B7-H4+巨噬细胞亚群参与构建肺腺癌胸膜预转移小生境及其作用机制的研究

Tumor metastasis is not only dependent upon the characteristics as well as invasion and migration potential of cancer cells but is also related to the interaction between tumor and metastatic niche cells. In our previous study, B7-H4-expressing macrophage (CD68+ cells) was in increased lung cancer compared among patients with tuberculosis and healthy donors. Intracellular levels of IL-10, TGF-β, VEGF and CCR2 expression in B7-H4+CD68+ macrophage were high than those in B7-H4-CD68+ macrophage, but a decrease secretion of IL-15 in B7-H4+CD68+ macrophage. In malignant pleural effusion (MPE) model mice, the infiltration of B7-H4+CD68+ macrophage in both abnormal and healthy pleural represented an increase compared with that in the corresponding spleen, liver and lung. We hypothesized that B7-H4+CD68+ macrophage was involved in the development of pleural pre-metastatic niche of lung adenocarcinoma. In this study, using cytology, immunology and animal methodology, we would define the formation of B7-H4+macrophage in tumor microenvironment of lung adenocarcinoma and its mechanism of infiltration of B7-H4+CD68+ macrophage in pleura. Then, it is to investigate the influence of B7-H4+CD68+ macrophage on the pleural vascular function and immune microenvironment. Furthermore, we would observe the therapeutic effect of local B7-H4 monoclonal antibody injection on MPE. Through the above research can improve the understanding of the immunological mechanism of metastasis of lung adenocarcinoma with pleural and provide valuable data for clinical treatment. Our data would support the possibility that B7-H4+ tumor macrophages contribute the process of pleural metastasis of lung adenocarcinoma, suggesting the B7-H4 mAb may be used as a reliable therapeutic reagent in MPE.

肿瘤的转移不仅和癌细胞生物学特性有关，也被认为和“肿瘤预转移小生境”发生有关。本课题组研究发现，肺癌患者存在一群以表达B7-H4为特点的巨噬细胞(M）亚群，和B7-H4-M相比具有不同的IL-10、TGF-β、IL-15、VEGF分泌和CCR2的表达，并能显著上调肺腺癌细胞CXCR4表达；在恶性胸腔积液小鼠模型中，不同于肝脏、肺脏和脾脏，健侧和患侧胸膜中均高水平浸润B7-H4+M。据此假说，B7-H4+M通过不同的免疫因子分泌和B7-H4固有免疫特性参与构建肺腺癌胸膜预转移小生境。鉴此，本项目将以肺腺癌胸膜转移为研究对象，基于前期研究，运用细胞学、免疫学、动物学方法，探讨B7-H4+M在肺腺癌微环境中的形成，分析其胸膜浸润机制及其对胸膜血管功能和免疫微环境的影响，并观察B7-H4单抗对恶性胸腔积液的治疗作用。通过以上研究可提高对肺腺癌胸膜转移免疫学机制的认识，为其临床治疗提供有价值的数据。

胸膜是肺部肿瘤常见转移部位，是临床肺癌治疗中需密切关注的并发症，也是良好的肺癌微环境研究对象。本项目以B7-H4、B7-H4+巨噬细胞为切入点对肺癌致胸腔积液、转移性肺癌进行了系列研究，并开展了B7-H4抗体治疗恶性胸腔积液的实验研究，同时对恶性胸腔积液中浸润T细胞亚群作了系列分析，取得的成果如下，1）发现B7-H4在癌细胞胞浆中普遍存在，但细胞核中B7-H4存在差异表达，提示B7-H4分子的细胞核/细胞浆的转位反映了肿瘤的发展过程。2）建立了恶性胸腔积液模型，恶性胸水和局部胸膜肿瘤组织均高水平浸润B7-H4+巨噬细胞。3）观察了B7-H4单抗对恶性胸腔积液的治疗作用，动物实验结果显示有效率80％。4）对恶性胸腔积液中CD39+CD8+T细胞的浸润及其意义进行了研究，发现了CD39+CD8+T细胞和伴或不伴驱动基因改变的肺腺癌性恶性胸水密切相关，并且表现为耗竭性T细胞表型。5）恶性胸腔积液中T细胞亚群的线粒体功能分析，与自身外周血相比，恶性胸水中的CD8+T细胞线粒体质量更少,膜电势更低。6）探讨了4-1BB信号增强PD-L1单抗增强抑制肺癌细胞生长的作用及其机制， PD-L1单抗不能发挥有效地抑制肿瘤生长的作用，但联合4-1BB单抗后，PD-L1单抗抑制肿瘤生长作用明显得到增强，肿瘤微环境分析发现，联合治疗能进一步增强局部Th1型免疫应答，尤其是能提高Resident T细胞的浸润水平，PD-L1和4-1BB单抗联合处理能进一步提高Resident T细胞杀伤肺癌细胞能力。项目建立了恶性胸腔积液研究技术，完善了肿瘤免疫治疗和微环境分析技术，采用胸腔内注射B7-H4抗体可为恶性胸腔积液的治疗提供新的途径，成果发表SCI收录论文5篇，已培养和在培养研究生共计10名，作为主要完成人获省部级科技奖励2项。