Figure 2: SARI inhibits angiogenesis in vivo and in vitro.

(a–c) Staining for CD31 (red) in control and SARI SW480 tumours. Vessel density (b; mm⁻²; n=5; **P<0.01; Student’s t-test) and vessel area (c; %; n=5; **P<0.01; Student’s t-test) were quantified. Scale bar, 100 μm. (d,e) H&E staining for SARI and control SW480 tumours after tumour cell injection for 25 days (d). Analysis of necrotic area (e, % of tumour area; n=5; **P<0.01; Student’s t-test). Scale bar, 100 μm. (f,g) Staining for pimonidazole (PIMO; green) in SARI and SW480-control tumours (f). Analysis of PIMO area (% of tumour area; g, n=5; **P<0.01; Student’s t-test). DAPI staining for the nucleus. Scale bar, 100 μm. (h,i) Staining for cleaved caspase-3 (green, CC3) and CD31 (red) in SARI and control SW480 tumours (h); apoptotic cells and vessel density were counted (i, n=5; **P<0.01; Student’s t-test). The correlation between CC3 and CD31 was analysed, and the red line indicates the correlation (Pearson correlation analysis). DAPI staining for the nucleus. Scale bar, 50 μm. (j–l) Staining for CD31 (red) in control and SARISW480 Matrigel plugs (j) revealed that SARI inhibits angiogenesis in SW480-matrigel plugs. Vessel density (k; mm⁻²; n=5; **P<0.01; Student’s t-test) and vessel area (l; %; n=5; **P<0.01; Student’s t-test) were quantified. Scale bar, 100 μm. (m) Endothelial tube formation was estimated following incubation of HUVECs with conditioned medium from SW480-control and SARI cancer cells. The number of branches were quantified (n=5; **P<0.01; Student’s t-test). Scale bar, 50 μm. (n) Representative photomicrographs of HUVECs that have invaded through Matrigel chambers after incubation with conditioned medium from SW480-control and SARI cancer cells for 72 h. Scale bar, 50 μm. Quantification of HUVECs that have invaded through Matrigel chambers (n=5; **P<0.01; Student’s t-test). Ctrl, control.