without observed widespread lumen formation. However, spher- oid culture for longer periods of time leads to the development of networks of capillary structures with lumen formation, as confirmed through the use of electron microscopy. Incubation in type-I collagen provides a controllable 3D milieu for spheroid incubation. The spheroid components are also shown to produce other matrix components they require for their invasion and sprout formation. Culturing for longer periods of time results in the formation of a more complex capillary-like network structure by the endothelial cells, with extensive matrix remodelling, within a homogenous scaffold of cancer cells and fibroblasts. 3D in vitro culture systems have been shown to reflect the in vivo response to therapeutic agents more accurately than conventional cell culture systems [27,61]. We have demonstrated that both functional blocking antibodies and small molecule inhibitors can be used in our model. This allows for detailed studies into the role of different proteins and signalling pathways in endothelial sprout formation in a 3D environment. It also suggests its suitability as a platform for testing potential therapeutic agents. Minitumour spheroids’ response to growth factor inhibitors and anti-angiogenic compounds correlates with the existing literature, showing dependence on a number of signalling pathways known to be important for tumour angiogenesis in vivo. These results can be obtained in a short time frame with high reproducibility, and indicate the Minitumour spheroid is a relevant model of the early stages of tumour angiogenesis. This model could therefore prove useful not only for studies into the mechanism of sprout formation, but also for preliminary studies of angiogenic inhibitors with therapeutic potential. In future, Minitumour spheroids could be developed into a high throughput format, maximising their usefulness as a drug-screening tool. This could be achieved with the use of liquid handling technology in order to keep the spheroids in a 96 well format during collagen incubation. The use of an automated imaging system could then enable the use of this model for high content screening of anti-angiogenic agents. This would be of particular interest since the need for physiologically relevant screening assays that take the third dimension into account has been identified as one of the current challenges in cell biology [27]. Of particular interest is the model’s response to Endostatin and Thalidomide. The addition of these compounds to Minitumour spheroids resulted in decreased inhibition of capillary sprout formation, suggesting the model could be used to investigate mechanisms of tumour resistance to these anti-angiogenic inhibitors. Data obtained using the gamma secretase inhibitor DAPT is also of relevance. It could open new avenues of investigation in the importance of heterotypical notch signalling in tumour angiogenesis, as this pathway has also been shown to be important in the communication between endothelial and mural cells, for example through activation of Notch3 in mural cells by endothelial cell-expressed Jagged-3 [33]. Using the Minitumour model, this mechanism could be studied in further depth, as mural and endothelial cells can be manipulated individually, leading to a better understanding of the relative importance of the notch-delta/ jagged components involved the different compartments in the regulation of sprout formation. A strong asset of this model is the fact that all separate components can be manipulated independently using common molecular techniques to dissect mechanisms regulating the sprouting process. Using this approach it was possible to identify new roles for fibroblasts in mediating sprouting angiogenesis, particularly through the expression of the metalloproteinase MT1- MMP. Its expression is essential in HUVECs to mediate their A 3D Spheroid Model of Tumour Angiogenesis migration process and angiogenesis in a number of systems. MT1- MMP has also been shown to be required for pericyte recruitment in vivo [57]. In our model, we demonstrate that 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 mural cells in mediating endothelial cell sprout formation. The novel observation that stromal derived proteinases are important for sprouting angiogenesis reveals the potential of the Minitumour model to identify new targets and mechanisms in tumour angiogenesis. These observations open new avenues of investiga- tion that can be explored in the future. While the Minitumour spheroid was developed primarily as a model of tumour angiogenesis, future work could be done in order to extend its scope towards the study of cancer cells. In this study we used luciferase-based technology for this purpose to study cancer cell proliferation and we were able to show MT1-MMP in the fibroblasts does not regulate cancer cell number in our system. The use of immunostaining techniques as well as the pre-dyeing of cancer cells could also be extended in the future in order to use this model to study the effects of the stroma in cancer cell invasion and proliferation. Our model can therefore provide for an advanta- geous tool where the behaviour of all included cells can be studied in a complex system. Cells constitutively expressing different fluorophores could potentially be used for a dynamic look into the invasive behaviour of fibroblasts and/or cancer cells under the influence of a heterogeneous environment. Allied to the potential high-throughput developments discussed, this could lead to a model where the invasive behaviour of all three different cell lines could be studied in an integrative systemic way, within the same complex environment. In summary, we present the first example of an in vitro model where the endothelial cells are cultured directly with cancer cells as well as a stromal component in a 3D setting. We demonstrate the model is readily analysed, manipulated and responds to inhibitors of angiogenesis and tumour growth in a manner that mimics in vivo observations. Initial studies using the Minitumour model have allowed us to unravel new roles for mesenchymal MT1-MMP in regulating endothelial sprout formation. Being highly reproducible and easily manipulated, we believe it is a powerful new tool for the study of tumour angiogenesis in vitro, opening the way for the development of innovative insights into this process. Supporting Information Figure S1 Validation of Minitumour spheroid out- growth quantification using a broad-spectrum metallo- proteinase inhibitor. A – Quantification of total endothelial cell sprout length from Minitumour spheroids after incubation with galardin or a vehicle control. B – Quantification of the total number of endothelial cell sprouts from Minitumour spheroids after incubation with galardin or a vehicle control. C – Analysis of number of endothelial cell sprouts counted manually from 1 mm step z-stacks from 10 different Minitumour spheroids analysed using the image analysis programme Volocity. D – Representative 3D reconstruction of a Minitumour z-stack using the programme Volocity. E – Linear regression analysis of the percentage inhibition of total spheroid sprouting by Galardin in 2D vs 3D. F – Linear regression analysis of the percentage inhibition of total spheroid sprouting by Galardin in 2 different experiments. (TIF) Figure S2 Minitumour spheroid pre-capillary sprouts have an endothelial phenotype. A – Minitumour spheroids containing endothelial cells pre-dyed with a CMFDA green tracker dye and incubated in collagen-I were immunostained with PLoS ONE | www.plosone.org 12 February 2012 | Volume 7 | Issue 2 | e30753