C5a-C5aR通路调控肝癌循环肿瘤细胞释放和干性表达的机制及其分子标志物价值

Circulating tumor cells (CTCs) play as the potential sources in cancer relapse and distant metastasis. Our previous work found that the expression level of complement component 5a receptor (C5aR) on HCC cell membrane correlated with the cell count of CTCs measured in 7.5 ml of blood before surgery by comparing the expression profiles among resected tumor tissues from different HCC patients. We distinctively found that activation of C5a-C5aR pathway in vitro enhanced the migration of Huh7 cells, promoted the epithelial-mesenchymal transition, actived the Wnt pathway and upregulated the expression of EpCAM. EpCAM+/C5aR+ cells were detected in the CD45-depleted peripheral blood mononuclear cells, and might represent a certain subgroup of CTCs with stem cell-like properties in HCC. We raise the hypothesis that the activation of C5a-C5aR pathway enhances the releasing of CTCs and the stem cell-like properties in HCC. Analyzing the C5aR expression on CTCs helps to recognize for a certain CTCs subtype with targeted value. We aimed to clarify the effect of C5a-C5aR pathway on the releasing and stem cell-like properties of CTCs in HCC by orthotopic transplantation models with in vivo flow cytometry CTCs assay, and probe the protential capacity of C5aR as a therapeutic target in HCC recurrence and molecular biomarker for liquid biopsy.

循环肿瘤细胞（Circulating tumor cell，CTC）是肝癌转移复发的种子。我们经前期工作发现：①患者肝癌组织中补体受体C5aR表达与术前CTC数量相关；②活化肝癌C5a-C5aR通路可增强细胞系的侵袭性、上皮-间充质转化、上调Wnt通路、并增加干性标志物EpCAM表达，而拮抗C5aR可阻断上述效应；③肝癌患者EpCAM+CTC表面亦表达有C5aR。我们由此推测C5a-C5aR通路是促肝癌CTC释放和干性表达的重要因素，检测CTC表面C5aR有助于识别其中具有特殊靶向意义的亚群。本研究将运用小鼠肝癌原位种植模型联合“活体流式”CTC技术，阐明C5a-C5aR通路对CTC释放及干性表达的影响和机制；运用人源肿瘤小鼠移植模型，阐明靶向C5a-C5aR通路的抗CTC释放、抗干性表达作用；结合临床患者资料，评价C5aR作为CTC分子标志物的价值，为肝癌转移复发的防治提供新思路和依据。

循环肿瘤细胞（Circulating tumor cell，CTC）是肝癌转移复发的种子。然而CTC在肝癌患者体内的释放机制，以及造成肝癌患者体内CTC数量差异的原因未明。我们通过对不同CTC数量肝癌患者组织测序，发现外周血CTC数量增多的患者肝癌组织中补体通路轴C5a-C5aR活化，补体受体C5aR表达与术前CTC数量相关。为进一步研究该通路轴在肝癌CTC释放中的作用，我们通过对不同转移潜能肝癌细胞株进行C5aR敲除／过表达的体外功能实验，观察验证C5a-C5aR轴在促进肝癌细胞侵袭转移中的作用。结果显示C5aR敲除后，肝癌细胞增殖能力及侵袭能力下降。而C5aR过表达后,其增殖能力及侵袭能力增强。为更好模拟C5a-C5aR轴在体内促进CTC释放中的重要作用，利用肝癌细胞系建立小鼠原位肝脏种植模型，我们发现C5aR敲除组小鼠肝脏肿瘤体积显著小于对照组（1.2±0.3cm^3 vs. 0.4±0.2cm^3），而C5aR过表达组的肿瘤体积却显著高于对照组（0.8±0.1cm^3 vs. 0.3±0.03cm^3）。同时C5aR敲除组小鼠肺部肿瘤灶数量显著低于对照组,而C5aR过表达组的肺转移数量显著高于对照组。通过比较干扰C5aR前后小鼠外周血CTC数量的变化，发现敲除C5aR后小鼠外周血CTC数量显著减少，而高表达C5aR后CTC数量则显著增加。为进一步探究C5a-C5aR轴调控CTC释放的机制，对C5aR敲除细胞系进行高通量测序并对其下游筛选分析。我们发现C5a-C5aR轴通过促进NF-κB通路核心分子p65的磷酸化诱导NF-κB入核，并促进INHBA表达。INHBA参与调控TGF-β下游多种核心分子表达，从而影响肝癌细胞发生EMT并促进其释放入血，使用靶向C5a-C5aR通路抑制剂可有效阻碍CTC释放。通过对187例肝癌患者的免疫组化结果验证C5aR可作为肝癌预后的分子标签。C5aR高表达患者组复发及死亡率显著高于C5aR低表达患者组。单、多因素风险回归分析也表明C5aR表达量是肝癌患者术后复发的独立风险因素。.本研究发现了补体相关信号通路C5a-C5aR在介导肝癌CTC释放中的重要作用，并解释了C5a-C5aR调控TGF-β通路诱导EMT的分子机制，找到了该通路的关键节点分子INHBA。靶向阻断C5a-C5aR通路可有效阻碍CTC释放，对肝癌治疗有着积极作用。