Figures & data
Figure 1. Effect of breast tumor cells conditioned media on morphology of endothelial cells. (A) Upper light microscopy micrographs were taken 24 h after treatments. Lower micrographs are confocal microscope images. Cells were incubated with fluorescent phalloidin (24 h after treatment) to detect polymerized actin. Analysis of area (B) and circularity (C) of endothelial cells after their exposure to conditioned media. This is a representative experiment of three performed independently. Statistical analysis was performed using one-way analysis of variance (ANOVA-test) with GraphPad Prism software version 5.01. *A p-value of <.05 was considered statistically significant.
Figure 2. Effect of breast tumor cells conditioned media on morphology and attachment of endothelial cells. (A) Endothelial cells treated with control medium (see video 1, Supplementary material) and (B) Endothelial cells treated with ZR-75-30 cells conditioned medium (see video 2, Supplementary material). Micrographs were obtained at 0, 6, 12, 18 and 24 h of treatment by time lapse microscopy. This assay is representative of the three independent experiments performed.
Figure 3. Effect of breast tumor cells conditioned media on the cell number. Cells were treated with the corresponding conditioned media for 72 h and cell proliferation was evaluated by crystal violet staining. Each value was estimated as a percentage of control (cells without any treatment). At least, three independent trials of the experiments were performed, and the data are expressed as the means ± standard deviation (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA-test) with GraphPad Prism software version 5.01. *A p-value of <.05 was considered statistically significant.
Table 1. Proteins detected in the secretome of breast tumor cell lines. Secretome was obtained according to methods. Expression of proteins was determined by Bio-Plex suspension array system.
Figure 4. Effect of neutralizing antibody anti-IL-8 on morphology of endothelial cells treated with ZR-75-30 cells secretome. (A) HUVEC treated with control medium, conditioned medium of ZR-75-30 cells and conditioned medium of ZR-75-30 cells plus neutralizing antibody anti IL-8 (0.4 μg/ml). Cells were treated for 24 h and then stained with crystal violet. (B) Positive control to test neutralizing activity of anti-IL-8. Cells were treated with IL-8 (100 ng/ml) and IL-8 plus neutralizing antibody against IL-8 (2 μg/ml) for 24, 48 and 96 h, then migration of MDA-MB-231 cells was evaluated mediating a wound assay. Analysis of the percentage of open wound area was performed with TScratch software. Data are shown as the percentage of open wound area in comparison with IL-8-treated cells. Results are expressed as the mean ± SD from three independent experiments. *p < .05 compared with IL-8-treated cells.
Figure 5. Effect of neutralizing antibody anti-VEGF on morphology of endothelial cells treated with ZR-75-30 cells secretome. HUVEC were treated with control medium, secretome of ZR-75-30 cells and secretome of ZR-75-30 cells plus neutralizing antibody anti-VEGF (150 ng/ml) for 24 h, and then stained with crystal violet.
