Abstract
Mortality in head and neck squamous cell carcinoma (HNSCC) is high due to emergence of therapy resistance which results in local and regional recurrences that may have their origin in resistant cancer stem cells (CSCs) or cells with an epithelial-mesenchymal transition (EMT) phenotype. In the present study, we investigate the possibility of using the cell surface expression of CD44 and epidermal growth factor receptor (EGFR), both of which have been used as stem cell markers, to identify subpopulations within HNSCC cell lines that differ with respect to phenotype and treatment sensitivity. Three subpopulations, consisting of CD44high/EGFRlow, CD44high/EGFRhigh and CD44low cells, respectively, were collected by fluorescence-activated cell sorting. The CD44high/EGFRlow population showed a spindle-shaped EMT-like morphology, while the CD44low population was dominated by cobblestone-shaped cells. The CD44high/EGFRlow population was enriched with cells in G0/G1 and showed a relatively low proliferation rate and a high plating efficiency. Using a real time PCR array, 27 genes, of which 14 were related to an EMT phenotype and two with stemness, were found to be differentially expressed in CD44high/EGFRlow cells in comparison to CD44low cells. Moreover, CD44high/EGFRlow cells showed a low sensitivity to radiation, cisplatin, cetuximab and gefitinib, and a high sensitivity to dasatinib relative to its CD44high/EGFRhigh and CD44low counterparts. In conclusion, our results show that the combination of CD44 (high) and EGFR (low) cell surface expression can be used to identify a treatment resistant subpopulation with an EMT phenotype in HNSCC cell lines.
Highlights
Head and neck cancer is a malignancy that despite advances in therapy still is associated with severe mortality
Cell cycle analysis 48 h after cell sorting by fluorescence-activated cell sorting (FACS), cells of the three subpopulations were detached using 0.25% trypsin and 0.02% EDTA, washed and resuspended in propidium iodide (PI) solution (0.4 mg/ml PI, 1 mg/ml sodium citrate, 60 mg/ml trizma base, 1.7 mg/ml spermine tetrahydrochloride and 0.01% nonidet P40; all from Sigma)
In order to identify subpopulations with different phenotypes, the surface expression of CD44 was investigated in three head and neck squamous cell carcinoma (HNSCC) cell lines (LK0923, LK0827 and LK0863) by flow cytometry
Summary
Head and neck cancer is a malignancy that despite advances in therapy still is associated with severe mortality. Mortality remains high due to emergence of local and regional recurrences and development of lymph node metastasis and, occasionally, distant metastasis. The standard treatment for head and neck squamous cell carcinoma (HNSCC) patients is radiotherapy, often in combination with surgery. Chemoradiotherapy has recently become part of the treatment of advanced tumors, and cisplatin is the most common agent in combination with radiation. Radio- and/or chemotherapy resistance and tumor recurrences are important clinical problems in the management of HNSCCs and the need for more effective treatment strategies is urgent [1]
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