【作者】 王明；

【导师】 蒋宇钢；

【作者基本信息】 中南大学 ， 外科学， 2022， 博士

【摘要】 研究目的:癌症的免疫检查点抑制剂疗法（Immune checkpoint inhibitor therapy,ICT）对于胶质母细胞瘤（Glioblastoma,GBM）常常难以带来有效的益处,这归结于GBM中枢抑制性的肿瘤免疫微环境（Tumor immune microenvironment,TIME）的形成,因此对GBM免疫抑制微环境形成的分子机制的深入研究有助于为GBM的ICT治疗提供理论基础。细胞外囊泡（Extracellular vesicles,EVs）或外泌体（Exosome,Exos）在GBM的诊断和治疗中备受瞩目,受限于脑活检的条件,脑脊液（Cerebrospinal fluid,CSF）中EVs/Exos在GBM的免疫动态监测和治疗干预策略中具有巨大的潜力。因此,本研究通过获取GBM患者脑脊液,对其EVs/Exos蛋白组学进行深入研究以筛选新的免疫监测或治疗靶点,为GBM患者的ICT治疗提供新的研究思路。研究方法:1.在临床研究水平,纳入GBM和GⅡ-Ⅲ胶质瘤（WHO grade Ⅱ and Ⅲ,GⅡ-Ⅲ）患者,并设置健康对照组。获取所有受试者的CSF,流式细胞术检测淋巴系和髓系免疫细胞比例和免疫表型,以确定GBM脑脊液中的免疫抑制性表型特征。超滤法提取和纯化GBM及GⅡ-Ⅲ患者CSF中的EVs/Exos,纳米颗粒跟踪分析（Nanoparticle Tracking Analysis,NTA）和生物透射电子显微镜对其进行表征,蛋白质免疫印记（Western blotting）鉴定EVs/Exos的表面标记物。液相色谱-质谱联用技术（liquid chromatography-mass spectrometry,LC-MS）对EVs/Exos进行蛋白组学分析,以筛选出与GBM免疫抑制表型相关的EVs/Exos携带货物蛋白。2.在细胞研究水平,使用CRISPR/Cas9技术敲除人恶性神经胶质瘤细胞U87-MG中LGALS9、Rab27a、n SMase2的表达,以抑制EVs/Exos-LGALS9的释放。将WT,n SMase2^-/-,Rab27a^-/-或LGALS9^-/-U87-MG与人树突状细胞（Dendritic Cells,DCs）和T细胞在体外进行共培养,流式细胞术和酶联免疫吸附技术（ELISA）检测DCs抗原呈递相关免疫表型和T细胞活化水平。此外,通过敲除DC细胞中TIM3受体基因以废除对LGALS9配体的识别,从而确定GBM来源EVs/Exos-LGALS9的释放对DC细胞抗原呈递抑制的影响。3.在动物研究水平,将构建的小鼠胶质母细胞瘤细胞GL-261WT,n SMase2^-/-,Rab27a^-/-,LGALS9^-/-细胞分别在C57BL/6J背景小鼠和免疫缺陷的NOD-SCID小鼠进行皮下和脑内原位荷瘤实验,观察小鼠肿瘤的体积和生存期（Overall Survival,OS）,通过流式细胞术分析CSF中相关免疫细胞的免疫表型,以确定EVs/Exos-LGALS9对小鼠脑脊液中TIME形成的影响。研究结果:1.相比于对健康对照组（n=4）,粒细胞和CD8+T细胞在GBM组患者（n=7）CSF中的比例下调（p<0.05）,单核细胞/巨噬细胞和树突状细胞在GBM和GⅡ-Ⅲ（n=7）组患者CSF中的比例上调（p<0.05）,而CD4+T细胞在GBM和GⅡ-Ⅲ组患者的CSF中的比例不具有显著性差异（p>0.05）。免疫表型分析结果显示,GBM-CSF中CD8+T细胞处于免疫抑制状态,包括低克隆活性和Type Ⅱ IFN响应失活。GBM患者的DCs的活性远低于GⅡ-Ⅲ患者,甚至低于无肿瘤抗原刺激的健康受试者,包括抗原识别的HLA-A的表达下降,共刺激分子CD40和CD86的表达以及抗原加工蛋白TAP1表达下调（p<0.05）。GBM和GⅡ-Ⅲ组肿瘤患者呈现出比健康对照者CSF中更高的浓度和更小的纳米粒径的EVs（p<0.05）。超滤获得的小型细胞外囊泡（SEVs;≦0.1μM）具有Exos的表面标记物（TSG101,CD63）和形态。GBM-SEVs/Exos可被DCs摄取并大幅度削减DCs的抗原呈递活性。蛋白组学分析显示,在所有CSF-EVs中共鉴定到2958个蛋白,GBM-SEVs/Exos中有487个蛋白货物水平要高于GⅡ-Ⅲ-SEVs/Exos组（p<0.05）。GO分析和Pathway分析显示其中,GBM-SEVs/Exos中18个蛋白分子参与了DCs抗原提呈信号通路,其中,LGALS9值得关注。2.与原代星状胶质细胞（HA）相比,U87 MG的LGALS9 m RNA转录水平显著上调（p<0.05）,而LGALS9蛋白翻译水平并无明显的改变（p>0.05）,U87 MG-derived Exos中LGALS9蛋白携带量却比HA-derived Exos高上百倍（p<0.05）。与U87 WT相比,U87 MG n SMase2^-/-Exos和U87 MG Rab27a^-/-Exos的分泌量显著的减少。DCs对m Cherry标记肿瘤抗原的摄取量在U87 MG n SMase2^-/-和U87 MG Rab27a^-/-共培养体系中显著增强。U87 MG n SMase2^-/-和U87 MG Rab27a^-/-肿瘤抗原激活的DCs的CD40,HLA-A和TAP1的表达要高于U87 MG WT（p<0.05）,而其Exos LGALS9的含量要低于U87 MG WT（p<0.05）。激活的DCs会促使与其共培养T细胞的克隆增殖（CSFE掺入）和Type Ⅱ IFN response（IFN-γ和Granzyme B释放）,这一过程也在U87 MG n SMase2-/-和U87 MG Rab27a-/-共培养体系中显著增强（p<0.05）。3.鼠源GL-261细胞LGALS9的敲除后对外泌体的影响也与人源的U87 MG细胞一致。n SMase2,Rab27a以及LGALS9敲除的GL-261细胞的增殖活性没有明显的抑制。皮下植入GL-261WT细胞的小鼠大约20天者可见到肿瘤生长和最长32天的OS。对比之下,GL-261n SMase2-/-,Rab27a-/-以及LGALS9-/-的荷瘤鼠肿瘤的生长速度显著下降以及更长的OS（p<0.05）。在NOD-SCID免疫缺陷背景下,GL-261 n SMase2-/-,Rab27a-/-以及LGALS9-/-荷瘤鼠的肿瘤生长速度和OS与GL-261WT荷瘤鼠并无明显差异（p>0.05）。在GL-261LGALS9-/-脑内原位荷瘤小鼠CSF中几乎检测不到LGALS9的表达。在GL-261 WT荷瘤小鼠CSF中,超过60%的DCs为LGALS9高表达(LGALS9^hiDCs),而GL-261 LGALS9-/-的LGALS9^hiDCs占总DCs的总数不足5%,重要的是,GL-261 LGALS9-/-荷瘤鼠CSF中DCs的MHC I,CD40,TAP1的表达均明显高于GL-261 WT荷瘤小鼠（p<0.05）。相比于GL-261 WT荷瘤鼠,GL-261 LGALS9-/-荷瘤鼠CSF中CD8+T细胞的抗肿瘤免疫活增强,包括Ki67阳性细胞增多和INF-γ/Gzm B表达增强。此外,初次接触GL-261 LGALS9-/-的小鼠对再次接种的GL-261 WT细胞产生抗性,表现为肿瘤生长抑制和生存率的提高。研究结论:1.GBM脑脊液中DCs处于抗原呈递失活状态,可能是GBM相比于GⅡ-Ⅲ更加恶性的原因。2.LGALS9在GBM脑脊液SEVs/Exos中独特存在,可能与DC细胞的抗原呈递失活有关。3.GBM-Exos来源的LGALS9通过抑制DC细胞抗原呈递的激活来促进肿瘤的生长。4.GBM-Exos来源的LGALS9通过TIM3受体途径抑制DC细胞的抗原递呈过程。5.GBM-derived exosomal LGALS9的减少恢复了GBM免疫微环境中的抗肿瘤活性。6.DCs在缺少GBM-derived exosomal LGALS9的微环境中接触TAA完成抗原呈递后,会对LGALS9的免疫抑制作用产生抗性。图44幅,表16个,参考文献83篇更多还原

【Abstract】 Background and purpose:Immune checkpoint inhibitor therapy（ICT）for Glioblastoma（GBM）often difficult to achieve effective benefits,which is attributed to the central inhibitory tumor microenvironment（TIME）in GBM,so in-depth research on the molecular mechanism of the formation of GBM immunosuppressive microenvironment is helpful to provide a theoretical basis for ICT therapy of GBM.Extracellular vesicles（EVs）or exosomes（Exos）have attracted much attention in the diagnosis and treatment of GBM,but limited by the conditions of brain biopsy,EVs/Exos in Cerebrospinal Fluid（CSF）has great potential for immunodynamic monitoring and therapeutic strategies making for GBM.Therefore,in this study,cerebrospinal fluid of GBM patients was obtained.EVs/Exos proteomics in CSF from GBM was further studied to screen new immune monitoring or therapeutic targets,and hope our results provide new research ideas for ICT treatment of GBM patients.Methods:1.For the clinical study,patients with GBM and GⅡ-Ⅲ glioma（WHO Grade Ⅱ and Ⅲ,GⅡ-Ⅲ）were included,and a healthy control group was set.CSF of all subjects were obtained,lymphoid and myeloid immune cell ratios and immunophenotypes were determined by flow cytometry in GBM cerebrospinal fluid.EVs/Exos in CSF of GBM and GⅡ-Ⅲ patients were extracted and purified by ultrafiltration,characterized by Nanoparticle Tracking Analysis（NTA）and biological transmission electron microscopy.Western blotting was used to identify the surface markers of EVs/Exos.Liquid chromatography-mass spectrometry（LC-MS）was used for proteomic analysis of EVs/Exos to screen out EVs/Exos cargo protein LGALS9,which was related to GBM immunosuppressive phenotype.2.In vitro study,the expression of LGALS9,Rab27a and n SMase2in human malignant glioma cells U87-MG was deleted using CRISPR/Cas9 technology to inhibit the release of EVs/Exos-LGALs9.WT,n SMase2-/-,Rab27a-/-or LGALS9-/-U87-MG were co-cultured with human Dendritic Cells（DCs）and T Cells in vitro.DCs antigen presentation related immunophenotypes and T cell activation levels were determined by flow cytometry and enzyme-linked immunosorbent assay（ELISA）.Furthermore,the antigen presentation inhibition effect of GBM-derived EVs/Exos-LGALS9 on DC cells was determined by knockout of TIM3 receptor gene in DC cells to disable the recognition function of LGALS9 li gand.3.In vivo study,the constructed mouse glioblastoma GL-261 WT,n SMase2-/-,Rab27a-/-and LGALS9-/-cells were transplanted subcutaneously or in the brain of C57BL/6J mice and immunodeficient NOD/SCID mice to construct tumor-bearing animals.The tumor volume and Overall Survival（OS）of mice were observed,and the immunophenotype of related immune cells in mice CSF was analyzed by flow cytometry to determine the effect of EVs/Exos-LGALS9 on TIME formation in CSF of mice.Results:1.Compared with the healthy control group（n=4）,the proportion of granulocytes and CD8+T cells in the CSF of GBM group（n=7）was down-regulated（P<0.05）,and the proportion of monocytes/macrophages and dendritic cells in the GBM and GⅡ-Ⅲ（n=7）was up-regulated（P<0.05）.There was no significant difference in the proportion of CD4+T cells in CSF between GBM and GⅡ-Ⅲ group（P>0.05）.Immunophenotypic analysis showed that CD8+T cells in GBM-CSF were immunosuppressed,including low clonal activity and Type Ⅱ IFN response inactivation.The DCs activity of GBM patients was significantly lower than that of GⅡ-Ⅲ patients,and even lower than that of healthy subjects without tumor antigen stimulation,including decreased expression of antigen-recognized HLA-A,costimulatory molecules CD40 and CD86,and antigen-processing protein TAP1（P<0.05）.Patients in the GBM and GⅡ-Ⅲ tumor groups showed higher concentrations of EVs and smaller nanosized particles in CSF than healthy controls（P<0.05）.Small extracellular vesicles obtained by ultrafiltration（SEVs;≦0.1μM）with Exos surface markers（TSG101,CD63）and morphology.GBM-SEVs/Exos can be ingested by DCs and significantly reduced the antigen presentation activity of DCs.Proteomic analysis showed that 2958 proteins were identified in all CSF-EVs,and the levels of 487 protein cargo in GBM-SEVs/Exos group were higher than those in GⅡ-Ⅲ-SEVs/Exos group（P<0.05）.GO and Pathway analysis showed that 18 protein molecules in GBM-SEVs/Exos were involved in antigen presentation signaling pathway of DCs,among which LGALS9 has attracted our attention.2.Compared with primary astrocytes（HA）,the m RNA transcription level of LGALS9 in U87 MG was significantly up-regulated（P<0.05）.However,there was no significant change in LGALS9 protein translation level（P>0.05）,and LGALS9 protein carrying amount in U87MG-derived Exos was 100 times higher than that in HA-derived Exos（P<0.05）.Compared with U87 WT,the secretion of U87 MG n SMase2-/-Exos and U87 MG Rab27a-/-Exos was significantly reduced.DCs uptake of m Cherry labeled tumor antigen was significantly increased in U87 MG n SMase2-/-and U87 MG Rab27a-/-co-culture systems.The expression of CD40,HLA-A and TAP1 in DCs activated by U87 MG n SMase2-/-and U87 MG Rab27a-/-was higher than that in DCs activated by U87 MG n SMase2-/-and U87 MG Rab27a-/-（P<0.05）.The content of exosomal LGALS9 was lower than U87 MG WT（P<0.05）.Activated DCs promoted clonal proliferation of co-cultured T cells（CSFE incorporation）and Type Ⅱ IFN Response（IFN-γand Granzyme B release）.This process was also significantly enhanced in the co-culture systems of U87 MG n SMase2-/-and U87 MG Rab27a-/-（P<0.05）.3.The effect of LGALS9 knockout on exosomes of mouse GL-261cells was similar to that of human U87 MG cells.n SMase2,Rab27a and LGALS9 knockout GL-261 cells showed no significant in the inhibition of cell proliferation activity.Tumor growth and OS up to 32 days were observed in mice with in-situ brain implantation of GL-261wt cells at about 20 days.In contrast,GL-261 n SMase2-/-,Rab27a-/-and LGALS9-/-tumor-bearing mice had significantly reduced the tumor growth rate and achieved longer OS（P<0.05）.Under NOD/SCID immunodeficiency background,there was no significant difference in tumor growth rate and OS between GL-261 n SMase2-/-,Rab27a-/-and LGALS9-/-mice and GL-261wt mice（P>0.05）.Almost no expression of LGALS9 was detected in the CSF of GL-261 LGALS9-/-tumor-bearing mice.In GL-261 WT tumor-bearing mice,more than 60%of DCs were LGALS9 highly expressed(LGALS9^hiDCs),while LGALS9^hiDCs accounted for less than 5%of the total DCs in GL-261LGALS9-/-.Importantly,The expressions of MHC I,CD40 and TAP1 in the CSF of GL-261 LGALS9-/-tumor-bearing mice were significantly higher than those of GL-261 WT tumor-bearing mice（P<0.05）.Compared with GL-261 WT tumor-bearing mice,CD8+T cells in the CSF of GL-261 LGALS9-/-tumor-bearing mice exhibited enhanced anti-tumor immune activity,including increased Ki67 positive cells and INF-γ/Gzm B expression.In addition,mice exposed to GL-261LGALS9-/-for the first time will developed resistance to re-inoculated GL-261 WT cells,as demonstrated by inhibition of tumor growth and improved survival.Conclusions:1.The antigen presentation function of DC cells in cerebrospinal fluid from GBM is inactivation,which may be the reason that GBM is more malignant than GⅡ-Ⅲ.2.LGALS9 is unique in GBM CSF SEVs/Exos and may be related to DCs antigen presentation inactivation.3.GBM-exos-derived LGALS9 promotes tumor growth by inhibiting DC cell antigen presentation.4.LGALS9 in GBM-Exos inhibits the antigen presentation process of DCs through TIM3 receptor pathway.5.Reduction of GBM-derived exosomal LGALS9 restores the anti-tumor immune activity in the GBM immune microenvironment.6.DCs that exposed to tumor-associated antigen in the microenvironment that absence of exosomal LGALS9 can successful complete the antigen presentation and resist to the immunosuppressive effect of exosomal LGALS9更多还原