Figure 6: Anti-VEGFR3 blocks TNF-α-lymphangiogenesis and metastasis.

(a) Confocal images of VEGF-C- and TNF-α-overexpressing LLC tumours. VEGF-C and TNF-α tumour-bearing mice were treated with an anti-VEGFR3 neutralizing antibody. Peri- and intratumoral lymphatics (PTLs and ITLs) were detected with LYVE-1 (red), and tumour cells expressed EGFP (green). Dashed line marks borders between tumour and neighbouring tissues. Scale bar, 100 μm. Quantification of LYVE-1⁺ lymphatic vessels in vehicle- and VEGFR3 blockade-treated VEGF-C and TNF-α tumours. Quantification data were obtained from eight randomized fields per group. (b) Confocal images of lymphatic vessels in VEGF-C- and TNF-α-overexpressing LLC tumours. Impairment of lymphatic tips of VEGF-C- and TNF-α-induced tumour lymphatic vessels by VEGFR3 blockade treatment. White arrowheads indicate sprouting lymphatic tips; yellow arrowheads indicate the ‘mushroom-like’ lymphatic structure without sprouting tips often seen in VEGFR3 blockade-treated TNF-α tumours. Scale bar, 100 μm. Quantification of lymphatic tips in vehicle- and VEGFR3 blockade-treated VEGF-C and TNF-α tumours. Quantification data were obtained from eight randomized fields per group. (c) Left: morphology (scale bar, 1 cm) and histological examination (scale bar, 100 μm) of LN. LNs of vehicle- and VEGFR3 blockade-treated TNF-α tumour-bearing mice were examined. Dashed line marks the border between a metastatic nodule and neighbouring LN tissues. T, tumour. Right: quantification of LN volume and weight of vehicle- and VEGFR3 blockade-treated TNF-α tumour-bearing mice (n=6 mice per group). *P<0.05; ***P<0.001. All error bars represent s.e.m. All P values were analysed according to Student’s t-test. H&E, hematoxylin and eosin stain.