Figure 4
From: Cell Spheroids with Enhanced Aggressiveness to Mimic Human Liver Cancer In Vitro and In Vivo
HUVECs promote proliferation and invasion by activation of MAPK pathway via cytokine-mediated signalling in vitro. (a) Distribution of 2% of HUVECs in the spheroid was analysed using ICC images of middle and bottom sections of cell spheroids co-cultured with 2% of GFP-tagged HUVECs. Scale bars, 200 μm. (b) ELISA of VEGF secretion in culture supernatant in spheroids co-cultured with the indicated concentrations of HUVECs as compared with Huh7 spheroids cultured alone, at 120 h. (c) Human cytokine array analysis of Huh7 spheroids cultured alone, with 2% HUVECs, and with 20% HUVECs. (d) Huh7-3D or Huh7/2% HUVEC-3D were transferred as single spheroids into each well of 96-well plate and treated with serum-free DMEM containing VEGF-neutralizing antibody (5, 10, 30 µg/ml) for 72 h. Spheroid areas were quantitated using ImageJ software before adding VEGF antibody (0 h) and after 72 h. Error bars display the standard deviation of five independent measurements. (e) At 72 h after VEGF antibody treatment, spheroids of each experimental group were harvested and analysed by western blotting with the indicated antibodies. (f) Huh7-3D or Huh7/2% HUVEC-3D were embedded into Matrigel in a 24-well plate and treated with serum-free DMEM containing 30 µg/ml VEGF-neutralizing antibody for 5 days. Mouse IgG isotype control antibody (30 µg/ml) was used as negative control. Representative images of each group are shown. (g) The sprouting area per experimental group was quantitated with ImageJ. Error bars display the standard deviation of six independent measurements. *P < 0.05; ***P < 0.001.
