Establishment Of Breast Cancer Mice Model And Analysis Of Subtypes Of Spleen MDSC Cells

Myeloid-derived suppressor cells(MDSCs)are considered as critical immune suppressor cells which belong to a group of immature myeloid cells with complex biological functions.MDSCs can suppress antitumor immunity,promote the tumoral immune escape through a variety of diverse mechanisms.Because MDSCs and a variety of different sources of myeloid cells such as tumor associated macrophage(TAM),tumor related neutrophils(TAN),M1,M2 macrophages,monocyte,granulocyte and dendritic cells have interwoven phenotypic characteristics,the relationship between certain subtypes and functions are not clear,limiting the further study of the molecular mechanism of its function.Objective:In this study,a breast cancer mice model was established to detect the expression of chemokine receptors and toll-like receptors of the single cells obtained from normal and tumor-bearing mouse spleen CD11b+Gr-1+ MDSCs.The multicolor flow cytometry analysis was used to type subtypes of spleen MDSCs which lay the foundation for further analyzing the function and molecular mechanism of MDSCs.Methods:(1)Establish the mice model using the method of orthotropic transplantation.(2)Flow cytometry analysis of chemokine receptors and Toll-like receptors expressed of 4T1 cell line(3)Spleen cells separation and flow cytometry analysis of spleen chemokine receptors and Toll-like receptors expressed on typical MDSCs,then the multicolor flow cytometry analysis was used to compare the differences between the spleen MDSCs subtypes of different group(normal group and 4T1 group)to determine the certain subtypes of spleen MDSCs in tumor-bearing mice.Results:(1)When the 4T1 cell line was transplanted into the mouse mammary fat pad after 1 week,a visible tumor mass like a rice grain was detected in the breast of the mouse.After 2 weeks,some of the mice was executed and the spleen index was calculated:the spleen index of normal group is 6.47± 3.06,the transplanted group is 33.1± 1.14,T-test of the two group showed that:P=2.24E-7<0.05.According to the date got in 4 weeks,the tumor growth curve was drew and there showed S-type tumor volume growth trend.After 6 weeks the leaving transplanted mice were dissection.there are also lung metastasis and liver metastasis.(2)The results of the flow data show that:almost higher than 50%of the breast cancer cell line-4T1 cells express TLR2,TLR8,CCR5.CCR7 and CXCR7.The expression rates of other chemokine receptors and Toll like receptors were very low or even with no expression.(3)According to the preliminary results obtained,several markers were chose:CCR1,CCR2,CCR6,CCR7,CXCR2,CXCR4,CXCR6 and CX3CR1.They were prepared for multicolor flow cytometric analysis and 28 different kinds of spleen MDSCs subtypes such as CD45+Lin-CD11 b+ ly6G+ ly6C-B7-Hx-CCR1 +were discovered.Conclusion:The mouse model of breast cancer which can metastasis to liver and lung was successfully established by the method of in-situ transplantation using 4T1 cells.The chemokine receptor and Toll-like receptor expressed on the surface of 4T1 cell line were analyzed using flow cytometry.The chemokine receptors,Toll like receptors and some other markers expressed on the surface of mouse spleen cells was successfully analyzed through the method of 3 color analysis and the subtypes of spleen MDSCs was identified by multicolor flow cytometry analysis.These series of experiments above can provide new look for the clinical treatment of breast cancer.