N-nitroso-tris-chloroethylurea Induces Premalignant Squamous Dysplasia in Mice

Abstract

Squamous cell carcinoma (SCC) and pre-malignant endobronchial lesions have been difficult to study in murine models. In this report, we evaluate the topical N-nitroso-tris-chloroethylurea (NTCU) murine SCC model, determine the extent to which resulting pre-malignant airway dysplasia develops, discuss clinicopathologic grading criteria in lesion progression, and confirm that immunohistochemical (IHC) staining patterns are consistent with those observed in human endobronchial dysplasia and SCC. Male and female FVB mice were treated biweekly with topical NTCU (4, 8, or 40mM) or vehicle for 32 weeks. Following sacrifice, squamous cell lesions were enumerated and categorized into the following groups: flat atypia, low-grade dysplasia, high-grade dysplasia, and invasive SCC. The 40mM NTCU concentration produced the entire spectrum of premalignant dysplasias and squamous cell carcinomas, but was associated with poor survival. Concentrations of 4mM and 8mM NTCU were better tolerated and produced only significant levels of flat atypia. Squamous origin of the range of observed lesions was confirmed with IHC staining for cytokeratin 5/6, p63, thyroid transcription factor-1 (TTF-1), and Napsin-A. This study demonstrates that topical application of high dose NTCU produces endobronchial pre-malignant lesions with classic squamous characteristics and should allow for improved pre-clinical evaluation of potential chemopreventive agents.

Inflammation and Lung Cancer: Prevention

“…Similar to primary models for ADC, strain differences were also observed using NTCU; SWR/J, A/J, BALB/cJ, NIH Swiss, and FVB/J were sensitive strains while AKR/J, 129/svJ, and C57BL/6 were resistant [32]. In FVB mice, NTCU leads to endobronchial premalignant airway dysplasia and SCC [33]. These lesions were verified as SCC using cytokeratin 5/6, expressed in squamous epithelia, and p63, a member of the p53 family and marker for SCC development [33][34][35].…”

“…In FVB mice, NTCU leads to endobronchial premalignant airway dysplasia and SCC [33]. These lesions were verified as SCC using cytokeratin 5/6, expressed in squamous epithelia, and p63, a member of the p53 family and marker for SCC development [33][34][35]. Recently, a Japanese group determined that a modified NTCU protocol in A/J mice could be used to study SCC tumor promotion, although currently none exist [36].…”

Vitamin D Repletion Reduces the Progression of Premalignant Squamous Lesions in the NTCU Lung Squamous Cell Carcinoma Mouse Model

“…For the intervention study, 12-week old female mice (n=180) were randomized into 2 groups to receive diets that were identical, with the exception of vitamin D3 content (0 IU/kg or2000 IU/kg vitamin D3) (Harlan Tekland). Female mice were chosen as all published studies demonstrate the efficacy of NTCU in female mice (2, 26). One thousand IU/kg is the amount of D3 recommended for mouse diets (27).…”

“…H&E staining was conducted using a standard protocol (28). Standard immunohistochemistry techniques were employed to stain for Ki-67, cytokeratin 5 & 6 (CK 5/6), thyroid transcription factor (TTF-1), p63, and F4/80 (26). The IHC technique used for VDR is similar to the IHC method cited above except that slides were processed with an ABC kit (Vector Labs) to enhance the visualization of VDR (18).…”

Epithelial cell migration as a potential therapeutic target in early lung cancer

“…Towards this end, methods have recently been developed to isolate and expand primary human bronchial epithelial cells that maintain a normal karyotype and multipotent differentiation capacity [52]. In addition, murine models of both human adeno and squamous NSCLCs, generated via targeted transgenesis or cutaneous application of chemical carcinogens, are available [53][54][55][56]. Taken together, these in vitro and in vivo models offer unique opportunities for monitoring the effects of Rho-GTPase activity on lung epithelial cell migration.…”