Isolation Of Cancer Stem Cells From Multiple Myeloma Cells Line U266 And Analyses Of Biological Characteristics

Objective:To compare the differencese of cancer stem cells isolated from multiple myeloma cells line U266 by immunemagnetic sorting and flow sorting side population cell analysis, We detected cell cycle, expression of drug resistance proteins and CD138 antigen, clone formation in vitro,and drug resistance genes expression to analyze the biological characteristics and multidrug resistance of cancer stem cell-like SP cells and CD138-cells.Methods:The percentages of SP cells and CD138+ cells in U266 cells were measured by flow cytometry. Fluorescence-activated cell sorting (FACS) were used to sort SP and non-SP (NSP) cells from U266 cells. Otherwise CD138+ cells and CD138-cells were separated by immunomagnetic sorting. Then the biological characteristics of these four cell populations, such as cell cycle, expression of drug resistance proteins and CD138 antigen, clone formation in vitro, and drug resistance genes ABCG2, MDR1, SMO, BCL-2, BAX gene expression. expression, were analyzed by flow cytometry and. using RQ-PCRResults:Before U266 cell sorting,the proportion of SP cells in U266 cells was 2.46±0.35%, and that of CD138+ cells was 77.68±2.14%. After sorting, the expression of CD138 in SP cell was significantly lower than NSP cells ((2.74±0.32% vs 98.08±0.48%), Flow-cytometric analysis of cell-cycle phase reavealed that after sorting most of SP cells and CD138-cells were in GO phase. The ratio of G0/G1 was respectively 81.50±5.42% and 57.85±3.16%, which were much higher than in NSP cells and CD138+ cells. The P-gp expression in SP cells was significantly higher than in NSP cells (77.74±0.70% vs 53.0±1.09%), and the percentage of P-gp+ cells in CD138-cell population was higher than in CD138+ cells (43.76±1.24% vs 12.66±0.75%). The percentage of ABCG2 expression in SP cells was 88.72±0.79%, which was significantly higher than in NSP cells (36.28±0.75%). And the ABCG2 expression in CD138 cells was also higher than in CD138+ cells (63.02±2.28% vs 23.05±1.55%). In colony formation analysis, colony formation rates of SP cells and CD138- cells were separately 39.45±4.02% and 31.05±6.82%, significantly higher than of NSP cells (8.21±3.85%) and CD138+cells (6.82±2.90%). The expression of gene ABCG2, MDR1, SMO, BCL-2 in SP cells and CD138- cells were significantly higher than in NSP cells and CD138+ cells, while the Bax gene expression was lower in SP cells and CD138- cells. The statistical analysis showed that these differences were statistically significant.Conclusion:U266 cells were present in a small group of SP cells and CD138 cell subsets, these cells had the biological characteristics of cancer stem cells, most of the quiescent and high-resistance protein expression and in vitro colony forming ability than strong, proved more than the biological characteristics of SP cells and CD138- cells with cancer stem cell properties. Most of SP cells and CD138- cells at the GO phase, and ABCG2, MDR1, SMO, Bcl-2 gene expression was significantly increased, BAX expression was decreased in the characteristics of MM stem cells,that participate in the multiple drug resistance and the complex mechanism of formation.