Sorting And Multidrug Resistance Research Of CD44+/CD24-Breast Cancer Stem Cells

Objective:To separate CD44+/CD24-breast cancer cells subpopulation from human breast cancer cells,then observe the capacity of self-renewal and multiplex differentiation potential of these subpopulation;analyze the expression levels of multidrug resistance-associated gene and protein of breast cancer stem cells;explore the relationship between breast cancer stem cells and multidrug resistance,which should provide ideas for further related researchMethods:We cultured human breast cancer cell line MCF-7 routinely,and then induced breast cancer multidrug resistant cell line MCF-7/ADR by low concentrations of chemotherapy drugs continued induction method.MACS immunomagnetic beads were used to sort CD44+/CD24-subsets of breast cancer cells.After that,we measured the capacity of self-renewal and multiplex differentiation potential of these subpopulation by the microspheres test and animal experiments in vitro.The sensitivity of these subsets of breast cancer cells to chemotherapeutic drugs was measured by MTT assay;Real Time PCR and flow cytometry were used to measure the expression level of multidrug resistance gene(MDR1)and multidrug resistance associated protein P-gp respectively;The accumulation of Doxorubicin in cell was detected by confocal laser scanning microscope.Results:(1)MCF-7/ADR cells induced by the low concentration of chemotherapeutic drugs can survive in culture-medium with lug/ml adriamycin.(2)The proportion of CD44+/CD24-cell subsets in MCF-7/ADR cell lines and MCF-7 cell lines is 6.36 ± 0.57%,1.50 ± 0.10%respectively,the difference was statistically significant(P<0.05)?(3)After sorting by MACS immunomagnetic beads,the purity of CD44+/CD24-breast cancer cell subsets is 93.85%.(4)CD44+/CD24-breast cancer cells subsets has stronger self-renemal capacity according the result of microspheres trial.Animal experiments in vitro show that CD44+/CD24-breast cancer cells subsets has stronger tumorigenic ability in vitro.(5)IC50 of MCF-7/ADR cells and CD44+/CD24-cells was 15.84±0.66mg and 17.37±0.46mg,respectively,the difference was statistically significant(P<0.05);The expression of MDR1 in CD44+/CD24-cells was 1.07 ± 0.02 folds than MCF-7/ADR,the difference was statistically significant(P<0.05);The expression of P-gp in MCF-7/ADR and CD44+/CD24-cells was(101177.10±2171.86)and(114906.70 ±2560.19)respectively,the difference was statistically significant,P<0.05;CD44+/CD24-cells accumulated less Doxorubicin than MCF-7/ADR cells,518.67 ± 11.93 and 571.67 ± 11.15 respectively,the difference was statistically significant(P<0.05).Conclusion:(1)Cancer stem cells have a stronger multidrug resistance capability,and breast cancer stem cells can be riched by a low concentration of the chemotherapy drugs.(2)MACS immunomagnetic beads is viable for the sorting of CD44+/CD24-breast cancer stem cells,which should get breast cancer stem cells with purity higher than 90%and a high degree of self-renewal capacity and differentiation potential.(3)Breast cancer stem cells have a higher resistance to chemotherapy drugs,express higher levels of drug resistance gene 1(MDR1)and resistance-associated protein P-gp,have stronger efflux capacity of chemotherapy drug,which should be a key factors for the fail of breast cancer chemotherapy.