Cao et al. BMC Cancer 2010, 10:68 http://www.biomedcentral.com/1471-2407/10/68 Page 5 of 11 Figure 1 FACS analysis of side population (SP) in the CNE-2 cell line labeled with Hoechst 33342. (A and B) SP cells exist in CNE-2 single clone cells at a various percentage; (C) One clone contains little SP cell; (D) SP cells are increased by cultured with macrophage-like cells. The bottom panel shows the reduction of SP after treatment with 50 μM of verapamil. The data are representative of experiments over three times with similar results. SP and the expression of CD133, one of the markers of neuroblastoma cancer stem cells. After cultured with activated macrophage medium, SK-N-CH cells were stained with CD133 antibody for FACS analyses. As shown in Fig. 2A, the condition cultured SK-N-CH cells contained 4.5% cancer stem cells, whereas the control SK-N-CH cells only contained 0.6%. Furthermore, we examined the expression of CD133 on SK-N-CH cells in response to the conditioned cul- ture induced by macrophage-like cells with immuno- fluorescence. The fluorescence density of CD133 positive cells was evidently stronger than that in con- trol (Fig. 2B). These results allowed us to conclude that human macrophages did play a role in maintain- ing cancer stem cell pool at least in several solid tumors. G1-S cell cycle and migration of CNE-2 single clone cell affected by conditioned culture Next, we analyzed cell cycle distribution in CNE-2-2 cells with conditioned medium. Cell cycle profiling of CNE-2-2 cells revealed that the conditioned culture induced reduction of the G1-S transition at 24 hours (Fig. 3A and 3B). By contrast, Control experiments showed no cell growth arrest features at 24 hours. Following culture with activated macrophage-like cells, derived from human THP-1 cells, in a Transwell Boyden chamber system without cell-to-cell contact, CNE-2-2 cells resulted in a robust increase (>4 folds) in cell moti- lity after 18 hours, whereas in control the normal cul- ture conditions did not enhance the migration ability of CNE-2-2 cells (Fig. 3C and 3D). Taken together, these results clearly indicated that the functional components in macrophage-like cells responsible for cancer stem cell were complicated. Differential gene expression profile of conditioned culture CNE-2 single clone cell The SP pool expansion, G1/S arrest and migration improvement in CNE-2-2 cells described above implied the cancer stem cell change in the cultured environment provided by macrophage-like cells. We further deter- mined gene pathways involved during this process and studied the differential gene expression profiles between condition cultured and non-condition cultured CNE-2-2 cells by substractive suppression hybridization (SSH). About 120 clones were obtained from the SSH library enriched for the conditioned cultured CNE-2-2 cells transcripts. Following PCR using vector primers M13F and M13R and DNA dot-blot screening, 45 clones exhi- biting expression differences were selected for DNA sequence analysis, which included 5 up-regulated (fold change >2) and 40 up-regulated (fold change >1.2) genes (Table 2).