Isolation,Culture And Identification Of Tumor Stem Cells From Primary Oral Squamous Cell

PURPOSE:To investigate the method of culture of squamous cell carcinoma cells derived from human fresh oral squamous cell carcinoma tissues and the method of isolation and identification of oral squamous cell carcinoma stem cells by immunomagnetic beads and flow cytometry.METHODS:1.Six fresh tumor specimens were taken from patients who underwent oral squamous cell carcinoma resection from 2016 to 2017 in the Affiliated Stomatology Hospital of Guangxi Medical University.Specimens were treated with anti-contamination to obtain primary oral squamous carcinoma cells by mechanical method and passaged by enzyme digestion.2.Primary squamous cell carcinoma cells were identified by cell morphology and immunohistochemical keratin antibody staining.3.Flow cytometry was used to detect the expression of CD133 and CD44 in primary squamous carcinoma cells and the corresponding CD133~+ and CD44+ cells were sorted by immunomagnetic beads method,and their sorting effects were identified at the same time.4.The cell cycle of the cells before and after sorting was measured by flow cytometry.5.Osteoblast and adipogenesis differentiation induced by oral squamous cell carcinoma cells and their subpopulations.6.The primary tumor cells,CD133~+ cells and CD44+ cells were injected into nude mice to perform tumorigenesis experiments in vivo to observe their tumorigenesis.7.HE staining was performed on the transplanted tumor tissues to observe histopathological changes.RESULTS:1.We cultivated and passaged purified oral squamous carcinoma cells successfully.2.The biological characteristics of oral squamous carcinoma cells can be reflected by cell morphology.Keratin stained positive in the cytoplasm of OSCC.3.The mean expression rates for CD133 and CD44,surface markers of stem cells in squamous carcinoma cells were 33.76% and 0.41% respectively.4.The purity of CD44+ cells and CD133~+ cells sorted by immunomagnetic beads were both more than 94% by flow cytometry.Their cell cycle is majorly in the G0/G1 phase.5.oral squamous cell carcinoma cells,CD44+ cells and CD133~+were successfully induced to differentiate into osteoblasts andadipocytes.6.On the 3rd day after subcutaneous injection,new tumors formed in the nude mouse inoculation area.The tumor volume in the CD133~+ cell group was greater than that in the CD44+ cell group and the unsorted oral squamous cell carcinoma group.A large number of cancerous structures can be clearly seen in the tumorigenic tissues by HE staining.The malignant grades of tumor tissues ranged from high to low: CD133~+ cells,CD44+ cells,and unsorted oral squamous cell carcinoma.CONCLUSION: A large number of primary oral squamous carcinoma cells can be successfully obtained from human fresh oral squamous cell carcinoma specimens by mechanical methods.And primary oral squamous cell carcinoma cells can be successfully passed.The expression of CD44,the surface marker of stem cell,was high in oral squamous cell carcinoma cells,and the expression of CD133 was significantly lower.High-purity CD133~+ cells and CD44+ cells,a group of cells that have long been in the G0/G1 phase of the cell cycle and have high tumorigenicity,could be successfully selected by immunomagnetic beads method.In addition,Oral squamous cell carcinoma cells,CD44 cells,and CD133 cells have the ability to differentiate into osteogenic and adipogenic cells,and CD133 cells are more differentiated than CD44 cells are stronger than oral squamous carcinoma cells.Finally,CD133~+ cells which had stronger stem cell properties were not only more tumorigenic than CD44+ cells,but also had a higher degree of malignancy in its xenografts than CD44+ cells.