Fig. 6: Combination treatment using antiVEGFA and antiCCL2 synergistically inhibits tumor growth and angiogenesis in CRC.

a Representative images of the formation of HUVEC tubules following incubation with supernatants collected from the indicated cells and treatment with recombinant human VEGFA and CCL2 protein or Bev and antiCCL2. Data are presented as mean ± SD of three independent experiments. b HUVECs were incubated with the indicated supernatants and treated with recombinant human VEGFA and CCL2 protein or Bev and antiCCL2. Activation of the VEGFR downstream signaling pathway was determined according to western blotting. GAPDH was used as a loading control. c The CAM assay was used to examine effect of a combination treatment using recombinant human VEGFA and CCL2 protein or a combination of Bevacizumab and antiCCL2 on blood vessel formation after stimulation with the supernatants from the indicated cells. Data are presented as mean ± SD of three independent experiments. d Tumor volumes were calculated to measure tumorigenesis ability of RKO/ETV5 cells after receiving antiVEGFA or/and antiCCL2 treatment. e Images of xenografted tumors derived from RKO/ETV5 cells in every group. The average tumor weight for each group (n = 5) was calculated. f Representative images of HE stains and IHC staining for CD31 and ki67 in subcutaneous tumor tissue in the RKO/ETV5, RKO/ETV5+Bev, and RKO/ETV5+Bev+anti-CCL2 groups. Bev Bevacizumab. “*” represents in comparison with the control. **p < 0.01, ***p < 0.001, ****p < 0.0001. ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001.