Abstract 4047: A novel in vivo model of breast cancer hypoxia reveals an epithelial-to-mesenchymal transition switch.
Authors: Tiezzi Daniel G. , Antonio Heriton MR , Sicchieri Renata D. , Milanezi Cristiane M. , Mandarano Larissa RM , Coelho Alan A. , de Andrade Jurandyr M. 
Published: August 2014

Background: Low levels of oxygen within cells are characteristic of many solid malignant tumors. This phenomenon has been described as an important trigger of tumor invasion and metastasis. Despite the importance of this association between hypoxia and more aggressive cancer phenotypes, experimental models to study the influence of hypoxia in cancer development and progression have been limited to in vitro models and in vivo models based on mice being subject to a systemic hypoxic state or drug-induced hypoxia. These models have provided inconclusive and controversial data. We developed a novel in vivo model of breast cancer hypoxia to study the influence of transitory hypoxia on epithelial to mesenchymal transition (EMT).

Methods: 4T1 and 67nr cell lines were engrafted into the kidney cortex of female BALB/c mice. Placing an aneurysm clip on the kidney hilum for 40 minutes, hypoxia was directed to tumor site after 7 days of engraftment. After 14, 21 and 28 (67nr only) days of engraftment, mice were sacrificed and the histological evaluation was used to analyze the morphological changes induced by ischemia in kidney cortex, and to verify the metastatic potential to lungs and liver. Tumors were sampled after 14 days of engraftment for gene expression assay. Real time RT-PCR was performed to analyze the relative expression of Cdh1, Vim, Fn1, Ctnnb1, Jag1, Notch1, Snai2, Sox9, Tgfb1, Twist1, HIF2A, Hif1a and Ca9 genes (TaqMan® Gene Expression Assay) in cell lines and in tumors in the control group and the hypoxia group. TBP and HPRT were used as housekeeping genes and 12 weeks-old normal breast mouse tissue was used as reference.

Results: All mice engrafted with 4T1 and 67nr into the renal cortex developed local tumors. We did not observed metastasis to the liver. In 4T1 group, metastases to the lungs were observed only in hypoxia group after 14 days after engraftment. After 21 days, all mice developed lung metastases. In 67nr, metastases to the lungs were observed only in hypoxia group after 28 days after engraftment. According to gene expression analyses, virtually all genes were different expressed after engraftment comparing to cell lines. When comparing the control and hypoxia groups, we observed a significant 2.6 fold and 3.1 fold change in VIM expression, and an 0.51 fold and 0.53 fold change in CDH1 expression in 4T1 and 67nr cell lines, respectively. In 67nr the SLUG expression changed 496 fold and the SLUG/SOX9 ratio changed 4.8-fold. The HIF1A/HIF2A expression ratio increased in 4T1 and 67nr tumors after hypoxia. The expression of TGFB1, TWIST1 and CTNNB1 genes was not significant changed in tumors subjected to hypoxia.

Conclusion: hypoxia directed to the tumor site in an in vivo model induces a EMT transition signature and accelerate the metastatic dissemination of murine breast cancer allograft. The expressive increase in SLUG expression suggests this transcription factor is crucial to hypoxia-induced EMT.

Citation Format: Daniel G. Tiezzi, Heriton MR Antonio, Renata D. Sicchieri, Cristiane M. Milanezi, Larissa RM Mandarano, Alan A. Coelho, Jurandyr M. de Andrade. A novel in vivo model of breast cancer hypoxia reveals an epithelial-to-mesenchymal transition switch. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4047. doi:10.1158/1538-7445.AM2013-4047