Measuring the response of human head and neck squamous cell carcinoma to irradiation in a microfluidic model allowing customized therapy

Abstract

Radiotherapy is the standard treatment for head and neck squamous cell carcinoma (HNSCC), however, radioresistance remains a major clinical problem despite significant improvements in treatment protocols. Therapeutic outcome could potentially be improved if a patient's tumour response to irradiation could be predicted ex vivo before clinical application. The present study employed a bespoke microfluidic device to maintain HNSCC tissue whilst subjecting it to external beam irradiation and measured the responses using a panel of cell death and proliferation markers. HNSCC biopsies from five newly-presenting patients [2 lymph node (LN); 3 primary tumour (PT)] were divided into parallel microfluidic devices and replicates of each tumour were subjected to single-dose irradiation (0, 5, 10, 15 and 20 Gy). Lactate dehydrogenase (LDH) release was measured and tissue sections were stained for cytokeratin (CK), cleaved-CK18 (cCK18), phosphorylated-H2AX (γH2AX) and Ki‑67 by immunohistochemistry. In addition, fragmented DNA was detected using terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). Compared with non‑irradiated controls, higher irradiation doses resulted in elevated CK18-labelling index in two lymph nodes [15 Gy; 34.8% on LN1 and 31.7% on LN2 (p=0.006)] and a single laryngeal primary tumour (20 Gy; 31.5%; p=0.014). Significantly higher levels of DNA fragmentation were also detected in both lymph node samples and one primary tumour but at varying doses of irradiation, i.e., LN1 (20 Gy; 27.6%; p=0.047), LN2 (15 Gy; 15.3%; p=0.038) and PT3 (10 Gy; 35.2%; p=0.01). The γH2AX expression was raised but not significantly in the majority of samples. The percentage of Ki‑67 positive nuclei reduced dose-dependently following irradiation. In contrast no significant difference in LDH release was observed between irradiated groups and controls. There is clear inter- and intra-patient variability in response to irradiation when measuring a variety of parameters, which offers the potential for the approach to provide clinically valuable information.

A patient tumour-on-a-chip system for personalised investigation of radiotherapy based treatment regimens

“…Further development to include the use of primary samples within on-chip platforms confers additional benefits pertaining to perfusion, with continuous delivery of nutrients, removal of waste products and the ability for repeated effluent sampling 19,20 . The maintenance of primary tissues in microfluidic devices has been successfully shown by ourselves and others over 2–14 days depending on the human tissue type 21–27 . The use of primary tissue on these platforms is paramount as the intricate structure and organisation of cells within tissues is crucial for correct function 28,29 .…”

Characterising a PDMS based 3D cell culturing microfluidic platform for screening chemotherapeutic drug cytotoxic activity

“…The LDH cytotoxicity assay was performed using the Pierce LDH Cytotoxicity Assay Kit (Thermo Fisher) and carried out as per the manufacturer’s instructions and guidelines. This method has been previously applied to evaluate the cell viability of tumour tissue specimens cultured on a microfluidic platform 51 – 53 .…”

Prospects and Opportunities for Microsystems and Microfluidic Devices in the Field of Otorhinolaryngology

“…In previous microfluidic irradiation models with metastatic lymph nodes and primary tumors from different patients, immunohistochemistry expression profiles relevant to cell death and proliferation markers were measured to determine an individual's tumor response to irradiation. The results showed clear inter-and intra-patient variability in response to irradiation when measuring a variety of parameters, which offered the potential to predict patient responses and could be helpful in predicting treatment effectiveness and quality of life, as well as offering cost savings and improved patient care [89].…”