FIGURE 6 from Establishment and Characterization of an Epstein-Barr Virus–positive Cell Line from a Non-keratinizing Differentiated Primary Nasopharyngeal Carcinoma

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posted on 2025-04-11, 11:03 authored by Annie Wai Yeeng Chai, Shi Mun Yee, Hui Mei Lee, Norazlin Abdul Aziz, Pei San Yee, Marini Marzuki, Ka Wo Wong, Alan K.S. Chiang, Larry Ka-Yue Chow, Wei Dai, Teng Fei Liu, Lu Ping Tan, Alan Soo Beng Khoo, Kwok Wai Lo, Paul V.H. Lim, Pathmanathan Rajadurai, Howard Lightfoot, Syd Barthorpe, Mathew J. Garnett, Sok Ching Cheong

NPC268 resembles the HypoNPC subtype and showed enrichment in immune-related gene expression. A, WGBS analysis revealed the methylation level of NPC268, resembling that of HypoNPC subtype. Heat map shows the global methylation levels of the human genome in nasopharyngeal epithelial cell lines and EBV-positive NPC cell lines. The human genome was equally segmented into consecutive tiles of 1 Mbps, and a methylation average is calculated for each window. The heat map color scale denotes the absolute methylation level. B, Ridge plot shows the global methylation levels of the human genome in the 1 Mbps windows (left plot) and the LINE1 methylation level (right plot). Normal, HypoNPC, and HypoNPC were the adjacent non-cancerous nasopharyngeal tissue, HypoNPC tumor, and HyperNPC tumor derived from patients with NPC, respectively (Chow et al. 2022). Like NPC43, NPC268 has regions with low methylation levels compared with the normal clinical specimen, normal immortalized cell lines and HyperNPC. The unmethylated peak (at “0”) of LINE1 surrogate marker further support the global hypomethylation status. C, Violin plots showing the methylation levels in specific differentially methylated regions (NPC-DMR) identified in Chow et al. 2022. These NPC DMRs were hypermethylated in the NPC clinical tissues compared with the non-cancerous adjacent tissues. D, Heat map shows the methylation average of the DMRs between HyperNPC and HypoNPC clinical specimens in the EBV genome. NPC268 and NPC43 showed lower methylation levels, similar to HypoNPC whose methylation levels in these regions were lower compared with the HyperNPC. The heat map color scale denotes the Z-score of the methylation levels. E, GSEA of NPC268 against NP550 revealed upregulation of immune-related hallmark pathways. F, Heat map snapshot of KEGG or Hallmark pathways where NPC268 was found to have the highest ssGSEA enrichment scores. G, RNA-seq expression of CD74 is markedly higher in HyperNPC cell lines (NPC268, NPC43) compared with HypoNPC cell lines (C666-1, C17).

Funding

Newton Fund (NF)

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ARTICLE ABSTRACT

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 revealed close similarities with the tissue of origin, including large chromosomal rearrangements, while whole-genome bisulfite sequencing 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. NPC268 is the first and only EBV-positive cell line derived from a primary non-keratinizing, differentiated nasopharyngeal carcinoma, an understudied but important subtype in Southeast Asian countries. This model adds to the limited number of authentic EBV-positive lines globally that will facilitate mechanistic studies and drug development for NPC.

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Keywords

Head, Neck & Oral Cancers Rare Cancers Genome Biology Silencing and reactivation of gene expression Preclinical Models In vitro models of cancer Xenograft models Drug Mechanisms Cellular responses to anticancer drugs

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CC BY