# A New Model to Investigate the Action of Radiation and Cigarette Smoke on Head and Neck Cancer Cells

## TL;DR

This paper introduces a new model to study how radiation and cigarette smoke affect head and neck cancer cells, showing combined effects on cell behavior.

## Contribution

The novel contribution is the development of an in vitro model to evaluate the combined effects of radiation and cigarette smoke on head and neck cancer cells.

## Key findings

- Radiation and cigarette smoke separately altered cancer cell behavior, with combined effects being more pronounced.
- Gene sequencing revealed a signature related to cell invasion, angiogenesis, and survival after treatments.
- CD44 and ALDH co-expression was reduced in the presence of cigarette smoke.

## Abstract

Smokers are at an increased risk of developing mucosal head and neck cancers. Moreover, they have worse oncological outcomes following treatment; the reasons for this are unknown. To address this, we developed a new experimental model investigating the effects of radiation and smoking on head and neck cancer cells. We found through that radiation and smoking separately altered cell behaviour, and when combined had a greater effect. Gene sequencing reflected the changes in cell behaviour after treatments. Our results show that this new experimental model is relevant in evaluating the combination of radiation and smoking on head and neck cancer cell behaviour.

Background/Objectives: Smokers are at an increased risk of developing mucosal head and neck squamous cell cancers (HNSCCs) and have a worse prognosis when treated. The cellular and molecular mechanisms underlying the latter has not been established. We therefore developed an in vitro model to investigate the effects of radiation and smoking on mucosal HNSCCs. FaDu hypopharyngeal cancer cells were subjected to daily fractionated radiation and cultured with and without cigarette-smoke-exposed media. Methods: The cells were characterised using assays for tumour sphere formation, proliferation, migration, invasion, CD44 and ALDH expression, and next generation sequencing. We also evaluated CD44 and ALDH1 expression in patient tumour samples. Results: Radiation and smoking separately reduced FaDu tumour sphere/Cancer Stem Cell (CSC) number and proliferation, and increased cell migration and invasion. Combined, they further reduced CSC number proliferation and promoted migration. CD44 and ALDH co-expression was reduced in conditions with cigarette smoke. Through next generation sequencing, radiation and smoking produced a gene signature related to cell invasion, angiogenesis, and survival. Immunohistochemistry for CD44 and ALDH1 on patient tumour specimens did not demonstrate a relationship with smoking status, supported our in vitro findings. Conclusions: The data show the utility of a new experimental model to test the combination of radiation and smoking on mucosal HNSCCs behaviour.