Fig 1.
32-week NTCU treatment induces high-grade bronchial dysplasia and lung SCC.
H&E stained images of (A) Normal epithelium (acetone vehicle), and NTCU-induced (B-D) premalignant lesions and (E&F) invasive carcinoma. (A) Normal epithelium: basally located nuclei and apical cilia (arrows and inset). (B) Flat-atypia: single cell layer, but enlarged hyperchromatic nuclei (arrow) and infrequent loss of cilia. (C) Low-grade dysplasia: stratified non-ciliated epithelium, horizontal orientation of nuclei and decreased nuclear:cytosolic ratio. (D) High-grade dysplasia: stratified squamous non-ciliated epithelium with lack of orientation and increased nuclear:cytosolic ratio. (E) Lung SCC: invasive squamous cell carcinoma with arrows showing central keratinaization in the nest of the tumor. (F) SCC is generated from the high-grade dysplastic airways (arrow-heads). Original magnifications are indicated in each panel. The pictures shown here are the representative images from 10 mice each for vehicle and NTCU group.
Fig 2.
Molecular phenotype of the cells present in NTCU-induced bronchial dysplasia.
Bronchial epithelium from mice treated for 32 weeks with vehicle (A-B) or NTCU (C-F). Tissues were stained with antibodies against (A) CCSP (green), ACT (red) and (B & C) K5 (green). Expression of other basal cell markers was detected by staining with (D) K14 (red), (E) p63 (red) and K5 (green), and (F) nerve growth factor receptor (NGFR, red). DAPI staining (blue) in all images depicts nuclei. Representative images from 10 mice treated with vehicle or NTCU.
Fig 3.
Development of bronchial dysplasia over time.
(A) Study design for time-course analysis of NTCU exposure. Arrows indicate the time-points when tissues were collected. (B-C) H & E stained bronchial tissues after 25 weeks of NTCU treatment. Immunofluorescence staining of dysplastic bronchial epithelium (D-G). (E) Amplified image of the boxed area shown in (D). Antigens used and scale bars are mentioned in the respective panel. Representative images are from 5 NTCU treated mice.
Fig 4.
NTCU induced squamous dysplasia of the tracheal epithelium: Tracheal epithelium of vehicle (left column) and NTCU treated (right column) mice at 32 weeks.
(A&B) H&E stained picture showing high-grade dysplasia of the tracheal epithelium induced by NTCU. (C&D) Dual staining with K5 (green) and K14 (red), (E&F) K5 (green) and p63 (red) and arrows show dual positive cells. Nonspecific staining under the epithelium are shown by white * symbol. (G&H) Staining for nerve growth factor receptor NGFR and (I &J) squamous differentiation marker, involucrin. DAPI staining (blue) in all images indicates nuclei and scale bar is indicated in each panel. Representative images from 10 mice each for vehicle and NTCU group.
Fig 5.
NTCU-induced increase in K5 and K14 expressing basal cells and decrease in CCSP and ACT positive cells in the tracheal epithelium.
(A-C) Immunostaining of the tracheal epithelium with antibodies to K5 (green), K14 (red) and DAPI (blue). Yellow arrows depict K5/K14-dual positive cells. (D&E) Quantification of K5, K14 cell frequency over time using morphometric analyses. (F-H) Immunostaining of the tracheal epithelium against antibodies to CCSP (green), ACT (red), DAPI (blue) and (I&J) quantification of CCSP and ACT positive cell frequency. Each point in the graphs D, E, I & J represent individual mouse, and n = 3–4 mice were used for each time-point. Vv/Sv represents the volume density of specific cell types (Vv) as a function of surface density (Sv) with the epithelium used as the reference volume. 1-Way ANOVA and post test using Bonferroni multiple comparison methods were used for statistical analysis. Significant difference (p<0.05) between the vehicle control and other comparison groups are shown by stars (****, p<0.0001, ***, p<0.001, ** p<0.01, and * p<0.05).
Fig 6.
NTCU treatment increased the mitotic index of tracheal basal cells.
(A-B) Tracheal tissue sections from mice treated with vehicle or NTCU for 32 weeks were stained for K5 (green), BrdU (red) and DAPI (blue). Representative pictures from n = 4 mice per group was used. (C) Quantification was performed by counting BrdU+ nuclei throughout the trachea from proximal to distal axis and represented as % of DAPI+ nuclei. Statistical analyses were performed as described in Fig 5.
Fig 7.
NTCU treatment increased the mitotic index of bronchial epithelial cells.
(A) Mitotic index of bronchial epithelial cells was measured by counting the numbers of BrdU+ nuclei as a % of DAPI+ nuclei (all epithelial cells). Quantification was limited to the main axial pathway and the images were taken at 200x magnification from 6 areas of the epithelium per tissue. (B) Lung tissues from mice treated with vehicle for 32 weeks were stained for CCSP (green), BrdU (red), and DAPI (blue). (C) 32 weeks NTCU treated tissues stained with K5 (green), BrdU (red), and DAPI (blue). Arrows in B & C show the BrdU+ nuclei. Inset in C shows the presence of BrdU+ cells in a hyperplastic area (D) Contribution of each of CCSP and K5 expressing cells in the mitotic pool was quantified as a percent of CCSP-BrdU dual positive/CCSP+ cells or K5-BrdU dual positive/K5+ cells. Statistical analyses were performed as described in Fig 5.
Fig 8.
A graphical representation of change in cell type frequencies in the trachea (solid line) and bronchial epithelium (dashed line) during NTCU time-course. Time points where changes are statistically significant are shown by * symbol.