A 3D Spheroid Model of Tumour Angiogenesis Figure 3. Immunostaining of Minitumour spheroids show deposition of extracellular matrix components. A – Minitumour spheroids incubated in collagen-I were immunostained with an anti-pan-laminin antibody and imaged by confocal microscopy showing the deposition of laminin around the endothelial sprouts after 40 h and D – a more widespread distribution after 7 days. B – Confocal microscopy of Minitumour spheroids immunostained with an anti-collagen IV antibody after 48 h shows a similar pattern, E – but after 7 days collagen-IV still localized around the endothelial cell sprouts. C – Confocal microscopy of Minitumour spheroids immunostained with an anti-Tenascin antibody show widespread distribution after 40 h, and F – after 7 days. All images were obtained using a 106 objective. Scale bars represent 100 mm. doi:10.1371/journal.pone.0030753.g003 pre-capillary sprout formation. For this purpose two different approaches were used, the use of function blocking antibodies and low molecular weight inhibitors. The use of function blocking antibodies in our model is of interest as recent years have seen a rise in the use of antibodies as anti-angiogenic therapeutic agents, with the notable case of VEGF antibody Bevacizumab [40]. Antibodies to the growth factors VEGF-A and PDGF-B were tested, due to their recognized roles in tumour angiogenesis and stromal activation [11,41,42]. The inclusion of control immuno- globulins within the collagen matrix did not disrupt normal sprout formation (data not shown). Both VEGF-A and PDGF-B blocking antibodies significantly inhibited sprouting (Figure 4A), confirming the important role of these two growth factors in tumour angiogenesis using the Minitumour model. This observation again confirms that our model can reliably reproduce results seen in other in vitro and in vivo systems. To further explore the results seen with the functional antibodies, low molecular weight receptor tyrosine kinase inhibitors were used to abrogate VEGFR and PDGFR functions. SU4312, an inhibitor of VEGFR signalling that also has a lower affinity for PDGFR [43], almost completely ablated sprout formation in spheroids in the presence and absence of cancer cells (Figure 4C). This is in accordance with the results obtained with the function-blocking antibody for VEGF. AG1296, an inhibitor of PDGFR [44], had an interesting effect on the spheroids. In spheroids without cancer cells (EndoFib spheroids), contrary to an expected reduction in sprout formation, inhibition of PDGFR appeared to increase sprout formation (Figure 4C). Closer examination however, showed this was due to scattering of endothelial cells rather than the formation of continuous sprouts, which consequently led to inconsistent quantification (Figure 4E). This scattering of endothelial cells was remarkably similar to that seen in spheroids constituted of HUVECs alone (Figure 1B). Considering PDGF is a well-documented stromal activator [4,19], we speculate that this could be due to a decrease in the mural cell- like phenotype of the fibroblasts, leading to a chaotic invasion of endothelial cells. In the Minitumour model, AG1296 caused a decrease in the number of endothelial sprouts, reducing them to levels similar to those seen in the control EndoFib co-cultures (data not shown), but it does not have a significant effect on sprout length (Figure 4C and E), contrary to the data shown with function blocking antibody (Figure 4A). The differences in the magnitude of the effect might be due to intrinsic differences between the use of immunoglobulins and small molecule inhibitors, which may penetrate better into the spheroids, show differential selectivity towards PDGF isoforms, or lack of selectivity towards PDGFR. PPP is a potent inhibitor of IGF-1R [45], another growth factor receptor that has also been implicated in tumour angiogenesis in colorectal cancer and multiple myeloma [46]. PPP significantly inhibited capillary sprout formation in the presence and absence of MDA-MB-231 cells (Figure 4C). Previous reports have shown that co-culturing MCF7 cells with macrophages markedly increased their efficiency in inducing endothelial cell tubule formation in vitro [47]. This effect was dependant on the macrophage release of inflammatory cytokines including IL-6 and IL-8. Estrogen-Receptor negative breast cancer cell lines, which include MDA-MB-231, have also been shown to overexpress IL-8, which is associated with a higher invasiveness potential [48]. We hypothesised that the increase in HUVEC sprouting in the Minitumour spheroids, compared to spheroids without cancer cells, might be due to the secretion of inflammatory cytokines by the MDA-MB-231 breast cancer cells. To investigate this hypothesis we used function-blocking antibod- ies to IL-6 and IL-8 in the Minitumour spheroids and compared their outgrowth with EndoFib spheroids. The function blocking antibodies against IL-6 and IL-8 significantly impaired endothelial cell sprouting from Minitumour spheroids. While the effect was not as marked as that seen previously with the anti-VEGF antibody, sprouting from the Minitumour spheroids were reduced to levels similar to those of EndoFib spheroids (Figure 4B). This PLoS ONE | www.plosone.org 7 February 2012 | Volume 7 | Issue 2 | e30753