Abstract: Objective To isolate lung cancer stem-like cells (LCSCs) from human large-cell lung cancer cell line NCI-H460
(H460) and explore their biological characteristics. Methods H460 cells were cultured in serum-free medium in the presence of
specific growth factors. Quantitative PCR (qPCR), flow cytometry and colony formation assay were performed to characterize
the stemness of H460 spheres. Adherent H460 cells and H460 cell spheres were inoculated subcutaneously in nude mice and
the tumor growth was assessed. Results The isolated LCSCs from H460 cells in serum-free medium grew as floating cell
spheres and exhibited stronger proliferative activity than H460 cells. Compared with H460 cells, H460 cells spheres showed
higher expressions of stem cell markers Sox2, Oct4, and especially Nanog, and possessed a stronger tumorgenicity in nude
mice. Conclusion The serum-free culture system can effectively enrich lung cancer stem cells from human lung cancer stem
cell line H460, and the high expression of Nanog may importantly contribute to the maintenance of cancer stem cell-like
properties of the isolated LCSCs.