Fig. 4

ERα reduced the CCL5 expression in CAF cells and consequently decreased  macrophages infiltration. a We compared gene profiles of macrophage attraction related chemokines/cytokines between CAF.ERα(+) and CAF.ERα(−) by QPCR. b The comparison of CCL5 production from CAF.ERα(+) and CAF.ERα(−) was determined by ELISA through measuring CCL5 concentration in the CM. c CCL5 promoter luciferase activity was used to determine whether ERα regulates CCL5 expression. CCL5 promoter (−83 bp)-luciferase reporter was transfected into CAF.ERα(+) and CAF.ERα(−) and cultured in CD-FBS media. 10 nM E2 was added and CCL5 promoter luciferase activity was analyzed using a dual luciferase kit. d CAF were cultured in bottom wells and incubated with CCL5 neutralizing Ab or IgG (control). After 24 h, macrophages were added into inserted upper transwells that also contained CCL5 neutralizing Ab or IgG (control). Migrated macrophages were counted and compared to CAF.ERα(−) with IgG as control. Quantification is in lower panel. e Adding recombinant CCL5 protein reversed CAF.ER(+) reduced macrophage migration. CAF cells were incubated with recombinant CCL5 protein or control for 24 h, and then macrophages with recombinant CCL5 protein or control were added to the inserted transwells for migration assay. All migrated macrophages were compared to CAF.ERα(−) with control protein. Quantification is in lower panel. f CCL5 expression was confirmed by IHC staining in the in vivo mouse PCa tumors co-implanted with/without both CAF/22Rv1-Luc cells. Arrows show positive CCL5 staining. *, P < 0.05 vs. CAF.ERα(−)/22Rv1 tumors