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. 2020 Nov 24;8(2):e001435. doi: 10.1136/jitc-2020-001435

Figure 1.

Figure 1

Dose-dependent effects of regorafenib on vascular normalization when used in combination with programmed cell death protein 1 (PD1) blockade in RIL-175 hepatocellular carcinoma (HCC) model. (A, B) Mice with established (4–5 mm in diameter) HCCs were treated with anti-PD1 therapy (P) alone or in combination with 5 mg/kg (R5), 10 mg/kg (R10) or 20 mg/kg (R20) regorafenib versus control (C) (n=8 mice per group, 1-week treatment). We used immunofluorescence (IF) for evaluation of tumor vessels by CD31 and α-smooth muscle actin (α-SMA) immunostaining. Quantitative analysis of IF data showed increased pericyte coverage only after R10+P treatment (plots in B). Representative IF of tumor sections after staining for CD31 (for endothelial cells in green) and α-SMA (for pericytes in red) and DAPI counterstaining (in blue). Five random fields from the tumor center (defined as no inclusion of the edge of the tumor) were evaluated for each sample. Data represent mean values. Scale bars=500 µm. (C) Representative IF confocal microscopy images of tumor vessels using and CD31 staining (green) and tissue hypoxia using CA-IX staining (red) of whole liver sections; scale bars=2 mm. (D) Tissue hypoxia was significantly reduced by 10 mg/kg regorafenib treatment alone or with anti-PD1 blockade (n=5 mice, 12 days of treatment, 5 images/tumor sample). (E) Representative IF confocal microscopy images of mature tumor vessels using CD31 staining (green) and α-SMA staining (red) of RIL-175 tumors; scale bars, 100 µm. (F) CD31+α-SMA+ mature vessel density was significantly increased in the combination treatment group (n=5 mice, 12 days of treatment, images/tumor sample). Data represent mean±SEM. *P<0.05; **p<0.01; ***p<0.001.