Figure 5: Defective lymphangiogenesis and metastasis in Tnfr1^−/− mice.

(a) Tumour growth rates of TNF-α- and vector-expressing LLC tumours implanted in wt and Tnfr1^−/− mice (n=12 mice per group). Tumour sizes were measured every other day and the experiments were terminated at day 23. (b) Gross appearance and hematoxylin and eosin stain (H&E) histology of TNF-α LLC tumours implanted in wt and Tnfr1^−/− mice. Dashed line marks the border between the necrotic and living tumour tissues. N, necrosis. Scale bar, 100 μm. (c) Tunel staining of TNF-α LLC tumours implanted in wt and Tnfr1^−/− mice and positive signals were quantified (12 randomized fields per group). Scale bar, 100 μm. (d) Primary TNF-α- and vector-expressing LLC tumours implanted in wt and Tnfr1^−/− mice were detected by LYVE-1 (red). LLC tumour cells expressed EGFP (green). Arrowheads point the lymphatic vessels. Scale bar, 100 μm. Quantification of LYVE-1⁺ tumour lymphatics in TNF-α- and vector-expressing LLC tumours implanted in wt and Tnfr1^−/− mice (eight randomized fields per group). (e) Morphology of LNs of vector and TNF-α tumour-bearing wt and Tnfr1^−/− mice. Tumour-free wt mice served as a control. Scale bar, 1 cm. Histological examination of EGFP⁺ metastasis in LNs (n=6 mice per group). Dashed line marks the border between a metastatic nodule and neighbouring LN tissues. T, tumour. Scale bar, 100 μm. Quantification of LN volume and weight in vector- and TNF-α tumour-bearing wt and Tnfr1^−/− mice (n=6 mice per group). (f) CD31⁺ microvessels (red) and EGFP⁺ tumour cells’ (green) double immunostaining of TNF-α- and vector-expressing LLC tumours implanted in wt and Tnfr1^−/− mice. Arrowheads point tumour blood vessels. Scale bar, 50 μm. Quantification of tumour CD31⁺ angiogenic vessels in wt and Tnfr1^−/− mice (n=6 mice per group). (g) Left: representative lung morphology of vector tumour implanted in wt mice and TNF-α tumours implanted in wt and Tnfr1^−/− mice. Scale bar, 1 cm. Quantification of metastasis (n=12 mice per group). Right: lung histology of vector and TNF-α tumour-bearing wt and Tnfr1^−/− mice. Dashed line encircles the metastatic nodules. T, tumour. Scale bar, 100 μm (upper panels); 400 μm (lower panels). *P<0.05; ***P<0.001; NS, not significant. All error bars represent s.e.m. All P values were analysed according to Student’s t-test. d, days.