1 SCIENTIFIC REPORTS | ͽ: 12372 | DOI:ͷͶ.ͷͶ;/sͺͷͻͿ;-Ͷͷͽ-ͷͶͺͻ-; www.nature.com/scientificreports CDͺ, CDͺͺ and EpCAM enrich for tumour-initiating cells in a newly established patient-derived xenograft of nasopharyngeal carcinoma Susan Ling Ling Hoe ͷ, , Lu Ping Tan ͷ , Norazlin Abdul Aziz ͷ , Kitson Liew ͷ , Sin-Yeang Teow , Fazlyn Reeny Abdul Razak ͷ , Yoon Ming Chin , Nurul Ashikin Mohamed Shahrehan ͷ , Tai Lin Chu ͷ , Noor Kaslina Mohd Kornain ͺ , Suat-Cheng Peh , Cheng Eng Koay ͻ , Kwok-Wai Lo ͼ , Munirah Ahmad ͷ , Ching-Ching Ng & Alan Soo-Beng Khoo ͷ Subpopulations of nasopharyngeal carcinoma (NPC) contain cells with diferential tumourigenic properties. Our study evaluates the tumourigenic potential of CDͺ, CDͺͺ, EpCAM and combination of EpCAM/CDͺͺ cells in NPC. CDͺͺbr and EpCAMbr cells enriched for higher S-phase cell content, faster- growing tumourigenic cells leading to tumours with larger volume and higher mitotic igures. Although CDͺͺbr and EpCAMbr cells signiicantly enriched for tumour-initiating cells (TICs), all cells could retain self-renewal property for at least four generations. Compared to CDͺͺ marker alone, EpCAM/CDͺͺdbr marker did not enhance for cells with faster-growing ability or higher TIC frequency. Cells expressing high CDͺͺ or EpCAM had lower KLFͺ and pͷ in NPC subpopulations. KLFͺ-overexpressed EpCAMbr cells had slower growth while Kenpaullone inhibition of KLF4 transcription increased in vitro cell proliferation. Compared to non-NPC, NPC specimens had increased expression of EPCAM, of which tumours from advanced stage of NPC had higher expression. Together, our study provides evidence that EpCAM is a potentially important marker in NPC. Nasopharyngeal carcinoma (NPC) is a type of head and neck cancer predominantly found in the southern Chinese population, several indigenous groups of the Southeast Asia, Amazigh- and Arabic-speaking popula- tions of North Africa, and the Inuits of North America and Greenland 1 . It is infamously associated with high die- tary intake of salted and/or preserved food, exposure to tobacco and formaldehyde as well as Epstein-Barr virus (EBV) infection 1 . Although it is a highly radiosensitive malignancy, its initial nonspeciic clinical presentations such as nasal blockage, blood-tainted sputum, ringing in the ears and mild hearing loss are the main obstacles to early diagnosis 2,3 and the 5-year overall survival rate for NPC patients can reduce remarkably in late-stage of the disease 4 . Studies on NPC tumour biology such as growth, stemness, invasion, metastasis, therapy resistance and EBV presence have been widely investigated mainly using NPC cell lines. Stem-like cells from NPC cell lines were commonly enriched by using functional assays such as the side population (SP) assay 5–7 , spheroid assay 8 and ALDH assay 9 . he SP cells in frequently used CNE-2 and HK1 cell lines were found to be more tumour-inducing in nude mice than bulk cells 6,7 . Cells from another NPC cell line, C666-1 which grew as free-loating spheres, displayed stemness characteristics such as a propensity for tumour formation, contained higher expression levels ͷ Molecular Pathology Unit, Cancer Research Centre, Institute for Medical Research, ͻͶͻ;;, Kuala, Lumpur, Malaysia. Institute of Biological Sciences, Faculty of Science, University of Malaya, ͻͶͼͶ, Kuala, Lumpur, Malaysia. Sunway Institute for Healthcare Development, Sunway University, ͺͽͻͶͶ, Bandar Sunway, Selangor, Malaysia. ͺ Department of Pathology, Faculty of Medicine, Universiti Teknologi MARA (UiTM), ͺͽͶͶͶ, Sungai Buloh, Selangor, Malaysia. ͻ Gleneagles Hospital (Kuala Lumpur) Sdn. Bhd., Jalan Ampang, ͻͶͺͻͶ, Kuala, Lumpur, Malaysia. ͼ Li Ka Shing Institute of Health Science, The Chinese University of Hong Kong, Hong Kong, Hong Kong SAR. Correspondence and requests for materials should be addressed to C.-C.N. (email: ccng@um.edu.my) or A.S.-B.K. (email: alankhoo@imr.gov.my) Received: 11 August 2016 Accepted: 25 August 2017 Published: xx xx xxxx OPEN