A 3D Spheroid Model of Tumour Angiogenesis Figure 6. MT1-MMP gene silencing in both HUVECs and fibroblasts decreases endothelial sprout formation. Cells were infected with lentiviral particles expressing 2 different shRNAs against MT1-MMP, selected with puromycin and used to make spheroids. A – Representative images of pre-dyed endothelial cell sprouting from Minitumour spheroids made with HUVECs transduced with the different lentiviral derived shRNAs and controls. B – Quantification of endothelial cell sprouting showing a decrease in sprout formation with HUVECs expressing MT1-MMP shRNAs. C - Western Blots confirming MT1-MMP knock down levels of about 50% in HUVECs, adjusted relative intensity was determined through the ImageJ software follwed by normalization against the loading and mock controls. D – Representative images of pre-dyed endothelial cell sprouting from Minitumour spheroids made with Fibroblasts transduced with different lentiviral derived shRNAs and controls. E – Quantification of endothelial cell sprouting showing a decrease in sprout formation with from Minitumour spheroids with Fibroblasts expressing MT1-MMP shRNAs. F - Western blots confirming MT1-MMP knock down levels of about 30% in the fibroblasts, adjusted relative intensity was determined through the ImageJ software followed by normalization against the loading and mock controls. doi:10.1371/journal.pone.0030753.g006 published data recording the importance of MT1-MMP in angiogenesis in several models, both in vitro and in vivo [57,59,60]. In fibroblasts, knock-down of MT1-MMP (about 30%) also significantly inhibited endothelial sprout formation (Figure 6D–F). However, knock-down of MT1-MMP in the MDA-MB-231 cells did not have a significant effect on endothelial cell sprouting (Figure S4). The expression of this protease, therefore, does not seem to be necessary in the cancer cells to activate tumour angiogenesis. However, the presence of this proteinase in fibroblasts seems to be essential for not only their invasion, but also that of the HUVECs, suggesting a role for the fibroblasts in mediating endothelial cell sprout formation. The observation that a mesenchymal derived proteinase is important for sprouting angiogenesis is a novel one, and reveals the potential of the Minitumour model to identify new targets and mechanisms in tumour angiogenesis. Having identified MT1-MMP as an important target in stromal-mediated tumour angiogenesis, it was subsequently our purpose to investigate if this effect could have repercussions on cancer cell proliferation. To measure changes in cancer cell PLoS ONE | www.plosone.org 10 February 2012 | Volume 7 | Issue 2 | e30753