NPC268 whole-genome bisulfite sequencing (WGBS)

Nasopharyngeal carcinoma (NPC), a cancer that is etiologically associated with the Epstein-Barr virus (EBV), is endemic in Southern China and Southeast Asia. The scarcity of representative NPC cell lines owing to the frequent loss of EBV episomes following prolonged propagation and compromised authenticity of previous models underscores the critical need for new EBV-positive NPC models. Herein, we describe the establishment of a new EBV-positive NPC cell line, designated NPC268 from a primary non-keratinizing, differentiated NPC tissue. NPC268 can undergo productive lytic reactivation of EBV and is highly tumorigenic in immunodeficient mice. Whole-genome sequencing (WGS) revealed close similarities with the tissue of origin, including large chromosomal rearrangements, while whole-genome bisulfite sequencing (WGBS) and RNA sequencing demonstrated a hypomethylated genome and enrichment in immune-related pathways, respectively. Drug screening of NPC268 together with six other NPC cell lines using 339 compounds, representing the largest high-throughput drug testing in NPC, revealed biomarkers associated with specific drug classes. NPC268 represents the first and only available EBV-positive non-keratinizing differentiated NPC model, and extensive genomic, methylomic, transcriptomic, and drug response data should facilitate research in EBV and NPC, where current models are limited. Overall design: The gDNA of NPC268 was processed for bisulfite conversion and library preparation, and WGBS was performed and analyzed. The methylation ratio of a CpG site/region is the number of cytosine reads divided by the sum of the cytosine and thymine reads. The human genome was segmented using a Methyl Kit (v.1.16.1) for global methylation pattern visualization.

鼻咽癌（Nasopharyngeal carcinoma, NPC）是一种病因学上与EB病毒（Epstein-Barr virus, EBV）相关的恶性肿瘤，在中国南方及东南亚地区呈地方性流行。由于长期传代培养过程中频繁发生EB病毒游离附加体丢失，且既往模型的真实性存在缺陷，目前具备代表性的NPC细胞系十分匮乏，这凸显了开发新型EB病毒阳性NPC模型的迫切需求。本文报道了一株新的EB病毒阳性NPC细胞系的建立，该细胞系源自原发性非角化分化型鼻咽癌组织，命名为NPC268。NPC268可介导EB病毒产毒性裂解激活，且在免疫缺陷小鼠中具有高成瘤性。全基因组测序（Whole-genome sequencing, WGS）结果显示，该细胞系与起源组织高度相似，包含大规模染色体重排事件；全基因组亚硫酸氢盐测序（Whole-genome bisulfite sequencing, WGBS）与RNA测序分析则分别揭示其基因组呈低甲基化状态，且免疫相关通路显著富集。研究团队使用339种化合物，对NPC268及另外6株NPC细胞系开展药物筛选——这是目前鼻咽癌领域规模最大的高通量药物测试实验，最终鉴定出与特定药物类别相关的生物标志物。NPC268是目前首个且唯一可用的EB病毒阳性非角化分化型NPC模型，其丰富的基因组、甲基化组、转录组及药物反应数据，将为当前模型有限的EB病毒相关鼻咽癌研究提供重要助力。实验整体设计：提取NPC268的基因组DNA（genomic DNA, gDNA），进行亚硫酸氢盐转化与文库构建，随后开展全基因组亚硫酸氢盐测序并完成数据分析。CpG位点/区域的甲基化比率为胞嘧啶读取数与胞嘧啶、胸腺嘧啶总读取数的比值。使用Methyl Kit（v.1.16.1）对人类基因组进行分段，以实现全局甲基化模式的可视化。