Tuomela et al. BMC Cancer 2010, 10:596 http://www.biomedcentral.com/1471-2407/10/596 [18F]EF5 (FGF8b n = 11, VEGF n = 12 and mock n = 29) and [18F]FDG (FGF8b n = 6, VEGF n = 6 and mock n = 5) were intravenously injected (5.6 ± 1.1 and 6.6 ± 1.4 MBq, respectively) into separate FGF8b (tumour weight 408 ± 33 mg), VEGF (tumour weight 506 ± 29 mg), and mock (tumour weight 408 ± 20 mg) tumour- bearing mice, and allowed to distribute 120 minutes before sacrifice. Blood, obtained by cardiac puncture, and tumour tissues were rapidly removed, counted for 18F- radioactivity in a well counter (3” × 3” NaI (TI) crystal, Bicron 3MW3/3P, Bicron Inc., Newbury, Ohio, USA) and weighed. The uptake of 18F-radioactivity in tissues was calculated as percentage of injected dose per gram of tis- sue weight, taking into account the background from the counter measurements and the radioactivity decay. Tumour-to-blood uptake ratios (T/B ratios) were then calculated for both [18F]EF5 and [18F]FDG. Plasma glu- cose levels were measured (Analox GM9, Analox Instru- ments Ltd., London, UK) in blood samples (FGF8b n = 10, VEGF n = 16 and mock n = 11). The intratumoral distribution of [18F]EF5 and [18F] FDG was studied in FGF8b, VEGF and mock tumour- bearing mice using digital autoradiography. After sacri- fice, tumours were removed, rapidly frozen in dry ice/ isopentane and cut with a cryomicrotome into 20 μm- thick sections. Tumour sections were then exposed to an imaging plate (Fuji BAS TR2025, Fuji Photo Film Co., Japan) for 3.5-4.5 hours. The spatial distribution of radioactivity from tumour sections was recorded with a phosphoimager (Fujifilm BAS-5000, Fuji Photo Film Co. Ltd., Tokyo, Japan). The dynamic linear range of this system is four decades, and the resolution (i.e. pixel size) of the images is 25 × 25 μm. The amount of 2-[18F]fluoro-2-deoxyglucose-6- phosphate ([18F]FDG-6-P), the main metabolite of [18F] FDG, was determined in tumour homogenates (FGF8b n = 6, VEGF n = 4 and mock n = 3) from animals injected with [18F]FDG by using radioHPLC equipment (Merck Hitachi, Peterlee, UK). Oxygen partial pressure (pO2) measurement In order to measure pO2 values in tumours, we used sterile, flexible polarographic electrodes (diameter 0.47 mm) of the Clark type (Licox® GMS, Kiel-Mielkendorf, Germany), supplied with a probe-specific microchip allowing automatic calibration. The probe was inserted into the tumour tissue by advancing it in a retrograde manner along the lumen of an insertion needle catheter, which was then removed. Tissue temperature was mea- sured with a needle probe and temperature-adjusted pO2 (mmHg) was graphically displayed and stored digi- tally. The whole length of the oxygen-sensitive part of the probe was at least 2 mm inside the tumour through- out the measurements to prevent contamination from Page 4 of 14 room-air O2. The duration of the pO2 measurement was sufficient to establish a stable pO2 level, which was then registered and stored. Three tumours from each group were measured over a time period of 20 min after a sta- bilisation period of approximately 5 min. The gluteus muscle of the experimental animal served as a control site after measurements to verify the proper function of the Licox® probe. Statistics All values are presented as mean ± SD unless otherwise stated. Non-parametric one-way ANOVA (Kruskal- Wallis test) with Dunn’s post hoc test was used for statistical evaluation of CD31, Ki67, GLUT1, HIF1, necrosis, [18F]FDG uptake, amount of cells and pO2 values. One-way ANOVA with Tukey’s post hoc test was used for statistical evaluation of plasma glucose levels and [18F]EF5 uptake. The above statistical tests were performed using GraphPad Prism version 5.01 for Win- dows (GraphPad Software, San Diego, CA, USA), based on the distribution of the data (normal or nonpara- metric) and sample size. A p-value less than 0.05 was considered statistically significant. Results Effects of FGF8b and VEGF on tumour growth and morphology In order to study the roles of FGF8b and VEGF in PC-3 tumours, we used xenografts of PC-3 cells ectopically expressing FGF8b and VEGF. Parental PC-3 cells do not express detectable levels of FGF8b [38] but they express VEGF at a low level (Figure 1). PC-3 cells transfected with FGF8b have been reported previously [38]. To obtain VEGF overexpressing PC-3 tumours, the cells were stably transfected with the expression vector pcDNA3.1(+) containing human VEGF cDNA at an ECORI site. PC-3 cells transfected with empty vectors were used as controls (mock). Expression of VEGF in the isolated clones was confirmed by Western blot ana- lyses (Figure 1) and Northern blot analyses (data not shown). Several clones positive for VEGF were obtained, and one clone (PC-3/VEGF3, later called VEGF) was selected for in vivo studies. Tumour growth associated with PC-3/FGF8b, PC-3/ VEGF and PC-3/mock cells was studied by inoculating them s.c. in the back of the necks of nude mice. The average growth (mean ± SEM) of FGF8b, VEGF and mock tumours is shown in Figure 2A. Mock-transfected cells produced only small tumours (749 ± 19 mm3), whereas FGF8b and VEGF tumours grew larger (3436 ± 41 mm3 and 4036 ± 51 mm3, respectively) during the 6 and 4-week study periods, respectively (p < 0.001). Both FGF8b and VEGF tumours showed strong angio- genic morphology. They were reddish in colour and