Figure 3

HUVEC migration, proliferation and tubulogenesis are suppressed by VEGF neutralizing antibody. Conditioned medium generated by culturing ML-1 thyroid cancer cells with ± 10^-8 M E₂ ± 10^-6 M fulvestrant was incubated with 1 μg of anti-human VEGF neutralizing antibody per mL of the conditioned media at 37°C for one hour. (A) HUVECs were allowed to migrate towards thyroid cancer cell conditioned medium with or without the VEGF neutralizing antibody used as chemoattractant. Data expressed as numbers of HUVECs counted (migrated cells) per 10X field micrograph for each sample well and normalized to the untreated control. The asterisk denotes statistically significant differences (p < 0.05) between experimental and untreated samples determined using a paired Student’s t - test. (B) HUVECs were cultured in presence of thyroid cancer cell conditioned medium followed by trypan blue exclusion cell count to calculate endothelial cell proliferation. Data expressed as % of HUVECs cell number normalized to the untreated control (no neutralizing antibody) set as 100%. The asterisk denotes statistically significant differences (p < 0.05) between experimental and untreated samples determined using a paired Student’s t - test. (C) HUVECs were seeded in ML-1 thyroid cancer cell conditioned medium with or without the VEGF neutralizing antibody on matrigel and monitored for tubulogenesis. The results are representative of three independent experiments performed in triplicates.