Figure 1.
Anti-tumor activity of LC06 and LC08 in s.c. Colo205 and orthotopic KPL-4 xenograft tumors.
(A) Colo205 tumor (mean 100 mm3) growth curves in female SCID beige mice receiving LC06 and LC08 (10 mg/kg) once weekly i.p. (n = 10, *p<0.0001, Student's t-test). (B) Total Colo205 tumor weight in tumor bearing mice treated with LC06 and LC08 (n = 10, *p<0.0001 compared to control, Student's t-test). (C) KPL-4 orthotopic tumor (mean 90 mm3) growth curves in mice receiving LC06 and LC08 (10 mg/kg) once weekly i.p. (n = 10, *p<0.05 compared to control, Student's t-test). Error bars represent ± SEM. Arrows indicate start of treatment. The results were confirmed in 3 independent experiments.
Figure 2.
Effect of LC06 and LC08 treatment on MVD (A and B), vascular coverage (C), vessel area (D), perfusion (E) and vessel branching (F) in Colo205 tumors.
(A) Representative pictures of CD34-stained Colo205 tumors (mean 800 mm3). (B) Quantitative analysis of tumor microvessel density (MVD). MVD was quantified in CD34-stained whole tumor slides. LC06 and LC08 treatment led to a significant reduction of intratumoral MVD (n = 5, *p<0.05 compared to control, Student's t-test). (C) Quantification of vessel coverage calculated as the percentage of desmin-positive vessels in relation to CD34-positive endothelial cells staining in six random regions of 1000×1000 µm per tumor slide. LC06 and LC08 treated tumors showed increased vessel coverage by desmin positive pericytes (n = 5 mice per group, *p<0.05 compared to control, Student's t-test). (D) The average vessel area of intratumoral microvessels were significantly reduced in tumors treated with LC06 and LC08 (n = 5, *p<0.05 compared to control, Student's t-test). (E) Perfusion was assessed based on analysis of TRITC-lectin perfusion and CD34-positive staining in six random regions of 1000×1000 µm per tumor slide. Almost all remaining intratumoral microvessels were perfused in LC06 (93%) and LC08 (97%) treated tumors compared to control (56%) (n = 5 mice per group, *p<0.01). (F) Number of branched intratumoral microvessels was counted in six random regions of 1000×1000 µm per tumor slide and calculated per mm2 and was significantly reduced in tumors treated with LC06 and LC08. LC06 treatment resulted in stronger inhibition of vessel branching compared to pan-Ang-2/-1 treatment via LC08 (n = 5, *p<0.01 compared to control; #p<0.01 compared to LC08, Student's t-test). Results are expressed as mean ± SEM. Scale bars represent 500 µm.
Figure 3.
Tumor necrosis in LC06 and LC08 treated Colo205 tumors.
(A) Quantitative image analysis shows 27% of necrotic area in vehicle treated, 51% in LC06 treated and 43% in LC08 treated tumors (n = 5, *p<0.05 compared to control, Student's t-test). Results are expressed as mean ± SEM. Two independent experiments were performed to confirm the results. (B) Representative mosaic images (10x) of vehicle, LC06 or LC08 treatments. Scale bar represents 1.3 mm.
Figure 4.
Tumor cell dissemination to the lungs in orthotopic KPL-4 xenograft tumors after LC06 and LC08 treatment.
Treatment with LC06 and LC08 resulted in a significant reduction of disseminated tumor cells to the lungs (n = 10, *p<0.05 compared to control, Student's t-test). The results were confirmed in two independent experiments. Results are expressed as mean ± SEM.
Figure 5.
Effect of LC06 and LC08 on quiescent vessels.
(A) Representative immunofluorescent pictures of CD31 stained tracheal whole mount sections of mice treated with control IgG1, LC06 and LC08. (B) Quantification of capillary branching points (in five random regions of 230×520 µm in each mouse whole mount trachea) reveals that pan-Ang-1/Ang-2 inhibition but not selective anti-Ang-2 treatment induced tracheal vessel regression (n = 3–5; *p<0.05 compared to control, Student's t-test). The results were confirmed in two independent experiments. Results are expressed as mean ± SEM. Scale bar represents 100 µm.
Figure 6.
Effect of LC06 and LC08 treatment on VEGF-induced corneal angiogenesis.
(A) Angiogenesis was induced by implanting VEGF (300 ng) or vehicle soaked nylaflo discs into the cornea of BALB/c mice. PBS control did not induce vessel outgrowth from the limbus to the disc compared to VEGF positive control. LC06, LC08 and bevacizumab were injected with 10 mg/kg i.v. at the day of disc implantation. Treatment with both anti-Ang-2 antibodies significantly inhibited neoangiogenesis in a comparable range as bevacizumab (n = 5, *p<0.001 compared to VEGF control, Student's t-test). (B) Representative pictures of vehicle, LC06 and LC08 treated mice. The results were confirmed in 3 additional independent experiments. Results are expressed as mean ± SEM.