Author contributions A.C., S.L. and C.B. designed the experiments and performed data analysis. A.C. and S.L. performed most of the biological experiments. E.T. performed the experiments and data analysis on prostate glands. A.Sifrim., M.M., Y.S., J.V.H. and T.V. performed the bioinformatic analysis. A.D. and G.B. provided technical help. C.D. performed FACS experiments. N.D. provided technical help with single-cell RNA sequencing. A.C., S.L., M.F., A.W. and A.V.K. performed immunostainings, blocking antibodies and small-molecule treatments and experiments with follow-up mice. A.Sahay contributed genetic tools. V.d.M. performed statistical analysis. C.W.S. provided the Notch antibodies. A.C., A.V.K. and C.B. wrote the manuscript. All authors read and approved the final manuscript.

Notch signalling is a causal determinant of cancer and efforts have been made to develop targeted therapies to inhibit the so-called canonical pathway. Here we describe an unexpected pro-apoptotic role of Notch3 in regulating tumour angiogenesis independently of the Notch canonical pathway. The Notch3 ligand Jagged-1 is upregulated in a fraction of human cancer and our data support the view that Jagged-1, produced by cancer cells, is inhibiting the apoptosis induced by the aberrant Notch3 expression in tumour vasculature. We thus present Notch3 as a dependence receptor inducing endothelial cell death while this pro-apoptotic activity is blocked by Jagged-1. Along this line, using Notch3 mutant mice, we demonstrate that tumour growth and angiogenesis are increased when Notch3 is silenced in the stroma. Consequently, we show that the well-documented anti-tumour effect mediated by γ-secretase inhibition is at least in part dependent on the apoptosis triggered by Notch3 in endothelial cells.