Isolation and identification of small cell lung cancer stem-like cells / 中国癌症杂志

ABSTRACT

Background and purpose:Small cell lung cancer(SCLC) is a highly aggressive malignancy with a 5-year survival rate of less than 10%.These features suggest the enrichment in cancer stem cells.Our study was aimed to establish a small cell lung cancer cell line from primary cultured from lung cancer tissue,and to identify and isolate cancer stem cells from the early generations of the established cell line.Methods:By using a serum-free medium,lung cancer primary cells were cultured from a small fresh lung cancer cell sample,and a small cell lung cancer cell line was established after passaging the cultured cells.Lung cancer stem cell markers were searched by using flow cytometry analysis to sort through the third or forth generation cells of the established cell line.Biological characteristics of lung cancer stem cells were studied by using the single cell clone formation test,plat colony formation test and cell sphere formation test.Results:A small cell lung cancer cell line was established by primarily culturing a fresh lung cancer sample.The cell line could easily passaged more than 25 generations.When the third or fourth generation cells of the established cell line were checked by flow cytometry,there was a small population of cells that were obviously with CD44 stronger positive(CD44++ cells,5.1%) than the main population cells,which were CD44 weak positive(CD44+ cells).CD44++ cells showed stronger colony formation ability than the CD44+ cells.Furthermore,only the CD44++ cells could form stem cells when a single cell was seeded in a well of 96 well plates.Most importantly,only the CD44++ cells could form cell spheres in ultra low attachment 96 well plates.These results indicated that the CD44++ cells enriched more cancer stem cells in the small cell lung cancer cell line.When CD44 and CD90 antibodies were co-stained,the cells of the established cell line could be separated into 4 populations,i.e.CD44+CD90-,CD44+ CD90+,CD44++ CD90+ and CD44++ CD90-cells.CD44+ CD90+ cells were the smallest population cells in the cell line,with a ratio of about 1.9%.When cultured in ultra low attachment 96 well plates,CD44++ CD90+ cells had the strongest cell sphere formation ability when compared with other population cells,indicating that CD90 might also be a small cell lung cancer stem cell marker.Conclusion:There were cancer stem-like cells in primary cultured small cell lung cancer cell lines,CD44 and CD90,which might mean that they could be lung cancer stem cell markers.