Analysis of radiation-induced cell death in head and neck squamous cell carcinoma and rat liver maintained in microfluidic devices

Objective The aim of this study was to investigate how head and neck squamous cell carcinoma (HNSCC) tissue biopsies maintained in a pseudo in vivo environment within a bespoke microfluidic device respond to radiation treatment. Study Design Feasibility study. Setting Tertiary referral center. Subjects and Methods Thirty-five patients with HNSCC were recruited, and liver tissue from 5 Wistar rats was obtained. A microfluidic device was used to maintain the tissue biopsy samples in a viable state. Rat liver was used to optimize the methodology. HNSCC was obtained from patients with T1-T3 laryngeal or oropharyngeal SCC; N1-N2 metastatic cervical lymph nodes were also obtained. Irradiation consisted of single doses of between 2 Gy and 40 Gy and a fractionated course of 5×2 Gy. Cell death was assessed in the tissue effluent using the soluble markers lactate dehydrogenase (LDH) and cytochrome c and in the tissue by immunohistochemical detection of cleaved cytokeratin18 (M30 antibody). Results A significant surge in LDH release was demonstrated in the rat liver after a single dose of 20 Gy; in HNSCC, it was seen after 40 Gy compared with the control. There was no significant difference in cytochrome c release after 5 Gy or 10 Gy. M30 demonstrated a dose-dependent increase in apoptotic index for a given increase in single-dose radiotherapy. There was a significant increase in apoptotic index between 1×2 Gy and 5×2 Gy. Conclusion M30 is a superior method compared with soluble markers in detecting low-dose radiation-induced cell death. This microfluidic technique can be used to assess radiation-induced cell death in HNSCC and therefore has the potential to be used to predict radiation response

Intermediate filaments of zebrafish retinal and optic nerve astrocytes and Müller glia: differential distribution of cytokeratin and GFAP

<p>Abstract</p> <p>Background</p> <p>Optic nerve regeneration (ONR) following injury is a model for central nervous system regeneration. In zebrafish, ONR is rapid - neurites cross the lesion and enter the optic tectum within 7 days; in mammals regeneration does not take place unless astrocytic reactivity is suppressed. Glial fibrillary acidic protein (GFAP) is used as a marker for retinal and optic nerve astrocytes in both fish and mammals, even though it has long been known that astrocytes of optic nerves in many fish, including zebrafish, express cytokeratins and not GFAP. We used immunofluorescence to localize GFAP and cytokeratin in wild-type zebrafish and transgenic zebrafish expressing green fluorescent protein (GFP) under control of a GFAP promoter to determine the pattern of expression of intermediate filaments in retina and optic nerve.</p> <p>Findings</p> <p>GFAP labeling and GFAP gene expression as indicated by GFP fluorescence was found only in the Müller glial cells of the retina. Within Müller cells, GFP fluorescence filled the entire cell while GFAP labelling was more restricted in distribution. No GFAP expression was observed in optic nerves. Cytokeratin labeling of astrocytes was observed throughout the optic nerve and less intensely in cells in the retinal inner plexiform layer. The retinal inner limiting membrane was strongly labeled by anti-cytokeratin.</p> <p>Conclusions</p> <p>Studies of astrocyte function during ONR in zebrafish cannot solely rely on GFAP as an astrocyte marker or indicator of reactivity. Future studies of ONR in zebrafish should include evaluation of changes in cytokeratin expression and localization in the optic nerve.</p

Integration of Biomarkers Including Molecular Targeted Therapies in Head and Neck Cancer

Head and neck tumors comprise a wide spectrum of heterogeneous neoplasms for which biomarkers are needed to aid in earlier diagnosis, risk assessment and therapy response. The search for biomarkers includes evaluation of tumor tissues and surrogate materials by molecular, genomic and phenotypic means. Ideal biomarkers should be accurate and easy to perform, highly specific, objective, quantitative, and cost effective. Because of the heterogeneity of head and neck tumors, the integration of multiple selected markers in association with the histopathologic features is advocated for risk assessment. For targeted therapy, however, a single key molecule must be identified. Key molecules and pathways for targeted therapy include growth factor receptors, MAPk/ERk pathway, angiogenesis, and epithelial to mesenchymal transition. Over-expression and mutations of genes in these pathways including EGFR, VEGF, HER2, BRAF and RET, contribute to tumorigenesis in head and neck cancers from squamous carcinomas, to salivary adenocarcinomas and thyroid carcinomas, both follicular and c-cell derived. Monoclonal antibodies to the EGFR receptor and oral tyrosine kinase inhibitors are currently being studied in multiple phase II and III clinical trials to determine their efficacy in head and neck cancers and correlative studies for biomarkers are on-going