Figure 4

In vivo effects of AM9928 on TNBC tumor growth and brain metastasis: (a) Expression levels of IL-6, IL-8, and VEGF in GFP-4T1-BrM5 cells untreated and following treatment with AM9928: Cell supernatant samples were prepared from GFP-4T1-BrM5 cells either treated with vehicle control or with AM9928. The levels of the murine cytokines/chemokines were measured using specific ELISA assays for IL-6, IL-8, and VEGF-A (R&D Systems ^®ELISAs). Data shown are representative of at least three independent assays performed on triplicate wells. The error bars indicate standard deviations. *p < 0.05 as compared to vehicle control, **p < 0.005 as compared to vehicle control and ***p < 0.0005 as compared to vehicle control. (b) Schematic presentation of time injection of AM9928 and GFP-4T1-BrM5 cells: AM9928 (at 10 mg/kg) or vehicle control were injected by I.V. twice a week for 3 weeks. (c) Tumor formation in mammary fat pads following treatment with AM9928: Murine tumor formation in mammary fat pads were measured at day 28: n = 10 mice/treatment; *p < 0.05, Mann–Whitney U‐test; **p < 0.005 as compared to vehicle control. (d) Detection of GFP-positive cells in brain tumors: GFP–4T1-BrM5 tumor cells were administered to the mammary fat pads as compared to control mice and were treated with vehicle control or with AM9928. Murine tumor cells in brain were detected by GFP immunostaining (GFP antibodies 1:50 dilutions; Abcam)(green) and their respective controls were used under the same standardized conditions. Brain nuclei were counterstained with DAPI (blue). n = 10 mice/treatment; These are representative images of over 50 images from three independent experiments. Scale bar = 20 μm. (e) Quantitative analysis of tumor cells in the brain: Murine tumor cells in brain were detected by GFP immunostaining as described above. The tumor areas at day 28 were detected by immunostaining with GFP antibody: n = 10 mice/treatment; *p < 0.05, Mann–Whitney U‐test; **p < 0.005 as compared to vehicle control.