ORIGINAL ARTICLE Cotargeting of VEGFR-1 and -3 and angiopoietin receptor Tie2 reduces the growth of solid human ovarian cancer in mice H Sallinen 1,2,3 , M Anttila 1,2,3 , O Gro ¨hn 4 , J Koponen 1 , K Ha ¨ma ¨la ¨inen 2,5 , I Kholova 1,2,5 , V-M Kosma 2,5 , S Heinonen 2,3 , K Alitalo 6 and S Yla ¨-Herttuala 1,7 1 Department of Molecular Medicine, AI Virtanen Institute, University of Eastern Finland, Kuopio, Finland; 2 Institute of Clinical Medicine, Gynaecology, and Pathology and Forensic Medicine, University of Eastern Finland, Kuopio, Finland; 3 Department of Gynaecology, Kuopio University Hospital, Kuopio, Finland; 4 National BIO-NMR Facility, AI Virtanen Institute, University of Eastern Finland, Kuopio, Finland; 5 Department of Pathology, Kuopio University Hospital, Kuopio, Finland; 6 Molecular/Cancer Biology Laboratory Biomedicum Helsinki and Haartman Institute, University of Helsinki, Helsinki, Finland and 7 Gene Therapy Unit and University Hospital Research Unit, Kuopio, Finland Despite optimal surgery and chemotherapy, the prognosis of ovarian cancer patients remains poor and new treatments are urgently needed. Solid tumors require the formation of new vessels for growth and metastasis. In the present study, we have used soluble vascular endothelial growth factor (sVEGF) receptors sVEGFR-1 and -3, soluble receptors Tie1 and Tie2 and their combinations in an ovarian cancer xenograft model. Human ovarian cancer cells were injected intraperitoneally into nude mice (n ¼ 42) and magnetic resonance imaging (MRI) was used for confirming tumors before gene delivery. Treatment with combined AdsVEGFR-1, AdsVEGFR-3 and AdsTie2 significantly decreased the size of the intraperitoneal tumors compared with the controls (AdLacZ; P ¼ 0.038) with significantly less microvessels and vascular area. Unexpectedly, treatment with combined AdsTie1 and AdsTie2 led to a dramatic shortening of the survival which was not observed in the groups receiving either of the soluble receptors alone (P ¼ 0.031). The only difference to other treatments was liver toxicity observed after the combined Tie receptor treatment. In conclusion, combined inhibition of VEGFR-1, VEGFR-3 and Tie2 pathways was safe and provided efficient therapy for ovarian cancer in mice. Cancer Gene Therapy (2011) 18, 100–109; doi:10.1038/cgt.2010.56; published online 24 September 2010 Keywords: antiangiogenesis; antilymphangiogenesis; ovary; carcinoma; MRI Introduction In a large majority of patients with ovarian carcinoma, the disease is diagnosed at an advanced stage with peritoneal carcinosis, ascites and poor prognosis. Surgical debulking followed by platinum-based chemotherapy has been the golden standard of managing with ovarian cancer. However, the long-term survival among the patients with an advanced disease (stage III or IV) is still low 1 and ovarian cancer has the highest mortality of gynecological malignancies. 2 Solid tumors require the formation of new vessels for growth and metastasis. 3 Angiogenesis, formation of new blood vessels, is controlled by proangiogenic growth factors and antiangiogenic molecules. In cancer, the balance of these factors is disturbed leading to excessive growth and branching of vessels. 4 Vascular endothelial growth factors (VEGFs) and angiopoietins have signifi- cant roles in tumor angiogenesis and they are mostly specific for vascular endothelial cells. 5 Members of the VEGF family (VEGF-A, -B, -C, -D and placental growth factor (PLGF) signal through three tyrosine kinase receptors VEGF receptor (VEGFR)-1, -2 and -3, also known as Flt-1, KDR/Flk-1 and Flt-4. 6 Both VEGFR-1 and -2 bind VEGF-A, which is the main regulator of blood vessel growth. 7 VEGFR-1 has higher affinity for VEGF-A but weaker tyrosine kinase activity than that of VEGFR-2. It also binds PLGF and VEGF- B, which do not bind to VEGFR-2. VEGFR-1 is expressed in endothelial cells and macrophages and promotes tumor growth, metastasis and inflammation. 8 A soluble alternatively sliced form of VEGFR-1 is a natural VEGF-A inhibitor 9 and soluble VEGFR-1 has been reported to be overexpressed in pre-eclamptic patients. 10 VEGFR-2 binds VEGF-A, -C and -D and has a key role in angiogenesis, vasculogenesis and vascular permeability. 11 Lymphatic vessels are essential Received 24 February 2010; revised 3 June 2010; accepted 19 July 2010; published online 24 September 2010 Correspondence: Professor S Yla¨ -Herttuala, Department of Molecular Medicine, AI Virtanen Institute, University of Eastern Finland, P.O.Box 1627, Kuopio FIN-70211, Finland. E-mail: seppo.ylaherttuala@uef.fi Cancer Gene Therapy (2011) 18, 100–109 r 2011 Nature America, Inc. All rights reserved 0929-1903/11 www.nature.com/cgt