Abstract 1433: Single cell RNA sequencing reveals smoking-associated alterations in bronchial airway epithelial subpopulations

Rationale: We have previously shown that bronchial airway epithelial gene expression reflects the physiologic response to cigarette smoke exposure. We have also shown that gene expression differences in cytologically normal airways cells can serve as a diagnostic biomarker for lung cancer. In this study, we use single cell RNA-seq to profile transcriptomes of individual bronchial epithelial cells from current and never smokers in order to detect smoking-associated alterations within specific epithelial cell types and to discover novel subpopulations that develop as a result of smoke exposure. This approach may be useful for identifying cell type-specific transcriptomic changes in the airways of cancer patients, which may lead to a better understanding of lung carcinogenesis and new approaches to early lung cancer detection. Methods: We obtained bronchial brushings from current smokers (n=6) and never smokers (n=6) and isolated single cells by FACS. The CEL-Seq RNA library preparation protocol was used to sequence the transcriptomes of 1,140 cells (n=95/donor). Results: Distinct populations of bronchial cells expressed known markers of basal (KRT5), ciliated (FOXJ1), secretory (SCGB1A1, MUC5AC) epithelial cells, as well as white blood cells (CD45). In the airways of smokers, we observed an increase in abundance of MUC5AC+ secretory cells as well as a decrease in abundance of KRT5+ basal cells and SCGB1A1+ secretory cells. A novel subset of KRT8+ cells that lacked expression of other known cell type markers was identified in the airways of smokers and may represent a population previously described as undifferentiated intermediate cells. Genes involved with metabolism of polycyclic aromatic hydrocarbons (CYP1B1) were detected in smoker secretory cells, whereas genes involved in the metabolic response to cigarette smoke toxins such as aldehydes (ALDH3A1) and quinones (NQO1), were most highly expressed by smoker ciliated cells. Interestingly, the novel KRT8+ cells identified in smokers expressed genes known to promote MUC5AC+ secretory cell differentiation (SPDEF), but did not express MUC5AC itself, suggesting that these may be pro-MUC5AC+ secretory intermediate cells. Furthermore, we found that genes previously associated with higher expression in the airways of lung cancer patients were enriched among genes most strongly associated with smoker ciliated and secretory cells, whereas genes with lower expression in lung cancer were enriched among genes most strongly associated with white blood cells. Conclusion: We have identified cell type-specific transcriptomic alterations and shifts in epithelial cell population abundance in smoker airways. In future studies, profiling the transcriptomes of single cells from bronchial airways of smokers with and without lung cancer may lead to the identification of specific cellular subpopulations contributing to the airway field of lung-cancer associated injury. Citation Format: Grant E. Duclos, Joshua D. Campbell, Yaron Gesthalter, Patrick Autissier, Yves M. Dumas, Robert Terrano, Gang Liu, Marc E. Lenburg, Avrum Spira, Jennifer Beane. Single cell RNA sequencing reveals smoking-associated alterations in bronchial airway epithelial subpopulations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1433. doi:10.1158/1538-7445.AM2017-1433