| BackgroundsCancer stem cells (CSCs) are small subpopulation of cells in tumor tissue characterized as stem cell properties with ability to self-renew and undirected differentiation potential. The existance of CSCs is one foundation of the tumor initialization, expansion and metastasis. The side-population cells (SP cells) isolation technique was established by Goodell by using Hoechst33342DNA labling fluorescence dye and flow cytometry. SP cells have stem cell-like biological characteristics, potential to self-renew and differentiation. Currently, majority of studies use fluorescence-activated flow cytometry to indentify and isolate SP cells and investigate as stem cells. Evidences suggest that SP cells present in many types of tumors. Cutaneous squamous cell carcinoma (SCC), a common type of cancer in dermatology, has the capability of invasiveness and metastasis. The CSCs of SCC are still unclear, no matter the functions or markers of SP cells. The aim of our study is to find whether CSCs exist in squamous cell line A431and Colon16, and to farther investigate the markers, characteristics and biological functions of CSCs in A431cell line. These will provide experimential foundation and a new field in the therapy of SCC.Contents and Methods1. Using SP cells isolation technique to detect and separate SP cells from human SCC cell line A431and Colon16individually with Hoechst33342fluorescence dye and flow cytometry. To investigate the presence of SP cells in A431and Colonl6.2. Identification and isolation of SP cells from A431by flow cytometry. To compare colony forming and proliferation capacity between SP cells and Non-SP cells in vitro. To detect the expression of stem cell marker ABCG2in SP cells and Non-SP cells using RT-PCR.3. To observe the different expression of retinoic acid receptors (RARα, RARβ, RARγ) and retinoid x receptors (RXRα, RXRβ, RXRγ) in isolated SP cells and Non-SP cells.4. To investigate the expression of stem cell markers ABCG2, CK19, P63in actinic keratosis, Bowen' disease and cutaneous SCC by immunohistochemistry.Results1. There exist a small subpopulation of SP cells in A431and Colon16cell line, with the percentage of1.1and1.0respectively.2. SP cells in human SCC showed greater capability of proliferation and colon-forming than that of Non-SP cells in vitro. The number of colony formation in SP cells was114.8±4.95compared with44.5±3.67of Non-SP cells(P<0.05).The expression level of ABCG2mRNA in SP cells was significantly higher than that in Non-SP cells (P <0.05) 3. The expression levels of RARα,RARβ,RARγmRNA were significantly different between SP cells and Non-SP cells in A431, which were greatly higher in SP cells than those in Non-SP cells (P<0.05). The mRNA expression of RXRα,RXRβ,RXRγmRNA showed no difference between SP cells and Non-SP cells in A431(P<0.05)4. Positive stained substance of ABCG2and CK19localized in cell cytoplasm and membrane. The number of ABCG2and CK19positively stained cells in SCC was significantly higher than that in AK and Bowen's disease (P<0.05). while the number of the latter two diseases has no difference. The positive cases of ABCG2and CK19increase with the development of pathological extents among AK, Bowen's disease and SCC,with ABCG2:40%,71.4%,84.6%and CK19:40%,50%,92.3%, respectively. Positive stained substance of P63localized in cell nucleolus. The number of P63positively stained cells among the three diseases has no difference, and all37cases present P63positive stained.Conclusions1. SP cells were present in A431and Colonl6. SP cells could be identified and isolated by flow cytometry with Hoechst33342labeling, and cultured in vitro to conduct cytobiology experiments.2. SP cells in A431had cancer stem-like characters. They can form holoclones which indicating the greater proliferative capability. The stem cell marker ABCG2was over-expressed in SP cells than Non-SP cells.3. The stem cell markers ABCG2, CK19, P63were detected in cutaneous SCC. The expressions levels of ABCG2and CK19were significantly different among actinic keratosis, Bowen'disease, and cutaneous SCC. They might be indicators for progression and prognosis of SCC.4. The expression levels of CSC marker ABCG2, RARα,RARβ,and RARγmRNA were significantly different between SP cells and Non-SP cells in A431which could be related to drug resistence. which could provide evidence for adjunctive therapy of retinoic acids.
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