[CANCER RESEARCH 63, 3919 –3922, July 15, 2003] Advances in Brief SiRNA-mediated Inhibition of Vascular Endothelial Growth Factor Severely Limits Tumor Resistance to Antiangiogenic Thrombospondin-1 and Slows Tumor Vascularization and Growth 1 Ste ´phanie Filleur, 2 Aure ´lie Courtin, 2 Slimane Ait-Si-Ali, Julien Guglielmi, Carole Merle, Annick Harel-Bellan, Philippe Cle ´zardin, and Florence Cabon 3 CNRS UPR 9079, 94801 Villejuif, France [S. F., A. C., S. A-S-A., C. M., A. H-B., F. C.], and INSERM U403, Faculte ´ Laennec, 69372 Lyon, France [J. G., P. C.] Abstract In the past few years, several laboratories have developed antiangio- genic molecules that starve tumors by targeting their vasculature and we have shown that, when produced in tumors, the antiangiogenic molecule thrombospondin-1 (TSP1) reduces the vascularization and delays tumor onset. Yet over time, tumor cells producing active TSP1 do eventually form exponentially growing tumors. These tumors are composed of cells secreting unusually high amounts of the angiogenic stimulator vascular endothelial growth factor (VEGF) that are sufficient to overcome the inhibitory TSP1. Here, we use short double-stranded RNA (siRNA) to trigger RNA interference and thereby impair the synthesis of VEGF and ask if this inability to produce VEGF prevents the development of TSP1 resistance. Systemic in vivo administration of crude anti-VEGF siRNA reduced the growth of unaltered fibrosarcoma tumor cells, and when the anti-VEGF siRNA was expressed from tumor cells themselves, such inhi- bition was synergistic with the inhibitory effects derived from TSP1 secretion by the tumor cells. Anti-VEGF siRNA delayed the emergence of TSP1-resistant tumors and strikingly reduced their subsequent growth rate. Introduction The observation that tumor growth is highly dependent on the ability of tumors to induce their own vascularization has led numerous laboratories to isolate or develop angiogenesis inhibitors such as TSP1 4 (1). This antivascular strategy that targets the normal, geneti- cally stable endothelial cells of the host rather than the genetically unstable tumor cell population was shown to be very efficient at reducing tumor growth and was not expected to trigger tumor resist- ance (2). However, recently we (3) and others (4) have demonstrated that changes in the tumor cells themselves, particularly sustained high-level secretion of the angiogenic stimulator VEGF, can enable tumors to bypass antiangiogenic treatments. It has recently been shown that the introduction in a mammalian cell of double-stranded oligoribonucleotides, also called siRNA, trig- gers the degradation of the endogenous mRNA to which the siRNA hybridizes (5). This mechanism is highly sequence specific and allows to turn off the expression of a target protein (6, 7). Many studies demonstrated the high efficiency and versatility of RNA interference in cell cultures. Some authors developed vectors or viruses to produce siRNA in cells (8). The in vivo regulation of a gene by RNA inter- ference has been obtained either using these vectors or viruses (9) or using the so-called hyperpressure technique (10, 11), which drives siRNA mainly in the liver and would not be possible to use in humans. In this work, we demonstrate that low doses of siRNA administrated by a systemic route penetrate into tumors and control the expression of target genes to produce phenotypic effects. The aim of the present work was to determine whether blocking the ability of tumor cells to secrete high levels of VEGF by the in vivo administration of siRNA could diminish or prevent the triggering of resistance to the antitumor effects of TSP1. Materials and Methods Cell Culture. The rat fibrosarcoma cJ4 cells (12) were grown as described previously. A bidirectional TSP1-luciferase-inducible expression vector was introduced in cJ4 cells to generate JT8 cells as described previously (3). Cells were grown in the presence of 100 ng/ml dox, a tetracycline analogue to repress TSP1 expression. siRNA. The siRNA (sense and antisense strands) were purchased from MWG Biotech (Ebersberg, Germany). The sense strands sequences were the following: VEGF, 5'-AUGUGAAUGCAGACCAAAGAA-TT; CONT, 5'- GAUAGCAAUGACGAAUGCGUA-TT; and LUC, 5'-AACGUACGCGG- AAUACUUCGA-TT. In vitro transfections were performed using the Transit- TKO polymer/lipid from Mirus (Madison, WI) as recommended. For 6  10 6 cells in 10 ml of medium, 2 g of siRNA were used. Cells were washed 24 h after transfection. Tumorigenicity Assays. cJ4 or JT8 cells were injected s.c. in PBS (10 6 cells/site) into the hind quarters of four to six female Swiss nu/nu mice, 4 – 6 weeks old (Iffa Credo, L’Arbresle, France) for each tested condition. Each experiment was repeated at least twice. When stated, dox (100 g/ml) was added to the drinking supply of the animals to repress TSP1 and luciferase expressions. The drinking supply was changed three times a week. Tumor volume was calculated as v = L  l 2  0.52, where L and l represent the larger and the smaller tumor diameter measured daily. For in vivo injections, each animal was injected daily with 50 (i.p., i.v., or s.c. injections) or 10 l (intratumoral injections) of PBS containing 3 g of siRNA (125 g/kg/day). The care of the animals was provided in the animal quarters of the Institut Andre ´ Lwoff in Villejuif according to the institutional guidelines. Immunohistochemistry, Scoring of Microvessel Density. TSP1, VEGF, and CD31 detection and scoring of blood vessels density in tumors were performed as described previously (3). Luciferase Activity. Tumors were homogenized with a polytron homoge- nizer in cell culture lysis reagent (Promega). Protein concentration was meas- ured using BSA as standard with the Bio-Rad DC protein assay. Luciferase activity was quantified in a luminometer (Analytical Luminescence Laborato- ries) using 1 mM luciferine as substrate. VEGF Quantification. VEGF was quantified in cell supernatants or in tumor homogenates using an ELISA kit for mouse VEGF from R&D. Cells were transfected with VEGF- or control-siRNA. Twenty-four h later, cells were replated at the same density and conditioned media collected on day 4 after transfection. Cells were replated at equal density on day 4 and media collected on day 6 and finally replated on day 11 and media collected on day Received 4/16/03; accepted 5/23/03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by grants from the Association pour la Recherche sur le Cancer (to F. C.), the Groupement des Entreprises Franc ¸aises dans la Lutte contre le Cancer (to F. C.), and the Fondation de l’Avenir (to F. C.). 2 These authors contributed equally to this work. 3 To whom requests for reprints should be addressed, at Institut Andre ´ Lwoff, CNRS UPR9079, 7 rue Guy Mo ˆquet, 94801 Villejuif, France. E-mail: fcabon@vjf.cnrs.fr. 4 The abbreviations used are: TSP1, thrombospondin-1; VEGF, vascular endothelial growth factor; siRNA, small interfering RNA; dox, doxycycline. 3919 Research. on October 27, 2015. © 2003 American Association for Cancer cancerres.aacrjournals.org Downloaded from