The Study Of Cancer Initiating Cells In Lung Cancer A549 And H446 Cell Lines

OBJECTIVE: This study was designed to investigate the fraction of cancer initiating cells in the non-small cell lung cancer cell line A549 and small cell lung cancer cell line H446 by cloning and tumorigenic analyses.METHODS: A clone formation assay was carried out to determine the clonogenicity and regeneration ability of a cell. The cells from a clone were injected to BALB/C-nude mice to test tumorigenicity. We isolated the cells by magnetic separation to determine whether both CD133~+ cells and CD133~- cells of A549 and H446 possess the characteristics of cancer stem cells. We also test whether Hoechst 33342 staining could affect the clonogenicity of A549 and H446 cells by clone formation assay.RESULTS: Both A549 and H446 single cells can form three kinds of clones in the serum-containing medium in one week, i.e. large clones (L, more than 100 cells), median clones (M, 50-100 cells) and small clones (S, less than 50 cells). After being dissociated and cultured similarly in new 96-well plates (1 cell/well), the cells from the small clones cannot form new clones and they underwent apoptosis instead in the second generation. The cells from the median clones formed a few median clones in the first generation and formed mostly small ones in the second generation; they underwent apoptosis and formed a few small clones in the third generation; all the cells underwent apoptosis in the fourth generation. The cells from the large clones formed the three kinds of clones even in the tenth generation.More than 45% of A549 and H446 cells formed large clones that are able to generate subclones and subsequently give rise to xenograft tumors in the BALB/C-nude mice. Both CD133~+ cells and CD133~- cells of A549 and H446 possessed stem cell characteristics and exposure to Hoechst 33342 affected the clonogenicity and proliferation of single A549 and H446 cell.The cycle phase distribution showed the percentages of A549 cells in G1, G2 and S phase were 43.39%, 13.65% and 42.96%, respectively. The percentages of H446 cells in G1, G2 and S phase were 62.75%, 13.29% and 23.96%, respectively. The time needed for A549 cells to expand from one single cell to 10~6 varied from 19 to 26 days. The time needed for H446 cells to expand from one single cell to 10~6 varied from 17 to 25 days. The growth curves showed that cells from different clones of A549 and H446 proliferated at the same rate when growing from one cell to 1×10~6 cells.CONCLUSION: More than 45% of cancer A549 and H446 cells can generate large clones, which were capable of self-renewal and giving rise to xenograft tumors in vivo. So our study showed: More than 45% of A549 and H446 cells were cancer initiating cells.