Fig. 7

SPRY2 knockdown in stromal fibroblasts promotes breast cancer stemness depending on glycolysis. A Sphere establishment in both primary and secondary passages compared with control CM and CM from shSpry2 stroma cells pretreated with 2-DG in sphere-formation assays in 4T1 cells. Graph showing sphere formation was increased in 4T1 cells treated with CM from shSpry2 stromal cells compared with that treated with control CM. 4T1 cells further formed bigger spheres in diameter under shSpry2 stromal cell CM treatment compared with controls. The enhanced sphere-forming abilities of 4T1 cells under treatment of shSpry2 stromal cell CM were inhibited when treated with CM from shSpry2 stroma cells pretreated with 2-DG. Scale bar = 100 μm. B Flow cytometry analysis showing the percentage in the CD44⁺ high; CD24⁻ low cell subpopulation in 4T1 cells treated with CM from shSpry2 fibroblasts compared to the control and that pretreated with 2-DG. C Relative mRNA expression of MCT1 and MCT4 in fibroblasts and 4T1 cells treated with CM from fibroblasts. D Protein expression of MCT1 and MCT4 in fibroblasts and 4T1 tumor cells treated with CM from fibroblasts. E Immunofluorescence staining of CK18 (green), CD44 (red) and DAPI (blue) in the tumor of mice co-injected with shNC or shSpry2 fibroblasts with tumor cells. Scale bar = 50 μm. F Model depicting the decreased stromal SPRY2 expression enhance the stem cell ability of breast cancer cells via glycolysis and promotes breast cancer progression. P values based on unpaired Student’s t test: *P < 0.05, **P < 0.01, ***P < 0.001