After 20 days of treatment, LC31 cells became elongated, central nucleus with fibroblast like shape and increased stress fiber reorganization. EMT takes 48 hours in A549 because it has stronger mesenchymal markers expression than LC31 needing 20 days to undergo EMT. Therefore, the LC31 cell line displays several features typical of EMT: reduction in cell-cell adhesion, flattening and scattering, expression of mesenchymal markers. Slug, Twist and b-catenin, main transcriptional factors involved in EMT, are up-regulated in the cells treated and these factors increase with increasing TGFb-1 treatment time, confirming their EMT phenotype. These results open the way to verify if LC31 primary lung cancer cell line, sensitive to TGFb-1 treatment, could increase stem cell characteristics. Interestingly, EMT-LC31 cells also display stemlike cell phenotype characterized by increased pneumosphereforming capacity compared with parental LC31 cell line with EMT pneumospheres greater than parental pneumopheres. Moreover EMT pneumospheres were positive for CD133 Phosphoramidon disodium salt marker reinforcing the stemness signature. Most importantly, it is well known that the co-expression of Sox-2, Nanog and Oct4 in human or mouse somatic cells can reprogram these cells into pluripotent embryonic stem-like cells. In our study, we found that the expressions of Sox-2, Nanog and Oct4 were dramatically up-regulated in EMT-LC31 cell line compared to parental cell line. Taken together these data, LC31 cell line with EMT signature showed an increase of stem-like cell characteristics associated with over-expression of Sox-2, Nanog and Oct4. Recent studies have shown that c-kit and its ligand are expressed in lung cancer. Immunohistological studies on the ckit expression showed that the protein is aberrantly expressed only in lung cancer cells and not in pneumocytes or normal bronchial epithelial cells. In addition, Levina et al. have showed that the treatment of lung PIM-1 Inhibitor 2 tumour cells with doxorubicin, cisplatin, or etoposide resulted in the selection of drug surviving cells expressing CD133, CD117, SSEA-3, TRA1-81, Oct-4, and nuclear beta-catenin with low expression of the differentiation markers cytokeratins 8/18. In our study, we demonstrate that TGFb-1 induces also an increase of c-kit m-RNA expression, another stemness marker, reinforcing the hypothesis that LC31 cell line with EMT signature showed an increase of stem phenotype. Another interesting result is the up-regulation of CD133 in EMT-LC31 cell line, main marker for CSCs identification in lung cancer as reported by Eramo et al. and Tirino et al.. In our study, we observed an increase of CD133 both in RT-PCR and Western blot, but not in cytometry and immunofluorescence on adherent cells.