巨细胞病毒感染与胶质母细胞瘤免疫微环境的关系

Relationship between cytomegalovirus infection and glioblastoma immune microenvironment

目的采用单细胞转录组测序技术分析巨细胞病毒(CMV)感染的胶质母细胞瘤(GBM)组织转录组图谱,探讨CMV感染与GBM免疫微环境的关系。方法2022年12月—2023年8月新疆医科大学第二附属医院诊治GBM患者20例,收集GBM组织,采用胶体金免疫电镜和微滴式数字PCR技术鉴定GBM组织中是否存在CMV颗粒及遗传物质,比较CMV阳性与阴性GBM组织病毒拷贝数。采用单细胞转录组测序技术分析6例CMV阳性和3例CMV阴性GBM组织的转录组图谱,鉴定共表达巨噬细胞标志物和肿瘤细胞标志物的免疫细胞亚群,对该免疫细胞亚群采用免疫荧光染色和流式细胞术验证,并分析差异表达基因及涉及的信号通路。结果胶体金免疫电镜结果显示,CMV阳性GBM组织存在稀少的金标记电子致密颗粒,与CMV颗粒形态一致。微滴式数字PCR结果显示,12例CMV阳性,8例CMV阴性;CMV阳性GBM组织病毒拷贝数[(150±30)copies/mL]高于CMV阴性GBM组织[(10±5)copies/mL](t=7.200,P<0.001)。单细胞转录组测序鉴定出CMV阳性和阴性GBM组织中均存在肿瘤细胞、巨噬细胞、少突胶质细胞、内皮细胞、基质细胞、T淋巴细胞6种主要细胞类型,以及1种新的免疫细胞亚群即共表达巨噬细胞标志物CD68和肿瘤细胞标志物SOX2的双阳性肿瘤相关巨噬细胞(TAM)(CD68^(+)SOX2^(+)TAM)。免疫荧光染色结果显示,CMV阳性GBM组织中CD68和SOX2共表达,CMV阴性GBM组织中CD68和SOX2共表达不明显。流式细胞术分析结果显示,CD68^(+)SOX2^(+)TAM在CMV阳性GBM组织中富集;CMV阳性GBM组织CD68^(+)SOX2^(+)TAM百分比[(4.86±0.25)%]高于CMV阴性GBM组织[(0.94±0.41)%](t=8.160,P<0.001)。功能分析结果显示,CMV阳性和阴性GBM组织CD68^(+)SOX2^(+)TAM的差异表达基因主要与CMV感染有关,涉及病毒蛋白与细胞因子及其受体相互作用的信号通路。结论GBM患者可能存在CMV感染,CMV感染可诱导共表达巨噬细胞和肿瘤细胞标志物的双阳性TAM(CD68^(+)SOX2^(+)TAM)产生从而重塑GBM免疫微环境。

Objective To analyze the transcriptome maps of cytomegalovirus(CMV)-infected glioblastoma tissue using single-cell transcriptome sequencing technology,and to explore the relationship between CMV infection and glioblastoma immune microenvironment.Methods Twenty patients with glioblastoma were diagnosed and treated in the Second Affiliated Hospital of Xinjiang Medical University from December 2022 to August 2023.The glioblastoma tissue was collected and the presence of CMV particles and genetic materials in glioblastoma tissue was identified by colloidal gold immunoelectron microscopy and droplet digital PCR.The viral copy number was compared between CMV-positive and CMV-negative glioblastoma tissues.Single-cell transcriptome sequencing technology was used to analyze the transcriptome maps of 6 CMV-positive and 3 CMV-negative glioblastoma patients,and to identify the immune cell subset co-expressing macrophage marker and tumor cell marker.Immunofluorescence staining and flow cytometry were used to validate this immune cell subset.The differentially expressed genes and involved signaling pathways were analyzed.Results The results of colloidal gold immunoelectron microscopy showed that there were few gold-labeled electron-dense granules in CMV-positive tissue,which were consistent with the morphology of CMV particles.The results of droplet digital PCR showed that there were 12 CMV-positive and 8 CMV-negative patients.The viral copy number of CMV-positive tissue[(150±30)copies/mL]was higher than that of CMV-negative tissue[(10±5)copies/mL](t=7.200,P<0.001).Single-cell transcriptome sequencing identified 6 major cell types in both CMV-positive and CMV-negative tissues,including tumor cell,macrophage,oligodendrocyte,endothelial cell,stromal cell and T lymphocyte,as well as a novel kind of immune cell subset,namely double-positive tumor-associated macrophage(TAM)co-expressing macrophage marker CD68 and tumor cell marker SOX2(CD68^(+)SOX2^(+)TAM).The results of immunofluorescence staining showed that there was a co-expression of CD68 and SOX2 in CMV-positive tissue,while the co-expression of CD68 and SOX2 was not obvious in CMV-negative tissue.The results of flow cytometry showed that CD68^(+)SOX2^(+)TAM was enriched in CMV-positive tissue.The percentage of CD68^(+)SOX2^(+)TAM in CMV-positive tissue[(4.86±0.25)%]was higher than that in CMV-negative tissue[(0.94±0.41)%](t=8.160,P<0.001).The results of function analysis showed that the differentially expressed genes of CD68^(+)SOX2^(+)TAM in CMV-positive and CMV-negative tissues were mainly associated with CMV infection,involving signaling pathways of the interaction of viral proteins with cytokines and their receptors.Conclusion CMV infection may exist in patients with glioblastoma,and it can induce the production of double-positive TAM co-expressing macrophage marker and tumor cell marker(CD68^(+)SOX2^(+)TAM),remodeling the glioblastoma immune microenvironment.