dry transfer system (BioRad, UK). Membranes were blocked in 5% (w/v) fat free milk powder (Marvel, UK) in PBS before incubation with the primary antibody. Membranes were subse- quently washed in PBS-Tween and incubated with the secon- dary antibody. After further washing in PBS-Tween the immunreactions were developed using the Amersham ECLTM (Enhanced Chemi-luminescence) Western Blotting Detection Reagents (GE Healthcare, UK). Band intensity was quantified using the ImageJ software and normalized against a loading control. Statistical analysis All graphs shown of spheroid outgrowth quantification represent the averaged parameters with the Standard Error of the Mean (SEM) as error bars. One-way ANOVA analysis with Student Newman-Keuls post-test was performed to calculate statistical significance, using the GraphPad Prim version 5.0 b (GraphPad software Inc, San Diego, CA, USA). P-values are specified in each experiment. Results The Minitumour spheroid model Previously published in vitro models developed for the study of angiogenesis regulated by cancer cells typically rely on the addition of cancer cell conditioned medium, or separation of cancer cells from endothelial cells by a matrix or membrane [31]. Accordingly, reports have shown direct cell-cell contact between tumour cells (including MDA-MB-231) and endothelial cells leads to an increase in endothelial cell apoptosis [13]. Evidence from our laboratory supports these observations, as the direct addition of tumour cells to a vasculogenesis model described by Bishop et al [18] decreased capillary formation (data not shown). We have also observed that Human Umbilical Vein Endothelial Cells (HU- VECs) do not form continuous sprouts solely in direct contact with the tumour cells (data not shown). To overcome these technical difficulties, we have co-cultured endothelial cells, Normal Human Dermal Fibroblasts (NHDF) and tumour cells in a spheroid before implantation in a collagen-I gel, an adaptation of the system described by Korff et al [29]. By pre- labelling the endothelial cells using a green cell tracker dye, it is possible to visualise and quantify the formation of endothelial pre- capillary sprouts from the model. The HUVECs initially mix with the other cell types in a multicellular spheroid (Figure 1A), which can then be implanted in a type-I collagen gel. After 40 h, confocal imaging of green-labelled HUVECs shows the formation of pre- capillary sprout-like structures (Figure 1B). This is, to our knowledge, the first model to include all three components in an in vitro system, allowing for the study of complex interactions underlying the early steps of tumour angiogenesis. We have named this system the Minitumour spheroid model. By comparing the Minitumour spheroids with simpler spheroid types, we observe that HUVECs alone form irregular projections into the collagen matrix, with high levels of scattering cells. However, in the presence of fibroblasts, HUVECs form contin- uous sprouts that can be analysed and quantified in terms of their length and number (Figure 1C and S1). Mesenchymal mural cells, such as pericytes or fibroblasts have been extensively shown to contribute to the development of more robust and continuous endothelial sprouts in other in vitro systems [17,22,32]. Particularly, the ability of fibroblasts to act as mural cells in vitro and drive the formation of endothelial cell networks has been described before [23,33]. Confocal imaging of Minitumour spheroids containing both pre-dyed fibroblasts and endothelial cells showed that the A 3D Spheroid Model of Tumour Angiogenesis fibroblasts develop a mural cell-like phenotype in this model, migrating in spindles adjacent to and around the endothelial cell sprouts (Figure 1E). The endothelial nature of the observed sprouts was confirmed by staining for endothelial markers CD31 and CD34 (Figure S2), showing a distribution comparable to that of the green tracker dye used to label the HUVECs. Endothelial cell sprouts showed, however, no staining for Lymphatic marker LYVE-1 (Figure S2), which had been previously shown to be expressed in HUVECs cultured in 3D but repressed in the presence of perivascular cells [34]. This confirmed the blood endothelial phenotype of these cells, as well as the perivascular/mural nature of the fibroblasts in our system. The MDA-MB-231 breast cancer cells in the model were shown to augment endothelial cell sprouting both in the presence and absence of exogenous angiogenic growth factors VEGF and bFGF (Figure 1B). This confirmed the Minitumour model as a reliable framework with which to observe the effects of tumour cells on endothelial outgrowth and sprout formation in vitro. The MDA- MB-231 cancer cells could also be imaged after pre-dyeing with the CMFDA Green Cell Tracker Dye and were shown to migrate uniformly around the spheroid within the collagen-I gel (Figure 1D). Minitumour spheroids and extracellular matrix structure To investigate the interaction between the Minitumour spheroids and their surrounding ExtraCellular Matrix (ECM), spheroids were imaged using Multiphoton Microscopy. This was used in order to detect the Second Harmonic Generation (SHG) signal emitted by collagen-I matrix fibrils as well as the endothelial cell sprout formation from the spheroids. On observing the spheroids immediately after their implantation in the collagen matrix, the SHG signal from the surrounding collagen is weak, consisting mostly of a low level homogeneous signal around the spheroids (Figure 2A and B). However, after incubation in the collagen matrix for 40 hours, an increase in the SHG signal was observed accumulating around the endothelial cell sprouts (Figure 2C–F). Furthermore, it was possible to distinguish empty paths in the SHG signal, corresponding to the areas of sprout formation, surrounded by areas of stronger intensity (Figure 2D). It is not clear currently if these differences in intensity are due to matrix rearrangements (matrix displacement, degradation, fibril formation), or due to production of new ECM (e.g. collagen-I production and processing by fibroblasts). Nevertheless the possibility of studying the interaction between endothelial sprout- formation and its surrounding matrix opens interesting new avenues of investigation, as recent work shows that the angiogenic process can be regulated by extracellular mechanical cues [35]. After 7 days of culture, the spheroids were observed to form more complex endothelial cell networks, which branch and interconnect within a denser layer of fibroblasts and tumour cells (Figure 2G–I). At this point the SHG signal from the collagen matrix is almost ablated, possibly reflecting the degradation and reorganisation of the matrix by the different cells in the model (Figure 2I). These more complex endothelial networks are also shown, though the use of transmission electron microscopy (TEM), to contain fully developed lumens (Figure S3), which are not detected after 40 h culture (data not shown). Optimized immunostaining techniques also allowed us to further dissect the deposition of additional ECM components with endothelial sprout formation. Immunostaining for compo- nents of the vascular basement membrane, such as Collagen IV and Laminin, showed that these localize mostly around the developing endothelial cell sprouts at 40 h (Figures 3A and B). PLoS ONE | www.plosone.org 4 February 2012 | Volume 7 | Issue 2 | e30753