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

Figure 3.

Figure 3

Combined treatment with regorafenib with antiprogrammed cell death protein 1 (anti-PD1) antibodies inhibits signal transducer and activator of transcription (STAT) activation and increases CXCL10 production by hepatocellular carcinoma (HCC) cells. (A) Changes induced by combination of regorafenib (10 mg/kg) with anti-PD1 antibody in immune-related pathways from the gene set enrichment analysis (GSEA) using curated gene sets in RIL-175 HCC model (see also online supplemental dataset S1 and figure S2E). (B, C) Changes induced by combination of regorafenib (10 mg/kg) with anti-PD1 antibody in STAT3 and STAT1 activity (B), showing an increased ratio of phosphorylated (p)-STAT1/p-STAT3 (normalized to total STAT1 and STAT3, respectively) by western blot analysis of whole RIL-175 HCC tissue (C) (n=4 mice). (D) Treatment with regorafenib increases CXCL10 expression by RIL-175 HCC cells in vitro after 36 hours, in a dose-dependent manner (n=6). Culture medium alone was used as a negative control (NC). (E, F) Changes induced by regorafenib (1 µM) in STAT3 and STAT1 activity (E), showing an increased ratio of p-STAT1/p-STAT3 (normalized to total STAT1 and STAT3, respectively) by western blot analysis of RIL-175 HCC cells (F). (G, H) Representative in situ hybridization (ISH) pictures of CXCL10 (red dots, arrow heads) and CXCR3 (green dots) on RIL-175 HCC tissue from the control group (G) versus combination therapy (RP) group (H). Scale bars=50 µm (G, H). (I) Combination therapy significantly increases in the number of HCC cells expressing CXCL10 compared with control. Data represent mean±SEM. (J, K) Combination therapy increases CXCL10 protein expression in whole RIL-175 tumor tissue 6 hours and 12 hours after in vivo when regorafenib treatment was used at doses of 10 mg/kg (R10) and 20 mg/kg (R20) (n=3) by western blot analysis (J); quantitative data of CXCL10/β-actin ratios are shown in (K). C, control; P, anti-PD1 antibody; R, regorafenib 10 mg/kg; R10-P, regorafenib 10 mg/kg+anti-PD1 antibody; n=3–4 per group. *P<0.05; **p<0.01; ***p<0.001.