Cao et al. BMC Cancer 2010, 10:68 http://www.biomedcentral.com/1471-2407/10/68 Page 7 of 11 Figure 3 Activated macrophage-like cells’ medium inhibits G1-S transition and enhances migration in CNE-2 single clone cells. (A) Cell cycle distribution of CNE-2 single clone cells after cultivation for 24 hours. (B) Percentage of G1, S and G2-M phases in CNE-2 single clone cells. Values are means ± SD (n = 3). * P < 0.01 vs control. (C) The migration of the CNE-2 single clone cells is enhanced by conditioned culture for 18 h in a transwell apparatus. Images shown are representative of three independent experiments. Nuclei were labeled with DAPI (blue), original magnification ×200. (D) Migration level is shown by quantified the migrated cells in 10 random fields per filter. Values are means ± SD (n = 3). *** P < 0.001 vs control. The SSH data was further validated by Northern blot- ting analysis. Results revealed that mRNAs of ADP-ribo- sylation factor-like 6 interacting protein (ARMER), poly (rC) binding protein 1 (PCBP1) and pyruvate dehydro- genase E1-b subunit (PDHB) were only expressed in conditioned culture CNE-2-2 (Fig. 4A, B and 4C), and the level of ubiquitin-like protein fubi and ribosomal protein S30 precursor (FUBI) mRNA transcript was higher in conditioned cultured cells compared with con- trol (Fig. 4D). Discussion In this study we have identified the non-SP cell clone in CNE-2 cells and found that conditioned culture non-SP cells were able to generate SP phenotype when microen- vironment were present. Non-SP cells cultured with macrophage-like cells could not only produce heterologous descendent cells but also showed cell cycle arrest and higher cell motility. Furthermore we have determined the gene pathway involved during these pro- cesses by the SSH. To our knowledge, this is the first report to demonstrate the correlationship between non- SP cells and microenvironment in a tumor. The impetus to CSC-theory has been only recently pro- posed due to the advances in stem cell biology although the concept of CSCs is not new. We showed here for the first time that non-SP cancer cells cultured with macro- phage-like cells were able to generate SP phenotype probably by asymmetric division or reprogramming. In addition, by using different cell line (SK-N-CH) and can- cer stem cell marker CD133, we have observed the con- sistent phenomena. It has been reported that brain CSCs are maintained within vascular niches [13]. Here we pro- vided evidences that CSCs depended upon aberrant