Abstract 1690: IGF-1 and TGF-β promote EMT and angiogenesis in 3D cultures of lung adenocarcinoma cells: A pilot study
Authors: Tarhoni Imad , Lobato Gabriela C. , Fhied Cristina , Borgia Jeffrey , Pergande Melissa , Chai Yi Wen 
Published: July 2016

Background

Lung cancer is leading cause of cancer related deaths worldwide, with metastasis underlying this high mortality rate. An epithelial to mesenchymal transition (EMT) and angiogenesis are processes well known to increase tumor cell invasive potential. Transforming growth factor - beta (TGF-β) and insulin-like growth factor (IGF-1) are well established inducers of EMT and promoters of angiogenesis. In this pilot study, we tested the effects of IGF-1 and TGF-β on the lung adenocarcinoma cell line, A549, using a novel 3D cell culture method.

Method

A549 lung adenocarcinoma cells were seeded (15 million cells/ 500 μL 3D cocoon) in a Cell Mate 3D matrix (BRTI). After 24 h of recovery the cocoons were maintained in DMEM containing 2.5% FBS and received either IGF-1 (100 ng/mL), TGF-beta (20 ng/mL) for up to 7 days. A fourth group receiving only DMEM medium with 2.5% FBS was used for control purposes. Cocoons were formalin-fixed and paraffin embedded; 5um sections were prepared and stained for vimentin and E-cadherin. Cell-conditioned media was evaluated for angiogenesis factors(MILLIPLEX® Human Angiogenesis Kits (EMD Millipore®, Billerica, MA)) using the Luminex immunobead platform (Luminex Corp., Austin, TX). All data were then analyzed using SPSS.

Results

80% of TGF-β treated cells expressed vimentin compared to 27.5% in the control group. Cell staining strongly positive for vimentin demonstrated increased migration to the periphery compared to cells that were positive for E-cadherin. Both of IGF-1 and TGF-β significantly upregulated VEGF-A, IL-8, sHer2. Tenascin-C and Follistatin were significantly increased with TGF-β only (p&lt;0.01). sVEGFR2 and sVEGFR3 were down regulated by IGF-1 and upregulated by TGF-beta relative to control (p&lt;0.05). G-CSF, FGF-2, sIL6R, sAXLs, HGFR, Neuropillin-1 and suPAR were significantly higher in IGF-1 and lower in TGF-β (p&lt;0.01) stimulated cultures. Other factors, such as VEGF-D, EGF, and Angiopoietin-2, showed significant decrease in both treated groups compared with control (p&lt;0.05).

Conclusion

A549 cells in this 3D model demonstrated increased metastatic potential with associated EMT features and increased expression of angiogenesis factors upon stimulation with IGF-1 and TGF-β. These findings will be contrasted to 2D conventional culture methods for the substantiation for the 3D scaffolds for routine culturing. Evaluation of EMT in a 3D format is a critical tool for cancer research as it emulates dynamic interactions of the cancer cells with surrounding matrix, the tumor microenvironment in a more practical fashion than conventional 2D culture methods.

Citation Format: Imad Tarhoni, Gabriela C. Lobato, Cristina Fhied, Jeffrey Borgia, Melissa Pergande, Yi Wen Chai. IGF-1 and TGF-β promote EMT and angiogenesis in 3D cultures of lung adenocarcinoma cells: A pilot study. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1690.