【作者】 张鑫；

【导师】 杨默；

【作者基本信息】 南方医科大学 ， 儿科学， 2019， 博士

【摘要】 肿瘤是一种病理性免疫耐受性疾病,肿瘤细胞往往可以利用机体复杂的异常失活或过度失活的免疫反应而获得免疫逃逸,进而得以持续生长和发生远处转移。近年来研究发现肿瘤细胞能够内化活的免疫细胞形成异质性cell-in-cell结构(heterotypic cell-in-cell,heCIC),免疫细胞(如 NK、CTL 细胞等)与肿瘤细胞可以利用免疫微环境的适应性特性,影响免疫自稳、肿瘤的发生发展以及肿瘤进化等多个生物学过程。Cell-in-cell结构(CIC structure,CICs)形成过程中,免疫细胞进入肿瘤细胞内部是一个主动过程,进入肿瘤细胞后免疫细胞的命运决定着肿瘤免疫调节的方向。目前已知的CICs有两类,除异质性CIC(heCIC)结构外还有同质性CIC(homotypic CIC,hoCIC)结构,即同种或异种肿瘤细胞之间形成的CICs。既往研究表明影响hoCIC形成的关键因素包括:E-钙黏蛋白介导的细胞间黏附连接和RhoA-ROCK-MLC信号通路的活化,而影响heCIC形成的具体机制尚待进一步研究。本研究通过对不同同源PLC/PRF/5单克隆细胞株分析,建立heCIC结构模型,利用基因芯片分析筛选出关键分子GOLM1。通过调节GOLM1在肿瘤细胞中的表达水平来检测heCIC的形成。结果提示降低GOLM1表达可促进heCIC的形成,而过表达GOLM1后抑制heCIC的形成。之后的实验发现,GOLM1与E-cadherin的表达量负相关,且发生在溶酶体通路,回升E-cadherin可有效提高heCIC的形成。体内外生物学功能研究发现,heCIC的形成可以产生肿瘤细胞免疫调节作用,提高heCIC的形成可以增强NK细胞对肿瘤细胞的杀伤作用。为进一步明确CIC结构与肿瘤患者临床预后与病理分型之间的关系,我们利用“EML”(E-cadherin、CD68和CD45)多色免疫荧光技术,对肿瘤组织芯片(TMA)进行分析并鉴定出不同CIC亚型,将不同CIC亚型同临床病理资料相结合进行单因素及多因素生存分析。结果表明,CIC结构及其亚型不仅显示出与TNM分期和癌症分型等传统病理参数相当的预后能力,甚至新亚型MiT对肿瘤患者临床预后分析具有更优越的价值。综上所述,本研究通过对heCIC形成的关键分子GOLM1进行验证和分析,发现GOLM1对heCIC的形成具有负向调控作用。并明确了 GOLM1调节heCIC的形成机制,即GOLM1通过的调节E-cadherin的表达量而影响heCIC的形成,这一过程和溶酶体通路相关。本研究进一步发现GOLM1通过调节heCIC的形成影响免疫细胞对肿瘤细胞的杀伤效力,CIC在此过程中贡献了一定的作用,增强了 NK细胞对肿瘤的杀伤作用。并且我们首次将CIC结构结合到TMA进行系统性预后分析,并提出了“功能性病理”这一新诊断指标,经验证CIC亚型应用于肿瘤患者临床预后判断具有极高的敏感性和特异性,是一种优越的生物学指标。我们的研究有望为肿瘤免疫治疗、临床诊断及预后分析提供新的靶点和策略。更多还原

【Abstract】 Tumor is a pathological immune-tolerant disease.Tumor cells can often use the complex abnormal inactivation or excessively inactivated immune response to obtain immune escape,which can continue to grow and distant metastasis.In recent years,it has been found that tumor cells can internalize living immune cells to form heterotypic cell-in-cell(heCIC),immune cells(such as NK,CTL cells,etc.)and tumor cells can be used for immunization.The adaptive properties of the microenvironment affect many biological processes such as immune homeostasis,tumor development and tumor evolution.During the formation of cell-in-cell structures(CICs),the entry of immune cells into tumor cells is an active process.The fate of immune cells after entering tumor cells determines the direction of tumor immune regulation.There are two types of CICs known at present,in addition to the heterotypic CIC(heCIC)structure,there is a homotypic CIC(hoCIC)structure,that is,CICs formed between homotypic or heterotypic tumor cells.Previous studies have shown that the key factors affecting the formation of hoCIC include:E-cadherin-mediated intercellular adhesion junction and activation of RhoA-ROCK-MLC signaling pathway,and the specific mechanism affecting the formation of heCIC remains to be further studied.In this study,the heCIC structural model was established by analyzing different homotypic PLC/PRF/5 monoclonal cell lines,and the key molecule GOLM1 was screened by gene chip analysis.The formation of heCIC was detected by modulating the expression level of GOLM1 in tumor cells.The results suggest that decreasing GOLM1 expression promotes the formation of heCIC,while overexpression of GOLM1 inhibits the formation of heCIC.Subsequent experiments found that GOLM1 is negatively correlated with the expression of E-cadherin and occurs in the lysosomal pathway.E-cadherin can effectively increase the formation of heCIC.In vitro and in vivo biological function studies have found that the formation of heCIC can produce immune regulation of tumor cells,and the formation of heCIC can enhance the killing effect of NK cells on tumor cells.To further clarify the relationship between CIC structure and clinical prognosis and pathological type of tumor patients,we used "EML"(E-cadherin,CD68 and CD45)multicolor immunofluorescence techniques to analyze and identify tumor tissue chips(TMA).Different CIC subtypes were combined,and different CIC subtypes were combined with clinical pathological data for single factor and multifactor survival analysis.The results showed that the CIC structure and its subtypes not only showed prognostic ability comparable to traditional pathological parameters such as TNM staging and cancer typing,but even the new subtype MiT has superior value for clinical prognosis analysis of tumor patients.In summary,this study verified and analyzed the key molecule GOLM1 formed by heCIC,and found that GOLM1 has a negative regulation effect on the formation of heCIC.It was also clarified that GOLM1 regulates the formation mechanism of heCIC,that is,the regulation of the expression of E-cadherin by GOLM1 affects the formation of heCIC,which is related to the lysosomal pathway.This study further found that GOLM1 affects the killing efficacy of immune cells on tumor cells by regulating the formation of heCIC.CIC contributes a certain role in this process and enhances the killing effect of NK cells on tumors.And we have combined the CIC structure into TMA for systemic prognosis analysis for the first time,and proposed a new diagnostic index of "functional pathology".It has been proved that CIC subtype has high sensitivity and specificity for clinical prognosis of tumor patients.Is a superior biological indicator.Our research is expected to provide new targets and strategies for tumor immunotherapy,clinical diagnosis and prognosis analysis.更多还原