Fig. 1.

Tumour growth, tumour angiogenesis and microvessel sprouting in β3-integrin-deficient heterozygous mice are sensitive to NRP1 perturbations. (A) Tumour growth was measured in animals of the indicated genotypes. Mice were given subcutaneous injections of CMT19T tumour cells. To generate NRP1-EC-KO (EC-null), 21-day slow-release OHT pellets were administered 3 days prior to tumour-cell injection. OHT-treated Cre-negative (NRP-EC-WT) littermates served as controls. Tumour volumes were measured after 12 days of growth (mean+s.e.m. of three independent experiments; n≥10 animals per genotype). Representative pictures of tumour macroscopic appearances are shown. Scale bar: 10 mm. (B) Microvessel sprouting of aortic ring explants of the indicated genotypes. NRP1-EC-KO was induced in culture with 1 μM OHT. OHT-treated Cre-negative (EC-NRP-WT) rings served as controls. The bar chart shows the total number of microvessel sprouts per aortic ring after 6 days of VEGF-stimulation (mean+s.e.m. from three independent experiments; n≥40 rings per genotype). (C) Blood-vessel density was assessed in tumours of the indicated genotypes by counting the total number of endomucin-positive vessels across tumour sections (mean+s.e.m.; n≥10 sections per genotype over three independent experiments). Representative micrographs of immunofluorescence staining for endomucin, an endothelial cell marker (Endo; red) and CD146, a pericyte marker (green) in tumour sections from each genotype are shown. DAPI (blue) was used as a nuclear counterstain. Scale bar: 100 μm. (D) CMT19T tumour growth and angiogenesis were measured in animals of the indicated genotypes. In addition to their β3-integrin genetic status, mice were negative (NRP1 WT) or positive (NRP1 Δcyto) for the loss of NRP1's cytoplasmic tail. Mice were given subcutaneous injections of CMT19T cells and tumour volumes were measured 12 days later. The bar chart shows tumour volumes (mean+s.e.m. of three independent experiments; n≥10 animals per genotype). (E) Microvessel sprouting of aortic ring explants of the indicated genotypes. The bar chart shows the total number of microvessel sprouts per aortic ring after 6 days of VEGF-stimulation (mean+s.e.m. from three independent experiments; n≥40 rings per genotype). (F) Blood-vessel density was assessed by endomucin (red) and CD146 (green) staining (mean+s.e.m.; n≥10 sections per genotype). DAPI was used as a nuclear counterstain (blue). Scale bar: 100 μm. Asterisks indicate statistical significance: *P<0.05; **P<0.01; ***P<0.001; nsd, not significantly different. Unpaired two-tailed t-test.