背景:非角化性鼻咽癌(NK-NPC)与EB病毒(EBV)感染密切相关。NK细胞在NK-NPC中的作用和肿瘤细胞的进化轨迹尚不清楚。在这项研究中,我们旨在通过单细胞转录组学分析来研究NK细胞的功能和NK-NPC中肿瘤细胞的进化轨迹,蛋白质组学和免疫组织化学。
方法:收集NK-NPC(n=3)和正常鼻咽粘膜(n=3)进行蛋白质组学分析。NK-NPC(n=10)和鼻咽淋巴增生(NLH,n=3)从基因表达Omnibus(GSE162025和GSE150825)获得。质量控制,降维和聚类基于Seurat软件(v4.0.2)过程,批量效应通过和谐(v0.1.1)软件去除。使用copykat软件(v1.0.8)鉴定鼻咽粘膜的正常细胞和NK-NPC的肿瘤细胞。使用CellChat软件(v1.4.0)探索细胞-细胞相互作用。使用SCORPIUS软件(v1.0.8)进行肿瘤细胞进化轨迹分析。使用clusterProfiler软件(v4.2.2)进行蛋白质和基因功能富集分析。
结果:通过蛋白质组学在NK-NPC(n=3)和正常鼻咽粘膜(n=3)之间共获得161个差异表达蛋白(log2倍变化>0.5,P值<0.05)。与自然杀伤细胞介导的细胞毒性途径相关的大多数蛋白质在NK-NPC组中被下调。在单细胞转录组学中,我们确定了三个NK细胞亚群(NK1-3),其中在NK-NPC中ZNF683高表达(组织驻留NK细胞的特征)的NK3亚群中鉴定出NK细胞耗竭。我们证明了这种ZNF683+NK细胞亚群存在于NK-NPC中,但不存在于NLH中。我们还使用TIGIT和LAG3进行了免疫组织化学实验,以确认NK-NPC中的NK细胞耗尽。此外,轨迹分析显示NK-NPC肿瘤细胞的进化轨迹与EBV感染状态(活跃或潜伏)相关.细胞间相互作用的分析揭示了NK-NPC中细胞相互作用的复杂网络。
结论:本研究揭示NK-NPC中NK细胞表面抑制性受体的上调可能导致NK细胞耗竭。逆转NK细胞耗竭的治疗可能是NK-NPC的有希望的策略。同时,我们首次确定了NK-NPC中EBV感染活跃状态的肿瘤细胞的独特进化轨迹.我们的研究可能提供新的免疫治疗靶点和涉及肿瘤发生的进化轨迹的新观点。NK-NPC的发展和转移。
Nonkeratinizing nasopharyngeal carcinoma (NK-NPC) has a strong association with Epstein-Barr virus (EBV) infection. The role of NK cells and the tumor cell evolutionary trajectory in NK-NPC remain unclear. In this study, we aim to investigate the function of NK cell and the evolutionary trajectory of tumor cells in NK-NPC by single-cell transcriptomic analysis, proteomics and immunohistochemistry.
NK-NPC (n = 3) and normal nasopharyngeal mucosa cases (n = 3) were collected for proteomic analysis. Single-cell transcriptomic data of NK-NPC (n = 10) and nasopharyngeal lymphatic hyperplasia (NLH, n = 3) were obtained from Gene Expression Omnibus (GSE162025 and GSE150825). Quality control, dimension reduction and clustering were based on Seurat software (v4.0.2) process and batch effects were removed by harmony (v0.1.1) software. Normal cells of nasopharyngeal mucosa and tumor cells of NK-NPC were identified using copykat software (v1.0.8). Cell-cell interactions were explored using CellChat software (v1.4.0). Tumor cell evolutionary trajectory analysis was performed using SCORPIUS software (v1.0.8). Protein and gene function enrichment analyses were performed using clusterProfiler software (v4.2.2).
A total of 161 differentially expressed proteins were obtained between NK-NPC (n = 3) and normal nasopharyngeal mucosa (n = 3) by proteomics (log2 fold change > 0.5 and P value < 0.05). Most of proteins associated with the nature killer cell mediated cytotoxicity pathway were downregulated in the NK-NPC group. In single cell transcriptomics, we identified three NK cell subsets (NK1-3), among which NK cell exhaustion was identified in the NK3 subset with high ZNF683 expression (a signature of tissue-resident NK cell) in NK-NPC. We demonstrated the presence of this ZNF683 + NK cell subset in NK-NPC but not in NLH. We also performed immunohistochemical experiments with TIGIT and LAG3 to confirm NK cell exhaustion in NK-NPC. Moreover, the trajectory analysis revealed that the evolutionary trajectory of NK-NPC tumor cells was associated with the status of EBV infection (active or latent). The analysis of cell-cell interactions uncovered a complex network of cellular interactions in NK-NPC.
This study revealed that the NK cell exhaustion might be induced by upregulation of inhibitory receptors on the surface of NK cells in NK-NPC. Treatments for the reversal of NK cell exhaustion may be a promising strategy for NK-NPC. Meanwhile, we identified a unique evolutionary trajectory of tumor cells with active status of EBV-infection in NK-NPC for the first time. Our study may provide new immunotherapeutic targets and new sight of evolutionary trajectory involving tumor genesis, development and metastasis in NK-NPC.