Abstract
Squamous cell lung cancer (SCC) is the second leading cause of lung cancer death in the US and has a 5-year survival rate of only 16%. Histological changes in the bronchial epithelium termed dysplasia are precursors to invasive SCC. However, the cellular mechanisms that cause dysplasia are unknown. To fill this knowledge gap, we used topical application of N-nitroso-tris chloroethylurea (NTCU) for 32 weeks to induce squamous dysplasia and SCC in mice. At 32 weeks the predominant cell type in the dysplastic airways was Keratin (K) 5 and K14 expressing basal cells. Notably, basal cells are extremely rare in the normal mouse bronchial epithelium but are abundant in the trachea. We therefore evaluated time-dependent changes in tracheal and bronchial histopathology after NTCU exposure (4, 8, 12, 16, 25 and 32 weeks). We show that tracheal dysplasia occurs significantly earlier than that of the bronchial epithelium (12 weeks vs. 25 weeks). This was associated with increased numbers of K5+/K14+ tracheal basal cells and a complete loss of secretory (Club cell secretory protein expressing CCSP+) and ciliated cells. TUNEL staining of NTCU treated tissues confirmed that the loss of CCSP+ and ciliated cells was not due to apoptosis. However, mitotic index (measured by bromodeoxyuridine incorporation) showed that NTCU treatment increased proliferation of K5+ basal cells in the trachea, and altered bronchial mitotic population from CCSP+ to K5+ basal cells. Thus, we demonstrate that NTCU-induced lung epithelial dysplasia starts in the tracheal epithelium, and is followed by basal cell metaplasia of the bronchial epithelium. This analysis extends our knowledge of the NTCU-SCC model by defining the early changes in epithelial cell phenotypes in distinct airway locations, and this may assist in identifying new targets for future chemoprevention studies.
Highlights
Squamous cell lung cancer (SCC) is the second most common type of lung cancer and accounted for approximately 40,000 deaths in the United States in 2013 [1]
We previously reported that bi-weekly topical application of 40 mM nitroso-tris chloroethylurea (NTCU) in FVB/n mice for 32 weeks caused a series of endobronchial squamous lesions classified as flat atypia, low and high grade dysplasia, as well as invasive SCC [6]
We utilize the NTCU model to determine the cellular processes that precede endobronchial squamous dysplasia and SCC. This analysis led to the following results: 1) Dysplastic changes in the tracheal epithelium happened significantly earlier than in the bronchial epithelium; 2) Tracheal dysplasia involved increased frequencies of K5, K14 and, p63 expressing basal cells and loss of Club (CCSP+) and ciliated (ACT+) cells; 3) Chronic NTCU treatment converts pseudostratified tracheal epithelium to an involucrin expressing stratified epithelium; 4) NTCU-induced tracheal dysplasia caused increased proliferation of tracheal basal cells; 5) Dysplasia of the bronchial epithelium was first observed at 25 weeks, and involved appearance of K5+/K14+ cells and the loss of resident CCSP+ and ACT+ cells; and, 6) NTCU exposure altered bronchial mitotic pool from CCSP+ to K5+ cells
Summary
Squamous cell lung cancer (SCC) is the second most common type of lung cancer and accounted for approximately 40,000 deaths in the United States in 2013 [1]. The goal of this study is to determine the sequence of cellular changes that leads to squamous dysplasia, the precursor to SCC. This effort requires a mouse model of SCC that faithfully recapitulates the human disease. NTCU exposure is the only model that generates squamous dysplasia of the mouse bronchial epithelium that is pathologically equivalent to the dysplasia encountered in human smokers [6]. Dose and time dependent generation of highgrade dysplasia and SCC makes the NTCU model an optimal system to investigate early phenotypic changes in central airway epithelial cells during dysplasia development
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