Fig. 3

SPRY2 knockdown in stroma promotes breast cancer progression. A Western blot analysis showing the protein expression of SPRY2 in fibroblasts after shRNA mediated SPRY2 knockdown. B Quantification of 4T1 cells treated with conditioned media (CM) from control or shSpry2 fibroblasts by CCK8 assay. C Colony formation assay of 4T1 tumor cells treated with CM from control or shSpry2 fibroblasts. Scale bar: 1 cm. D Mice were subcutaneously implanted with EO771 cells in combination with shNC or shSpry2 fibroblasts. Representative tumors images from each group. Right graph showing the tumor weights from each group at the experimental endpoint. E Representative HE, Masson staining and immunohistochemical images of Ki67 staining from shNC and shSpry2 subcutaneous xenograft tissues. Scale bar:50 µm. F, G Mice were implanted with EO771 cells in combination with shNC or shSpry2 fibroblasts into tail vein and the lung was resected after 5 weeks. Representative HE staining images in sections of lung tissue in (F). Scale bar:100 µm. Graph showing the number of metastatic nodules and the percentage of lung area with tumor in the lung of mice in (G). Data are presented as mean ± SD. (n = 5 independent samples). H Luciferase tagged 4T1 cells mixed with shNC or shSpry2 fibroblasts were transplanted in the fourth left mammary fat pad of female nude mice. Tumor growth in the mammary fat pad was monitored using bioluminescence imaging on day 7, 14 and 20 (image scales in p/s/cm²/sr, n = 5 mice). The right graph showing the corresponding quantification of bioluminescence imaging signal (total flux) in the fat pad area. I Gross morphology of liver showing metastases on day 20. J bioluminescence imaging of lungs on day 20. K Immunofluorescence staining of α-SMA (green), SPRY2 (red) and DAPI (blue) in primary breast tumor (upper panel) and lung metastases (lower panel). P values based on unpaired Student’s t test (B, C and G) or Mann–Whitney test (D, H): *P < 0.05, **P < 0.01, ***P < 0.001