Fig 1: ETV5 induces Bevacizumab resistance in CRC in vivo.a The CAM assay was used to examine effect of Bevacizumab and VEGFA on blood vessel formation after stimulation with the supernatants from the indicated cells. Data are presented as mean ± SD of three independent experiments. b Tumor volumes were calculated to measure tumorigenesis ability. c Images of xenografted tumors after subcutaneous injection of mice with the indicated cells (n = 5). d The average tumor weight for each group was calculated. e Representative images of HE staining and IHC staining for CD31 and VEGFA in subcutaneous tumor tissues of the RKO/Vector, RKO/Vector+Bev(2 mg/kg), RKO/ETV5, and RKO/ETV5+Bev(2 mg/kg) groups. “*” represents in comparison with the control. Bev Bevacizumab. **p < 0.01, ***p < 0.001, ****p < 0.0001. ##p < 0.01, ###p < 0.001, ####p < 0.0001.
Fig 2: Tumor cell-derived CCL2 partially contributes to ETV5-mediated angiogenesis in CRC.a ELISA analysis of CCL2 and VEGFA secretion after treatment with recombinant human VEGFA, CCL2, Bev, or anti-CCL2 in ETV5 downregulated/overexpressing cells. Bev Bevacizumab. b Representative images of the formation of HUVEC tubules following incubation with supernatants collected from the indicated cells and treatment with recombinant human CCL2 protein or CCL2 antibody. c The CAM assay was used to examine the effect of recombinant human CCL2 protein or CCL2 antibody on blood vessel formation after stimulation with the supernatants from the indicated cells. Data are presented as mean ± SD of three independent experiments. “*” represents in comparison with the control. **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 3: ETV5, VEGFA, and CCL2 show positive expression correlations with angiogenesis and are positively correlated with poor prognosis in CRC.a Representative images of IHC staining for ETV5, VEGFA, CCL2, and CD31 in the 75-patient cohort. b Expression of ETV5, VEGFA, CCL2, and CD31 was up-regulated in CRC tissues compared to levels in normal tissues (all p < 0.0001). c Expression of ETV5 was positively related to expression of VEGFA (p < 0.0001), CCL2 (p = 0.0342), and CD31 (p < 0.0001) in CRC tissues. d VEGFA (p < 0.0001) and CCL2 (p = 0.0002) were significantly associated with CD31 in CRC tissues. e, f Disease-free survival (DFS) and overall survival (OS) curves of the 75 patients in the cohort, as stratified by ETV5 and VEGFA expression patterns or by ETV5 and CCL2 expression patterns. *p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 4: Schematic of ETV5-mediated anti-VEGF therapy resistance in ETV5+ CRC.In CRC, ETV5 could promote angiogenesis via the secretion of VEGFA and CCL2. When ETV5+ CRC was treated with Bevacizumab, paracrine CCL2 could induce angiogenesis by activating the MAPK and AKT pathways in human umbilical vein endothelial cells, ultimately resulting in Bevacizumab resistance. Therefore, a combination using Bevacizumab and antiCCL2 treatment could synergistically suppress angiogenesis by simultaneously neutralizing ETV5-induced VEGFA and CCL2 in CRC, and this combined therapy might provide promising anti-angiogenic strategy for ETV5+ CRCs.
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