Fig 1.
Tumor progression and histopathological phenotype in a transgenic mouse model.
Hematoxylin and eosin (H&E)-stained tumors from Tg mice at various stages of age: (a) an atypical adenomatous hyperplasia (AAH) lesion progressing to adenocarcinoma in situ (AIS). The scale bar showed 200 μm in distance. (b) Uniform nuclei are shown in an AIS. The scale bar showed 200 μm in distance. (c) Pleomorphic nuclei are shown in an adenocarcinoma. The scale bar showed 200 μm in distance. (d) Formation of lung adenocarcinomas. The scale bar showed 400 μm in distance. (e) Adenocarcinoma with glandular/acinar architecture and desmoplasia. The scale bar showed 200 μm in distance. (f) Invasive adenocarcinoma displaying mixed cellular phenotypes. Cells of the invasive component were more columnar and of higher nuclear grade (inset). The scale bar showed 200 μm in distance. Panels a and d were at 100x magnification; panels b, c, e, and the inset were at 400x magnification; panel f was at 200x magnification.
Fig 2.
IHC analysis reveals lung tumors are displaying markers related to adenocarcinoma.
IHC evaluation of multidrug resistant protein 3 (MRP3), thyroid transcription factor (TTF)-1, and periodic acid-Schiff (PAS) stain in tumors obtained at 6 (Tg-6m) and 3 months (Tg-3m). MRP3 expression was only diffused in Tg-3m lung tumors. Nuclei from tumor cells of both Tg-6m and Tg-3m were stained with TTF-1; it was more intense in Tg-3m than Tg-6m tumor cells. Both Tg-6m and Tg-3m tumor samples were PAS-positive. Panels are at 200x magnification. The scale bar showed 200 μm in distance.
Fig 3.
Tumor growths and metastasis of transgenic mice.
(a) Normal lung (wild-type; WT) and lung tumors at 3 (Tg-3m) and 8 months (Tg-8m) were evaluated using micro-CT. Arrows and circles indicate where solitary tumor nodules were observed. (b-c) Bar charts represent analytical results of the growing lung volume (mm3) and the reduced lung volume percentage compared to wild-type mouse (%). Statistical analysis between each stage of lung tumors and wild-type lung (Tg-8m v.s. WT and Tg-3m v.s. WT) was significant (P value <0.05) in t-test; group statistics of Tg-8m, Tg-3m, and WT lungs were also significant (P value <0.001) as verified using ANOVA (data not shown). The lung images shown of each age were representative of more than 3 animals that been scanned. (d-e) Macroscopic features of tumors in the chest wall and inguinal region. (f) Macroscopic and histological (g) image of formalin-fixed lung tumor multiplicity (100x magnification, H&E stained). (h) Histological examination of metastatic tumors in the chest wall (CW) and inguinal region (IR) (H&E stained). Histology of the metastatic nodules revealed typical ADCs features of rounded aggregates of tumor cells or acinar pattern with a glandular formation and cribriform arrangements. Scattered tumor cells within myofibroblastic stroma were also noted (400x magnification).
Fig 4.
Six months (Tg-6m) mice displayed the epithelial-mesenchymal transition (EMT) phenomenon.
(a) Hierarchical cluster analysis of normal lung and lung tumors from Tg-3m and Tg-6m mice. (b) GSEA data showing enrichment of EMT signatures in both Tg-6m and Tg-3m tumors. (c) Real-time qPCR of key EMT regulators, Snail, Twist, and Zeb, indicates they were upregulated in Tg-6m tumor cells. Data shown were representative of 3 independent experiment. (d, e) Western blot analysis of E-cadherin and fibronectin proteins in Tg-3m and Tg-6m tumor cells. The amount of the epithelial marker, E-cadherin, had decreased in Tg-6m cells compared to Tg-3m cells (d). In contrast, the amount of the mesenchymal marker, fibronectin, had increased in Tg-6m cells (e). Shown were representative blots of 3 independent experiments.
Fig 5.
Holographic microscopy analysis of cellular movement.
Six-month (Tg-6m) cells were more versatile compared to Tg-3m cells in the captured image. Surface areas of Tg-3m and Tg-6m cells were measured and statistically plotted in a bar chart (μm2). (b) The paths of cell migration are presented as individual colored lines from Tg-3m and Tg-6m cells, and the average motility was calculated in the bar chart (μm). The Fig showed representative results of 3 independent experiments.
Fig 6.
Candidate genes associated with the epithelial-mesenchymal transition (EMT).
(a) IPA-analyzed EMT-relevant genes from 6 months (Tg-6m) lung tumors and their interactive network according to cellular locations. (b) Expression levels of EMT-related genes from Tg-3m and Tg-6m tumor cells were examined using a real-time qPCR. Data shown were representative of 3 independent experiment.