Progress in cancer biology knowledge is not satisfactorily followed by development of new anti-cancer therapies. One of the possible reasons is the presently used preclinical animal models that do not reflect physiological cancer development observed in clinical practice. Here, we propose a novel murine model of tumor development that better imitates complexity of cancer biology and therefore can be used to design new anti-cancer strategies with possibly higher rate of efficiency after translating results into clinical phase. The novel model implies in vitro formation of tumor spheroids that consist of approximately 1500 cells. The formed tumor spheroids (~250 μm diameter) are injected subcutaneously into a mouse within liquid Matrigel that polymerises after injection. This strategy results in relatively fast and simple formation of subcutaneous Matrigel plug that contains single tumor spheroid that mimics the initial stage of primary tumor development. A potential anti-cancer drug can be also mixed with the liquid Matrigel. In the present study a luciferase expressing B16–F10 melanoma cell line was used to form tumor spheroids. An in vivo biolumines- cence imaging showed an intact single spheroid in a Matrigel plug after injection and the following tumor growth and spreading within the plug during the next 10–14 days. A prolongation of an observation time resulted in distant metastasis detection. Important differences between the novel model and classical method of subcutaneous tumor cells injection are observed when analyzing angiogenesis process in tumor microenvironment. An ultrasonography (USG) blood perfusion examination evidenced that in case of classical way of tumor induction a necrotic, non-perfused core quickly occurred in the center of the growing tumor. In contrast, the proposed model allows fast tumor growth paralleled by angiogenesis that does not lead to the necrotic core formation. Vessels formed in tumor microenvironment are connected to the host vasculature, as shown by perfusion of intrave- nously injected FITC-dextran, however the kinetics of blood flow is impaired when analyzed by USG with microbubbles contrast, which is typical for tumor vessels. Concluding, the present study proposes a new spheroid–plug model to study tumor angiogenesis and development. The model provides a simple method to induce tumor growth from low initial number of cells organized in spheroid that better reflects the pathophysiological tumor development. Presented advantages of the model make it a valuable research tool to study tumor angiogenesis, tumor development and dissemination among other cancer biology processes. Supported by grants 347/N-INCA/2008 and 311/N-COST/2008/ from the Ministry of Science and Higher Education. doi:10.1016/j.vph.2011.08.173 P.12.9 Proangiogenic enzymes in non-small cell lung carcinoma — An interplay between thymidine phosphorylase and heme oxygenase-1? Magdalena Tertil a,b , Klaudia Skrzypek a,b , Kazimierz Weglarczyk b , Chantal Pichon b , Claudine Kieda b , Halina Was a , Alicja Jozkowicz a , Jozef Dulak a a Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, ul. Gronostajowa 7, 30-387 Krakow, Poland b Centre de Biophysique Moléculaire, CNRS, Rue Charles-Sadron, Orléans, France E-mail addresses: magdalena.tertil@uj.edu.pl (M. Tertil), jozef.dulak@uj.edu.pl (J. Dulak) Thymidine phosphorylase (TP) is an enzyme catalysing conver- sion of thymidine (Thd) into thymine and 2-deoxy-d-ribose-1- phosphate (dRP), which is further dephosphorylated to 2-deoxy-d- ribose (dR). Heme oxygenase-1 (HO-1) is an inducible isoform of an enzyme degrading heme. Apart from their roles in basic metabolism, both proteins exert proangiogenic activities and are considered as cancer therapeutic targets. While the role of HO-1 in angiogenesis is well established, the detailed mechanism of proangiogenic action of TP remains unknown. It was shown previously in bladder carcinoma cells that TP induces HO-1. Here we report results of a study on the involvement of HO-1 in proangiogenic activity of TP. In endothelial cells, when we overexpressed TP, an upregulation of HO-1 and a concomitant increase in VEGF expression was observed, confirming its proangiogenic property. Nevertheless, treatment of cells with a major TP pathway product, dR, failed to induce HO-1. Moreover, dR-induced migration and sprouting of ECs were not modulated by HO-1 inhibitor. In non-small cell lung carcinoma cells, we investigated regulation of production of angiogenic factors by the tumor cells under conditions mimicking the in vivo situation. In normoxia, interleukin-8 (IL-8) was upregulated in NCI-H292 cells overexpressing TP (NCI-TP). When the cells were exposed to hypoxia (1% oxygen) in the presence of Thd, HO-1 and VEGF expression were higher in NCI-TP cells than in the control. Stimulation with high dose of dR increased HO-1 mRNA level but the upregulation was non-detectable on protein level. dR and dRP did not influence IL-8, VEGF and matrix metalloproteinase-1, which was described as another TP-regulated gene. This report shows that the regulation of angiogenic factors by TP, including the HO-1, cannot be limited to the action of its main product and can be modulated by hypoxia and substrate availability. The findings will be verified in the in vivo tumor xenograft model. Supported by grants 347/N-INCA/2008, 311/N-COST/2008/ from the Ministry of Science and Higher Education and CNRS-INCA-MSHE Polish French conv. 2009-011. doi:10.1016/j.vph.2011.08.174 P.12.10 Heme oxygenase 1 (HO-1) challenges the angiogenic switch in prostate cancer Elba S. Vazquez, Geraldine Gueron, Mercedes Ferrando, Belen Elguero, Angeles Salles, Adriana De Siervi Department of Biological Chemistry, School of Sciences, University of Buenos Aires, CONICET, Argentina E-mail address: elba@qb.fcen.uba.ar (E.S. Vazquez) Prostate cancer (PCa) is the second leading cause of cancer- associated death in men. Once a tumor is established it may attain further characteristics via mutations or hypoxia, which stimulate new blood vessels. Angiogenesis is a hallmark in the pathogenesis of cancer and inflammatory diseases that may predispose to cancer. Heme oxygenase-1 (HO-1) counteracts oxidative and inflammatory damage and was previously reported to play a key role in prostate carcinogenesis. To gain insight into the anti-tumoral properties of HO-1, we investigated its capability to modulate PCa associated-angiogenesis. In the present study, we identified in PC3 cells a set of inflammatory and pro- angiogenic genes down-regulated in response to HO-1 overexpression, in particular VEGFA, VEGFC, HIF1α and α5β1 integrin. Our results indicated that HO-1 counteracts oxidative imbalance reducing ROS levels. An in vivo angiogenic assay showed that intradermal inoculation of PC3 cells stably transfected with HO-1 (PC3HO-1) generated tumors less vascularised than controls, with decreased microvessel density and reduced CD34 and MMP9 positive staining. Interestingly, longer term grown PC3HO-1 xenografts displayed reduced neovascularization with the subsequent down-regulation of VEGFR2 expression. Additionally, HO-1 repressed nuclear factor κB (NF-κB)-mediated transcription from an NF-κB responsive luciferase reporter construct, which strongly suggests that HO-1 may regulate angiogenesis through this pathway. Abstracts 370