Tuomela et al. BMC Cancer 2010, 10:596 http://www.biomedcentral.com/1471-2407/10/596 pathogen-free environmental conditions (20-21°C, 30-60% relative humidity and a 12-hour lighting cycle). FGF8b, VEGF and mock cells were inoculated subcuta- neously (1 × 106 cells in 100 μL PBS) into the back of the neck. The animals were monitored daily for clinical signs. Tumour measurements were performed once a week and tumour volume was calculated according to the formula V = (π/6)(d1 × d2)3/2 [43], where d1 and d2 are perpendicular tumour diameters. The tumour volume was expressed as mean ± SEM. The animal experiments were carried out according to the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes, plus Statutes 1076/85 § and 1360/90 of The Animal Protection Law in Finland, and EU Directive 86/609. The experiment procedures were reviewed by the local Ethics Committee on Animal Experimenta- tion at the University of Turku and approved by the local Provincial State Office of Western Finland. Tumour morphology and immunohistochemical analyses Morphological evaluation of tumours was determined from frozen sections stained with haematoxylin and eosin (H&E) using standard techniques. Frozen sections (10 μM) were cut, and fixed with ice-cold acetone for 10 minutes at 4°C. Sequential frozen sections were incu- bated with antibodies against CD31 (BD Biosciences, Pharmingen, CA, USA), Ki67 (Novocastra Laboratories Ltd., Newcastle upon Tyne, UK) and GLUT1 (Alphadiag- nostics, TX, USA) o/n at 4°C. HIF1 (BD Biosciences, Pharmingen, CA, USA) antibody was used on formalin- fixed, paraffin-embedded tumour slides o/n at 4°C. The samples were then treated with biotin-labelled rabbit anti-mouse (DAKO Denmark A/S, Glostrup, Denmark or Vectastain CA, USA) secondary antibodies. A mouse-on- mouse kit (Vector Laboratories, Burlingame, CA, USA) was used in the Ki67 antibody staining procedure in order to inhibit non-specific staining of anti-mouse sec- ondary antibodies. Visualisation of the primary antibodies was carried out by using Vectastain ABC reagent and a diaminobenzidine substrate kit (Vector Laboratories, Burlingame, CA, USA), the methodology which is based on the indirect streptavidin-biotin method. The slides were later counterstained with Meyer’s haematoxylin. Negative controls (sections of every sample stained with- out the primary antibody) were used to verify the specifi- city of staining. Three representative non-overlapping fields inside each tumour were analysed microscopically (Leica, DMRB, Leica Microsystems, Heerburg, Germany) and images were taken with a digital camera (Leica DC 300F, Leica Microsystems, Heerburg, Germany). The lengths of CD31-positive vessels were counted in each tumour by drawing lines following stained vessels and measuring the length of the lines using ImageJ software Page 3 of 14 (ImageJ, 1.37v, Wayne Rasband, National Institutes of Health, USA) [44]. The relative numbers of Ki67- and GLUT1-positive cells were counted in three non- overlapping sections at 500-μm intervals. Altogether, 1.000-3.000 cells/tumour were counted [44]. The results were blind-tested by two independent analysers compar- ing study versus control samples. The relative proportion of necrotic tissue was deter- mined in representative H&E-stained tumour slides using the ImageJ software. The evaluation was based on morphological features of the tissue. The perfusion marker Hoechst 33342 was intrave- nously injected into tumour-bearing nude mice two minutes before sacrifice in order to study the intratu- moral perfusion pattern. Images were acquired from fro- zen sections (10 μm) using a Zeiss AxioVert 200 M fluorescence microscope (Zeiss GmbH, Jena, Germany). Flow cytometry Three tumours from each group were collected in ice- cold PBS supplemented with pepsin (0.5%, pH 1.4, Merck, Darmstadt, Germany) immediately after sacrifice of the mice. Tumour tissue was homogenised with Ultra-Turrax equipment (Rose Scientific Ltd., Canada) and incubated at 37°C for 1 h. The suspension was col- lected and filtered prior to analysis. For cell cycle analy- sis, tumour homogenate was incubated in hypotonic buffer (PBS with 1% Triton X-100 and propidium iodide at 0.05 mg/mL) for 20 min at 4°C. The fractions of cell cycle phases (G0/G1, S and G2/M) were calculated with ModFit cell cycle modelling software (Verity Software House, Inc., Topsham, ME, USA). For cell enumeration, cells were analysed using TrueCount tubes (Becton Dickinson, CA, USA). Briefly, the number of cells in the tube was calculated by comparing a known number of beads in the tube with cell number in a known volume. All flow cytometry was performed using a FACSCalibur (Becton Dickinson, CA, USA) flow cytometer and Cell- QuestPro software (Becton Dickinson, CA, USA). [18F]EF5 and [18F]FDG uptake in tumours 18F-labelled EF5 (2-(2-nitro-1H-imidazol-1-yl)-N- (2,2,3,3,3-pentafluoropropyl)-acetamide) was synthesised from 2-(2-nitro-1H-imidazol-1-yl)-N-(2,3,3-trifluoroal- lyl)-acetamide using high specific radioactivity 18F-F2 as the labelling reagent [45]. The specific radioactivity of [18F]EF5, decay corrected to the end of synthesis, exceeded 3.7 GBq/μmol. Radiochemical purity was higher than 98.5% in every production batch. 18F- labelled FDG (2-Deoxy-2-fluoro-D-glucose) was synthe- sised from mannosyl triflate using a nucleophilic method. Radiochemical purity exceeded 95% and speci- fic radioactivity was approximately 74 GBq/μmol at the end of synthesis.