suggests the MDA-MB-231 contribution to increased endothelial sprouting in the Minitumour spheroids is dependent on the inflammatory cytokines IL-6 and IL-8. The presenilin family of aspartyl proteinases plays a significant role in cellular signalling by processing transmembrane receptors [49]. This includes the processing of the Notch-Delta, Notch- Jagged and Eph-Ephrin receptor systems that have been shown to be important in the regulation of angiogenesis. The Notch-Delta system has largely been described in the initial processes of endothelial tip-cell formation, namely through Delta-like ligand 4 (Dll4) signalling [11,50]. This supports results seen with the use of the presenilin inhibitor DAPT in our spheroid systems, which led to a significant increase in endothelial cell sprouting Minitumour spheroids as well as EndoFib spheroids (Figure 4D). Minitumour spheroid response to anti-angiogenic inhibitors Further characterization of the model was carried out using known anti-angiogenic agents, which have already been used in clinical trials with mixed results (Figure 5). This had the purpose of enabling us to understand if the model’s drug response is closer to the pre-clinical trial results in rodents and simpler in vitro systems, or the drug effects seen in human clinical trials. Metalloproteinase activity is extremely important to endothelial cell migration and tube formation [51]. Galardin (GM6001), a broad-spectrum inhibitor of metalloproteinases [52], had a very significant effect on spheroid sprouting, both in the Minitumour model and spheroids with no cancer cells (Figure 5A and B). Thalidomide was first used clinically to relieve symptoms of morning sickness when significant developmental side effects were observed in foetuses, causing defects in limb development caused in part by poor development of the limb bud. This was not detected in rodents [53]. More recently Thalidomide was shown to be an inhibitor of angiogenesis [53]. Although it was effective in pre-clinical and in vitro models, it failed to show a significant effect in the treatment of some solid tumours including metastatic colorectal, melanoma and breast cancers [54]. Thalidomide inhibited capillary sprout formation in EndoFib spheroids, but not in Minitumour spheroids containing MDA-MB-231 breast cancer cells (Figure 5A and B), which agrees with previously existing clinical data. A 3D Spheroid Model of Tumour Angiogenesis Endostatin is a proteolytic fragment of collagen-XVIII that has been shown to inhibit angiogenesis through binding to integrin a5b1 [55]. Endostatin was found to be a very effective inhibitor in simple in vitro models and murine in vivo models but failed to show such significant effect in human trials [56]. Endostatin inhibited capillary sprout formation in the EndoFib spheroids, but was no longer an inhibitor in Minitumour spheroids (Figure 5A and B). The role of MT1-MMP in the Minitumour spheroid model Being an in vitro model comprising different human cells lines – primary and tumourigenic – the Minitumour spheroid model can be used for more detailed mechanistic studies. This can be done through independent manipulation of the different components of the spheroids using common molecular techniques. To illustrate this aspect of the model, the role of the metalloproteinase MT1- MMP (Membrane-Type 1 Matrix MetalloProteinase) was ad- dressed in the different cell types in terms of its ability to regulate sprout formation. MT1-MMP (or MMP14) is widely expressed by multiple cell types within the tumour microenvironment, including endothelial cells, fibroblasts and in some instances tumour cells [51]. MT1-MMP has also long been recognized as a major regulator of angiogenesis [57]. It is involved in endothelial cell migration and sprouting, and it has also been shown to up-regulate VEGF expression, and therefore tumour angiogenesis, through increased transcriptional activation [58]. However, the relative contribution of this proteinase to the angiogenic process from different cells in a tumour has yet to be clarified. In order to address this using our model, cells stably transduced using lentiviral delivery of short hairpin RNA (shRNA) targeting MT1-MMP were prepared for each individual cell type. Two different shRNAs targeting MT1-MMP were used and their efficacy confirmed by western blot analysis (Figure 6C and F) and qRT-PCR (data not shown), in comparison to a non-targeting control shRNA (shCont) and the puromycin resistance expression cassette alone (pLKO.1). All three different cell types were independently transduced in order to determine the importance of MT1-MMP in each of the spheroid components separately in capillary sprout formation. In HUVECs (Figure 6A–C), the expression of the shRNA targeting MT1-MMP resulted in a knock down of around 50% of the protein, leading to impaired endothelial cell sprouting. This is in accordance with previously Figure 5. Minitumour spheroids have a different drug response than spheroids consisiting of only HUVEC and fibroblasts. A – Incubation of EndoFib spheroids with different anti-angiogenic agents for 40 h shows inhibition of sprouting with Thalidomide, Endostatin and Galardin. B – Incubation of Minitumour spheroids with different anti-angiogenic agents for 40 h shows a response to Galardin but a non-significant effect of both Thalidomide and Endostatin. doi:10.1371/journal.pone.0030753.g005 PLoS ONE | www.plosone.org 9 February 2012 | Volume 7 | Issue 2 | e30753