The Feasibility Analysis Of ALDH As A Marker Of Human Tongue Cancer Stem Cells

Objective: To investigate the expression of ALDH1 in human tongue squamous cell carcinoma cell line Tca8113, and compare the biological characteristics of ALDH^hi cells, ALDH^lo cells and unsorted Tca8113 cells; To investigate the expression of ALDH1A1 in the lesion and lymph node specimen of human tongue squamous cell carcinoma, and discuss its clinical significance. In order to investigate the ability that whether ALDH can be adopted as one of the phenotypes of tumor stem cells or not.Methods: 1. The in vitro survival ability of single cell was determined using monoclonal experiment; the expression of ALDH1 in human tongue squamous cell carcinoma cell line Tca8113 was determined using cell immunofluorescence assay; the positive rate of ALDH1 was determined using flow cytometer after ALDHFLUOR staining; the ALDH^hi and ALDH^lo cell subsets were sorted using flow cytometer according to the expression level of ADLH in cells, the in vitro proliferation ability of two cell subsets and unsorted cells was determined using CCK-8 method, the in vitro invasion ability of three groups of cells was determined using Transwell assay, and the independent tumor formation ability of three groups of cells was determined using nude mouse tumor formation experiment; 2. Using CCK-8 method to determine the ability of anti- chemotherapy of the sorted ALDH^hi cells, ALDH^lo cells and unsorted Tca8113 cells; 3. A total of 50 paraffin specimens of human tongue squamous cell carcinoma and 31 specimens of lymph nodes were randomly selected, and the complete data of all the patients were available. The expression of ALDH1A1 in above tissues was determined using immunohistochemistry, and the relationship between its expression and clinical factors was analyzed.Results: 1.After single cell culture for 120 h, 49.59% of cells survived, and without medium change some cells still survived 12 days after seeding, which suggested that these cells had strong proliferation and survival ability and might be associated with tumor stem cells; immunofluorescence assay showed that most of ALDH was expressed in cytoplasm and little of ALDH was expressed in nucleus, and ALDH^hi and ALDH^lo cells could be distinguished according to fluorescence; flow cytometer showed that the ratio of ALDH^hi cells was 5.21%; the in vitro proliferation experiment showed that the order of three groups of cells based on the in vitro proliferation ability was ALDH^hi>Tca8113 primary cells >ALDH^lo; Transwell assay showed that the number of ALDH^hi cells crossing the membrane was 84.13±31.75, that of ADLHlo cells was 56.57±19.37 and that of unsorted cells was 73.93±28.42, which suggested that the invasion ability of ALDH^hi cells was higher than that of ADLHlo cells and unsorted cells; ALDH^hi cells of different concentrations all formed tumor, only 1 tumor was formed in the 1x106 group of ADLHlo cells, unsorted cells of 1x106 concentration all formed tumor but only 1 tumor was formed in 1x105 group and no tumor was formed in 1x104 group. All the groups showed no significant difference in tumor size. 2. The result of analysis of variance and LSD pairwise comparison shows that the medical sensitivity of three kinds of cells is that Vincristine > TAX > Cisplatin > 5-FU. The medical sensitivity of three types of cells of 5- FU and cisplatin is that ALDH^hi cells?unsorted Tca8113 cells?ALDH^lo cells?P?0.05?. But there is no siginificant difference between the sensitivity of paclitaxel?P=0.343? and vincristine?P=0.181? to ALDH^hi cells and ALDH^lo cells.The medical sensitivity of the four drugs for unsorted Tca8113 cells?ALDH^hi cells and ALDH^lo cells have significant difference. P<0.05. 3. Immunohistochemistry showed that ALDH1A1 was mainly expressed in cytoplasm. Its expression in human tongue squamous cell carcinoma specimen was higher than that in normal mucosa, and the positive rate of ALDH1A1 in 50 lesion specimens was 58%. The expression of ALDH1A1 in lesion was correlated with clinical stage?P=0.038?, lymph node metastasis?P=0.003?, lesion size?P=0.021? and histological grade?P=0.019?, but not correlated with gender?P=0.413?, age?P=0.441? and smoking amount?P=0.126??P>0.05?. The expression ALDH1A1 in lymphocytes was not high. In metastatic lymph nodes, it was mainly expressed in the cytoplasm of cancer nest cells. The expression of ALDH1A1 lymph nodes was correlated with clinical stage?P=0.001?, lymph node metastasis?P=0.000? and lesion size?P=0.001?, but not correlated with gender?P=0.422?, age?P=0.975?, smoking amount?P=0.658? and histological grade?P=0.786?.Conclusion: 1. The ALDH^hi subset of human tongue squamous cell carcinoma Tca8113 cell line has strong in vitro proliferation ability, invasion ability and independent tumor formation ability, accounts for a very low proportion in the cell line, and has characteristics like tumor stem cells. 2. ALDH^hi cells have a strong anti-chemotherapy characteristic, and the drug resistance of tumor cells may be associated the intracellular content of ALDH1. 3. ALDH1A1 protein is associated with the progression of human tongue squamous cell carcinoma, and its high expression may suggest poor prognosis. 4. ALDH can be adopted as one of the phenotypes of tumor stem cells.