Tuomela et al. BMC Cancer 2010, 10:596 http://www.biomedcentral.com/1471-2407/10/596 ratio of the hypoxia tracer [18F]EF5 in VEGF tumour- bearing animals, indicating that these tumours are indeed more perfused and oxygenated than the others (Figure 4). The intratumoral distribution of [18F]EF5 was studied autoradiographically. Uptake of [18F]EF5 was generally in accordance with the results of the perfusion study, show- ing a relatively high level of [18F]EF5 uptake in large peripheral regions of FGF8b and mock tumours, whereas VEGF tumours showed a more uniform uptake, seen as smaller clusters throughout the tumour. The partial pressure of oxygen (pO2) seen in healthy tissues varies from 95 mmHg in arterial blood to a mean value of about 40 mmHg in tissues. Usually hypoxia is defined as an O2 concentration below 10 mmHg and pO2 values less than 5 mmHg are generally considered as severe hypoxia [2]. According to our oxy- gen measurements, both the FGF8b and mock tumours were hypoxic, showing pO2 values of 2.3 ± 0.6 mmHg and 1.3 ± 0.3 mmHg, respectively, whereas VEGF tumours were well oxygenated (17.4 ± 2.2 mmHg). Even if there were intratumoral necrotic areas, the difference seemed to be significant. Importantly, tumour hypoxia is generally considered as a poor prognostic factor [54]. In order to evaluate the hypoxia status of FGF8b and mock tumours further, we immunostained tumours against HIF1a, which mediates acute molecular responses to hypoxia [49]. The HIF1a-positive cells in FGF8b and mock tumours were seen as hypoxic clusters. When these clusters were visually evaluated from microscope images and compared to the expression of Ki67, we found a negatively related expression pattern. In order to evaluate the metabolic activity of the tumours, we used [18F]FDG, a glucose analogue, that is trapped inside metabolically active cells in the form of [18F]FDG-6-P [55]. As expected, relatively high uptake of [18F]FDG was seen in all three tumour models. Surpris- ingly, the lowest T/B ratio was seen in FGF8b tumours. This can be partly explained by the lower cell density and increased intracellular space seen in the FGF8b tumours compared with the mock and VEGF tumours (Figure 2B and 6C). When [18F]FDG uptake was balanced against cell number, the level of uptake in FGF8b tumours was similar to that in the mock tumours. Expression of the glucose transporter GLUT1 was also increased in FGF8b and mock tumours, which indicates that hypoxic tumours compensate for their hypoxia by increased glu- cose consumption [6]. Hypoxic cells often show enhanced glycolysis to maintain production of energy in the form of ATP without requiring O2 [6]. The results of several studies have shown a relationship between [18F] FDG accumulation and GLUT1 expression in cancer [56]. The relationship between hypoxia and glucose metabolism might, however, be more complicated than is generally believed [57]. In a study by Rajendran et al. the Page 11 of 14 authors found a wide variation in the relationship between hypoxia and energy metabolism in patients with different forms of cancer [58]. Acute hypoxia can stimu- late anaerobic glycolysis in normal tissues and also in some tumours, but it is not a prime cause of glycolysis, which is the preferred means of energy metabolism in many tumours under aerobic conditions as well [6]. Glu- cose metabolism is also influenced by factors other than hypoxia, such as the number of tumour-infiltrating immunoreactive cells. Cellular proliferation might be reduced in chronically hypoxic regions, whereas the pro- liferation rate can be elevated in non-hypoxic tumour areas. This kind of cellular stress often activates other metabolic pathways as well, such as fatty acid synthetase (FAS) pathways [59]. Enhanced glycerolipid/free fatty acid cycling, on the other hand, favours tumour cell growth in environment poor in nutrients [60]. In addi- tion, the expression of mitogenic FGF8b itself increases tumour growth. In clinical practice, high metabolic activ- ity and hypoxia are considered to be characteristics of aggressive tumours [58]. Increased expression of FGF8b is known to predict poor clinical outcome of the patient [17,32] but it remains to be studied whether hypoxic tumour microenvironment is associated with or contri- butes to FGF8b-driven tumour progression. The limitation of our experimental study is that subcu- taneous tumours instead of orthotopic tumours were used, which means heterotopic tumour microenviron- ment for tumour cells. The PC-3 control tumours formed were, however, hypoxic as clinical prostate tumours are. Another limitation is that the expression of FGF8b in PC-3 cells was not endogenous but produced by transfec- tion of the ectopic gene which did not allow studies on the possible effects of silencing the gene on tumour microenvironment. Unfortunately, no prostate cancer cell line expressing FGF8 is available. Silencing endogen- ous VEGF would also add to the results of the role of VEGF in tumour oxygenation and tumour growth in our model. Additionally, studies of correlations of VEGF, FGF8 and HIF1 with each other and characteristics of clinical tumour samples and outcome of prostate cancer patients would clarify the role and significance of FGF8 in regulation of prostate cancer progression. Our study revealed that our prostate tumour models have different profiles as regards hypoxia, perfusion and metabolism. The relationship between hypoxia and blood flow seems to be complex, and our results thus support the theory of variable oxygenation status coupled to a highly vascular morphology but differential numbers of functional vessels. Our results also support earlier evidence that hypoxia and accelerated glycolysis are common but independent phenomena in a malig- nant tumour phenotype. Our results are in line with those of studies on human prostate cancer, where the