FIGURE 4 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 is highly tumorigenic and demonstrate lytic activity in vivo. A,In vivo growth curve of NPC268 mouse xenografts (early passage xenograft, E-X-1, E-X-2, E-X-3 and late passage xenograft, L-X-1, L-X-2, L-X-3). B, H&E and IHC staining of pan-cytokeratin of the mouse xenografts showing the non-keratinizing morphology of the xenografts, with some mixture of well-differentiated and poorly differentiated cells. C, Representative images of the EBER-ISH and IHC staining of LMP1 and BZLF1 on the xenografts. Positivity of these EBV markers confirmed the preservation of EBV in the late passage-derived xenografts. EBER and LMP1 staining were generally seen in the non-keratinizing, less differentiated cells while the BZLF1 were found in the more squamous-like, differentiated and keratinizing cells (black arrows), in which EBV are likely in permissive infection. D, Average EBV CNs per cell increased markedly in the xenograft tissue in vivo, compared with the in vitro cultured cells before (p131) and after [L-X-2 (p1), L-X-3 (p1)] xenograft transplantation. E, qPCR analysis of the EBV latent and lytic genes showing significant upregulation in the xenografts compared with those from the in vitro cultured cells. Data are shown as mean ± SD (n = 2 biological repeats). Two-tailed, unpaired Student t tests were performed for L-X xenografts, comparing with p131 in vitro cell culture; for L-X-2 (p1)/L-X-3 (p1) in vitro cell culture, comparisons were made with corresponding xenografts. *, P < 0.05; **, P < 0.005; ***, P < 0.001.

Funding

Newton Fund (NF)

History

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