In-vitro Pre-Treatment of Cancer Cells with TGF-β1: A Novel Approach of Tail Vein Lung Cancer Metastasis Mouse Model for Anti-Metastatic Studies

Abstract

Background: Aggressive behavior of tumor metastasis comes from certain mutations, changes in cellular metabolic and signaling pathways that are majorly altered by tumor microenvironment (TME), its other components and growth factors like transforming growth factor-β1 (TGF-β1) which is chiefly known for its epithelial to mesenchymal transformation (EMT). EMT is a critical step of metastasis cascade in actual human lung cancer scenario. Objective: Our present study is focused on unveiling the in-vivo metastatic behavior of TGF-β1 treated lung cancer cells that undergo EMT. Methods: The lung cancer epithelial A549 cells were treated in-vitro with TGF-β1 (3-5ng/ml for 72 h) for EMT. After confirming the transformation of cells by phenotype modifications, wound healing and cell migration assay and qRT-PCR analyses of EMT biomarkers including E. Cadherin, Vimentin, Snail, Slug, MMP2 and MMP9; those TGF-β1 modified cells were probed with fluorescent trackers and were injected into the tail vein of BALB/c nude mice for metastatic dissemination studies. Results: Our findings indicate that the distribution of TGF-β1 treated A549 cells as compared to W.T A549 towards lungs is less in terms of total relative fluorescent cluster count, however, the difference is insignificant (52±4, 60±5 respectively). Additionally, we show that TGF-β1 treated cells tend to metastasize almost 2, 3, 1.5, 2 and 1.7 times more than W.T towards liver, brain, ovaries, bones and adrenal gland, respectively, which is very much like human lung cancer metastasis. Conclusion: Conclusively, it is the first study ever reporting that a pre-treatment of cells with TGF-β1 for experimental lung cancer metastasis mouse model may portray a more precise approach for the development of potential therapeutic treatments. Additional pre-treatment studies with the application of other TME conditions like hypoxia and factors like NFκB, VEGF etc. may be a future prospect to develop a better understanding.