Characterization of a transgenic mouse model exhibiting spontaneous lung adenocarcinomas with a metastatic phenotype

<div><p>Developing lung cancer in mouse models that display similarities of both phenotype and genotype will undoubtedly provide further and better insights into lung tumor biology. Moreover, a high degree of pathophysiological similarity between lung tumors from mouse models and their human counterparts will make it possible to use these mouse models for preclinical tests. Ovine pulmonary adenocarcinomas (OPAs) present the same symptoms as adenocarcinomas in humans and are caused by a betaretrovirus. OPAs have served as an exquisite model of carcinogenesis for human lung adenocarcinomas. In this study, we characterized the histopathology and transcriptome profiles of a jaagsiekte sheep retrovirus (JSRV)-envelope protein (Env) transgenic mouse model with spontaneous lung tumors, and associations of the transcriptome profiles with tumor invasion/metastasis, especially the phenomenon of the epithelial-mesenchymal transition (EMT). Genetic information obtained from an expression array was analyzed using an ingenuity pathways analysis (IPA) and human disease database (MalaCards). By careful examination, several novel EMT-related genes were identified from tumor cells using RT-qPCR, and these genes also scored high in MalaCards. We concluded that the JSRV-Env mouse model could serve as a spontaneous lung adenocarcinoma model with a metastatic phenotype, which will benefit the study of early-onset and progression of lung adenocarcinoma. In addition, it can also be a valuable tool for biomarkers and drug screening, which will be helpful in developing intervention therapies.</p></div

Presentation_1_Therapeutic Effect of Repurposed Temsirolimus in Lung Adenocarcinoma Model.zip

<p>Lung cancer is one of the major cause of cancer-related deaths worldwide. The poor prognosis and resistance to both radiation and chemotherapy urged the development of potential targets for lung cancer treatment. In this study, using a network-based cellular signature bioinformatics approach, we repurposed a clinically approved mTOR inhibitor for renal cell carcinomans, temsirolimus, as the potential therapeutic candidate for lung adenocarcinoma. The PI3K-AKT-mTOR pathway is known as one of the most frequently dysregulated pathway in cancers, including non-small-cell lung cancer. By using a well-documented lung adenocarcinoma mouse model of human pathophysiology, we examined the effect of temsirolimus on the growth of lung adenocarcinoma in vitro and in vivo. In addition, temsirolimus combined with reduced doses of cisplatin and gemcitabine significantly inhibited the lung tumor growth in the lung adenocarcinoma mouse model compared with the temsirolimus alone or the conventional cisplatin–gemcitabine combination. Functional imaging techniques and microscopic analyses were used to reveal the response mechanisms. Extensive immunohistochemical analyses were used to demonstrate the apparent effects of combined treatments on tumor architecture, vasculature, apoptosis, and the mTOR-pathway. The present findings urge the further exploration of temsirolimus in combination with chemotherapy for treating lung adenocarcinoma.</p

Tumor growths and metastasis of transgenic mice.

<p>(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 (<i>P</i> value <0.05) in <i>t</i>-test; group statistics of Tg-8m, Tg-3m, and WT lungs were also significant (<i>P</i> 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).</p

Protective Efficacy of VP1-Specific Neutralizing Antibody Associated with a Reduction of Viral Load and Pro-Inflammatory Cytokines in Human SCARB2-Transgenic Mice

<div><p>Hand-foot-mouth diseases (HFMD) caused by enterovirus 71 (EV71) and coxsackievirus 16 (CVA16) in children have now become a severe public health issue in the Asian-Pacific region. Recently we have successfully developed transgenic mice expressing human scavenger receptor class B member 2 (hSCARB2, a receptor of EV71 and CVA16) as an animal model for evaluating the pathogenesis of enterovirus infections. In this study, hSCARB2-transgenic mice were used to investigate the efficacy conferred by a previously described EV71 neutralizing antibody, N3. A single injection of N3 effectively inhibited the HFMD-like skin scurfs in mice pre-infected with clinical isolate of EV71 E59 (B4 genotype) or prevented severe limb paralysis and death in mice pre-inoculated with 5746 (C2 genotype). This protection was correlated with remarkable reduction of viral loads in the brain, spinal cord and limb muscles. Accumulated viral loads and the associated pro-inflammatory cytokines were all reduced. The protective efficacy of N3 was not observed in animals challenged with CVA16. This could be due to dissimilarity sequences of the neutralizing epitope found in CVA16. These results indicate N3 could be useful in treating severe EV71 infections and the hSCARB2-transgenic mouse could be used to evaluate the protective efficacy of potential anti-enterovirus agent candidates.</p></div

Tumor progression and histopathological phenotype in a transgenic mouse model.

<p>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 <i>in situ</i> (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.</p

Candidate genes associated with the epithelial-mesenchymal transition (EMT).

<p>(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.</p

Six months (Tg-6m) mice displayed the epithelial-mesenchymal transition (EMT) phenomenon.

<p>(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, <i>Snail</i>, <i>Twist</i>, and <i>Zeb</i>, 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.</p

Survival of N3 treated hSCARB2-transgenic mice preinfected with 5746.

<p>Seven-day old mice preinfected with 1×10⁵ pfu of 5746 were given 200 µg N3 at time points of 3 h (▾), 24 h (▴), 48 h (▪), and 48 h twice (•) post infection. Mice injected without N3 (O), with the same amounts of isotype antibody (♦), and mice treated with a low dose of N3 (70 µg) (□) 3 h post infection were included. Mice were monitored daily and survival rates were recorded. Each group consisted of 7 to 10 mice and the results were representative of 2 independent experiments. The Logrank test was used for statistical analysis.</p

IHC analysis reveals lung tumors are displaying markers related to adenocarcinoma.

<p>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.</p

N3 conferred the reduction of hair loss and scurf on E59-preinfected hSCARB2-transgenic mice.

<p>(<b>A</b>) and (<b>B</b>) 1-day old hSCARB2-transgenic mice (n = 9–10/group) were preinfected with a HFMD symptom-causing dose of E59 at 1×10⁷ pfu subcutaneously. After 3 h of infection, 200 µg of N3 or isotype antibody was injected i.p. Mice without any antibody treatment were included. The Experiments were repeated twice, and 100× magnification of pictures (<b>A</b>) shown the representative data were taken. The graph represents the scoring of severe hair loss and skin scurf (<b>B</b>) from each group of mice. The scores were monitored on a daily basis. One-way ANOVA and the Kruskal-Wallis test were used for statistical analysis (*<i>p</i>< = 0.05, **<i>p</i>< = 0.01).</p