Abstract
Abstract Mortality in head and neck squamous cell carcinoma (HNSCC) is high due to the emergence of therapy resistance which led to local and regional recurrences that may originate from resistant cancer stem cells (CSCs). The aim of the present study was to identify cells with CSC characteristics in a HNSCC cell line and evaluate differences in CSC proteins, invasion, migration and in treatment sensitivity to radiation, cisplatin and cetuximab (a antibody blocking epithelial growth factor receptor, EGFR) between subpopulations of cancer cells. A HNSCC cell line established from a larynx cancer at the division of otorhinolaryngology at Linköping University Hospital was used in passages 8-15. Cells were cultured in Keratinocyte Media supplemented with 1% fetal calf serum. Subpopulations of cells were detected after direct immunofluorescense staning of surface CD44 and EGFR. Three different populations (CD44high/EGFRlow, CD44high/EGFRhigh and CD44low/EGFRhigh) were sorted for further investigations using a BD FACSAriaTM II Cytometer. Culturing of sorted cells demonstrated diverse morphology between the populations, where CD44high/EGFRlow cells displayed a spindle shaped morphology compared to CD44low/EGFRhigh. The subpopulations sensitivity to radiation (4Gy), cisplatin (2μg/ml, 1h) or cetuximab (30nM) was analysed 10 days after sorting by a crystal violet assay. Here, CD44high/EGFRlow cells showed the lowest sensitivity to both radiation and cisplatin. A real time PCR array was performed in order to screen for differences in gene expression between the subpopulations. Comparison of the cells with a high contra low CD44 surface expression, showed a change in mRNA levels in 19 genes, 14 up regulated and 5 down regulated. Of these 19 differently expressed genes 13 can be associated to an epithelial to mesenchymal transition (EMT) phenotype (VIM, MMP2, TIMP1, TWIST1, COL3A1, CDH1, CDH2, ITGA5, FN1, FOXC2, WNT5A, WNT5B and SPARC). Two stem cell marker genes (NANOG and SOX1) were slightly up regulated in the CD44high/EGFRlow population compared to CD44low/EGFRhigh. These populations ability for migration and invasion should now be investigated as well as differences in proliferation and clonogenic efficiency. We here identify, in a recently established HNSCC cell line, subpopulations of cells using markers suggested to characterize CSCs. More importantly, the CSC-like subpopulation displayed a lower sensitivity to radiation and cisplatin compared to the control population. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 486. doi:10.1158/1538-7445.AM2011-486
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