Tuomela et al. BMC Cancer 2010, 10:596 http://www.biomedcentral.com/1471-2407/10/596 Page 5 of 14 Figure 1 Expression of VEGF after transfection of PC-3 cells with VEGF or mock expression vectors. Western blot analysis of conditioned medium of PC-3/VEGF cell clones revealed several clones positive for 23 kD VEGF protein. The larger bands at approximately 60 kD may represent cleaved formes of larger splice variants in these cells. The expression level in mock cells was very low. One PC-3/VEGF clone (PC-3/VEGF3) and one PC-3/mock clone (PC-3/mock2) were selected for in vivo studies. many of the FGF8b tumours were fragile and haemor- rhagic. The morphology of mock tumours was solid, homogeneous and similar to parental PC-3 tumours. Morphological examination of the H&E-stained sections showed a rich capillary network and large sinusoid-like vessels, especially in VEGF tumours (Figure 2B). Capil- laries in FGF8b tumours looked more aberrant, contain- ing large cisternal spaces filled with blood (Figure 2B). The number of capillaries was modest in mock tumours (Figure 2B). Vascularisation was verified using immuno- histochemical staining for the endothelial marker CD31 and this procedure confirmed the differences in blood capillary density between these three different tumour types (Figure 2C). Significantly increased density of capillaries (mean ± SEM, p < 0.001) was seen in both FGF8b (51 ± 27 μm/mm2) and VEGF (97 ± 4.51 μm/ mm2) tumours compared with that found in the mock tumours (36 ± 21 μm/mm2, Figure 2C-D). The relative area of necrosis was measured in H&E- stained tumour sections. The data demonstrated that the relative area of necrosis was larger in the FGF8b tumours than in the VEGF (p < 0.05) or mock (p < 0.01) tumours (Figure 2E). Cell cycle analysis and proliferation Cell cycle phases of FGF8b, VEGF and mock tumours were determined using flow cytometry. These analyses revealed a higher number of S-phase cells in both FGF8b (21%) and VEGF (16%) tumours compared with mock tumours (12%) (data not shown). Immunohistochemical staining of tumour sections for Ki67 confirmed these find- ings (Figure 3), showing a significantly higher proliferation rate of FGF8b and VEGF tumours compared with that of mock tumours (p < 0.001). The percentage of Ki67-posi- tive cells per mm2 was 26 ± 11, 33 ± 10 and 10 ± 7 in FGF8b, VEGF and mock tumours, respectively (Figure 3). Perfusion and oxygenation status of tumours The labelling intensity of the fluorescent perfusion mar- ker Hoechst 33342 displayed distinct differences in per- fusion patterns among the tumours. Labelling intensity was very weak in FGF8b tumours, indicating poor perfu- sion. As expected, the intensity was strongest in the VEGF tumours (Figure 4A), where signals were detected in both central and peripheral areas, suggesting that VEGF tumours are well perfused. Labelling intensity was modest in the mock tumours. The nitroimidazole family compound EF5 forms cova- lent bonds with cellular macromolecules under hypoxic conditions, and therefore tends to accumulate at hypoxic sites [45,46]. The intratumoral distribution of [18F]EF5 determined by digital autoradiography in tumour sec- tions showed a wide variation in the hotspot areas inside tumours (Figure 4B). The uptake pattern was very similar with that we have earlier detected and reported in regard to other hypoxia tracers [47]. It was not possible to ascer- tain clear divisions based on the intratumoral uptake pat- tern of [18F]EF5 according to tumour size, or the expression levels of the studied growth factors. In gen- eral, however, some distinguishable trends were detected. Variation in the intratumoral distribution of [18F]EF5 in smaller tumours (~200 mg) was similar (data not shown) in all groups. Larger tumours (~500 mg) tend to show more uniformly distributed hotspots of [18F]EF5 uptake in VEGF-expressing tumours, whereas uptake of [18F]EF5 was primary located in peripheral parts of FGF8b- and mock-tumours (Figure 4B). The accumulation of [18F] EF5, in whole tumours was measured after a distribution time of 120 min. As shown in Figure 4C, a significantly lower T/B uptake ratio (p < 0.05) of [18F]EF5 was found in VEGF tumours (1.45 ± 0.37) compared with that mea- sured in mock tumours (1.81 ± 0.45). The mean T/B uptake ratio (1.97 ± 0.73) of [18F]EF5 in FGF8b tumours was not significantly different compared to VEGF or mock tumours (Figure 4C). In order to confirm these findings, we measured the intratumoral partial pressure of oxygen (pO2) using a Licox® Revoxode CC1.2 polarographic probe (Figure 4D). Significantly lower (p < 0.001) mean pO2 values were measured in both FGF8b and mock tumours (2.3 ± 0.6 mmHg and 1.3 ± 0.3 mmHg, respectively) compared with that in VEGF tumours (17.4 ± 2.2 mmHg). The pO2 value of the gluteus muscle, measured as a control, was 45 mmHg (data not shown).