A subpopulation of CD133+ cancer stem-like cells characterized in human oral squamous cell carcinoma confer resistance to chemotherapy
Introduction
Head and neck cancer/oral pharyngeal carcinoma is one of the 10 most common cancers worldwide [1]. In 2002, over 500,000 cases of head and neck squamous cell carcinoma (HNSCC) were diagnosed, and more than 300,000 deaths from this disease were reported worldwide [1]. In 2008, about 47,500 cases of head and neck cancer were diagnosed in the United States, accounting for approximately 3% of new cancer diagnoses, and about 11,260 people died from this disease [2]. Although the overall incidence of HNSCC has been declining in the United States over the last two decades [3], a most recent analysis of the National Cancer Institute’s Surveillance, Epidemiology and End Results (SEER) data from 1973 to 2001 indicated that the incidence of oral tongue, palatine tonsil, and base-of-tongue cancers increased annually by 2.1%, 3.9%, and 1.7%, respectively, in 20- to 44-year-old white patients [4]. The high recurrence rate and development of second primary tumors in advanced clinical stage is the most important factor determining the survival of head and neck cancer patients. Despite the progress in the treatment modalities of HNSCC, the overall survival has not improved substantially for the last three decades due to the resistance to conventional chemotherapy or radiation therapy [5] because most of the current treatment strategies for the malignant disease still attempt to attack tumors as a homogenous tissue. However, in recent years, there has been overwhelming evidence in some malignancies supporting the notion that tumors are organized in a hierarchy of heterogeneous cell populations [6]. The capability to sustain tumor formation and growth is exclusively due to a small proportion of tumor cells termed cancer stem cells (CSCs) or tumor-initiating cells (TICs) [7].
The cancer stem cell hypothesis has recently been tested in some of the head and neck carcinomas [8], [9], including laryngeal carcinoma and nasopharyngeal carcinoma [10], [11]. Using ‘organotypic’ in vitro culture models, cells derived from human oral squamous cell carcinoma (OSCC) contain a small subpopulation with clonogenic characteristics [12] and capable of a range of growth potentials that might be related to a stem and amplifying cell pattern present in the primary OSCC [12]. Previous studies have shown that, even years after their isolation and extensive in vitro propagation, human OSCC-derived cell lines generate marked heterogeneity and retain a range of cell types corresponding to the stem and amplifying cells of normal oral epithelium, as well as tumor-initiating cells, and therefore possess the essential defined properties of CSCs [13]. These OSCC-derived cell lines with distinct colonies of holoclone morphologies express higher levels of stem cell-related molecules such as β1-integrin, E-cadherin, β-catenin and epithelial specific antigen (ESA) [13]. However, due to the lack of reliable cell surface markers to identify these stem cell-like cells in OSCC both in vitro and in vivo, the roles of these CSCs in tumor initiation and drug resistance in OSCC still remain largely unknown [14].
To date, CSCs or TICs have been successfully isolated from solid tumors via different approaches. Firstly, CSCs or TICs were initially identified as a small side population (SP) of cells that can actively exclude fluorescent DNA staining dye, Hoechst 33342 [15], [16]. This SP phenotype is found to be closely associated with the expression of ABCG-2 or breast cancer resistance protein (BCRP), one of the ATP-binding cassette (ABC) family of transporter proteins [15]. Secondly, CSCs of different tissue origins were isolated based on the expression of specific cell surface markers. For example, prostate cancer CSCs or TICs are identified as small population of cells with different phenotypes, including CD133+[17], CD44+[18], CD44+α2β1+[19], or the population [20]. In breast tissue, a small subpopulation was characterized by the phenotype Lin−/CD29hi/CD24+[21], while a CD44+/CD24−/low side-population exists in breast cancer cells [22], [23]. In head and neck squamous cell carcinoma, a subpopulation of CD44+ cells was identified to possess the unique properties of CSCs [9]. In pancreatic cancer cells, about 0.2–0.8% of the cells with CD44+CD24+ESA+ phenotype have been found to possess a 100-fold increased tumorigenic potential compared with nontumorigenic cancer cells [24]. In brain tumors, TICs usually express CD133+/musashi-1+/nestin+ or similar phenotypes [6], [25]. These findings have indicated the highly heterogeneity of CSCs or TICs of different tissue origins. Thirdly, CSCs or TICs can be enriched from individual tumors or cancer cells by sphere body formation under the cultivation of defined serum-free medium with growth factors whereby the serum-free condition favors the maintenance of the undifferentiated status of CSCs [6], [18], [26].
In the present study, we have identified a small subpopulation of CD133+ cells (1–2%) in human OSCC, and have demonstrated for the first time that this subpopulation of CD133+ cells possess distinct properties of cancer stem-like cells, including increased expression of specific stem cell markers, higher potential for clonogenicity, invasion and in vivo tumorigenicity as well as increased chemoresistance as compared with their CD133− counterparts. Our findings suggest that these unique CD133+ cells represent a small subpopulation of cancer stem-like cells or tumor-initiating cells that may contribute to tumor formation and recurrence in human oral squamous cell carcinomas.
Section snippets
Cell lines and culture
UPCI: SCC-016 and UPCI: SCC-076 (two human tongue SCCs) and UPCI: SCC-29B (a buccal SCC), were kindly provided by Professor Susanne M. Gollin (Head and Neck Spore Grant at University of Pittsburgh Cancer Institute). All these cell lines were cultured in MEM medium supplemented with MEM-non-essential amino acid (NEAA) (Invitrogen), gentamycin, and 10% FBS. UM: SCC-1, a floor-of-the mouth squamous cell carcinoma derived from tumor recurrence, was kindly provided by Professor Cun-yu Wang
Characterization of CD133+ cells in OSCC in vitro and in vivo
To determine whether a small subpopulation of CD133+ cells also exist in human oral squamous cell carcinoma (OSCC), we examined the expression of CD133 in a panel of OSCC cell lines (see Section 2) by flow cytometry or immunocytochemistry after the cells were stained with a PE-conjugated antibody specifically for human CD133 (PE-CD133/1). Our results showed that about 1.0–2.0% of CD133+ cells exist in these OSCC cell lines (Fig. 1A and B).
To determine whether CD133 cells are present in human
Discussion
Tumor tissues have long been recognized to be composed of heterogeneous populations of cancer cells. Recently, accumulating evidence supports the notion that tumors are generated and sustained by a small phenotypic subset of cancer cells, currently defined as “cancer stem cell” (CSC) or “tumor-initiating cell” (TICs) subset, that are endowed with the tumorigenic capacity to self-renew and differentiate into the bulk tumor populations [6], [7]. Up to date, CSCs or TICs with proposed role in
Conflict of interest
None of the authors has any financial or other interest with regards to the submitted manuscript that might be construed as a conflict of interest.
Acknowledgements
This work was supported in part by National Institute of Health Research Grant, AR47359 (A. Le) and Oral and Maxillofacial Surgery Foundation (OMSF) Research Grant (Qunzhou Zhang and A. Le).
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