survival [11]. The following endothelial cells acquire a stalk cell phenotype, stabilizing the vessel through the recruitment of mural cells and deposition of a basement membrane [12]. A number of methods have been developed recently in which engineered tumours capture aspects of in vivo processes, allowing for the study of these processes in a controlled environment. However few have been successfully applied to the study of tumour sprouting angiogenesis. The majority of existing models of in vitro angiogenesis tend to involve the separation of endothelial cells from cancer cells by a barrier of matrix or membrane, as cancer cells have been described to induce cell death in endothelial cells when in direct contact [13]. Several of these models also consist of variations of the tube formation assay, where endothelial cells are cultured in different matrix compositions, such as matrigel, fibrin or collagen, to form cord like structures in vitro [14,15,16]. Whilst allowing for a more detailed look into endothelial cell differenti- ation, these models are still somewhat simplistic in their nature, as they do not account for heterogeneous cell interactions important for this process. More recently these models have been growing in complexity. Earlier work has suggested a role for fibroblasts in inducing tubule formation using 3-dimensional systems where endothelial cells are separated from fibroblasts by collagen disks [17]. More complex in vitro models have since been developed where the fibroblasts are added in direct contact with the endothelial cells, most notably in a monolayer co-culture of dermal fibroblasts and human umbilical cord endothelial cells that allows formation of endothelial cell tubules in vitro [18]. These observations are in accordance to the increasingly recognised role of fibroblasts, mostly under the form of carcinoma-associated fibroblasts (CAFs), in cancer development. While the full complexity of their role is still not fully understood, the importance of these cells in tumour invasion, progression and metastasis has been widely documented [19,20]. This role is partly enacted through the secretion of several growth factors and cytokines that stimulate tumour growth. CAFs also stimulate tumour progression through the remodelling of the extracellular matrix (ECM), notably by the expression of ECM-degrading proteases, such as Matrix Metalloproteinases (MMPs) [19]. Their importance for the angiogenic process has also been described, and is thought to be regulated partly through the secretion of growth factors including Vascular Endothelial Growth Factor (VEGF) [4,20]. The importance of the development of 3 dimensional (3D) models for the study of complex cellular process has been extensively discussed in the literature. Recent studies have seen the development of 3D co-culture models for the study of angiogenesis in vitro, which have suggested the role of mesenchy- mal cells in vessel formation, as well their requirement to form endothelial tubules in vitro [21,22,23]. However, to our knowledge, no such model has yet been developed that allows for the study of this process in direct cell-cell contact with cancer cells. The development of 3D in vitro engineered human tumours which can mimic the complexities of cancer-stromal interactions, be readily manipulated and quantified and allow for the study of tumour angiogenesis, bridging the gap between 2D monoculture and in vivo systems, would be of enormous potential [24,25,26,27]. Previous work by Korff and Augustin has resulted in the development of a method for culturing endothelial cells as 3- dimensional spheroids in vitro, which induces endothelial cell differentiation [28]. Further developments of this technique have included the introduction of mesenchymal mural components to support endothelial tubule formation [21,29]. Based on this work, we have developed the first 3-dimensional in vitro model of tumour angiogenesis, consisting of a spheroidal co-culture of endothelial A 3D Spheroid Model of Tumour Angiogenesis cells, fibroblasts and the tumour cell line MDA-MB-231. Incubation of these spheroids in type-I collagen leads to the formation of capillary-like sprouts, which are shown to be a quantifiable and reproducible model of the early stages of tumour angiogenesis. This model is further shown to be amenable to genetic manipulation of individual cell types, which allows for the identification of new roles for specific genes in cell-cell interactions leading to endothelial sprout formation, in a cancer environment. Materials and Methods Antibodies and reagents Function blocking antibodies for human VEGF, PDGF-B, IL-6 and IL-8 were purchased from R&D systems (Oxford, UK). The antibodies used for Western Blotting were as follows: sheep anti- human MT1-MMP ectodomain polyclonal antibody (clone N175/ 6) [30], monoclonal mouse anti-human MT1-MMP catalytic domain antibody (Clone Lem2/15.8, Millipore, UK), polyclonal rabbit anti-human antibody to b-actin (Abcam, UK). Secondary HRP-conjugated antibodies were obtained from Jackson Immu- noresearch Laboratories (Stratech, UK). Antibodies for immuno- staining were as follows: monoclonal mouse anti-human tenascin, polyclonal rabbit anti-human CD34, polyclonal rabbit anti-human pan-laminin (Abcam, UK), monoclonal mouse anti-human CD31, monoclonal mouse anti-human collagen-IV (Dako, Ely, UK) and polyclonal goat anti-human LYVE-1 (R&D Systems, UK). Secondary donkey anti-mouse and anti-rabbit antibodies conju- gated with FITC, Texas Red or Cyanine Cy5 fluorophores were obtained from Jackson ImmunoResearch Laboratories (Stratech, UK). Endostatin and SU4312 were purchased from Sigma- Aldrich, UK. Thalidomide, Galardin (GM6001), AG1296 and PPP were obtained from Merck Biosciences, UK. Cell culture Human Umbilical Vein Endothelial Cells (HUVECs) and Normal Human Dermal Fibroblasts (NHDF) were obtained from Promocell GmbH (Heidelberg, Germany). The MDA-MB-231 breast cancer cell line was purchased form the European Collection of Cell Cultures (Dorset, UK). HUVECs were cultured in Endothelial Cell Growth Medium (ECGM, Promocell), containing a final concentration of 1 ng/ml basic Fibroblast Growth Factor, 4 ml/ml Endothelial Growth Supplement/ Heparin, 0.1 ng/ml Epidermal Growth Factor, 1 mg/ml Hydro- cortisone, 0.62 ng/ml phenol red and 2% (v/v) Fetal Calf Serum. NHDFs and MDA-MB-231 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Invitrogen, UK) with 10% FCS (v/v) (Hyclone, Thermo Fisher Scientific, UK), 100 units/ml Penicilin (Invitrogen), 100 mg/ml Streptomycin (Invitrogen) and 500 mg/ml L-Glutamine (Invitrogen). Minitumour 3D spheroid co-culture and sprouting assay The 3-dimensional (3D) spheroid co-culture assays were performed in Endothelial Cell Growth Medium-2 (EGM-2) (Lonza, Basel, Switzerland), supplemented with 5% FCS (v/v), Hydrocortisone, Epithelial Growth Factor (EGF), Insulin-like Growth Factor-1 (IGF-1), ascorbic acid, GA-100, Heparin and with or without bFGF and VEGF. A stock methocel solution was prepared by dissolving 6 g of methylcellulose in 500 ml of EGM-2 medium. Cells were previously incubated in a 2 mM solution of CellTrackerTM green CMFDA or CellTrackerTM orange CMRA (Molecular probes, Invitrogen, UK). 750 HUVECs, 375 NHDFs and 750 MDA-MB-231 cells were added to each well of a 96 U- well suspension plate (Greiner BioOne, UK) in a 150 mL of EGM- 2 with 20% methocel (v/v). The cells were allowed to form PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e30753