Cao et al. BMC Cancer 2010, 10:68 http://www.biomedcentral.com/1471-2407/10/68 Since this question was first raised in 2005 [12], soon in 2007 evidence was shown that the vascular niches in brain tumors are abnormal and contribute directly to the generation of CSCs and tumor growth [13]. There- fore, CSCs might arise from normal stem cells that have acquired mutations that enable them to escape from niche control. Alternatively, deregulation of extrinsic factors within the niche might lead to uncontrolled pro- liferation of stem cells and tumorigenesis [14]. The fate of CSCs depends upon aberrant niche microenviron- ments to some extent, thus these niches might represent targets for the treatment of cancer. We paid particular attention to the source of CSCs and possible regulatory pathway in nasopharyngeal carcinoma (NPC) microen- vironment herein. NPC is a malignancy of the head and neck region that arises from the epithelial cells that cover the surface and line of the nasopharynx. This disease was initially reported in 1901, and characterized clinically in 1922 [15]. It is endemic in many geographical regions, includ- ing Southern China and Southeast Asia, where the observed incidence rates range from 15 to 50 per 100,000 persons. Previous studies have reported that squamous cell carcinoma (former WHO type 1) accounts for approximately 25% of all NPC; whereas undifferentiated carcinoma (former WHO type 3) accounts for 95% of all cases in high incidence areas [15-18]. The management of recurrent cervical lymph node metastases in NPC after radiation and chemother- apy is a radical surgery of the lymph nodes of the neck with postoperative brachytherapy. The salvage surgical procedure for persistent or recurrent neck disease shows a 5-year control rate of 66% and a 5-year actuar- ial survival of 38% [18]. Thus, the control of the differ- entiation and metastases may be the key to understanding this carcinoma. Our study combining the NPC CSCs with microenvironment aimed to address this key question. Some markers and phenotype of CSCs were similar to that of normal neural stem cells. For example, they express CD133 and nestin in brain cancer, CD44 in breast cancer and CD34 in leukemia [4,5,7,8]. Recent evidences demonstrate that ABCG5 and USP22 may be used as cancer stem cell marker [19,20]. On the other hand, a character of stem cells termed side population (SP) can be identified due to the cellular exclusion of Hoechst 33342 dye. The SP cells have been isolated by flow cytometric analysis from various types of adult tumor tissue where they possess stem cell activity, including lung adenocarcinoma, gastrointestinal cancers, head and neck squamous cell carcinoma, ovarian cancer, thyroid cancer, and hepatocellular carcinoma [21-27]. Here, we performed our work on human NPC CNE-2 cells, of which SP phenotype revealed several stem cell Page 2 of 11 properties [28], to investigate whether NPC CSCs are tightly regulated by the immediate microenvironment, to evaluate the differential gene expression profile of the CSCs with microenvironment, and to find more specific stem cell marker for NPC CSCs. In this study, we tried to identify cancer non-SP cell clone from human NPC cancer cell line CNE-2. Such non-SP cell clone was isolated and cultured with macro- phage-like cells. We found that conditioned culture could induce generation of SP cells by the non-SP cells derived from both NPC and neuroblastoma cell lines. Moreover, we evaluated the cell cycle and migration ability of condition cultured non-SP cells, which cells showed G1/S arrest and enhanced motility compared with control. We further identified the differential gene profile in condition cultured non-SP cells by the sub- traction suppression hybridization (SSH). Methods Cell lines and reagents Human acute monocytic leukemia cell line THP-1, human nasopharyngeal cancer cell line CNE-2 and human neuroblastoma cell line SK-N-CH were obtained from American Tissue Culture Collection (Rockville, MD, USA). Antibodies including CD133 and CD133-PE were pur- chased from Miltenyi Biotech (Bergisch Gladbach, Ger- many). All the other reagents were purchased from Sigma (St Louis, MO, USA) unless otherwise noted. Single clone selection Cells were seeded in a 24-well plate with 1 cell/ml and single cell clones were isolated from wells with only a solitary colony. Selecting 50 single cell clones were sub- jected to expansion culture until sufficient amounts of cells were obtained. The 50 single cell clones were detected by the flow cytometric analysis of SP and non- SP distribution respectively. One CNE-2 single clone is named CNE-2-2 with little SP phenotype. Macrophage-conditioned medium THP-1 cells at a density of 0.5 × 106/ml were cultured in RPMI 1640 (Invitrogen, San Diego, CA, USA) supple- mented with 10% FBS (Hyclone, Logan, UT, USA) and 20 ng/ml phorbol 12-myristate 13-acetate (PMA) for 48 h at 37°C in a humidified atmosphere of 5% CO2 incu- bator. After nonadherent cells were discarded, remaining cells were washed twice in PBS and once in RPMI 1640 with 15-min incubation at 37°C. These PMA induced macrophage-like cells were cultivated for 24 h with fresh RPMI 1640 containing 10% FBS (8 ml per 75-cm3 flask). The supernatant after culture was subsequently filtered and used as the macrophage-conditioned medium.