脾脏中性粒细胞通过“N2”型极化促进肝癌进展的作用机制研究

Polarization of tumor-associated neutrophils (TANs) towards protumorigenic "N2" phenotype plays critical roles in the progression of hepatocellular carcinoma (HCC), but the origin of TANs in HCC remains obscure. It has been reported that the spleen is one of the origins of TANs in transplanted tumor model. Using murine HCC model, our previous study also showed that the frequency of splenic and hepatic neutrophils was significantly increased in tumor-bearing mice, while splenectomy resulted in lowered hepatic neutrophils infiltration, and accompanied by decreased tumor growth. So the previous study suggested that the splenic neutrophils might accumulate to the tumor site and promote HCC growth via a protumorigenic "N2" phenotype polarization. In the present study, we proposed to establish orthotopic and metastatic murine HCC model. Immunohistochemical analysis and flow cytometry analysis will be used to illustrate the frequency and phenotype of splenic neutrophils during the progression of HCC. Proliferation assay and cytotoxicity assays will be used to determine the function of splenic neutrophils. In some experiments, the murine models will receive splenectomy treatment or adoptive transfer of splenic neutrophils to investigate the role of splenic neutrophils on HCC growth and metastasis. Finally, using RNA expression assay and multiplex protein profiling assay, we would try to explore the regulatory mechanisms involved in the accumulation and protumorigenic polarization of splenic neutrophils in HCC progression. The present study expects to provide new information for understanding the regional immunity of liver-spleen site during HCC progression.

肿瘤相关中性粒细胞（TANs）向“N2”型极化可促进肝癌进展，但TANs的来源尚待明确。脾脏在肝脾区域免疫中具有重要作用。本课题前期研究示，肝癌模型小鼠脾脏及肝脏内中性粒细胞数量显著升高，切脾可降低肝脏内中性粒细胞数量并抑制肿瘤生长。由此我们假设肝癌负荷下脾脏中性粒细胞向肝内及肿瘤局部聚集，并向“N2”型极化促进肿瘤进展。本课题拟建立肝癌原位瘤/转移瘤动物模型，分析脾脏、肝脏及肿瘤组织内中性粒细胞的表型和功能，明确中性粒细胞向“N2”型极化的作用；其次通过切脾及脾脏中性粒细胞过继回输研究，比较不同处理组小鼠肿瘤生长情况，明确脾脏中性粒细胞参与肝癌进展的作用；进一步通过转录组分析、细胞因子芯片及体内外功能研究，对参与调控TANs趋化的相关因子进行筛选、鉴定及功能验证，阐明脾脏中性粒细胞促进肝癌进展的作用机制。本课题拟为认识肝脾区域免疫在肝癌进展中的作用提供新的理论基础。

肿瘤相关中性粒细胞(TANs)向“N2”型极化可促进肝癌进展，但TANs的来源尚待明确。 脾脏在肝脾区域免疫中具有重要作用。本课题前期研究示，肝癌模型小鼠脾脏及肝脏内中性粒细胞数量显著升高，切脾可降低肝脏内中性粒细胞数量并抑制肿瘤生长。由此我们假设肝癌负荷下脾脏中性粒细胞向肝内及肿瘤局部聚集，并向“N2”型极化促进肿瘤进展。本课题在前期研究的基础上，建立了稳定的肝癌原位瘤小鼠模型，对肝癌负荷下脾脏内中性粒细胞的表型和功能进行系统分析，发现肝癌负荷下脾脏内中性粒细胞亚群存在动态变化，在肿瘤负荷下脾脏内脾脏内中性粒细胞浸润水平升高、伴随CD101-中性粒细胞亚群比例升高。体外功能分析结合转录组测序技术研究显示，脾脏内CD101-中性粒细胞亚群基因表达与经典中性粒细胞有差异，差异表达基因功能富集分析显示CD101-粒细胞亚群在正性免疫调节及免疫应答的功能减弱，如T细胞活化和细胞因子及受体互作反应降低，在染色体复制和细胞周期调控方面功能增强，推测CD101-中性粒细胞亚群在抗肿瘤免疫应答中的作用相对减弱。体外功能检测显示CD101-中性粒细胞亚群吞噬功能减弱、ROS释放水平降低，提示其免疫应答能力相对减弱；同时与肿瘤细胞体内共注射研究证实，CD101-中性粒细胞亚群可促进肿瘤细胞生长，且肿瘤条件培养基可促进CD101-中性粒细胞向CD101+中性粒细胞表型转化。综上，肿瘤负荷下脾脏内促进肿瘤进展的中性粒细胞亚群增多，后者成为肝癌负荷下肿瘤相关中性粒细胞的重要来源，并可在在肿瘤微环境作用下可发生表型转化，本研究揭示了脾脏通过中性粒细胞表型转化参与肝癌进展的重要作用，为认识肝脾区域免疫在肝癌进展中的作用提供了新的理论基础。