Airway brushing as a new experimental methodology to detect airway gene expression signatures in mouse lung squamous cell carcinoma

Jing Pan,Eva Szabo,Ronald A Lubet,Mark Steven Miller,Yian Wang,Ming You,Qi Zhang,Donghai Xiong

doi:10.1038/s41598-018-26902-7

Abstract

As a consequence of exposure to environmental toxicants, a “field cancerization” effect occurs in the lung, resulting in the development of a field of initiated, but morphologically normal appearing cells within a damaged epithelium containing mutations in oncogene or tumor suppressor genes. Unlike humans, whose airway field of injury associated with lung cancer has long been investigated with airway brushings obtained via bronchoscopy, no methods are available for similar studies in the mouse due to the small size of the murine airways. In this protocol, we describe a detailed method for performing airway brushing from a live mouse, which enables repeated sampling from the same mouse and thus, mimicking the bronchoscopy protocol used in humans. Using this approach in the N-nitroso-tris-chloroethylurea (NTCU)-induced mouse lung squamous cell carcinoma (SCC) model, we isolated airway epithelial cells with intact cell membrane structure and then performed transcriptome sequencing (RNA-Seq). We found activation of the PI3K signaling network to be the most significant in cytologically normal bronchial airway epithelial cells of mice with preneoplastic lung SCC lesions. Prolonged exposure to NTCU also induced activation of NF-kappaB (NFƙB), the downstream pathway of PI3K; this NTCU-induced lung SCC progression can be reversed by blocking the NFƙB pathway.

Highlights

Lung cancer is the leading causes of cancer death worldwide
We developed a non-lethal method of collecting airway epithelial cells from mice and conducted RNA-sequencing analysis using bronchial airway samples from a NTCU-induced lung squamous cell carcinoma (SCC) model to test the relevance of this method against data obtained from human datasets
We found that PI3K/NFƙB pathway activation upregulated a gene expression signature which was significantly downregulated by XL-147 treatment (Fig. 7)

Summary

Introduction

Lung cancer is the leading causes of cancer death worldwide. Of the two main histopathologic classifications, non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), approximately 85% of lung cancer falls into the NSCLC category and can be further subclassified as adenocarcinoma, squamous cell carcinoma (SCC), and large cell carcinoma. Prolonged exposure to cigarette smoke or other carcinogens creates a field of injury in the cytologically normal epithelium that lines the respiratory tract, reflecting processes associated with the precancerous disease state[2], such as mutations in oncogenes or tumor suppressor genes induced by inhaled environmental toxicants[3,4,5] Molecular characterization of this field of injury in the upper airways can provide novel insights into lung carcinogenesis and identify biomarkers to guide early detection of, and early intervention in, lung cancer[2]. We identified significant activation of oncogenic pathways, especially the PI3K/Akt pathway, as early events during murine lung SCC development We further validated these RNA-seq data with NFƙB reporter mice and found that prolonged NTCU exposure induced significant NFƙB activation in vivo. Mice will be placed in a warm, clean, dry, new cage for recovery, and will be returned to the animal facility once they can maintain an upright posture and walk normally about the cage

Methods

Results

Conclusion