Single-Cell RNA Sequencing of the Bronchial Epithelium in Smokers With Lung Cancer

Cigarette smoking, the major cause of lung cancer, creates a field of injury throughout the respiratory tract. We have previously shown that gene expression from bronchial epithelial cells reflects the physiologic response to cigarette smoke exposure and can serve as a diagnostic biomarker for lung cancer. The purpose of this Idea Development Award is to conduct single cell RNA sequencing on airway epithelial cells obtained from smokers with and without lung cancer to identify cell-type dependent gene expression alterations in the lung cancer field of injury. Cells are being collected by brushing the right mainstem bronchus of smokers undergoing bronchoscopy for the suspicion of lung cancer. A protocol has been developed to isolate single cells from these bronchial brushings using fluorescence-activated cell sorting (FACS). Next, we have implemented an adapted version of the CEL-Seq RNA library preparation protocol that includes plate-, well-, and transcript-specific barcodes allowing hundreds of cells to be pooled together and sequenced. We have also developed a computational pipeline to process the sequencing data into gene level counts for each cell. In order to demonstrate that the methodologies described are working, a pilot experiment was conducted in which bronchial epithelial cells from 1 former smoker and 1 current smoker were profiled (n=24 cells per donor). Data generated from this experiment illustrated that there is low cell-to-cell technical variation and known smoking-associated gene expression alterations can be detected. The success of this initial experiment is significant because it demonstrates that all the protocols are currently in place to begin processing samples collected from smokers with and without lung cancer. Over the next year we plan to sequence hundreds of cells per donor from about 30 smokers with and without lung cancer to discover known and novel cell types with gene expression changes associated with the presence of lung cancer The original document contains color images.