Fig 2: CHMFL-VEGFR2-002 inhibits VEGFR2 activity. (A) IC50 determination of CHMFL-VEGFR2-002 with purified VEGFR2 kinase protein using the ADP-Glo assay. (B) GI50 determination of CHMFL-VEGFR2-002 with TEL-VEGFR2-BaF3 cells using the CCK8 assay. (C) EC50 determination of CHMFL-VEGFR2-002 on auto-phosphorylation of VEGFR2 in TEL-VEGFR2-BaF3 cells. (D) The cellular on-target effect of CHMFL-VEGFR2-002 using the Cellular Thermal Shift Assay. (E) Docking of CHMFL-VEGFR2-002 into VEGFR2 kinase X-ray crystal structure (PDB ID: 4AG8).

Fig 3: Schematic flow diagram of the study design and method. After surgery and tissue processing of OPSCC samples, the HPV-status was determined by GP5+/GP6+ polymerase chain reaction (PCR) and p16INK4A-immunohistochemistry (IHC). To test the specificity of the VEGFR2-antibodies, liver, melanoma, papillary thyroid carcinoma, and cervix squamous cell carcinoma tissues were selected as positive controls. First, hematoxylin and eosin (H&E) staining was performed to determine the regions relevant for further analyses. This was followed by staining of consecutive sections with VEGFR2-AB using the avidin-biotin-peroxidase complex (ABC). Subsequently, densitometric analyses of the VEGFR2-staining intensity of blood vessels in tumor regions and tumor-free regions, as well as tumor cells, were performed. The number of VEGFR2-expressing capillaries in a defined microscopic field was counted and statistically analyzed. Consecutive sections of OPSCC were selected for double immunofluorescence analysis to show colocalization of VEGFR2 and CD31 in endothelial cells (EC), to show the localization of VEGFR2 in tumor cells (TC) by double staining for p16INK4A- (in HPV-positive tumors) or for p53 (in HPV-negative tumors). Confocal double immunofluorescence analysis was performed for VEGFR2 and the stem cell marker ALDH1A1.

Fig 4: CBZ intravitreal injection alleviates CNV leakage and the CNV lesion area and downregulates the phosphorylation of MET and VEGFR2. (a) Schematic diagram of the experimental procedure. The mice were left untreated (with CBZ treatment, negative control) or were treated (control group) with a laser at day 0. At day 3, 0.1% DMSO (vehicle group), 2 μg/μl CBZ (CBZ group), or 10 μg/μl RBZ (RBZ group) (1 μl for each) was injected intravitreally. The mouse eye tissues were collected and analyzed at day 7. (b) FFA was performed, and (e) fluorescein leakage in CNV lesions was graded at 7 d after CNV in negative control (n = 30 lesions), control (n = 32 lesions), vehicle (n = 30 lesions), CBZ (n = 32 lesions), and RBZ (n = 32 lesions) groups. Scale bar = 200 μm. (c) ICGA was performed in the negative control (n = 30 lesions), control (n = 32 lesions), vehicle (n = 30 lesions), CBZ (n = 32 lesions), and RBZ (n = 32 lesions) groups. Scale bar = 200 μm. (d, f) The mouse CNV lesion area at 7 d after CNV induction was assessed by the staining of choroidal flat-mounts with fluorescent IB4 in the negative control, control, vehicle, CBZ, and RBZ groups. Scale bar = 100 μm. n = 8/group. ∗∗P < 0.01 vs. the control group. (g) The HGF, p-MET, MET, VEGF, p-VEGFR2, and VEGFR2 protein levels in the negative control, control, vehicle, CBZ, and RBZ groups were detected by western blotting. (h) Quantification of the HGF, p-MET, VEGF, and p-VEGFR2 protein levels in each group. n = 6/group. ∗∗∗P < 0.005 vs. the negative control group; ##P < 0.01 and %%P < 0.01 vs. the control group.

Fig 5: Specific blockade of VEGF-A activation of VEGFR2 suppresses tumor lymphangiogenesis, lymphatic endothelial cell proliferation/viability and migration. A: Intratumoral lymphatics were identified by immunofluorescence staining of frozen sections of control IgG and r84 treated MDA-MB-231 tumors for the lymphatic marker LYVE-1 (red). Scale bars = 100 µm. B: The entire area of each LYVE-1 stained tumor section was examined at low magnification and the percent of LYVE-1 positive area was determined for each field using NIS-Elements imaging software. Ten fields with the highest LYVE-1 positive percent area were averaged together to yield a final score for each tumor and group means were tested for significance by an unpaired student's t-test. The percent of LYVE-1 positive area of control tumors (7.03±1.013) was significantly greater than r84 treated tumors (2.23±0.986). Asterisk = P = 0.0042.