Monitoring the response of head and neck tumour tissue to irradiation using a microfluidic-based approach

Radiotherapy remains the standard treat74ment for head and neck squamous cell carcinoma (HNSCC), however, resistance remains a real clinical problem despite the improvements and development of new treatment strategies. Patient outcome could potentially be improved if the response to irradiation could be predicted allowing alternative treatments to be implemented. The current project has investigated the ability to maintain HNSCC tissue under pseudo in vivo-like conditions using a bespoke microfluidic device. The interrogation of the tissue with irradiation has also been performed with a view to predicting outcome and ultimately personalising medicinal treatment. Following extensive optimisation of key steps in device establishment, e.g. flow rates, serum concentration, oxygen perfusion, using rat liver and human tumour tissue, a series of HNSCC biopsies were divided and placed into parallel microfluidic devices and pieces of each tumour were subjected to single-dose irradiation (5, 10, 15, and 20Gy). Lactate dehydrogenase (LDH) release was measured in effluent collected every 2hr. In five of the tumours (primary n=3; metastatic lymph node n=2), frozen tissue sections were stained for cytokeratin (CK), M30 (detects cleaved CK-18), γH2AX, Ki-67 and for TUNEL. A CK18-labelling index (CK18-LI) was developed by expressing the percentage of apoptotic area (M30) over the total tumour area (CK+ staining). The positive γH2AX, Ki-67 expression and TUNEL were evaluated as positive nuclei within selected tumour areas. Single-dose irradiation induced variable, yet higher, CK18-LI compared with nonirradiated controls. In contrast no statistically significant differences in LDH release were observed between irradiated samples and controls. The percentage of Ki-67 expression reduced dose-dependently but not significantly following on-chip irradiation. Variation in expression profiles of these markers was identified between patients when using the same irradiation regimen demonstrating the potential of current microfluidic irradiation experimentation in monitoring patient’s radiotherapeutic response. In conclusion, microfluidics offers a potential tool in personalised medicine, capable of predicting a tumours response to irradiation prior to clinical administration although further validation experiments are required. The approach has equal applicability to all solid tumours and multiple types of treatment, i.e. chemo-radiation. Thesis associated presentations and published abstracts Cheah R. , Srivastava R. , Kazmierow H. , Patel R. , Stafford N. , Green V. , and Greenman J. 2013. Optimisation of culture conditions for the maintenance of viable tissue samples in a microfluidic device. Hull York Medical School. (Poster) 2013 HYMS Postgraduate Research Conference (June 2013, Hull). Cheah R. , Srivastava R. , Kazmierow H. , Patel R. , Stafford N. , Green V. , and Greenman J. 2013. Optimisation of culture conditions for the maintenance of viable tissue samples in a microfluidic device and the study of tissue response to irradiation. The National Cancer Research Institute. (Poster) 2013 NCRI Cancer Conference (November 2013, Liverpool). Patel R. , Green V. , Srivastava R. , Cheah R. , Hunter, I. A. , and Greenman J. 2013. The use of apoptotic markers to predict rectal cancer response to neo-adjuvant therapy. The National Cancer Research Institute. (Poster) 2013 NCRI Cancer Conference (November 2013, Liverpool). Cheah R. , Srivastava R. , Stafford N. , Green V. , and Greenman J. 2014. Investigation of the response of head and neck squamous cell carcinoma (HNSCC) to irradiation using a microfluidic device. Hull York Medical School (HYMS). (Poster) 2014 HYMS Postgraduate Research Conference (June 2014, York). Cheah R. , Srivastava R. , Stafford N. , Green V. , and Greenman J. 2014. Analysis of cell death in head and neck squamous cell carcinoma (HNSCC) tissue in response to irradiation during microfluidic culture. The National Cancer Research Institute. (Poster) 2014 NCRI Cancer Conference (November 2014, Liverpool). Cheah R. , Srivastava R. , Stafford N. , Green V. , and Greenman J. 2015. Can microfluidic culture be used to determine the response of head and neck squamous cell carcinoma tissue to irradiation using different markers of cell death? European Association for Cancer Research (EACR). (Poster) EACR Conference Series 2015. (February 2015, Essen) Cheah R. , Srivastava R. , Stafford N. , Green V. , and Greenman J. 2015. Exploiting microfluidic culture to determine the radio-sensitivity of head and neck squamous cell carcinoma tissue. Hull York Medical School (HYMS). (Poster) 2015 HYMS Postgraduate Research Conference (June 2015, Hull). Thesis associated journal publication (under reviewed) Cheah R. , Srivastava R. , Stafford N. , Beavis AW, Green V. , and Greenman J. 2015. A microfluidic approach to determine the radio-sensitivity of head and neck squamous cell carcinoma biopsies. Radiotherapy and Oncology. Elsevier Publisher. (Under reviewed)