Figure 7.

In vivo blocking of integrin α₆ impairs endothelial podosome rosette formation and reduces vessel branching in tumours. (a) Rapid accumulation of anti-α₆ integrin antibody into endothelial podosome rosettes of RipTag2 tumour vessels. xyz-sections of confocal micrographs of the distribution of immunoreactivity in RipTag2 tumours 10 min after intravenous injection of 25 µg of anti-α₆ integrin antibody. Vessels are delimited by white dotted lines; white arrows indicate the podosome rosette. Inset, high magnification of the podosome rosette. Scale bar, 5 µm. (b) Measurements of rosette density in vessels of RipTag2 mouse tumours, treated with rat IgG or anti-α₆ blocking antibody. Mean ± s.e.m. of n = 30 fields, 5 fields per pancreatic islet from 6 mice per treatment group. Statistical significance was calculated using an unpaired non-parametric Mann-Whitney test (**P<0.01 versus rat IgG.). (c) Branching density in blocking antiα₆-treated RipTag2 tumours. Mean ± s.e.m. of n = 30 fields, 5 fields per mouse from 6 mice per treatment group. Statistical significance was calculated using an unpaired non-parametric Mann-Whitney test (*P<0.05 versus rat IgG). (d) Measurements of rosette density in vessels of gastrocnemius muscles from unilateral hindlimb ischaemia experiments in WT (α₆fl/fl-Tie2Cre−) or endothelial α₆ null (α₆fl/fl-Tie2Cre+) mice. Mean ± s.e.m. of n = 9 fields, 3 fields per muscle from 3 mice. Statistical significance was calculated using an unpaired non-parametric Mann-Whitney test (°°P < 0.01 versus normal α₆fl/fl-Tie2Cre−; *P<0.05 versus α₆fl/fl-Tie2Cre+). (e) Measurements of rosette density in vessels of subcutaneous B16-F10 tumours in WT (α₆fl/fl-Tie2Cre−) or endothelial α₆ null (α₆fl/fl-Tie2Cre+) mice. Mean ± s.e.m. of n = 21 fields, 3 fields per tumour from 7 mice per treatment group. Statistical significance was calculated using an unpaired non-parametric Mann-Whitney test (**P< 0.01 versus α₆fl/fl-Tie2Cre−). (f) Branching density in B16F10 melanoma subcutaneously injected in Tie2-dependent α₆ KO mice. Mean ± s.e.m. of n = 42 fields, 5 fields per tumour from 7 mice per treatment group. Statistical significance was calculated using an unpaired non-parametric Mann-Whitney test (**P<0.01 versus α₆fl/fl-Tie2Cre− mice). (g) Cartoon showing α₆ integrin/laminin molecular mechanisms involved in sprouting angiogenesis. (1) Quiescent EC have low levels of α₆β₁ integrin, which binds vBM laminin, is recruited in FAs, and results in blood vessel stabilization. (2) When the tumour produces VEGF, the VEGF induces upregulation of the α₆ integrin subunit in ECs. The increased availability of α₆β₁ integrin then allows the formation and stabilization of endothelial podosome rosettes and the ensuing MMP-driven degradation of ECM that, in turn, (3) allows vBM invasion by ECs and sprouting angiogenesis.