Characteristics Of CD133+ Cancer Stem Cells In Primary Oral Squamous Cell Carcinoma

Objective: To investigate the biological characteristics of CD133+ cancer stem cells in primary oral squamous cell carcinoma and compare the biological characteristics of CD133+ cells and CD133-cell subsets.The clinical significance of CD133 as a surface marker of oral squamous cell carcinoma stem cells was clarified.To explore the culture conditions for maintaining or improving the expression level of CD133.Methods: Primary oral squamous carcinoma cells were obtained from oral squamous cell carcinoma tissues by mechanical methods.Flow cytometry analysis and immunofluorescence staining were used to determine the expression of CD133 in primary oral squamous carcinoma cells.The immunomagnetic bead sorting method was used to separate the CD133+ cell subsets and CD133-cell subsets in primary oral squamous cell carcinoma cells.The CCK-8 method was used to detect the CD133+ cell subpopulation and CD133-cell subpopulation proliferation ability and cisplatin chemotherapy resistance.Transwell method was used to detect the effect of cisplatin on the invasion ability of CD133 + cell subsets and CD133-cell subsets.Serum-free culture method was used to detect the sphere-forming ability of CD133 + cell subsets and CD133-cell subsets.Serumfree culture method and serum-containing culture method were used to culture CD133 + cell subsets,respectively,and flow cytometry analysis was used to detect the change of CD133 cell ratio.RT-PCR analysis was used to determine the difference in expression of stem cell factors between CD133 + cell subsets and CD133-cell subsets,and to verify the difference in tumorigenicity between CD133 + cells and CD133-cells in animal models.Finally,the transplanted tumor was removed for HE staining and immunohistochemistry.Results: In this study,primary oral squamous cell carcinoma cells were successfully obtained from oral squamous cell carcinoma tissues.Flow cytometry analysis detected that primary oral squamous cell carcinoma cells contained a very small proportion of CD133 cell subsets(0.37 ± 0.08%),immunofluorescence staining the analysis showed that both CD133 + cell subsets and primary oral squamous cell carcinoma cells expressed CD133,which was statistically different(p <0.01).By immunomagnetic bead sorting,a relatively pure CD133 cell subset(93.79 ± 5.01%)can be obtained.Compared with CD133-cell subsets,CD133 +possesses stronger in vitro proliferation capacity(p <0.05)and cisplatin chemotherapy tolerance(p <0.001).Compared with CD133 + cell subsets,cisplatin had a stronger effect on the invasion ability of CD133-cell subsets(p<0.01).Under serum-free culture conditions,CD133 + cell subsets showed stronger stem cell sphere-forming ability.Compared with the serum-containing culture method,the serum-free culture method can maintain the ratio of CD133(p <0.05).RT-PCR results showed that CD133 + cells expressed higher stem cellrelated genes such as NANOG,SOX2,ALDH1A1 and OCT4 than CD133-cells(p <0.001).At the same time,in the in vivo tumorigenesis experiment of nude mice,the CD133 + cell subpopulation showed a strong tumorigenicity on a smaller order of magnitude(p <0.05).Finally,the CD133 + xenograft slices were proved to be well-differentiated squamous cell carcinoma by HE staining.Immunohistochemistry detected positive expression of CK in CD133 + xenograft tumor sections.Conclusions: There are indeed a small number of cancer stem cells in primary oral squamous carcinoma cells.CD133-positive cells have the characteristics of cancer stem cells.CD133 may be one of the markers of oral squamous cell carcinoma stem cells.Serum-free culture method can maintain the ratio of CD133.