Expansion and characterization of cancer stem-like cells in squamous cell carcinoma of the head and neck
Introduction
Evidence has accumulated indicating that only a minority of cancer cells with stem cell properties, cancer stem cells (CSCs), are responsible for maintenance and growth of the tumor.1, 2, 3 Recent advances in stem cell biology enable the identification of CSCs in solid tumors as well as putative stem cells in normal organs.4, 5, 6, 7 CD44 is currently used to identify CSCs as one of the cell surface markers for solid tumors.4, 5, 6 With respect to squamous cell carcinoma of the head and neck (SCCHN), Prince et al. demonstrated that a small population of CD44+ cancer cells obtained from fresh tumor tissues, but not CD44− cancer cells, gave rise to new tumors in immunodeficient mice.8 Interestingly, CSCs have been also identified in cultured SCCHN cell lines. Pries et al. demonstrated that permanent SCCHN cell lines constitutively expressed CD44.9 Alternatively, Zhou et al. have shown that CD133+ cells in a laryngeal carcinoma cell line possess the capacity for self-renewal, extensive proliferation, and multilineal differentiation potency in vitro.10 These findings suggest that a subpopulation of CSCs can persist even with long-term culture in vitro; however, CSCs are not only a small or rare subpopulation in tumors, but are also maintained in a hierarchy together with a spectrum of cells at different stages of differentiation; therefore, it is too difficult to maintain an enriched status of CSCs in long-term culture. Recent reports have demonstrated that CSCs from epithelial organs can be expanded as sphere-like cellular aggregates in serum-free medium (SFM) containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF).11, 12, 13 In the current study, we first sought to detect CD44+ cells in the established SCCHN cell line, the Gun-1 cell line and, as expected, CD44+ cells represented a minority of the tumor cell population. Surprisingly, CD44+ cells were able to be propagated in vitro under SFM containing EGF and bFGF culture conditions. We then focused on whether such expanded CD44+ cells have stem cell properties by comparing with CD44− cells.
Section snippets
Cell line and culture conditions
A SCCHN cell line, Gun-1, was established from a squamous cell carcinoma of the hypopharynx.14 Gun-1 was cultured in either RPMI-1640 (Sigma-Aldrich, St. Louis, MO) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 2 mM l-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin (all reagents were from Invitrogen, Grand Island, NY), or Serum Free Expansion Medium (StemCell Technologies, Inc., Vancouver, Canada) supplemented with EGF (Calbiochem, Darmstadt, Germany) and
Identification and expansion of CD44+ cell in Gun-1 cell line
We first examined CD44 expression in a SCCHN cell line, Gun-1, using flow cytometry, and it was detected in 2.1%, as shown in Figure 1A. Gun-1 was then cultured in SFM containing EGF and bFGF. After 5 weeks of culture, the CD44+ population was increased up to around 40%, as shown in Figure 1B.
Expression of CSC-related markers on CD44+ and CD44− cells
To examine the difference between CD44+ and CD44− subpopulations, the expression of CSC-related markers was analyzed by flow cytometry (Fig. 2). The expression of CD133 and ABCG2 on CD44+ cells was higher
Discussion
The objective of this study was the characterization of CSCs in the SCCHN cell line. We succeeded in achieving this objective, but three points are of particular importance: (1) The serum-free culture condition was capable of expanding CD44+ cells from the permanent SCCHN cell line; (2) These CD44+ cells also expressed both CD133 and ABCG2, and possessed a marked capacity for forming tumor spheres, proliferation, migration, and invasion in vitro; (3) CD44+ cells were more resistant to
Conflicts of interest statement
None declared.
Acknowledgments
This work was supported in part by grants-in-aid (19791201 to AO, 20592013 to KC, 20592014 to KM) from the Ministry of Education, Cultures, Sports, Science and Technology, Japan.
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