Tuomela et al. BMC Cancer 2010, 10:596 http://www.biomedcentral.com/1471-2407/10/596 Page 7 of 14 Figure 3 The effect of ectopic FGF8b and VEGF on proliferation. Ki67 immunostaining of FGF8b (n = 40), VEGF (n = 5) and mock (n = 23) tumours (Bar 200 μm). The relative number of Ki67-positive cells in tumours was counted in a blinded manner in 3 fields per tumour and the results were expressed as percentage of positive cells per mm2. There were significantly more proliferative cells (p < 0.001) in both FGF8b and VEGF tumours compared with mock tumours. In addition to autoradiography, hypoxic clusters in FGF8b and mock tumors were also detected by immunos- taining of hypoxia-inducible factor (HIF1a) [48,49]. As shown in Figure 5, the areas staining strongly for HIF1a showed little immunopositivity for Ki67 whereas the areas with Ki67 positive cells did not markedly stain for HIF1. Metabolic activity of tumours In order to evaluate the metabolic activity of FGF8b- and VEGF-expressing tumours, we measured the uptake of [18F]FDG-derived radioactivity. The intratumoral dis- tribution of [18F]FDG determined by digital autoradio- graphy showed uniformly distributed uptake in tumours (Figure 6A). We found a significantly lower (p < 0.05) T/B uptake ratio of [18F]FDG in FGF8b tumours (7.4 ± 0.7) compared with that seen in VEGF and mock tumours (14.3 ± 8.9 and 17.8 ± 9.3, respectively), as shown in Figure 6B. The intracellular [18F]FDG-6-P (the main metabolite of [18F]FDG, which is trapped inside the cell) was detected by radioHPLC. The amount did not differ between the tumour groups (data not shown). The mean plasma glucose level varied from 7.9 to 10.6 mmol/L and was not significantly different in the three groups of tumour-bearing mice. Since morphological studies indicated a lower num- ber of cells in FGF8b tumours, which would affect the relative level of [18F]FDG uptake, we evaluated cellular density in the different tumour models by flow cyto- metry. Enumeration analysis showed that indeed FGF8b tumours contained fewer cells/cm3 than VEGF and mock tumours (Figure 6B). A similar result was obtained by counting nuclei per field in H&E-stained sections (data not shown). When the [18F]FDG uptake (percentage of injected dose per gram tissue) was nor- malised to cell number, the differences between FGF8b and mock tumours disappeared, and according to the normalised data, the uptake was smallest in VEGF tumours (Figure 6B). Immunohistochemical staining of GLUT1 (Figure 6C) correlated with the uptake of [18F] FDG, showing a trend towards a lower expression pat- tern of GLUT1 in VEGF tumours compared to mock tumours (p < 0.05).