胃癌细胞源性外泌体对肿瘤相关中性粒细胞的募集和极化作用及其机制研究

Neutrophils are the important component of innate immune system and are the first line of defense during infections. Neutrophils are also found infiltrating many types of tumors, and named as tumor-associated neutrophils (TANs). TANs can have an antitumorigenic (N1) phenotype but also a protumorigenic (N2) phenotype. Up to now, the mechanism for the recruitment and polarization of TANs has not been clarified. Our previous studies found that the number of N2 type TANs increased in gastric cancer, and neutrophils coming from normal peripheral blood could be polarized to N2 type by culture supernatant of gastric cancer cells. Exosomes are the perfect carriers of information transmission between cells. Therefore, we hypothesize that the gastric cancer cells can recruit and polarize TANs through exosomes. In this project, we will compare the levels of chemokines and polarization factors (TGF-beta, IFN-beta) of neutrophil in the exosomes derived from gastric cancer and adjacent tissues, gastric cancer cells and gastric mucosa cells. After treated with different exosomes, the capacity of neutrophil migration and the expression of chemokine receptor on neutrophil will be observed, the polarization and MAPK signaling pathway activation will be detected. The mice model of gastric cancer will be established and intervened by exosomes, to explore the effects of exosomes on recruitment and polarization of TANs. This project will provide new understanding of mechanism of tumor growth and metastasis, and provide new therapeutic options for cancer treatment.

中性粒细胞是固有免疫系统的重要组成，是抗感染的主力军。但浸润在肿瘤中的“肿瘤相关中性粒细胞”(TANs)有两种极化方向，既有抗肿瘤的N1型, 又有促肿瘤的N2型。目前对TANs的募集和极化的机制尚未阐明。我们前期发现胃癌中N2型TANs增多，正常人外周血中性粒细胞可被胃癌细胞培养上清极化为N2型。而外泌体是细胞间传递信息的载体。由此提出胃癌细胞通过外泌体募集和极化TANs。本项目拟比较胃癌和癌旁组织、胃癌细胞和胃黏膜细胞的外泌体所含的中性粒细胞趋化因子和极化因子（TGF-β、IFN-β）含量；用外泌体干预正常人中性粒细胞，观察中性粒细胞迁移能力和趋化因子受体表达的变化、中性粒细胞极化情况及MAPK信号通路激活情况；用外泌体干预荷胃癌鼠模型，观察外泌体对移植瘤TANs的募集和极化作用。阐明胃癌细胞源性外泌体对TANs的募集和极化作用及其机制，为了解胃癌生长转移机理，及抗肿瘤治疗提供新的线索。

肿瘤微环境的变化直接影响肿瘤的发展和预后，其中炎症被认为是最重要的因素之一。恶性肿瘤中浸润的炎症细胞“肿瘤相关中性粒细胞(TANs)”, 既有抑制肿瘤生长的N1型, 又有促进肿瘤生长和转移的N2型，其募集和极化的机制尚未阐明。细胞外囊泡（EVs）可以携带各种生物分子释放至细胞外影响环境和其他细胞功能。其中外泌体具有内含物丰富、在血液中的稳定性高、向细胞运输生物信息的效率高等特点，是理想的细胞因子和趋化因子运载工具。本项目通过比较人胃癌和癌旁组织、胃癌细胞和胃黏膜细胞分泌的外泌体所含的中性粒细胞趋化因子和极化因子；用外泌体干预正常人中性粒细胞，观察中性粒细胞迁移能力和趋化因子受体表达的变化和中性粒细胞极化情况；人多通路磷酸化芯片检测外泌体干预中性粒细胞后相关信号通路的变化；用外泌体干预荷胃癌鼠模型，观察外泌体对移植瘤中TANs的募集和极化作用。结果表明，胃癌细胞源性的外泌体包含较多的对中性粒细胞有趋化作用的CXCL1、2、5、8和有极化作用的TGF-β。可通过趋化因子，并上调中性粒细胞趋化因子受体CXCR1、CXCR2，增强其迁徙能力，以募集中性粒细胞到肿瘤组织中浸润。同时胃癌细胞源性的外泌体中的极化因子可诱导肿瘤组织中的TANs上调NE、LOX、MMP2、MMP9等，下调TNF-α、ICAM-1、ROS，使TANs向促肿瘤的N2型极化，促进血管生成，进而加速胃癌进展。外泌体的作用可能是通过活化中性粒细胞的PI3K-Akt、MAPK等信号通路产生的。我们的研究结果在一定程度上阐明了胃癌细胞源性外泌体对TANs的募集和极化作用及其机制，为了解胃癌的生长和侵袭转移机理，及抗肿瘤治疗提供了新的线索。具有重要的科学意义和潜在的临床应用前景，具有重要的社会效益。