Elsevier

Oral Oncology

Volume 47, Issue 9, September 2011, Pages 855-860
Oral Oncology

Isolation and characterization of cancer stem-like side population cells in human oral cancer cells

https://doi.org/10.1016/j.oraloncology.2011.06.501Get rights and content

Summary

Recent studies suggest that cancer stem cells may be responsible for tumorigenesis and contribute to some individuals’ resistance to cancer therapy. Some studies demonstrate that side population (SP) cells isolated from diverse cancer cell lines harbor stem cell-like properties; however, there are few reports examining the role of SP cells in human oral cancer. To determine whether human oral cancer cell lines contain a SP cell fraction, we first isolated SP cells by fluorescence activated cell sorting, followed by culturing in serum-free medium (SFM) using the SCC25 tongue cancer cell line, so that SP cells were able to be propagated to maintain the CSC property. Differential expression profile of stem cell markers (ABCG2, Oct-4 and EpCAM) was examined by RT-PCR in either SP cells or non-SP cells. Growth inhibition by 5-FU was determined by the MTT assay. Clonogenic ability was evaluated by colony formation assay. SCC25 cells contained 0.23% SP cells. The fraction of SP cells was available to grow in SFM cultures. SP cells showed higher mRNA expression of stem cell markers (ABCG2, Oct-4 and EpCAM) as compared with non-SP cells. Moreover, SP cells demonstrated more drug resistance to 5-FU, as compared with non-SP cells. The clone formation efficiency of SP cells was significantly higher than non-SP cells at an equal cell number (P < 0.01). We isolated cancer stem-like SP cells from an oral cancer cell line. SP cells possessed the characteristics of cancer stem cells, chemoresistance, and high proliferation ability. Further characterization of cancer stem-like SP cells may provide new insights for novel therapeutic targets.

Introduction

Although monoclonal in origin, most tumors appear to contain a heterogeneous population of cancer cells.1 The concept of cancer stem cells (CSC) was introduced to explain this heterogeneity.[2], [3] Recent studies suggest that CSC may be responsible for tumorigenesis and contribute to some individuals’ resistance to cancer therapy.[2], [3] Some studies demonstrate that side population (SP) cells isolated from diverse cancer cell lines harbor stem cell-like properties[4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21]; however, there are few reports examining the role of SP cells in human oral cancer.[5], [6], [15], [16], [17], [18], [20], [21] Isolation of CSC-like SP cells from cancer cell lines has been successful using two distinct methods based on the properties of CSC. First, isolation of CSC is made possible by flow cytometry according to CSC characteristics.[7], [8], [9], [14], [16], [17], [20] Plotting fluorescence intensity on blue versus red wavelengths, the SP fraction appears as a low-fluorescent tail-shaped cell population. The SP phenotype is determined by the ability to efflux Hoechst 33342 dye through an ATP-binding cassette (ABC) membrane transporter. Second, the sphere formation of CSC is enriched under the cultivation of defined serum-free medium (SFM) with growth factors.[15], [18], [22], [23] In these methods, however, the CSC population from the cancer cell line is not only small, but it is also difficult to maintain an enriched status of CSCs in long-term culture.[7], [8], [18], [24], [25] In our experiments, we first isolated SP cells by fluorescence activated cell sorting (FACS), followed by culturing in SFM containing basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), so that SP cells were able to be propagated to maintain the CSC property.

The purpose of this study was the characterization of CSC in the oral cancer cell line. The novel therapeutic strategies that selectively target the CSC subset might nonetheless achieve long-term disease eradication by exhausting self-renewal and growth potential of cancer tissues.

Section snippets

Cells

The human tongue cancer cell line SCC25, obtained from the American Type Culture Collection (Manassas, VA), was cultured in a 1:1 mixture of Ham’s F-12/DMEM supplemented with 10% fetal bovine serum (FBS) at 37 °C in the presence of 5% CO2.

SP analysis and cell sorting

Cells were labeled with 2.5 μg/ml Hoechst 33342 (Sigma–Aldrich, St. Louis, MO) for 30 min at 37 °C. The control cells were incubated in the presence of 50 μM verapamil (Sigma–Aldrich). Propidium iodine (PI) 1 μg/ml was added to discriminate dead cells. Analysis and

SP analysis

SCC25 Hoechst-low cells were sorted from the SCC25 cell line after excluding dead cells and cellular debris based on scatter signals and propidium iodide fluorescence. SP cells have been shown to exhibit a distinct projection pattern by actively effluxing Hoechst 33342 dye from cytoplasm. The SP cell fraction comprised 0.23% of the total cell population, but totally disappeared after treatment with the selective ABC transporter inhibitor Verapamil (Fig. 1).

Sphere formation

Isolated SP cells and non-SP cells of

Discussion

Since the concept of CSC has been proposed to explain tumor cell heterogeneity, some research has suggested that current therapies fail to prevent cancer relapse and metastasis because of a small, surviving population of CSC.[2], [3], [24], [25] It is assumed that the most effective therapy should target CSC. Recent researches on various solid tumors revealed the existence of CSCs, providing strong evidence for the presence of functional heterogeneity within the tumor population;[4], [16], [26]

Conflicts of interest statement

None declared.

Acknowledgements

This study was partially supported by Grant 20791534 from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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