Hypoxia induces radioresistance, epithelial­mesenchymal transition, cancer stem cell­like phenotype and changes in genes possessing multiple biological functions in head and neck squamous cell carcinoma.

Hypoxia has been linked with increased resistance to treatment in various solid tumors, including head and neck squamous cell carcinoma (HNSCC). The aim of the present study was to identify genes involved in hypoxia­mediated responses to radiotherapy in HNSCC. A total of three HNSCC cell lines with an epithelial phenotype were selected for this study and cultured under normoxic (21% O2) or hypoxic (1% O2) conditions. The sensitivity of the HNSCC cells to radiotherapy was assessed by a crystal violet assay. Western blotting (for protein expression), cDNA microarrays and reverse transcription­quantitative PCR (for gene expression) were also applied. Small interfering RNA silencing was used to knock down target genes. The results revealed that hypoxia negatively affected the response of HNSCC cells to radiotherapy. Of note, increased levels of N­cadherin, vimentin and fibronectin, as well as stem cell­associated transcription factors, were observed under hypoxia. The microarray analysis revealed a number of hypoxia­regulated genes that were involved in multiple biological functions. However, downregulation of hypoxia­regulated genes did not affect sensitivity to radiotherapy of the investigated cell lines. Taken together, the present findings indicated several important pathways and genes that were involved in hypoxia and radiotherapy resistance. It is hypothesized that panels of reported hypoxia­regulated genes may be useful for the prediction of radiotherapy responses in patients with HNSCC.

Cancer-Associated Fibroblasts Modulate Transcriptional Signatures Involved in Proliferation, Differentiation and Metastasis in Head and Neck Squamous Cell Carcinoma.

Cancer-associated fibroblasts (CAFs) are known to increase tumor growth and to stimulate invasion and metastasis. Increasing evidence suggests that CAFs mediate response to various treatments. HNSCC cell lines were co-cultured with their patient-matched CAFs in 2D and 3D in vitro models, and the tumor cell gene expression profiles were investigated by cDNA microarray and qRT-PCR. The mRNA expression of eight candidate genes was examined in tumor biopsies from 32 HNSCC patients and in five biopsies from normal oral tissue. Differences in overall survival (OS) were tested with Kaplan-Meier long-rank analysis. Thirteen protein coding genes were found to be differentially expressed in tumor cells co-cultured with CAFs in 2D and 81 in 3D when compared to tumor cells cultured without CAFs. Six of these genes were upregulated both in 2D and 3D (POSTN, GREM1, BGN, COL1A2, COL6A3, and COL1A1). Moreover, two genes upregulated in 3D, MMP9 and FMOD, were significantly associated with the OS. In conclusion, we demonstrated in vitro that CAF-derived signals alter the tumor cell expression of multiple genes, several of which are associated with differentiation, epithelial-to-mesenchymal transition (EMT) phenotype, and metastasis. Moreover, six of the most highly upregulated genes were found to be overexpressed in tumor tissue compared to normal tissue.

In vitro measurement of glucose uptake after radiation and cetuximab treatment in head and neck cancer cell lines using 18F-FDG, gamma spectrometry and PET/CT.

The standard treatment for head and neck squamous cell carcinoma (HNSCC) is radiotherapy, often in combination with chemotherapy or surgery. However, a novel monoclonal antibody, cetuximab (Erbitux®), has also been approved for patient therapy. The aim of present study was to develop an in vitro method for the measurement of 18F-fluoro-2deoxy-D-glucose (FDG) to determine if cellular 18F-FDG uptake is associated with response to radiotherapy or cetuximab treatment. In the current study, HNSCC cell lines were treated with radiation or with cetuximab. Next, the uptake of 18F-FDG was measured using a gamma spectrometer (GS). Thereafter, uptake after radiation was measured first with GS and then compared with positron emission tomography (PET)/computed tomography (CT) imaging. Furthermore, the mRNA expression of glucose transporter 1 (GLUT1) was measured following cetuximab treatment via reverse transcription-quantitative PCR. A study protocol was developed to measure the cellular uptake of 18F-FDG via gamma-ray spectrometry and comparable results were obtained with those of clinical PET/CT. The results revealed a decrease in 18F-FDG after radiation and cetuximab treatment. The uptake of 18F-FDG following cetuximab treatment was significantly lower in the cetuximab-sensitive cell line UT-SCC-14 compared with the cetuximab-resistant cell lines UT-SCC-2 and UT-SCC-45. Furthermore, after treatment with cetuximab for 24 and 48 h, a significant increase in GLUT1 expression was detected in the sensitive cell line compared with the two resistant cell lines. In conclusion, a novel yet reliable method for the measurement of intracellular 18F-FDG via GS has been developed, and our results indicate that 18F-FDG uptake is associated with radiation and cetuximab response in HNSCC.