Tuomela et al. BMC Cancer 2010, 10:596 http://www.biomedcentral.com/1471-2407/10/596 Tumour hypoxia is also related with increased angiogen- esis which is primarily stimulated by HIF1-induced vascu- lar endothelial growth factor (VEGF) but also by other tumour cell produced cytokines and growth factors including fibroblast growth factors (FGFs) [10,11]. Regard- less of neovascularisation, tumours are often poorly oxyge- nated due to disorganised and leaky vessels [2]. VEGF is a mitogen for vascular endothelial cells but not for other cell types [12,13]. It induces a strong angiogenic response in different in vivo models and increases vascular perme- ability, which is essential to angiogenesis associated with tumour growth and wound healing [14]. In human pros- tate cancer patients, the expression of VEGF correlates with Gleason score and occurrence of lymph node metas- tasis [15,16] as well as the poor outcome of radical treat- ment of localised prostate cancer [17]. The family of FGFs plays an important role in many physiological processes including development [18], wound healing [19], angiogenesis [20], bone formation and osteoblast differentiation [21]. Studies of our own and others on FGF8 have shown that reciprocal FGF/ FGF receptor mediated interactions between tumour cells and stromal cells play important roles in prostate cancer progression and angiogenesis [20,22-26]. Fibro- blast growth factors 1, 2, 6, 9 and 17 are also expressed at high levels in prostate cancer, where they may func- tion as paracrine and/or autocrine mediators [22,27]. Fibroblast growth factor 8 was originally cloned from conditioned medium of mouse mammary tumour-derived SC-3 cells and was identified as androgen-induced growth factor (AIGF) [28]. Four human FGF8 isoforms, named FGF8a, FGF8b, FGF8e and FGF8f, are formed by alterna- tive splicing [29]. FGF8b has been found to be the most transforming of these isoforms and FGF8b targeted to prostate epithelium causes prostatic intraepithelial neopla- sia (PIN) lesions in transgenic mice [30]. FGF8b is also the major isoform expressed in prostate cancer [17,26,31,32]. FGFs mediate their effects by binding to specific tyrosine kinase receptors (FGFR1-4), which all are expressed in prostate cancer [18,33]. The in vitro and in vivo studies have shown that FGF8b increases growth, invasion, tumor- igenesis, angiogenesis and bone metastasis in experimental breast [23,34,35] and prostate cell lines and tumours [20,36-38]. FGF8b is a strongly angiogenic factor, which property has been considered to contribute to increased tumour growth [20,23]. In human prostate cancer, the expression of FGF8b has been shown to predict a poor prognosis [17,32]. We aimed to study whether FGF8b-induced rich neo- vascularisation is able to influence oxygenation of pros- tate tumour microenvironment and influence tumour growth by these mechanisms. PC-3 cells were used to create models for prostate cancer expressing VEGF or FGF8b, which both are known to be angiogenic and to Page 2 of 14 be increased in prostate cancer. Nude mice were subcu- taneously inoculated with PC-3 cells transfected with FGF8b, VEGF or empty vectors. PC-3/VEGF cells were used as “positive” controls because they were expected to produce well-vascularised and oxygenised tumours. The FGF8b tumours were studied for growth rate, vascularisation, energy metabolism as well as tumour hypoxia and oxygenation, and the findings were com- pared with those from VEGF and mock tumours, which served as positive and negative controls, respectively. Methods Cell culture and transfection The human hormone-resistant prostate cancer cell line PC-3 was obtained from the American Tissue-Type Cul- ture Collection (Rockville, MD, USA). PC-3 cells, which were stably transfected with FGF8b (PC-3/FGF8b) were used as previously described [38]. Stable VEGF transfec- tion was carried out by using the expression vector pcDNA3.1(+) (Invitrogen, CA, USA) containing human VEGF cDNA in an ECORI site (kindly provided by Prof. Kari Alitalo, University of Helsinki, Finland, [39]) and empty pcDNA3.1(+) vector-transfected cells (mock) were used as controls. Neomycin (G418, 500 μg/mL) was added to culture media of transfected PC-3 cells for the selection of clones. At near confluence, the cells were harvested in trypsin/EDTA (Biochrom AG, Germany), washed with culture medium and finally suspended at a concentration of 1 × 106/100 μL in sterile phosphate-buf- fered saline solution (PBS, Biochrom AG, Germany). The cells were kept on ice until inoculation. Northern and Western blot analysis Total RNA was extracted from PC-3 cells using the gua- nidinium isothiocyanate method [40]. Northern blotting was performed as previously described [41]. Serum-free DMEM conditioned by FGF8b, VEGF and mock cells was harvested from the cultures as previously described [42]. Heparin-sepharose-bound proteins were extracted by 5-min incubation in Laemmli sample buffer at 95°C and separated by sodium dodecyl sulphate-polyacryla- mide gel electrophoresis. After transfer to nitrocellulose membranes (Bio-Rad), proteins were detected by using goat polyclonal anti-FGF8b neutralising antibodies [20] and anti-human VEGF antibodies (both from R&D sys- tems, Minneapolis, MN, USA). Horseradish peroxidase- labelled anti-goat IgG (DAKO, Denmark) was used as a secondary antibody. Protein bands were visualised by using an ECL chemiluminescence detection system (GE, Healthcare Biosciences, Uppsala, Sweden). Animals and tumour models Eight-week-old male athymic nu/nu mice (Harlan, the Netherlands) were maintained under controlled