ISOLATION, CHARACTERISATION AND FUNCTIONAL ANALYSIS OF CANCER STEM CELLS FROM NASOPHARYNGEAL CARCINOMA

Abstract

Background: Nasopharyngeal carcinoma (NPC) is a malignant disease originated from the epithelial cells of the nasopharynx. NPC is prevalent in southern China and South East Asia (Canton province, Taiwan, Hong Kong, Malaysia, Singapore and Thailand) with male to female ratio of 3:1. The major aetiological factors of NPC are Epstein-Barr virus (EBV) infections, genetics, diet and environmental. Radiotherapy with or without chemotherapy is the primary treatment modality for NPC. Treatment generally produce good locoregional control and five year overall survival rate of 80%. However, as high as 30% of patients will eventually develop distant metastasis as treatment failure and this is the main contributor to NPC mortality. Cancer stem cells (CSC) are a subset of cells in tumour that possess stem cell characteristics such as the ability to proliferate indefinitely and differentiate into specialised cells. It is believed that the deregulation of normal quiescence stem cells will give rise to cancer stem cells and they are responsible for the metastasis, relapse and treatment failure of cancers. Objective: The objective of the study is to isolate and characterise cancer stem cells from nasopharyngeal carcinoma. Methodology The expression level of the putative cancer stem cell markers (CD44, CD24, ABCG2, CD133 and ALDH) on selected NPC cell lines CNE1, TW01, HK1 and NP69 were analysed using flow cytometer. The expression of the same markers on NPC biopsy samples were also studied by immunohistochemistry staining, and markers expression in cell lines and biopsy samples were compared. Subsequently, the putative cancer stem cells and non-cancer stem cells, the ALDH+ and ALDH- CNE1 cells were sorted by using fluorescence activated cell sorting (FACS). The biological characteristics of the sorted ALDH+ and ALDH- cells were studied and compared using sphere formation assay, soft agar colony formation assay, invasion assay and cisplatin sensitivity test. The in vivo tumour initiation efficiency of sorted ALDH+ vs. ALDHcells were also studied by xenograft transplantation to Balb/c nude mice. mRNA expression and miRNA expression of ALDH+ vs. ALDH- cells were carried out and subsequent mRNA-miRNA integrated analysis was performed. The resultant list of differentially expressed genes were subjected to gene ontology and KEGG pathway analyses. The regulation interactions of the mRNA and miRNA were viewed using Cytoscape software. The expression level of a subset of genes and miRNA from microarray were verified using quantitative real time-PCR (qRT-PCR). Results: The expression level of putative cancer stem cell markers (CD44, CD24, ABCG2, CD133 and ALDH) were analysed in selected NPC cell lines. Both CD44 and CD24 were found to be highly expressed in cell lines, ABCG2 was expressed at varying levels, ALDH and CD133 had very low expression. In NPC biopsy samples tested with immunohistochemistry, CD44, CD24, ABCG2 and CD133 had moderate to high expression levels, while ALDH had low expression level. The putative cancer stem cells, the ALDH+ and ALDH- CNE1 cells were sorted by using fluorescence activated cell sorting (FACS). ALDH+ cells showed higher sphere formation and soft agar colony formation efficiency indicating higher self-renewal activity and higher in vitro carcinogenicity. ALDH+ cells also exhibited higher invasion activity and higher resistance to cisplatin treatment compared to ALDH- cells. The xenograft transplantation assay showed that ALDH+ had greater tumour formation propensity and resulted in larger tumour size as compared to the ALDH- cells. However, the limiting dilution analysis showed there were no difference in stem cell frequency between them. Microarray results showed 58 differentially expressed miRNAs and 635 differentially expressed genes. miRNA-mRNA integrated analysis and subsequent functional enrichment studies showed biological processes and pathways related to cell cycle, invasion, drug resistance and stem cells being enriched. Subsequent bioinformatics analyses identified LAMC2, TNC, ELF3, MUC1, SPRY4, ITGA3, miR-23c, miR-589-3p and miR-1301-3p as important genes and miRNAs that are potentially important for the growth and progression of ALDH+ cells. Lastly, qRT-PCR for the validation for microarray data showed results were in consistent trend with the microarray data. Conclusion: ALDH+ cells from NPC exhibited characteristics of CSCs, including increased self-renewal, clonogenic activity, invasion and higher resistance to cisplatin treatment. ALDH+ cells also showed higher tumourigenicity in vivo but it was not statistically significant. Microarray and subsequent functional enrichment studies showed some biological processes and pathway related to cell cycle, invasion, drug resistance and stem cells being enriched. Deregulated genes and miRNAs like LAMC2, TNC, ELF3, MUC1, SPRY4, ITGA3, miR-23c, miR-589- 3p and miR-1301-3p may play important roles in the function of NPC CSCs. More studies into the mechanistic functions of these genes and miRNAs might be useful for the development of new treatment strategy against NPC CSCs.