Isolation and characterization of cancer stem-like side population cells in human oral cancer cells
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.
References (51)
- et al.
Characterization of a stem cell population in lung cancer A549 cells
Biochem Biophys Res Commun
(2008) - et al.
Expansion and characterization of cancer stem-like cells in squamous cell carcinoma of the head and neck
Oral Oncol
(2009) - et al.
Aldehyde dehydrogenase 1 is a putative marker for cancer stem cells in head and neck squamous cancer
Biochem Biophys Res Commun
(2009) - et al.
Cancer stem cell traits in squamospheres derived from primary head and neck squamous cell carcinomas
Oral Oncol
(2011) - et al.
Characterization of stem cell-like cancer cells in immune-competent mice
Blood
(2006) - et al.
ABCG2: a potential marker of stem cells and novel target in stem cell and cancer therapy
Life Sci
(2010) - et al.
The epithelial cell adhesion molecule (Ep-CAM) as a morphoregulatory molecule is a tool in surgical pathology
Am J Pathol
(2003) - et al.
Ep-CAM overexpression in breast cancer as a predictor of survival
Lancet
(2000) - et al.
Clinicopathologic significance of EpCAM expression in squamous cell carcinoma of the tongue and its possibility as a potential target for tongue cancer gene therapy
Oral Oncol
(2007) - et al.
Tumor heterogeneity: biological implications and therapeutic consequences
Cancer Metastasis Rev
(1983)
Cancer stem cells: models and concepts
Annu Rev Med
Cancer stem cells in solid tumours: accumulating evidence and unresolved questions
Nat Rev Cancer
Glioma stem cells promote radioresistance by preferential activation of the DNA damage response
Nature
Identification of a subpopulation of cells with cancer stem properties in head and neck squamous cell carcinoma
Proc Natl Acad Sci U S A
Stem cell patterns in cell lines derived from head and neck squamous cell carcinoma
J Oral Pathol Med
Identification of cancer stem cell-like side population cells in human nasopharyngeal carcinoma cell line
Cancer Res
Persistence of side population cells with high drug efflux capacity in pancreatic cancer
World J Gastroenterol
The polycomb gene product BMI1 contributes to the maintenance of tumor-initiating side population cells in hepatocellular carcinoma
Caner Res
MCF7 side population cells with characteristics of cancer stem/progenitor cells express the tumor antigen MUC1
Cancer Res
HER2 regulates the mammary stem/progenitor cell population driving tumorigenesis and invasion
Oncogene
Identification and characterization of ovarian cancer-initiating cells from primary human tumors
Cancer Res
Squamous cell cancers contain a side population of stem-like cells that are made chemosensitive by ABC transporter blockade
Brit J Cancer
Positive correlations of Oct-4 and Nanog in oral cancer stem-like cells and high-grade oral squamous cell carcinoma
Clin Cancer Res
Side population in oral squamous cell carcinoma possesses tumor stem cell phenotypes
Cancer Lett
Resistance to cytotoxic chemotherapy-induced apoptosis in side population cells of human oral squamous cell carcinoma cell line Ho-1-N-1
Int J Oncol
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2019, Saudi Dental JournalCitation Excerpt :Side population (SP) is a term describing a subset of cancer cells, that is considered CSCs, which possess the ability to efflux Hoechst DNA binding dye and chemotherapeutic drugs using ABC transporters (Dou and Gu, 2010). SP cells isolated from HNC are more tumorigenic, chemo-resistant and demonstrate self-renewal ability in-vivo (Tabor et al., 2011; Yanamoto et al., 2011; Zhang et al., 2009). Interestingly, a study reported an increase in the SP cells in HNC by the activation of EGFR, a receptor tyrosine kinase often overexpressed in HNC, and this phenotype was reversed by addition of EGFR inhibitor (Chen et al., 2006).
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