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Head and neck squamous cell carcinoma (HN-SCC) is an immunosuppressive disease with functional impairment of tumour-infiltrating lymphocytes including natural killer (NK) cells, which are critical in cancer immune surveillance.

2D co-culture models can be utilised to examine inter-relationships between different groups of cells in the tumour and normal tissue microenvironment, such as between cancer and immune cells. These cell culture models are also often used to study the effects of radiation in cancers and normal tissues. However, in these settings, radiation is commonly delivered using orthovoltage irradiators in single dose fractionation schedules, which is not reflective of clinical radiotherapy applications. We have developed a 2D co-culture model of HN-SCC and NK cells using a clinically operational linear accelerator and evaluated the impact of NK cells on radiation-mediated cancer cell death.  

The 2D co-culture model consists of human buccal SCC cells (TR146 cell line) and human leukaemic NK cells (KHYG-1 cell line) added either before or after irradiation at 5:1 to 10:1 NK:HN-SCC ratios. Tumour cell death was assessed using an ATP-based cell viability assay at D4-6 post-irradiation. Irradiation was carried out using a clinically-operational linear accelerator at a range of doses including 2Gy, 5Gy and 10Gy in a single fraction. Fractionated radiation regimens were also carried out including 6Gy over 3 fractions (2Gy per fraction) and 15Gy over 3 fractions (5Gy per fraction).  Significance was assessed with the ANOVA statistical test.

The addition of NK cells prior to irradiation contributed more significantly to radiation-mediated HN-SCC killing, at effector to target ratios of 5:1 (p<0.0001). SCC viability was significantly reduced in 5:1 and 10:1 groups compared with the TR146 alone group irrespective of single fraction radiation dose (p<0.0001). There was no significant difference in SCC viability between the 5:1 and 10:1 groups. Additionally, there was no significant difference in SCC viability with the different single fraction radiation doses within 5:1 group and the 10:1 group.

With fractionated irradiation, SCC viability was significantly reduced in the 5:1 group compared with the TR146 alone group at both 6Gy and 15Gy (p<0.0001). There was no significant difference in cell viability between the TR146 groups irradiated with 6Gy or 15Gy. There was significantly reduced SCC viability in the 5:1 group at 6Gy over 3 fractions compared with 15Gy over 3 fractions (p=0.008).

We demonstrate that NK cells increase radiation-mediated cell death of radioresistant HN-SCCs. Furthermore, our findings suggest that NK cells present in the tumour microenvironment prior to irradiation are more integral to radiation-mediated SCC cell death than NK cells recruited to the tumour post-irradiation. This co-culture model will also enable the study of interactions between cancer, NK cells and the wider tumour micro-environment.