number spheroids were made with MDA-MB-231-luc2 cells, expressing luciferase from the Ubiquitin C promoter, allowing the measurement of changes in cell number using bioluminescence (Figure 7A and B). A sequential dilution of cancer cells was used to establish the linear relationship between cell number and bioluminescence signal (Figure 7A). Nocodazole was used as a positive inhibition control and as expected decreased biolumines- cence significantly (Figure 7C). It should be noted that, due to the presence of cancer cells in the spheroid core, the maximum level of bioluminescence signal reduction detectable is 50%, as seen in the Nocodazole control (Figure 7C). No significant effect on bioluminescence was detected after co-culturing MB231luc21H4 cells in a Minitumour spheroid with MT1-MMP depleted fibroblasts (Figure 7D). This was confirmed by the addition of the broad-spectrum metalloproteinase inhibitor Galardin to the Minitumour spheroids, which also resulted in no significant change in luminescence signal (Figure 7C). Discussion The use of 3D in vitro models for the study of tumour progression is becoming established as a bona fide way to mimic its cellular context, consequently increasing the physiological significance of cell-based assays [24,26,27,61]. The use of multicellular spheroids in particular has become an established way to mimic cellular interactions in the tumour microenviron- ment in a 3D setting when embedded in a biological scaffold A 3D Spheroid Model of Tumour Angiogenesis [27,62,63]. One historical limitation of this approach has been the restriction in cell types included in the spheroid. The published literature mainly contains examples of homotypic cancer cell spheroids, or cancer cells in co-culture with one other type of cell, mostly fibroblasts. This will inevitably mean several processes associated with tumour progression will not be represented in these models, including angiogenesis. Attempts at using multicellular models for tumour angiogenesis studies have included cancer cell spheroid incubation with endothelial monolayers, often resulting in damage to the endothelial cells [64,65], or the measurement of angiogenic factors from spheroid conditioned medium and their use in angiogenic studies [61]. Alternatively 3D models of angiogenesis tend to focus on the process itself, including only endothelial cells or co-cultures with mesenchymal mural cells, and do not include direct contact with a tumour component. In this study, we have developed an in vitro model where stromal-driven angiogenesis can be investigated under the direct influence of the tumour microenvironment. To our knowledge, the Minitumour model represents the first time endothelial cells, fibroblasts and cancer cells are cultured in direct cell-cell contact to activate endothelial tubule formation. After 48 h culture, the fibroblasts are seen to behave as mural cells, as described in the literature [17,22,23,32,33]. The MDA-MB-231 breast cancer cells are shown to induce pre-capillary sprout formation, with or without the addition of exogenous angiogenic growth factors such as VEGF-A and bFGF. These pre-capillary sprouts correspond to early stages of sprouting angiogenesis, Figure 7. Bioluminescence imaging of Minitumour spheroids reveals no difference in cancer cell proliferation with MMP inhibition. A – Quantification of bioluminescence levels from MB231luc21H4 cell dilutions showing a linear relation between cell number and luciferase signal. B – Representative images showing the bioluminescence signals from sequential concentration dilutions of MB231luc21H4 cells. C – Quantification of total luminescence signal of Minitumour spheroids including MDA-MB-231-luc2 after 40 h incubation in collagen-I with galardin, a vector control and Nocodazole as a positive control for proliferation inhibition (p-value,0.05). D – Quantification of bioluminescence signal from Minitumour spheroids made with MB231luc21H4 and fibroblasts expressing lentiviral derived shRNAs for MT1-MMP and non-targeting controls. doi:10.1371/journal.pone.0030753.g007 PLoS ONE | www.plosone.org 11 February 2012 | Volume 7 | Issue 2 | e30753