Enrichment Of Cancer Stem Cells In Mouse Breast Cancer Cell Line 4T1 With Chemotherapeutic Drug And Its Mechanism In Drug Resistance

Objective: To enrich and identify cancer stem cells in mouse breast cancer cell line 4T1 with chemotherapeutic drug , and to investigate the mechanisms of these cells in drug resistance.The goal is to establish the foundation for further exploring the regulation mechanisms of breast cancer stem cells and developping molecular praeparatum targetting it.Methods: 1 Mouse transplanted tumor model enriching for breast cancer stem cells was established with chemotherapeutic drug. Construct mouse breast cancer model by injecting 4T1 cells subcutaneouly into BALB/c mice in the right shoulder. 20 mice were randomly divided into control group and 5-Fu-treated group. Normal sodium was injected to mice intraperitoneally in control group. Different concentrations of 5-Fu(0.05mg/ml,0.1mg/ml,0.2mg/ml)were injected intraperitoneally to mice once a day according to different groups, once a week after 7days. Mice were executed after 4 weeks. The tumor tissue were divided into three portions, one was fixed with neutral formalin to detect expression of ALDH1 and CD55 by immunohistochemistry, one was used to extract RNA and another was dissociated to cell suspension to detect the proportion of CD44+CD24-/low by flow cytometry and the proportion of SP cells by Hoechst33342 staining. Construct the second generation of mouse model enriching cancer stem cell with cells derived from mouse tumor tissue treated with 0.1mg/ml 5-Fu, in which the proportion of CD44+CD24-/low cells was the highest. Mice was treated with 0.1mg/ml 5-Fu again as above. Four generations of mice model were established in all.2 Flow cytometry (FCM) was used to measure the proportion of CD44+CD24-/low cells in 4T1 cells derived from all generations of mouse transplanted tumor tissue in control group and 5-Fu treated group. 3 Hoechst33342 staining was used to measure the proportion of SP cells in 4T1 cells derived from all generations of mouse transplanted tumor tissue in control group and 5-Fu treated group.4 Expression of ALDH1 and CD55 in every generation of mouse transplanted tumor tissue in control group and 5-Fu treated group were detected by immunohistochemistry.5 Serum-free suspension culture tumor tissue cells derived from all generations of mouse model in control group and 5-Fu treated group, the formation of mammospheres was observed, the number of mammospheres was counted and the proportion of mammospheres formation was caculated.6 The differentiation of the third generation of 4T1-3rd mammospheres was observed when cultrued in serum-suspplenmented medium.7 Tumorigenic ability of different 4T1 cells derived from the third generation of mouse model in control group and 5-Fu treated group was detected by mouse tumorgenesis experiment.8 Morphology of mouse transplanted tumor tissue were observed under optical microscope after HE staining.9 Expression of MDR1, BCRP, ALDH1 andβ-catenin mRNA in the third generation of mouse transplanted tumor cells were detected by semi-quantitative RT-PCR and Real-time PCR.Results: 1 Mouse transplanted tumor model enriching for breast cancer stem cells was established through passaging 4T1 cell line in mouse induced by chemotherapeutic drug 5-Fu. The cells derived from every generation of mouse transplanted tumor tissue in 5-Fu treated group was named 4T1-1st cells, 4T1-2nd cells, 4T1-3rd cells, and 4T1-4th cells respectively, and the cells derived from generation of mouse transplanted tumor tissue in control group was named parental 4T1 cells.2 The results of FCM showed that the proportion of CD44+CD24-/lowcells in the first generation of transplanted tumor tissue in control group was 11.5±0.9%, while it was 40.1±3.4%, 45.6±1.6% and 49.8±1.2% respectively in 5-Fu treated group which was significantly higher than in control group (P<0.05). It was 49.8±1.26%, 56.8±1.76%, 66.4±1.5% and 69.0±1.6% respectively in every generation of transplanted tumor tissue in 5-Fu treated group, significantly higher than it in control group(P<0.01). However, there was no statistically significant differences between the third and fourth of generations(P>0.05).3 The results of Hoechst33342 staining showed that the proportion of SP cells in every generation of transplanted tumor tissue in 5-Fu treated group was respectively 25.0±1.21%, 42.6±2.8%, 58.4±2.2%, 61.3±2.6%, which was significantly higher than it in control group(P<0.01). However, there was no statistically significant differences between the third and fourth of generations(P>0.05).4 The results of immunohistochemistry showed that the expression level of ALDH1 in the transplanted tumor tissue in control group was negative, while weakly positive to positive in mouse transplanted tumor tissue in 5-Fu treated group, the latter was significantly higher than the former.The proportion of cells in which CD55 was highly expressioning in the transplanted tumor tissue in control group was 0.6±0.3%, while 7.8±1.6%, 10.1±2.0%, 15.6±1.4% and 17.3±1.9% in mouse transplanted tumor in 5-Fu treated group, the latter was significantly higher than the former(P<0.01).5 The results of serum-free suspension culture revealed that the proportion of mammospheres formation in 4T1-3rd cells was 5.9±0.4 %, but 0.5±0.2 % in parental 4T1cells. The former was as about 12-fold as the latter(P<0.01). Moreover, dissociated 4T1-3rd cells from the first generation of mammospheres generated an equivalent proportion of the second and third generation of spheres. 4T1-3rd mammospheres cultures could be maitained for 5 passages, while within three passages, parental 4T1 mammospheres failed to generate spheres, became adherent, and differentiated. The proportion of mammospheres formation in 4T1-4th cells was 6.1±0.3 %, and there was no statistically significant differences between 4T1-3rd and 4T1-4th cells (P>0.05).6 The results of serum-suspplenmented culture revealed that the 4T1-3rd mammospheres was becoming gradually adherent and differenciated when cultured in serum-suspplenmented medium.7 The results of mouse tumorgenesis experiment revealed that 4T1-3rd cells were more tumorigenic than 4T1 parental cells.8 All transplanted tumor tissue showed the morphology of breast cancer pathologically through HE staining under optical microscope,and there was no significant differences between untreated and treated mice.9 The results analyzed by semi-quantitative RT-PCR showed that expression level of MDR1, BCRP and ALDH1 mRNA in 5-Fu treated group was up regulated compared to the control group(P<0.01). However,β-catenin mRNA did not significantly changed after treated with 5-Fu(P>0.05).The results analyzed by Rt-PCR showed that expression level of MDR1, BCRP and ALDH1 mRNA in 5-Fu treated group was as 4.35, 6.14, 3.78 fold as the control group respectively(P<0.01),β-catenin mRNA was as 1.75 fold as the control group(P<0.05).Conclusion: 1 The transplanted tumor model enriching for breast cancer stem cells was established with chemotherapeutic drug 5-Fu, and highly malignant breast cancer cell line 4T1-3rd enriching for breast cancer stem cells was constructed, which suggested that use of chemotherapeutic drug is a effective way to sort breast cancer stem cell2 Expression level of MDR1 and BCRP were up regulated in 5-Fu treated group, which demonstrated that breast cancer stem cells might be resistent to chemotherapeutic agents through high expression of MDR1 and BCRP.3 Expression level ofβ-catenin was up regulated in 5-Fu treated group, which demonstrated that the activation of Wnt signaling pathway might participate in the regulation of breast cancer stem cell.4 Expression level of CD55 and ALDH1 was significantly higher in the transplanted tumor tissue in 5-Fu treated group, which suggested that CD55 and ALDH1 might act as tumor markers of breast cancer stem cell.