A 3D Spheroid Model of Tumour Angiogenesis Figure 2. Multiphoton microscopy images of Minitumour spheroids after 40 h or 7 days culture. A - HUVECs dyed with a CMFDA Green CellTracker dye were imaged within the Minitumour spheroid immediately following their embedding into the type-I collagen matrix using a Multiphoton microscope with a 206 objective. B – Immediately after collagen embedding, the collagen-I gel emits a weak homogenous Second Harmonic Generation (SHG) signal. C – Multiphoton imaging from of spheroids after 40 h incubation in the collagen-I gel shows the formation of green endothelial sprouts into the collagen matrix. D - The SHG signal from the collagen reveals an increase in matrix intensity around the endothelial sprouts. E – Merged image between CMFDA Green CellTracker dye and SHG signals after 40 h incubation. F – A higher amplification (406) image of an endothelial cell sprout from a Minitumour spheroid after 40 h shows the alignment of collagen fibrils along the endothelial cell sprout (white arrows). G – Phase contrast images after 7 days incubation in the collagen-I gel showing a homogenous layer of cells. H – Multiphoton imaging after 7 days incubation shows the formation of a network of pre-dyed endothelial cells within the layer of cells. I – SHG signal from the collagen matrix after 7 days spheroid incubation. Scale bars represent 50 mm in F and 100 mm in all others. doi:10.1371/journal.pone.0030753.g002 from endothelial cells in collagen I gels [14], suggesting the establishment of a pro-angiogenic environment within the long term growth of Minitumour spheroids. The pattern of collagen IV staining after 7 days, however, still localized around endothelial cell sprouts, providing for a suitable long-term indirect endothelial cell marker in the model (Figure 3E). The immunoreactivity signal for tenascin was also widespread after 7 days, similar to laminin (Figure 3F), possibly due not only to their production by fibroblasts, but also by the MDA-MB-231 cells, which has also been previously documented [39]. Angiogenic signalling pathway characterization of Minitumour spheroids To further establish the model as a suitable tool for the study of angiogenesis in a tumour microenvironment we characterized it in terms of previously established signalling pathways that govern PLoS ONE | www.plosone.org 6 February 2012 | Volume 7 | Issue 2 | e30753