SDF-1α Secreted By Cancer-associated Fibroblasts In The Tumor Microenvironment Promotes The Progreesion Of Breast Cancer

[Objective]To investigate the effect of estrogen on estrogen receptor-negative breast cancer and elucidate the mechanism of microenvironmental factors in estrogen-induced progression of estrogen receptor-negative breast cancer.[Method]1.Ovariectomized Balb/C mouse were used to construct an animal model and subcutaneously implanted homologous estrogen receptor-negative breast cancer cells.2.To elucidate the mechanisms of the different tumorigenic abilities between the estrogen group and the control group,we compared several tumor-promoting factors produced by CAFs in 4T1 and EMT6 xenografts in these two groups,and attempted to find out the most interesting factors that lead to the difference of tumorigenesis.3.In order to investigate whether SDF-la was derived from CAFs,tissue immunofluorescence analysis was used to detect the co-localization of CAF-specific markers and SDF-la.The CAFs isolated from patients were identified by immunocytochemistry,and the tumor epithelial cells were screened by CD326+ magnetic beads.Then,ELISA analysis was used to detect the effect of estrogen on the secretion of SDF-laby CAFs or tumor epithelial cells.4.Colony formation assay was used determine whether the different tumorigenic abilities between the estrogen group and the control group was caused by the effect of SDF-la on cell proliferation.Flow cytometry was used to compare the amount of MDSC in the two groups,and the chemotaxis effect of recombinant SDF-la on bone marrow-derived MDSC was analyzed by Transwell chamber.We still compared the different effect of the control group,CAF group,estrogen group and Estrogen + CAF)group on MDSC chemotaxis.5.After inhibited SDF-1/CXCR4 pathway by AMD3100 or deleted tumor-infiltrating MDSC by gemcitabine,we observed the recruitment of MDSC and the effect of estrogen on tumorigenesis in the ovariectomized model[Result]1.Estrogen promotes the tumorigenesis of estrogen receptor-negative breast cancer cells 4T1 and EMT6 in vivo.2.After continuous injection of estrogen in tumor-bear ovariectomized mice,the mRNA levels of several tumor-promoting factors produced by CAFs in 4T1 and EMT6 xenografts were increased.After a comprehensive assessment,we found that the levels of SDF-la(CXCL12)in the estrogen group were significantly increased.3.The a-SMA protein as the marker of CAFs present in the tumor stroma was largely co-localized with the SDF-la protein.Comparing the SDF-la secreted by CAFs and tumor epithelial cells with or without estrogen,we found that the main sources of SDF-la were CAFs after stimulated by estrogen.4.Recombinant SDF-la had no effect on the proliferation of estrogen receptor-negative breast cancer cells 4T1 and EMT6.Estrogen-treated mice had a higher number of Gr-1+CD11b+MDSC within CD45+cells than control mice(Control group VS Estrogen group,4T1:(11.1±3.3)%VS(49.5±7.4)%and EMT6:(9.7±1.8)%VS(39.6±8.3)%.What’s more,we found that recombinant SDF-la could recruit MDSC isolated from bone marrow.When co-culturing CAFs and MDSC with estrogen,we found that the numbers of MDSC per field were larger than culturing with CAFs or estrogen alone.5.Using AMD3100 to block the SDF-la/CXCR4 axis could decrease the recruitment of MDSC in tumor microenvironment(Estrogen group VS(Estrogen+AMD3100)group,4T1:(47.4±5.2)%VS(19.5±1.9)%;EMT6:(42.7±7.3)%VS(18.7±1.2)%)and neutralize the effect of estrogen on tumorigenesis;meanwhile,using gemcitabine to delete MDSC(Estrogen group VS(Estrogen+ gemcitabine)group,4T1:(47.4±5.2)%VS(22.0±3.4)%;EMT6:(42.7±7.3)%VS(21.7±3.1)%)could also neutralize the effect of estrogen on tumorigenesis.[Conclusion]Estrogen may promote the progression of ER-negative BC by stimulating CAFs to secrete SDF-la,which can recruit MDSC to the tumor microenvironment to cause tumor-associated immune abnormalities and contribute to tumor-mediated immune escape.[Objective]To compare the effect of CAFs on the invasion and migration of breast cancer cells under hypoxia and normoxia,and to explore the effect and mechanism of SDF-la secreted by CAFs on breast cancer metastasis.[Methods]1.Immunofluorescence was used to identify the purity of CAFs isolated from patients.The effect of CAFs on the migration and invasion of breast cancer cells in hypoxia and normoxia was compared in the Transwell chamber.2.Using ELISA analysis to detect the production of SDF-la by CAFs under hypoxia.At the same time,the recombinant SDF-1α protein was used to observe the effect of SDF-la on the migration and invasion of breast cancer cells in vitro.Tail vein metastasis assays was conducted to observe the lung metastases of breast cancer cells treated by recombinant SDF-la.3.The effect of recombinant SDF-la on PI3K/AKT and MAPK/Erk pathway was detected by Wetsern blot.Inhibitor was used to suppress the PI3K/AKT or MAPK/Erk pathway.The effect of these inhibitors on the migration and invasion of breast cancer cells were analyzed by Transwell chamber.[Results]1.Comparing hypoxia with normoxia,we found that CAF could promote breast cancer cell migration and invasion under hypoxic condition.2.Hypoxia could promote the secretion of SDF-la from primary CAF.Recombinant SDF-1α enhanced the migration and invasion of breast cancer cells.Using specific inhibitors to inhibit SDF-la/CXCR4 axis could attenuate the migration and invasion of tumor cells.Meanwhile,breast cancer cells pretreated with recombinant SDF-la are more likely to develop lung metastases in nude mice.3.SDF-la activated PI3K/AKT and MAPK/Erk pathway in a time-dependent manner which could be inhibited by blocking SDF-1α/CXCR4 axis using receptor antagonists AMD3100.Inhibitting PI3K/AKT and MAPK/Erk by LY294002 and PD98059 respectively could reverse the effect of SDF-1 on the migration and invasion of breast cancer cells.[Conclusion]Hypoxia can promote cancer-associated fibroblasts to secrete SDF-1α,which can affect the migration and invasion of breast cancer cells through PI3K/AKT and MAPK/Erk pathways.