应用单细胞测序转录组分析骨巨细胞瘤的免疫环境特征及免疫逃逸机制

Single-cell transcriptome reveals features of immune environment and mechanisms of immune escape in giant cell tumor of bone

目的:通过单细胞转录组测序揭示骨巨细胞瘤特殊的免疫浸润环境以及可能存在的免疫逃逸机制。方法:收集2018年1月至2021年12月4例原发性骨巨细胞瘤患者手术获得的肿瘤新鲜标本,采用10X平台进行单细胞转录组测序,使用t-分布领域嵌入算法(t-distributed stochastic neighbor embedding,t-SNE)降维分析将单细胞数据可视化及定量。对4例标本的35643个单细胞数据进行细胞聚类、基本细胞类型的定义、免疫细胞的分类以及细胞类型之间的相互调控关系分析,探究骨巨细胞瘤免疫环境的特征。通过细胞通讯分析,观察骨巨细胞瘤中免疫细胞调节及作用的主要细胞类型及主要作用的信号通路。结果:骨巨细胞瘤由9种基本细胞类型组成,其中免疫细胞以CD8^(+)T细胞为主约占51%,非免疫细胞以成纤维细胞样梭形基质细胞为主,约占74%。在骨巨细胞瘤的免疫浸润中细胞毒性的CD8^(+)T细胞占比最高(49%),而耗竭状态的CD8^(+)T细胞占比最低(5%)。CD4^(+)T细胞以高表达免疫检查点基因CTLA4和TIGIT为特征。骨巨细胞瘤中CD8^(+)T细胞及NK细胞主要通过PARs和CCL两条信号通路的调控作用于多核破骨样巨细胞,而非基质细胞。结论:通过单细胞测序数据定义了骨巨细胞瘤的主要组成细胞类型,并进一步揭示了其免疫浸润的组成特征,且发现其免疫细胞的作用对象主要为多核破骨样巨细胞,为更加深入了解骨巨细胞瘤发生、发展提供了有效信息。

Objective This study aims to reveal the special immune infiltrating environment and possible immune escape mechanism of giant cell tumor of bone through single-cell sequencing data.Methods The fresh samples obtained from 4 patients with primary giant cell tumor of bone from January 2018 to December 2021 were collected,and single-cell transcriptome sequencing was performed on the 10X platform to explore the characteristics and immune environment of giant cell tumor of bone by using t-distributed stochastic neighbor embedding(t-SNE).The main cell types and signal pathways of immune cell regulation and function in giant cell tumor of bone were observed by cell communication analysis.Results Cell clustering,the definition of basic cell types,the classification of immune cells,and the mutual regulatory relationship between cell types were analyzed for 35643 single-cell data from 4 giant cell tumor samples of bone.It was found that giant cell tumor of bone was composed of 9 basic cell types,in which the immune cells were mainly CD8^(+)T cells(51%)and the non-immune cells were mainly fibroblast like spindle stromal cells(74%).The immune infiltration of giant cell tumor of bone is dominated by cytotoxic CD8^(+)T cells and lacks exhausted CD8^(+)T cells.CD4^(+)T cells are characterized by high expression of immune checkpoint genes CTLA4 and TIGIT.In giant cell tumor of bone,immune cells mainly act on multinucleated osteoclast like giant cells through PARs and CCL signaling pathways,but not stromal cells.Conclusion This study defined the main cell types of giant cell tumor of bone through single cell sequencing data,and further revealed the composition characteristics of its immune infiltration,and found that the target of its immune cells was mainly multinuclear osteoclast like giant cells,which provided effective information for further understanding the occurrence and development of giant cell tumor of bone.