PO-335 The effect of three-dimensional (3D) cell cultures on phenotype and gene expression of EMT- and CSC-associated genes in head and neck squamous cell carcinoma cell lines

Abstract

IntroductionHead and Neck Squamous Cell Carcinoma (HNSCC) tumours are often resistant to therapies. Therefore searching for predictive markers and new targets for treatment in clinically relevant in vitrotumour models is essential. The aim of this study was to develop a 3D HNSCC tumour model to investigate key targets implicated in treatment response such as epithelial-to-mesenchymal transition (EMT) and cancer stem cell (CSC)-associated proteins. Furthermore, we aimed to assess phenotypic differences between two dimensional (2D) monolayer cells and 3D cell cultures as well as inter-tumoral differences.Material and methodsIn this study, five HNSCC cell lines from University of Linköping, LK0858B, LK0902, LK0917, LK1108 and LK1122, were cultured in 3D in ultra-low attachment 96-well plates. Spheroid morphology was investigated by phase-contrast microscopy. mRNA expression profile of EMT (E-cadherin, N-cadherin, vimentin, fibronectin, Twist1, Foxc2) and CSC (CD44, SOX2, Nanog)-associated genes was determined using quantitative real-time PCR. Protein expression was determined by Western blot or immunohistochemistry.Results and discussionsAll analysed cell lines formed spheroids but with different size and density. After three and seven days of culture expression of the proliferation marker Ki67 was decreased in all spheroids and after seven days Ki67 positive cells were mainly found in the outer layer of spheroids. All the cell lines exhibited an upregulation of E-cadherin along with downregulation of N-cadherin and vimentin. Furthermore, an increased expression of the CSC-associated genes, Nanog and SOX2 was observed in 3D model in comparison to 2D monolayers. We could also verify the expression of these genes on protein level using Western blot.ConclusionHNSCC cells present a high rate of phenotypic heterogeneity when cultured in 3D associated with size and density of multicellular spheroids as well as upregulation of E-cadherin, Nanog and SOX2. Further studies including cellular response to anti-cancer treatment will help us to develop a reliable model for biomarker screening.