| ObjectiveTriple-nagative(TN)breast cancer is a special kind of breast caner,the ER, PR and HER2 are all negative, but lack of the therapy target,The prognosis of TN tumor is poor. In recent reports shows that the cancer stem cells play a important role in the generation and metasis of breast cancer.Cancer stem cells is a special group cells in the cancer cells,cancer stem cells are defined as rare cells and have the potential to selfrenewal and differentiation.More and more evidence shows there is a closely linked between CSCs and EMT.EMT(epithelial-mesenchymal transition) is a common embryonic development and tumor metastasis process,Vasculogenic mimicry(VM) is found a new model of tumor blood supply. VM is a endothelial-indenpendent tumor microcirculation model, and it formed by tumor cells, the red blood cells can be found in lumen, VM is closely linked with tumor malignant degree and metastasis. In the recent study, we found that the VM is associated with the metastasis of tumors. And the cancer stem cells may influence the formation of VM. In this study, we try to find the mechanism of cancer stem cells influence the formation of VM,provide a new way for the treatment of breast cancer stem cells.Methods1. To study the relationship between tumor stem cells and VM in TN breast cancer.Collcet 100 breast cancer patients' clinical speciments with complete follow-up data were obtained from Tianjin Medical University General Hospital. Use immuohistochemical ananlysis to annalyse the relationship of CD44,CD24 and VM, age, tumor size, gender, stage, metastasis,VE-cadherin.2. Effect of cancer stem cells on EMT and VM formation in TN breast cancer in vitro1) Conventional cultivation triple negative breast cancer cell line MDA-MB-231 and non triple negative breast cancer cell line MCF-7 and detection of MDA-MB-231 and MCF-7 channel instructure formation, migration and invasion ability of differences, using Western blot assay the EMT related protein expression difference; analysis of MDA-MB-231 and MCF-7 cancer stem cell markers ALDH1 + and CD133 + cell phenotype of content and in MDA-MB-231 cells were sorted with ALDH1 + and ALDH1- phenotype of the cells using flow cytometry. The ALDH1+ phenotype of MDA-MB-231 cells and ALDH1-phenotypes were referred to as ALDH1+231 and ALDH1-231, respectively.2) Use serum-free suspensiion culture the isolated ALDH1+ and ALDH1-cells,observe the differnence of the ability to form breast caner cells spheres between between ALDH1+231 and ALDH1-231 cells.3) Innvasion and migration assay were used to observe the migration and invasion ability between ALDH1+231 and ALDH1-231 cells.4) Use three-dimensional culture to detect the tube structure formation between ALDH1+231 and ALDH1-231 cells.5) Western blot assay were used to detect the EMT-related protein(E-cadherin,Twist1), VE-cadherin and ALDH1 between ALDH1+231 and ALDH1-231 cells.3. Effect of tumor stem cells on EMT, VM formation and biological behavior of high metastatic breast cancer in TA2 mice1) Select the triple negative breast cancer TA2 mice highly metastatic breast cancer which were bred by the University Animal House.Make TA2 high metastatic tumor tissue into single cell suspension. Flow cytometry was used to detect and sorted the cells with the marker of ALDH1 and CD133.2) Inject the sorted cells with the marker of ALDH1 and CD133 cells into TA2 mice. Observe the influence of Biological behavior in TA2 mice.3) Collect in tumor from TA2 mice,and made the tumor into paraffin sections and use immunofluorescence to detecet the relationship between cancer stem cells marker ALDH1 and CD133 with VM, E-cadhern,VE-cadherin Twist1 and VM formation.Results1 The expression of CD44 was higher than that of none-TN breast cancer in TN breast cancer. The expression of CD44 in VM patients was higher than that in VM negative patients, whereas the expression of CD24 was lower than that of VM negative patients.Immunohistochmeical analysis shows that 56 samples presented positive CD44 expression and 44 samples presents negative CD44 expression.In contrast with the CD44 positive(n=56) group and CD44 nagative(n=44) group shows that CD44 was positive associated with VM(c2=3.89, P=0.049), and in the clinicopathological data,in triple-nagitive group the expresson in CD44 is higher than none-triple-nagitive group(c2=5.884, P=0.016), and TNM stage types the?, ? group expressed more CD44 than?,? group(c2=3.89, P=0.049), and the VE-cadherin immunohistochmeical analysis shows that in VE-cadherin positive group express more CD44(c2=4.127, P=0.042). Kaplan-Meier analysis shows that compared with the overallsurivall time of patiens with CD44 nagitive group(P=0.083), the CD44 positive group is shorter. However, in other clinicopathological data, for example age,tumor size,degree.metastasis, the presence of CD44 shows no obvious association with the control group(P>0.05). Immunohistochmeical analysis of CD24 shows that CD24 positive samples is 66, and the nagitive samples is 34, shows that CD24 is inversely associated with VM.(c2=4.397, P=0.036). Other clinicopathological data have no statistically significance with CD24.2. The content of breast cancer stem cells in TN breast cancer cell line MDA-MB-231 was higher than that in none-TN breast cancer cell line MCF-7. ALDH1 +231 cells in vitro into cells Spheroid forming ability, invasion and migration,tube formation capacity, EMT associated proteins were higher than those of the ALDH1-231 cells.1) Flow cytometry shows that in MDA-MB-231 cells,the percentage of ALDH1+group was 26.87%. The percentage of CD133 group is 9.75%. In MCF-7 cells,the percentage of ALDH1 group is 1.27%. The percentage of CD133 group is3.4%.2) In the migration and invasion assay shows that the ALDH1+groupshows a higher ability in migraion and invasion.3) Three-dimensional culture shows that the ALDH1+ group the ability of the tube structure formation is significantly higher than ALDH1- group.4) Westerbolt shows that ALDH1+express higher ALDH1,the expressed higher VE-cadherin and Twist1 than ALDH1- group. The E-cadherin expressed is lower expressed in ALDH1+ group.3 TA2 high metastatic TNbreast cancer tissue in the presence of cancer stem cells,ALDH1+and CD133+phenotype of cancer stem cells have a strong ability to tumor growth, and affect the expression of EMT related proteins.1) Flow cytometry in TA2 high metastasis tumor shows that the percentage of ALDH1 is 31.2%. The CD133 is 6.5%.2) After injected the cells into the TA2 mice,the ALDH1+and CD133+group have a more tumorigenesis ability than that in ALDH1- and CD133- group.3) The tumor cells in TA2 high metastatic breast cancer expressed ALDH1(31.2%) and CD133(6.5%).Compare with the ALDH1-and CD133-group, the ALDH1+and CD133+ group have a higher tumorigenicity, and the average tumor volume weight is higher than the control group(P<0.05).The results of immunohistochemical staining shows that the expression of ALDH1 in ALDH1+group is higher than that of ALDH1-group(P<0.05), the difference have statistical significance(P<0.05) while the expression of E-cadherin was significantly lower,whereas Twist1 and VE-cadherin expression was significantlly higher in ALDH1+(P<0.05). In the CD133+ group, the immunohistochemical staning shows that the expression of CD133 was significantly higher than CD133-(P<0.05), E-cadherin was significantly lower than CD133-group, VE-cadherin is significantly higher than CD133- group.Conclusions1. The expression of CD44 in human triple negative breast cancer was higher than that of the non triple negative breast cancer, CD44 positive patients with a worse prognosis, CD44 expression in breast cancer and whether there VM and ve cadherin expression and TNM stage were positively correlated, CD24 expression with VM formation was negatively correlated.2. The contents of ALDH1+ and CD133+ phenotypes in the TN breast cancer cell line MDA-MB-231 were higher than that in the none-TN breast cancer cell line MCF-7. With respect to the cells ALDH1-231, ALDH1+231 cell migration,invasion, and form a stronger capacity of pipeline structure, EMT related protein expression increased, indicating that breast cancer stem cells are more VM forming ability, and promote the expression of EMT related protein.3. The ability of tumorigenesis ALDH1+and CD133+phenotypes of tumor stem cells in TA2 high metastatic breast cancer were stronger in TA2 mice, and affect the expression of EMT related proteins. ALDH1+ phenotype of breast cancer stem cells can promote the formation of VM in TN breast cancer. |
PDF Full Text Request