The hypothesis of cancer stem cell (CSC) suggests that neoplastic clones are maintained exclusively by a small subpopulation of cells that give rise to phenotypically diverse cancer cells [1–3]. CSCs were first identified in 1990s in hematological malignancies, mainly acute myelogenous leukemia (AML) and also in other subtypes like AML M0, M1, M2, M4, and M5, chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and multiple myeloma [4, 5]. This subset of cancer cells was also found in solid tumors in the breast, brain, lung, prostrate, testis, ovary, stomach, colon, skin, liver and pancreas [6–11]. Thus, there is overwhelming evidence that certain pathways which regulate or maintain the extensive proliferative and self-renewal potential of the tumor clone are present in CSC.
It is known that niches control stem cell function, thus it may seem counterintuitive that CSCs would be located within these regulatory microenvironments. Since this question was first raised in 2005 , 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 . Therefore, 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 proliferation of stem cells and tumorigenesis . The fate of CSCs depends upon aberrant niche microenvironments 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) microenvironment 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 . It is endemic in many geographical regions, including 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 chemotherapy 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 actuarial survival of 38% . Thus, the control of the differentiation 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 properties , 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 macrophage-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 subtraction suppression hybridization (SSH).