| Popis: |
Introduction Recent work has shown that tobacco smoke exposure increases the mutational burden and cell-to-cell mutational heterogeneity of the normal bronchial epithelium. However, smoking cessation allows the preferential expansion of low-mutant epithelial cells, which could be responsible for the decline in lung cancer risk on quitting smoking. Although less is understood about the nasal epithelium, the incidence of squamous cell cancer (SCC) in the nose is much lower than in the bronchi. Whether a difference in function or mutational burden underlies this variation is unknown. Methods Nasal brushings and endobronchial biopsies were enzymatically dissociated and underwent initial analysis by flow cytometry for expression of CK5, ITGA6, PDPN and NGFR (basal cell markers). Subsequently, single bronchial (n=152) and nasal epithelial (n=47) cells were flow-sorted and cultured from an ex-smoker to permit more detailed study of regional inter-cell variability and functionality. Index sorting was performed on the most relevant basal cell subpopulation marker PDPN. Single cells were clonally expanded in vitro and colony-forming efficiency and colony size were calculated. After expansion, DNA and RNA were isolated for downstream analysis. Results Flow cytometric analysis showed that CK5-positive basal cells constituted a similar proportion of the epithelial cell population in the nose and the bronchus (35% v. 30%, respectively, p=0.8). CK5-positive nasal basal cells compared with CK5-positive bronchial basal cells differed most in their expression of PDPN (6% v. 64% PDPN-positive, respectively, p=0.2). This was coupled with a lower median intensity of PDPN expression in nasal basal cells compared with bronchial basal cells (2437 v. 8434, respectively). Nasal epithelial cells showed lower colony-forming efficiency (15%) than bronchial epithelial cells (35%). In addition, the nasal clones that grew were significantly smaller in area than the bronchial clones (9.84 mm2 v. 13.26 mm2, respectively, p=0.0002). Conclusions The lower colony-forming efficiency, smaller colony size and lower expression of PDPN in the nasal epithelial population suggests nasal basal cells may have a lower stem cell capacity than their bronchial counterparts. Analysis of the mutational burden and transcriptomic profile of both populations is a crucial next step to understand the difference in incidence of SCC in these compartments. |
