A 3D cell culture system to study epithelial-mesenchymal transition and drug resistance of lung cancer

AIM To explore the molecular mechanism of transforming growth factor-β1 （TGF-β1）-induced epithelial-mesenchymal transition （EMT） and cisplatin resistance in three-dimensionally cultured lung cancer cells. METHODS Under three-dimensional culture condition， the morphological changes and protein expression changes of human non-small-cell lung cancer 95D cells were observed by inversed fluorescence microscopy， scanning electron microscopy， laser scanning confocal microscopy and Western blot before or after TGF-β1 stimulation. The cisplatin sensitivity was determined by MTT assay. RESULTS Under the three-dimensional culture condition， the structure of 95D cell spheroids after TGF-β1 stimulation collapsed， the cells were dispersed and migrating， and the spheroids merged with each other. The results of laser confocal microscopy showed that E-cadherin protein expression in the 95D cells did not changed after TGF-β1 stimulation， and the protein expression of N-cadherin and vimentin was significantly up-regulated. The results of Western blot showed that the expression of E-cadherin was down-regulated after TGF-β1 stimulation， and the protein levels of N-cadherin， vimentin， phosphorylated AKT and phosphorylated mTOR were up-regulated. LY294002 and rapamycin reversed TGF-β1-induced expression of the above proteins. The results of MTT assay showed that TGF-β1 reduced the sensitivity of three-dimensionally cultured 95D cells to cisplatin， while LY294002 and rapamycin reversed the cisplatin resistance of the 95D cells stimulated by TGF-β1. CONCLUSION TGF-β1 induces the EMT and cisplatin resistance of three-dimensionally cultured lung cancer cells through the activation of PI3K/AKT/mTOR signaling pathway.