Research In The Stem Cell Characteristics Of Esophageal Cancer Eca109Monoclonal Cell Line

Objective: To isolate and identify cancer stem-like cell in esophageal carcinoma cell Eca109lines.Methods: The monoclonal esophageal Eca109cell lines had been Isolated and cultured using thelimiting dilution method, then observed the cell morphology by the HE staining; The expressionand localization of P75^NTR、Oct-4、SOX₂、Lin28and Nanog were detected by immunocytochemicaltechnique, and the cell’s proliferation in vitro with MTT method and plate clone formation inEca109monoclonal cell lines,respectively.Using transwell chamber assay to detect invasive abilityof each cell line; andcomparing the survival percentage of esophageal Eca109monoclonal cellsafter in different concentrations of chemotherapy drugs for48hours.Results: Successfully obtain two types of the monoclonal cell line, small round and short fusiform,by the limiting dilution method, respectively.HE staining show that small round cell with scantcytoplasm and nuclear is round; the short spindle cell is abundant cytoplasm with a relativelysmall nucleus. The expression of P75^NTR,Oct-4,SOX₂,Lin28and NANOG are all positive invarious cell lines,respectively.Among them,the positive expression SOX₂were localized in thecell cytoplasm in all cell lines,and Lin28were all expressed in the cell nuleus.The P75^NTRandOct-4, localized in the nuclei of round cell and the cytoplasm and cell membrance of spindle cells,but NANOG,in the cytoplasm of round cell and the cell nuleus of the spindles cells.According toMTT results, the round cell line proliferation rate is obviously higher than the other cell line, andthe difference is statistical significance （P<0.001）. Round,fusiform,unsorted cells were seeded inculture plate for15days,count the clone mission until the cell proliferation than50, and thecolony forming rate were38%,6%,22%,respectively.The cell clone formed by round cell line wassignificantly greater than that by the other two cell lines. After treated with chemotherapy drugs,the survival rate of round cell line is higher than that of spindle cells and unsorted cells.The resultsof Transwell chamber assay showed that the invasive ability is little difference among themonoclonal cell lines.Conclusion:（1） Esophageal cancer stem cell may exist in the esophageal cancer cell;（2）according to the expressions of stem cell markers P75^NTR,Oct4and NANOG were in differentparts of cells, speculated that the three antibodies may be the marker sorting esophageal cancerstem cell;（3） The round esophageal Eca109cell may be the esophageal cancer stem cells.