KI of the BRAFV600E allele in hTERT-HME1 cells enhances angiogenesis in CAM assay and increases VEGF-mediated migration of ECs. (A) Representative images of hTERT-HME1 cells plated on the CAM (a and b). (Scale bar: 1 mm.) Immunofluorescence analysis of hTERT-HME1 cells plated on the CAM. Human epithelial cells were stained with the HLA marker (green) whereas blood vessels were labeled with the vWF, which is specific for ECs (red) (c–f). Arrows indicate blood vessels. (Scale bar: 100 μm.) (B) Qualitative assessment of angiogenesis (angiogenic index) in the CAM assay (Materials and Methods). Bars represent the number of CAM samples that exhibit low, medium, or high angiogenesis in parental or BRAF^V600E mutant cells. Measurements were performed by two observers in blinded fashion. (C) Quantitative analysis of vWF intensity fluorescence mean in hTERT-HME1 plugs. Data are expressed as fold change mean ± SEM (^§P < 0.05, Student t test; WT hTERT-HME1, n = 6; BRAF^V600E KI hTERT-HME1, n = 9 samples). (D) Quantification of secreted VEGF-A in hTERT-HME1 cells by ELISA. Data are expressed as mean ± SD of a technical replicate and are representative of three independent experiments. (E) Migration of human ECs is enhanced by the supernatant of hTERT-HME1 BRAF^V600E KI cells compared with the WT counterpart. Two different clones of BRAF^V600E KI were analyzed (KI-A and KI-B). SF, serum free medium. VEGF blocking antibody (VEGF-Ab), but not unrelated Ig, abolished BRAF^V600E -induced cell migration. Bars represent the mean ± SEM of the number of migrated cells of three samples (^§P < 0.05, Student t test). The representative of three independent experiments is shown.

PLX4720 inhibits proliferation and turns off MAPK signaling in tumor cells carrying BRAF^V600E. (A) Proliferation of COLO205 and SK-MEL-28, harboring BRAF^V600E, and BRAF WT DiFi cells, was assessed with increased PLX4720 concentration. Data are expressed as percentage of viability compared with vehicle by the ATP assay. Mean ± SD of two independent experiments performed in quadruplicate is shown. (B) Biochemical analysis of phospho-ERK and phospho-AKT (Ser473 and Thr308) in COLO205, SK-MEL-28, and DiFi cells. Protein loading was normalized by vinculin. Starved cells were stimulated for 30 min with 2.5% FBS plus 1 μM PLX4720 or vehicle. (C) Tumor growth curve of COLO205 and SK-MEL-28 xenografts. COLO205 and SK-MEL-28 xenografts were treated with PLX4720 60 mg/kg or vehicle for 2 wk after tumor volume reached an average of 150 to 200 mm³. Arrows indicate the time point at which treatment was started. Data are presented as mean tumor volume ± SEM. (D) Representative images of COLO205 tumors (a and b). (Scale bar: 4 mm.) Representative images of Ki-67 staining in COLO205 (c and d) and SK-MEL-28 (e and f) xenografts. (Scale bar: 100 μm.) (E) Quantification of proliferating cells by Ki-67 staining in COLO205 and SK-MEL-28 xenografts. Bars show the mean of positive cells per 10× field ± SD (^§P < 0.0001, Student t test). For COLO205, vehicle, n = 4 mice; PLX4720, n = 6 mice; for SK-MEL-28, vehicle, n = 4 mice; PLX4720, n = 5 mice. (F) Biochemical analysis of cleaved caspase 3 in protein extract of COLO205 and SK-MEL-28 xenografts. Protein loading was normalized by vinculin. At least four independent samples were evaluated.

PLX4720 treatment stabilizes blood vessels in COLO205 and SK-MEL-28 xenograft models. (A) Representative images of immunofluorescence analysis with the CD31 endothelial marker (green) in COLO205 (a–c) and SK-MEL-28 (d–f) tumors. (Scale bar: 100 μm.) (B) Percentage of surface area occupied by vessels quantified by CD31 staining. Bars show mean ± SEM. (^§§P < 0.001 and ^§P < 0.01, Student t test). For COLO205, start point, n = 8 mice; vehicle, n = 9 mice; PLX4720, n = 14 mice; for SK-MEL-28, start point, n = 6 mice; vehicle, n = 5 mice; PLX4720, n = 5 mice. (C) Quantification of blood vessel lumen diameter. Red bars represent the median for each group (^§§P < 0.0001, Student t test). Dots indicate the individual vessels measured for the indicated cell lines. (D) Representative images of colocalization analysis between perfused beads (red) and CD31 (green) in COLO205. (Scale bar: 50 μm.) Quantification is shown as mean ± SEM of colocalization area (in μm²; ^§§P < 0.01 and ^§P < 0.05, Student t test). COLO205, start point, n = 39 blood vessels; vehicle, n = 30 blood vessels; PLX4720, n = 35 blood vessels. S.p., start point; V, vehicle; P, PLX4720.

PLX4720 treatment abolishes tumor hypoxia and decreases necrosis in COLO205 and SK-MEL-28 xenograft models. Histological analysis of COLO205 and SK-MEL-28 xenografts before and after PLX4720 treatment. (A) Representative images of pimonidazole and H&E staining in COLO205 (a–c and d–f) and SK-MEL-28 (g–i and j–l) tumors. (Scale bar: 100 μm.) (B) Quantification of the hypoxic area (percentage) by pimonidazole staining. Bars show mean ± SEM (^§§P < 0.005 and ^§P < 0.05, Student t test). For COLO205, start point, n = 7 mice; vehicle, n = 8 mice; PLX4720, n = 8 mice; for SK-MEL-28, start point, n = 6 mice; vehicle, n = 5 mice; PLX4720, n = 5 mice. (C) Quantification of necrotic area after H&E staining in COLO205 and SK-MEL-28 xenografts. Bars represent the percentage mean area ± SEM (^§§P < 0.01 and ^§P < 0.05, Student t test). For COLO205, start point, n = 8 mice; vehicle, n = 9 mice; PLX4720, n = 14 mice; for SK-MEL-28, start point, n = 6 mice; vehicle, n = 5 mice; PLX4720, n = 5 mice.

Effect of bevacizumab on tumor growth and vasculature. COLO205 xenografts were treated with bevacizumab 10 mg/kg or vehicle for 2 wk after tumor volume reached an average of 150 to 200 mm³. (A) Tumor growth curve of COLO205; arrow indicates time point at which treatments were started. Data are presented as mean tumor volume ± SEM of five mice. (B) Representative images of H&E (a and b) and pimonidazole staining (c and d) in COLO205. (Scale bar: 100 μm.) Colocalization analysis between perfused beads (red) and CD31 (green) in COLO205 (e–h). (Scale bar: 50 μm.) (C) Percentage of surface area occupied by vessels quantified by CD31 staining. Bars show mean ± SEM (^§§P < 0.01, Student t test). Vehicle, n = 5 mice; bevacizumab, n = 6 mice. (D) Analysis of colocalization area between perfused beads and CD31. Quantification is shown as mean ± SEM of colocalization area (in μm²; ^§§P < 0.01). Vehicle, n = 35 blood vessels; bevacizumab, n = 15 blood vessels. (E) Quantification of the percentage hypoxic area by pimonidazole staining. Bars show mean ± SEM. Start point, n = 4 mice; vehicle, n = 5 mice; bevacizumab, n = 6 mice. (F) Quantification of necrotic area by H&E staining. Bars represent the percentage mean area ± SEM. Start point, n = 4 mice; vehicle, n = 5 mice; bevacizumab, n = 6 mice.

PLX4720 treatment down-regulates the expression of proangiogenic factors. (A) Expression of proangiogenic factors was evaluated by real-time PCR. Cells carrying BRAF^V600E, COLO205, and SK-MEL-28, and BRAF WT DiFi cells were treated for 24 h with PLX4720. Data are expressed as relative quantity (RQ) of PLX4720 compared with vehicle-treated samples. Bars show mean ± SD of triplicate measurements. (B) Quantification of secreted VEGF-A in cells supernatant by ELISA after 24 h of the indicated treatments. Data are expressed in pg/mL and bars represent mean ± SEM of duplicate observations. The representative of two experiments is shown. (C) Biochemical analysis of phospho-p90RSK1, -eIF4E, and -4EBP1 in COLO205, SK-MEL-28, and DiFi cells. Protein loading was normalized by vinculin. Starved cells were stimulated for 30 min with 2.5% FBS plus 1 μM PLX4720 or vehicle.

Drug inhibition of oncogenic (i.e., cancer-causing) BRAF inhibits tumor hypoxia (i.e., oxygen deprivation) by stabilizing vascular architecture and improving the perfusion of nutrients to tissues. PLX4720 affects proangiogenic program in cancer cells carrying BRAF^V600E by turning off the MAPK signaling pathway.