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Suppression of β3-integrin in mice triggers a neuropilin-1-dependent change in focal adhesion remodelling that can be targeted to block pathological angiogenesis.

Ellison TS, Atkinson SJ, Steri V, Kirkup BM, Preedy ME, Johnson RT, Ruhrberg C, Edwards DR, Schneider JG, Weilbaecher K, Robinson SD - Dis Model Mech (2015)

Bottom Line: Anti-angiogenic treatments against αvβ3-integrin fail to block tumour growth in the long term, which suggests that the tumour vasculature escapes from angiogenesis inhibition through αvβ3-integrin-independent mechanisms.The simultaneous genetic targeting of both molecules significantly impairs paxillin-1 activation and focal adhesion remodelling in endothelial cells, and therefore inhibits tumour angiogenesis and the growth of already established tumours.These findings provide a firm foundation for testing drugs against these molecules in combination to treat patients with advanced cancers.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK.

No MeSH data available.


Related in: MedlinePlus

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-), 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.
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DMM019927F1: 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-), 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.

Mentions: We crossed β3-integrin-wild-type (β3-WT) and β3-HET mice to tamoxifen (OHT)-inducible Pdgfb-iCreERT2/NRP1-floxed mice (Claxton et al., 2008; Gu et al., 2003) and examined the effect of an acute EC-specific depletion of NRP1 (EC-NRP1-KO) on subcutaneous allograft tumour growth with both CMT19T cells (Fig. 1A) and B16F0 cells (supplementary material Fig. S1), as well as on aortic ring sprouting (Fig. 1B). Depleting EC-NRP1 expression in this way had no effect on β3-WT responses, but significantly inhibited tumour growth and VEGF-induced microvessel sprouting in β3-HET mice. Tumour angiogenesis was significantly inhibited in β3-HET mice by depleting EC-NRP1, although vessel morphology and pericyte coverage were normal (Fig. 1C). These studies are reminiscent of the changes observed in β3-KO mice, but, importantly, suggest that NRP1 function is already perturbed by subtle changes in β3-integrin expression levels.Fig. 1.


Suppression of β3-integrin in mice triggers a neuropilin-1-dependent change in focal adhesion remodelling that can be targeted to block pathological angiogenesis.

Ellison TS, Atkinson SJ, Steri V, Kirkup BM, Preedy ME, Johnson RT, Ruhrberg C, Edwards DR, Schneider JG, Weilbaecher K, Robinson SD - Dis Model Mech (2015)

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-), 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.
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DMM019927F1: 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-), 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.
Mentions: We crossed β3-integrin-wild-type (β3-WT) and β3-HET mice to tamoxifen (OHT)-inducible Pdgfb-iCreERT2/NRP1-floxed mice (Claxton et al., 2008; Gu et al., 2003) and examined the effect of an acute EC-specific depletion of NRP1 (EC-NRP1-KO) on subcutaneous allograft tumour growth with both CMT19T cells (Fig. 1A) and B16F0 cells (supplementary material Fig. S1), as well as on aortic ring sprouting (Fig. 1B). Depleting EC-NRP1 expression in this way had no effect on β3-WT responses, but significantly inhibited tumour growth and VEGF-induced microvessel sprouting in β3-HET mice. Tumour angiogenesis was significantly inhibited in β3-HET mice by depleting EC-NRP1, although vessel morphology and pericyte coverage were normal (Fig. 1C). These studies are reminiscent of the changes observed in β3-KO mice, but, importantly, suggest that NRP1 function is already perturbed by subtle changes in β3-integrin expression levels.Fig. 1.

Bottom Line: Anti-angiogenic treatments against αvβ3-integrin fail to block tumour growth in the long term, which suggests that the tumour vasculature escapes from angiogenesis inhibition through αvβ3-integrin-independent mechanisms.The simultaneous genetic targeting of both molecules significantly impairs paxillin-1 activation and focal adhesion remodelling in endothelial cells, and therefore inhibits tumour angiogenesis and the growth of already established tumours.These findings provide a firm foundation for testing drugs against these molecules in combination to treat patients with advanced cancers.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK.

No MeSH data available.


Related in: MedlinePlus