和实验上依据。

预转移龛形成可加速、促成转移在靶器官的最终实现，是肿瘤转移中具有“限速”作用的关键节点。从基质硬度生物力学角度，解析原发瘤调控靶器官肺预转移龛形成相关机制目前尚未见报道。项目组前期研究证实基质硬度增加可显著增强肝癌细胞恶性特征，包括上调侵袭转移关联基因表达，增强肝癌细胞干性特征，独立诱导肝癌细胞EMT发生，本项目则进一步聚焦基质硬度增加诱导肝癌肺预转移龛形成机制，同时探索肺基质硬度对肺预转移龛形成影响。项目通过建立有创新意义的肝、肺硬度基底体外细胞培养平台，无瘤荷裸鼠肺预转移龛模型及LOXL2过表达裸鼠原位肝癌模型，对LOXL2参与基质硬度诱导肝癌肺预转移龛形成机制进行了多角度解析，发现基质硬度增加既能活化肝癌细胞integrinβ1/α5/JNK/c-JUN信号通路上调LOXL2表达，又能增强肝癌细胞周边M2型巨噬细胞极化，通过激活integrinβ5–FAK-MEK1/2-ERK1/2-HIF-1α途径促进巨噬细胞LOXL2表达，同时也阐明了原发瘤来源的LOXL2参与基质硬度诱导肝癌肺预转移龛形成的相关机制。原发瘤释放可溶性因子LOXL2随血流到达转移靶器官肺组织募集BMDCs，同时作用肺成纤维细胞，使其fibronectin，MMP9均上调表达，导致肺基质土壤重塑。此外，LOXL2处理后的肺成纤维细胞可明显增加肝癌细胞粘附数量。LOXL2处理后的肺成纤维细胞CXCL12表达也明显升高，趋化肝癌细胞靶向肺预转移龛。LOXL2也可增加肝癌细胞迁移和侵袭。数据同时也显示，LOXL2诱导肺基质变硬可进一步增强肝癌细胞靶向肺预转移龛。LOXL2加速肺预转移龛形成促转移在动物模型及临床肝癌组织标本也获得验证。项目形成了有创新意义的肝、肺硬度研究实验平台，发现了一些硬度调控肝癌预转移龛形成的新机制，对充实、更新基质硬度力学信号调控肝癌侵袭转移理论知识，及针对预转移龛、硬度微环境干预研究奠定了良好的理论实验基础。