Fig. 3: GREM1 regulates the growth of breast cancer cells in vitro and in vivo.

a Relative viability of transformed and cancerous breast cells. Cells were seeded in 96-well plates and incubated for 72 h, followed by the MTT assay. b Colony-forming activity of transformed and cancerous breast cells. Cells were seeded in 6-wells with low density (2 × 10³/well) and incubated for 7 to 10 days. c Effects of GREM1 silencing on spheroid formation. CCD-1068sk breast fibroblasts and SKBR3 human breast cancer cells were stained with green and red fluorescent cell linker dyes, respectively, and seeded in 96-well hanging-drop plates for 5 days. The size of each spheroid was measured by fluorescence microscopy, and the cell viability was examined by the MTT assay. d Effects of ectopic overexpression of GREM1 on viability of MCF-7 and T47D cells. Corresponding cells were seeded in 96-well plates, incubated for 72 h, and subjected to the MTT assay. e Suppressive effects of GREM1 silencing in a 3D spheroid invasion assay. CCD-1068sk and SKBR3 cells were stained with fluorescent dyes as described in  c, suspended in the spheroid formation extracellular matrix and seeded in spheroid formation plate. After 3-day incubation, cell culture medium containing invasion matrix was added and further incubated at 37 °C for additional 7 days. The spheroids were visualised under a fluorescence microscope and each image was analysed using ImageJ to measure changes in the area of the invasive structures. f Effect of exogenous GREM1 on the 3D spheroid invasion of SKBR3 cells. SKBR3 cells were suspended in the spheroid formation extracellular matrix and incubated for 3 days. The invasion matrix and cell culture medium containing recombinant human EGF or GREM1 (50 ng/ml, each) were then added, and the mixtures were incubated at 37 °C for additional 7 days. The images were analysed using ImageJ. g, h Representative images of dissected xenogeneic tumours in nude mice inoculated with GREM1-silenced (g) or overexpressing (h) SKBR3 breast cancer cells. These cells were inoculated subcutaneously into the right dorsal flanks of female nude mice while their respective control cells were inoculated into the left dorsal flanks as described in ‘Methods’ section. Representative images of dissected xenogeneic tumours from nude mice in each group. i, j Quantification of tumours. Female nude mice were treated as described for g, h. All values in the graphs represent mean ± SD of three independent experiments except for i, j, where n = 6 and 4 per group, respectively. Two-sided t test. *P < 0.05; **P < 0.01; ***P < 0.001.