A 3D Spheroid Model of Tumour Angiogenesis Figure 1. Characterization of the Minitumour spheroid model. A - Fluorescent (left) and phase contrast (right) images of HUVEC, EndoFib and Minitumour spheroids before incubation in the collagen gel; endothelial cells pre-dyed with a CMFDA Green CellTracker dye are seen in each different spheroid type. B – Representative fluorescent images of spheroids after 48 h incubation in collagen gels, in the presence of complete medium, showing pre-dyed endothelial cells organized into pre-capillary sprouts. C – Quantification of endothelial sprout length from different spheroids show that MDA-MB-231 cells stimulate sprout formation even in the absence of exogenous growth factors VEGF and bFGF. D – Confocal (upper) and phase contrast (lower) images of MDA-MB231 cells pre-dyed with the green CellTracker dye in the Minitumour spheroid after 48 h incubation in complete medium. E - A 3D reconstruction of a Minitumour spheroid where the HUVECs have been dyed with a CMRA Orange CellTracker dye and the fibroblasts with a CMFDA Green Cell Tracker side panels show optical x and y sections of sprouts showing the deposition of HUVECs and Fibroblasts relative to sprout formation. doi:10.1371/journal.pone.0030753.g001 Tenascin has been shown to be largely associated with mesenchymal regions in tissues such as normal breast, and secreted by fibroblasts. Its secretion is increased in neoplastic tissue, where stromal fibroblasts are thought to be its major source [36,37]. Likewise, pan-tenascin staining of Minitumour spheroids showed a diffuse pattern (Figure 3C), reminiscent of the pattern of invading fibroblasts within the model (Figure 1E). After allowing the spheroids to grow for 7 days, the pattern of pan-laminin staining was altered, acquiring a more widespread distribution, with the formation of a network of fibrils along the spheroid outgrowth area (Figure 3D). Extensive laminin produc- tion has been reported in breast carcinomas, correlating particularly with areas of vessel formation [38]. Laminin has also been shown to stimulate the production of capillary-like tubules PLoS ONE | www.plosone.org 5 February 2012 | Volume 7 | Issue 2 | e30753