1695P - Fibroblast Induced Epithelial to Mesenchymal Transition (EMT) in a Novel Non-Small Cell Lung Cancer (NSCLC) Model

Abstract

ABSTRACT Introduction Different molecular processes lead to metastatic spread and the occurrence of tumour cell resistance to therapeutic interventions. Among them, the epithelial to mesenchymal transition (EMT) process plays a key role. During EMT, epithelial tumour cells lose the expression of specific proteins and adopt the phenotype of mesenchymal cells. These structural conversions are substantially dependent on the tumour microenvironment. EMT of tumour cells can induce drug resistance and metastasis. Thus, EMT inhibition may offer a new strategy for overcoming tumour progression. Methods To evaluate EMT in a non-small cell lung cancer (NSCLC) model a 2D and a 3D- cell culture system was applied using both the human lung cancer cell line (A549) and the human lung fibroblast cell line (SV-80). For generating 3D cell spheroids, a novel system was established consisting of 96-well hanging drop microtiter plates (InSphero AG, Zurich, Switzerland). 2D co-culture assays were performed in transwell filter inserts (Costar). EMT was induced with transforming growth factor-β (TGF-β) or co-cultivation with fibroblasts in 2D/3D. The switch from epithelial to mesenchymal cells was monitored by Western Blot (WB) analyses of e-cadherin, vimentin and n-cadherin. Furthermore, immunohistochemical analyses of e-cadherin, vimentin, α-smooth muscle actin, fibronectin, KI-67 and CA-IX were done on paraffin embedded spheroids. Results EMT could be induced in the 2D not only by incubating tumour cells with TGF-β but also by co-culturing them with fibroblasts in transwell filter inserts. In A549 cells a change in morphology as well as in protein expression defined by WB analysis (down regulation: E-cadherin; up regulation; Vimentin, n-cadherin) could be detected. When cultivated in the 3D system, A549 cells showed an up regulation of the mesenchymal protein vimentin without TGF-s stimulation. Furthermore, a significant up regulation of vimentin, KI-67, CA-IX, and a slight down regulation of e-cadherin could be measured compared to monocultures. Conclusion 3D culture represents a model to study EMT in tumour cell lines without addition of growth factors and thus reflects in vivo conditions closer than 2D culture. Disclosure All authors have declared no conflicts of interest.