Cotargeting of VEGFR-1 and -3 and angiopoietin receptor Tie2 reduces the growth of solid human ovarian cancer in mice AbstractDespite 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=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=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=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. IntroductionIn 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 low1 and ovarian cancer has the highest mortality of gynecological malignancies.2Solid 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 significant roles in tumor angiogenesis and they are mostly specific for vascular endothelial cells.5Members 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 inhibitor9 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 for draining interstitial fluid from tissues and returning it to the blood circulation. They also participate in immune defence and are important for metastatic tumor spread.12 VEGF-C and-D stimulate lymphangiogenesis through VEGFR-3 which is predominantly expressed not only in lymphatic endothelium13, 14 but also in angiogenic sprouts.15Angiopoietins (Ang1鈥?) are ligands for the tyrosine kinase receptor Tie2.16, 17, 18 Ang1 activates Tie2 signaling pathways and promotes the recruitment of pericytes and smooth muscle cells to the developing vessels16, 19 and contributes to tumor dissemination and metastasis.20 Ang2, on the contrary, functions in a context-dependent manner as an antagonist promoting either blood vessel growth or regression depending on the levels of other growth factors, such as VEGF-A.21 Tie2 is expressed in lymphatic capillaries and Ang1 has been shown to induce lymphangiogenesis in parallel to upregulation of VEGFR-3.22 Ang2 is also required for normal lymphatic vessel formation.23 Mouse Ang3 and human Ang4 are interspecies orthologues,18 whose functions have not yet been clarified. The function of Tie1 is less well characterized than that of Tie2 because of the lack of its own specific ligands, although it has been recently shown that the Tie1 receptor can interact with Tie2 and signal as a heterodimeric complex. Furthermore, Ang1 and Ang4 can activate Tie1.24In our earlier study, targeting VEGF pathways by soluble VEGFR 1, 2- and -3 showed efficacy as assessed by reduced tumor volume and weight, but they did not significantly prolong survival.25 To further test new treatment options in this mouse model closely mimicking human ovarian cancer,26 we have now targeted both endothelial cells and pericytes using soluble VEGFR (sVEGFR)-1 and -3, sTie1 and sTie2 and their combinations. Soluble receptors lack transmembrane domain and intracellular tyrosine kinase parts and therefore they do not initiate signal transduction. As the high-affinity VEGF binding and signaling requires dimerization of the VEGF receptor monomers, all constructs contain an immunoglobulin Fc domain to ensure an efficient dimerization of the soluble receptors. Magnetic resonance imaging (MRI) was applied for the timing of gene therapy to treat solid sizeable tumors, instead of micrometastatic disease, and to follow-up tumor progression non-invasively in vivo. This is the first ovarian cancer study where angiogenesis and lymphangiogenesis are restricted by both soluble VEGF and angiopoietin decoy receptors.Materials and methodsCell lineSKOV-3m cell line has been characterized.26 Cells were cultured in McCoy\'s 5A medium (Sigma, Steinheim, Germany). Before in vivo inoculation, the cells were trypsinized and counted.Viral vectorsAdenoviral vectors encoding sVEGFR-1-IgG (immunoglobulin) fusion protein (AdsVEGFR-1),27, 28, 29 sVEGFR-3-IgG fusion protein (AdsVEGFR-3),30, 31 soluble Tie1-IgG (AdsTie1), soluble Tie2-IgG (AdsTie2)32, and LacZ (AdLacZ) as a control vector were used for the study. Replication-deficient E1鈥揈3-deleted clinical good manufacturing practice (GMP)-grade adenoviruses were produced in 293T cells. Adenoviruses were analyzed to be free from helper viruses, lipopolysaccharides and bacteriological contaminants.33, 34Animal modelBalb/cA-nu female nude mice (n=42), 8鈥?0-weeks old, were used for the studies. Ovarian carcinoma was produced by inoculating 1 脳 107 SKOV-3m cells into the peritoneal cavity of the nude mice with a 22 G needle. Development of the ovarian carcinoma tumors was followed by sequential MRI. When the first solid, measurable tumor was detected in MRI, gene transfer was done the following day. Mice were randomly divided into five groups: five animals received AdsTie1 (1 脳 109 plaque-forming units (PFU)), six animals received AdsTie2 (1 脳 109鈥塒FU), eight animals received AdsTie1 and AdsTie2 (1 脳 109鈥塒FU each vector), eight animals received AdsVEGFR-1, AdsVEGFR-3 and AdsTie2 (0.7 脳 109鈥塒FU each vector), and eight control animals received AdLacZ (2 脳 109鈥塒FU; Table 1). Gene transfer was performed intravenously via tail vein in the final volume of 200鈥壩糽 in 0.9% saline. MRI was done weekly after gene transfer and tumor volumes were assessed. The overall follow-up time lasted until the appearance of significant symptoms necessitating killing or till the death. At the time of death, all tumor tissue, liver, spleen, kidneys and lungs were harvested and tumor masses were weighed. Ascites fluid was collected with a syringe. The mice were kept in a pathogen-free isolated unit at the National Experimental Animal Center of the University of Kuopio. Food, water and sawdust bedding were autoclaved and the mice received chow and water ad libitum. All animal studies were accepted by the Experimental Animal Committee of the University of Kuopio according to the Finnish and European Union legislation directives and recommendations (86/609/EEC and 2007/526/EC).Table 1 Description of the study groupsFull size tableHistology, immunohistochemistry and microvessel measurementsTissue samples were immersed in 4% paraformaldehyde for 4鈥?鈥塰, followed by overnight immersion in 15% sucrose.35 The specimens were embedded in paraffin and 5渭m-thick sections were processed for hematoxylin-eosin, Ki-67 (DakoCytomation, Glostrup, Denmark), CD-34 (HyCult biotechnology b.v., AA Uden, The Netherlands), lymphatic vessel hyalyronan receptor-1 (LYVE-1) (ReliaTech GmbH, Braunschweig, Germany) and pericyte marker 伪-smooth muscle actin (伪-SMA) (DakoCytomation).Photographs of histological sections were taken and processed using an Olympus AX70 microscope (Olympus Optical, Tokyo, Japan), and analySIS (Soft Imaging System, GmbH, Germany) and PhotoShop (Adobe) softwares. Mean microvessel area (渭m2), microvessel density and total microvascular area (%) of the tumors (tumor vascular area) were measured from CD34-immunostained sections using analySIS software at 脳 100 in a blinded manner. In all, 10 different fields that represented maximum microvessel areas were selected from each tumor. Necrotic areas were avoided. The pericyte coverage was assessed as missing (0%), covering 50% of the vessel wall circumference (low pericyte covering), more than 50% of the vessel wall circumference (high pericyte covering) and fully (100%) covered. All vessels in five different fields from tumor serial sections were evaluated under a 脳 20 objective in CD34 and 伪-SMA immunostained sections. The total number of LYVE-1-positive lymphatic vessels per section was counted. Ki-67 was quantified semiquantitatively by two observers who counted the number (%) of Ki-67-positive cells in the epithelial tumor tissue in a blinded manner. Means卤s.e.m. of the measurements are reported.MRITo follow the development of ovarian carcinoma and to measure tumor volumes, 9.4 T vertical magnet (Oxford Instruments, Oxford, UK) equipped with actively shielded field gradients (Magnex Scientific, Abdington, UK) interfaced to an s.m.i.s. console (Surrey Medical Imaging Systems, Guolford, UK) was used. Details of MRI imaging have been described previously.26 MRI was performed weekly after the first tumors were detectable.Reverse transcription PCRReverse transcription PCR was used to confirm the adenoviral transgene expression in mouse liver samples. Total RNA was extracted using Trizol Reagent (Gibco BRL, Grand Island, NE) according to manufacturer\'s instructions. Total RNA was treated with DNaseI (Promega, Madison, WI) to remove any contaminating DNA, and complementary DNA synthesis was performed with 2鈥壩糶 of RNA with random hexamers (Promega). Primers for the amplification of sTie1 were forward: 5鈥?IndexTermTCCCCATCCTCTTCTTGGC-3鈥? reverse: 5鈥?IndexTermTTGTCTGGAAGCAGGTGGG-3鈥? for sTie2, forward: 5鈥?IndexTermAAGCTTGGTACCGAGCTCG-3鈥? reverse: 5鈥?IndexTermTTATCTCCCCTGTCCACGG-3鈥? for sVEGFR-1, forward: 5鈥?IndexTermAGGCCAGACACTGCATCTCC-3鈥? reverse: 5鈥?IndexTermGCTTCACAGG TCAGAAGCCC-3鈥? for sVEGFR-3 complementary DNA, forward: 5鈥?IndexTermTGAAGGCACAGAAGCTAGGCC-3鈥? reverse: 5鈥?IndexTermACCTGAGTCGAACTCAGCCC-3鈥?producing amplicons of 363, 442, 500 and 530鈥塨p, respectively. PCR reaction mixtures consisted of 20鈥塸mol of each primer, 0.2鈥塵M dNTPs (Promega), 1.5鈥塵M MgCl2, Dynazyme EXT PCR buffer, 1.5鈥塙 of Dynazyme EXT DNA polymerase (Finnzymes, Helsinki, Finland) and 500鈥塶g of complementary DNA sample. Amplifications were carried out with the following conditions: the first denaturation step at 95鈥壜癈 for 3鈥塵in, followed by 35 cycles with 45鈥塻 at 95鈥壜癈, 45鈥塻 at 58鈥壜癈 for sTie1 and sTie2, 60鈥壜癈 for sVEGFR-1 or at 62鈥壜癈 for sVEGFR-3, 45鈥塻 at 72鈥壜癈, with the final step at 72鈥壜癈 for 15鈥塵in.Clinical chemistry from plasma samplesPlasma samples were collected at day 3, 6 and 13 after the gene transfer and when the mice were killed. Alanine aminotransferase (ALT) was monitored using routine clinical chemistry assay at Kuopio University Hospital Central Laboratory.Statistical analysesStatistical significance was evaluated using Kruskal鈥揥allis test, followed by Mann鈥揥hitney U-test with correction for multiple comparisons. Kaplan鈥揗eier plots and log rank test was used for the analysis of survival. Results are expressed as mean卤s.e.m. A value of P 0.05 was considered as statistically significant.ResultsIntraperitoneal tumor growth and formation of ascitesIntraperitoneal tumors were detected within 2 weeks after the SKOV-3m cell inoculation in all mice. Treatment groups are shown in Table 1. Reverse transcription PCR showed mRNA expression of all transgenes in liver samples 6 days after the gene transfer (results not shown). MRI was repeated weekly after the gene transfer (Figure 1a). At the time of the first and the second MRI, there were no significant differences in tumor volumes between the control AdLacZ group and the gene therapy groups. In the third MRI, tumor volumes of the group V (sVEGFR-1, -3 and sTie2) were only one-third of that of the control mice (763卤222 versus 2227卤532鈥塵m3, P=0.032; Figures 1b and c). The final tumor weights at the end of the follow-up of the same animals were only half of that of the control mice (2.3卤0.25 versus 5.0卤0.77鈥塯, P=0.020; Figure 2a). A trend towards smaller tumor weights was also noted in other treatment groups, but without statistical significance (Figure 2a). Ascites formation did not significantly differ between the study groups. However, targeting both VEGF and Tie pathways, a tendency towards a greater amount of ascitic fluid was seen (Figure 2b).Figure 1(a) Outline of the study. Tumors developed within 15 days after inoculation of the SKOV-3m tumor cells. The presence of all tumors was verified by MRI before starting gene therapy. Tumors were observed weekly until the death of the mice. (b) Measured by MRI, the mean tumor volumes (mm3) were significantly smaller in group V (sVEGFR-1, -3 and sTie2) versus controls 2 weeks after the gene therapy (III MRI), P 0.05. (c) MRI pictures of the development of ovarian tumors in group V (sVEGFR-1, -3 and sTie2) compared with group I (AdLacZ). Tumors are marked with arrows and broken lines. Ascites is marked with arrowhead.Full size imageFigure 2(a) At the end of the follow-up, the weights of the tumors were significantly smaller in the combination group V (sVEGFR-1, -3 and Tie2). (b) Although a tendency towards a greater amount of ascites in combination group V was noted, this was not statistically significant. *P 0.05 versus LacZ, error bars, s.e.m. (c) Hematoxylin-eosin staining of serous adenocarcinoma in group I (AdLacZ). (d) Focal necrosis and connective tissue were present in the tumor tissue in group V (sVEGFR-1, -3 and Tie2). Hematoxylin-eosin staining. (e) More proliferating tumor cells were seen in control group I (e) than in group V (f), P=0.001. Ki-67 staining in e and f. Bar=100鈥壩糾.Full size imageHistologyIntraperioneal tumors were poorly differentiated (grade 3) serous cystadenocarcinomas, which consist of variable size of nucleus and limited stroma (Figure 2c). Active mitoses were also noticed. Morphology of the tumors was disturbed and the tumor tissue was partly substituted by connective tissue in the combination group V (sVEGFR-1, -3 and sTie2; Figure 2d) in addition to lower cell proliferation measured by Ki-67 staining (27.9卤9.8 versus 76.0卤2.7% in the controls, P=0.001; Figures 2e and f). Necrotic areas in the tumors were significantly larger in combination group of sTie1 and sTie2 compared with the control group (26.7卤11.2 versus 0%, P=0.015).Microvessel measurementsMean microvascular area (渭m2), microvessel density and tumor vascular area were measured to detect the effect of gene therapy on intratumoral microvessels. In the combined group of sVEGFR-1, -3 and sTie2, the mean areas of CD34-stained microvessels (248卤29鈥壩糾2) and tumor vascular area (1.42卤0.09%) were significantly smaller than that in controls (512卤178鈥壩糾2, P=0.036 and 3.48卤1.4%, P=0.040; Figures 3a, b and 4a, b). In addition, in these tumors, we observed a significant decrease in pericyte coverage (that is, pericytes covered 50% of the vessel circumference) in comparison to LacZ group (P=0.008; Figures 3c, 4c and d). Furthermore, also in the group of sTie1+sTie2 together and sTie1 alone, low pericyte coverage was detected in most cases. On the contrary, in LacZ and sTie2 groups, high pericyte coverage was detected (Figure 3c). The majority of LYVE-1-positive intratumoral lymphatic vessels were located in the periphery of the tumors and only a minority were present in the central part (Figures 4e and f). In sVEGFR-1, -3 and sTie2 group, the mean number of lymphatic vessels was 3卤0.7 compared with controls which had 6.3卤2.3 vessels per section, P=0.077.Figure 3(a) Mean microvessel area (渭m2) was significantly reduced in mice treated with the combination of sVEGFR-1, -3 and Tie2. (b) Combination gene therapy with sVEGFR-1, -3 and sTie2 significantly reduced the area of tumors covered by microvessels (TVA, tumor vascular area). (c) The highest number of vessels without pericytes was found in group V (sVEGFR-1, -3 and sTie2). Decrease in the pericyte coverage (that is, pericytes covered 50% of the vessel wall circumference) was statistically significant in comparison with LacZ group (P=0.008). In group IV (sTie1+sTie2) and group V (sVEGFR-1, -3 and Tie2), there was the highest number of vessels covered with 50% of the circumference. In control LacZ group and sTie2 group, no vessels without pericytes were detected. *P 0.05 versus LacZ, error bars, s.e.m.Full size imageFigure 4(a) CD34-positive microvessels in tumor tissue of control group I. In group I (a), mean microvessel area and total vascular area of tumors were higher than in group V (sVEGFR-1, -3 and sTie2) (b). Magnification, 脳 200. Bar=100鈥壩糾. (c) CD34 staining of microvessels in group V (arrowheads). (d) A serial section of the same tumor as in c. Microvessel covered by pericytes with 50% of the vessel circumference (arrowhead) and microvessel covered with more than 50% of the vessel circumference (arrow), 伪-SMA staining. Magnification, 脳 400. Bar=100鈥壩糾. (e) More LYVE-1-positive lymphatic vessels were seen in the periphery of the tumors in group I than in the treatment group V (f). Magnification, 脳 200. Bar=100鈥壩糾.Full size imageFigure 5Kaplan鈥揗eier survival plots of all study groups. A significant difference was found between control group I (LacZ) versus group IV (sTie1+sTie2), P 0.05.Full size imageSurvival and safetyThe mean survival (days) in the study groups was as follows: 28卤1 in control group I, 26卤2 in group II (sTie1), 29卤1 in group III (sTie2), 19卤3 in group IV (sTie1 and sTie2) and 30卤2 in combination group V (sVEGFR-1, -3 and sTie2). Most importantly, in group IV, survival was significantly shorter than in controls (P=0.031; Figure 5).Safety was evaluated by clinical examination, investigating histological samples of liver, spleen, kidneys and lungs, and by the analysis of plasma ALT levels. In groups where only a single gene was transferred, the therapy was well tolerated and the mice did not show any side effects, and histological samples of the organs harvested were within the normal range (Figures 6b鈥揹). However, at the end of the follow-up, 38% of the mice in group IV (sTie1 and sTie2) showed macroscopic alterations in the liver, which histologically consisted of local necrosis, regenerative changes in hepatocytes and lymphocytic infiltrations (Figures 6f and g). One mouse in this group had bleeding from rectum and another had blood in the peritoneal cavity. In the group V, (sVEGFR-1, -3 and sTie2) liver cell architecture was well preserved and only mild lymphocytic infiltrates around hepatocytes in portal tract were noticed (Figure 6h). In all, 63% of the mice had edema beneath the skin. Plasma ALT levels are shown in Table 2. Levels were elevated in the later stages of the disease due to liver metastases in treated and control groups. However, ALT values were clearly increased already at earlier time points in combined sTie1- and Tie2-treated animals compared with others.Figure 6Histologically normal liver tissues of healthy mice (a), in control group I (b) and in groups II (c) and III (d). (e) In group IV (sTie1+sTie2), mild regenerative changes in hepatocytes around central vein and well-preserved liver cell architecture were noticed 7 days after gene transfer. (f) At the end of the follow-up in group IV (sTie1+sTie2), strong confluent hepatocellular necrosis was seen around central vein (arrow) and strong regenerative changes were present in hepatocytes (arrowhead). (g) Another example of liver tissue in group IV (sTie1+sTie2) at the end of the follow-up. Strong confluent hepatocellular necrosis in portal tract (arrow) and lymphocytic infiltrates around hepatocytes (arrowhead) were seen. (h) In group V (sVEGFR-1, -3 and sTie2), only mild lymphocytic infiltrates around hepatocytes in portal tract were noticed (arrow) although liver cell architecture was well preserved. Magnification, 脳 100. Bar=100鈥壩糾.Full size imageTable 2 ALT values after the gene transfer (mean卤s.e.m.)Full size tableDiscussionWe present here the first combined antiangiogenic and antilymphangiogenic cancer gene therapy study with soluble decoy VEGFR-1, -3 and Tie2. These soluble receptors competitively inhibit the binding of VEGFs and angiopoietins to their full-length receptors and therefore prevent signal transduction via these pathways. To evaluate the efficacy of the therapies, we have utilized an intraperitoneal ovarian cancer xenograft model which resembles the clinical situation of patients with advanced disease, that is, large solid tumors, carcinosis and ascites.26 MRI confirmed the presence of intraperitoneal macroscopic tumors before the gene therapy and provided a non-invasive imaging of tumors during the follow-up. As assessed by sequential MRI, histology, immunohistochemistry and survival, combined adenoviral gene therapy with sVEGFR-1, -3 and sTie2 was able to reduce the growth of intraperitoneal solid ovarian carcinoma. Specifically, the therapeutic advantage of the combination therapy was seen at the cellular level, where a marked effect on pericytes was achieved by simultaneous blocking of VEGFR-1, -3 and Tie2.Vessels consist of endothelial cells and covering mural cells, which are called pericytes in capillaries and smooth muscle cells in larger arteries and venules. Pericytes provide structural support to endothelial cells and it has been hypothesized that protecting signals from pericytes limit the efficacy of antiangiogenic therapies targeting only the endothelial cells. As the expression of both VEGF-A36 and Ang1/Ang237 has been detected in ovarian cancer, we decided to block both pathways. In our present study, the most effective inhibition of tumor growth was achieved in the combination treatment group V, in which the mice received a combination of sVEGFR-1, -3 and sTie2. Indeed, in this group, tumors were 51% smaller than in controls at the end of the follow-up. MRI also confirmed this result, since 2 weeks after gene therapy, we detected smaller tumors in mice of the group V than in the control group. The treatment effect was also seen in CD34-stained microvessels, where the mean area of microvessels was significantly smaller in group V than in controls. We also counted the total tumor area covered by microvessels, which was significantly smaller in group V than in controls. Unfortunately, in our aggressive model of ovarian carcinoma, survival of this treatment group did not reach statistical significance compared with controls.Only a few previous reports are available from cancer studies where tumor angiogenesis has been blocked by simultaneous inhibition of both VEGFs/VEGFRs and Ang/Tie pathways. In a melanoma xenograft model, VEGFR-2 and Tie2 intradiabodies have reduced subcutaneous tumor growth.38 In a prostate tumor model, combined local delivery of endostatin-angiostatin and sTie2 revealed increased treatment effect.39 Inhibition of Tie2 has been utilized in cancer models including mammary tumors,32 melanoma32, 40 and glioblastoma multiforme41 and reduced tumor growth has been achieved. To our knowledge, no preclinical studies in ovarian cancer with blocking of either Tie1 or Tie2 have been published. Expression of Tie2 has been associated with microvessel density in human ovarian cancers.37, 42, 43 In the present study, mice received either single sTie1 or sTie2 therapy or the combinations of these agents. According to our results, it seems that both the soluble angiopoietin receptors Tie1 and Tie2 have some minor effects on ovarian cancer growth and formation of ascites. These findings are in line with the melanoma xenograft study by Siemeister et al.40 in which they used extracellular ligand-binding domain of human endothelial receptor Tie1, but they did not observe any significant effects on tumor growth.A dramatic shortening of the survival was observed in the group IV (sTie1 and sTie2) as compared with the other groups. A likely reason for this shorter survival was unexpected liver toxicity. These mice had macroscopic alterations in liver, which histologically resembled local necrosis. Also, one mouse had bleeding from rectum and another one into peritoneal cavity 4 days after gene therapy. The reason for these adverse events is unclear, as sTie1 and sTie2 given alone did not lead to such side effects. Additionally, the total dose of the adenoviral vector was similar (2 脳 109鈥塒FU) to control LacZ mice. The mechanism may involve an interaction of these receptors and needs further studies. It seems that ALT values appear to reflect the total adenoviral titer. In this study, we have used the maximal dose of adenoviruses. It is likely that with lesser amount of viruses, liver toxicity might have been lower without reducing the treatment effects. However, ALT values might have been high also due to the aggressive ovarian carcinoma with metastases in the liver. Previous studies have shown that Tie1-deficient mice die because of edema and hemorrages resulting from poor structural integrity of endothelial cells.44 Tie2-deficient mice have immature vessels that lack branching networks and periendothelial support cells.44, 45 In our earlier study with sVEGFR-1, -2 and -3,25 we did not notice such liver toxicity. In the combination group V (sVEGFR-1, -3 and sTie2), a tendency towards a greater amount of ascites was found and 63% of the mice presented edema under the skin. VEGFR-3 is the main mediator of lymphangiogenesis and Tie2 is also involved in lymphatic vessel growth.15 Thus, the increased amount of ascites might be due to simultaneous block of these pathways. The highest number of vessels without pericytes and a decrease in pericyte coverage was observed in group V (sVEGFR-1, -3 and sTie2) compared with controls. On the contrary, in control LacZ group and sTie2 group, no vessels without mural cells were detected. It seems that dual blocking of the functions of both VEGFs and angiopoietins resulted in the most powerful effect on pericytes compared with blocking only Tie2. This is in line with previous results from a Lewis lung carcinoma study, where Tie2-Fc alone did not have any effects on the recruitment of pericytes.46We achieved a significant antitumor effect in mice using sVEGFR-1, -3 and sTie2. Thus, inhibition of VEGF and angiopoietin pathways of angiogenesis seems to be more efficient than focusing on the single pathways. 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This study was supported by the Finnish Academy, EU Lymphangiogenomics network (LSHG-CT-2004-503573), Kuopio University Hospital (EVO grant 5185), the Finnish Medical Foundation, the Foundation of Finnish Cancer Institute, the Cancer Foundation of Northern Savo, the Finnish Cultural Foundation of Northern Savo, the Research Foundation of Orion Corporation and the Emil Aaltonen Foundation.Author informationAffiliationsDepartment of Molecular Medicine, AI Virtanen Institute, University of Eastern Finland, Kuopio, FinlandH Sallinen,聽M Anttila,聽J Koponen,聽I Kholova聽 聽S Yl盲-HerttualaInstitute of Clinical Medicine, Gynaecology, and Pathology and Forensic Medicine, University of Eastern Finland, Kuopio, FinlandH Sallinen,聽M Anttila,聽K H盲m盲l盲inen,聽I Kholova,聽V-M Kosma聽 聽S HeinonenDepartment of Gynaecology, Kuopio University Hospital, Kuopio, FinlandH Sallinen,聽M Anttila聽 聽S HeinonenNational BIO-NMR Facility, AI Virtanen Institute, University of Eastern Finland, Kuopio, FinlandO Gr枚hnDepartment of Pathology, Kuopio University Hospital, Kuopio, FinlandK H盲m盲l盲inen,聽I Kholova聽 聽V-M KosmaMolecular/Cancer Biology Laboratory Biomedicum Helsinki and Haartman Institute, University of Helsinki, Helsinki, FinlandK AlitaloGene Therapy Unit and University Hospital Research Unit, Kuopio, FinlandS Yl盲-HerttualaAuthorsH SallinenView author publicationsYou can also search for this author in PubMed聽Google ScholarM AnttilaView author publicationsYou can also search for this author in PubMed聽Google ScholarO Gr枚hnView author publicationsYou can also search for this author in PubMed聽Google ScholarJ KoponenView author publicationsYou can also search for this author in PubMed聽Google ScholarK H盲m盲l盲inenView author publicationsYou can also search for this author in PubMed聽Google ScholarI KholovaView author publicationsYou can also search for this author in PubMed聽Google ScholarV-M KosmaView author publicationsYou can also search for this author in PubMed聽Google ScholarS HeinonenView author publicationsYou can also search for this author in PubMed聽Google ScholarK AlitaloView author publicationsYou can also search for this author in PubMed聽Google ScholarS Yl盲-HerttualaView author publicationsYou can also search for this author in PubMed聽Google ScholarCorresponding authorCorrespondence to S Yl盲-Herttuala.Ethics declarations Competing interests The authors declare no conflict of interest. Rights and permissionsReprints and PermissionsAbout this articleCite this articleSallinen, H., Anttila, M., Gr枚hn, O. et al. Cotargeting of VEGFR-1 and -3 and angiopoietin receptor Tie2 reduces the growth of solid human ovarian cancer in mice. Cancer Gene Ther 18, 100鈥?09 (2011). https://doi.org/10.1038/cgt.2010.56Download citationReceived: 24 February 2010Revised: 03 June 2010Accepted: 19 July 2010Published: 24 September 2010Issue Date: February 2011DOI: https://doi.org/10.1038/cgt.2010.56KeywordsantiangiogenesisantilymphangiogenesisovarycarcinomaMRI Anita Kort, Selvi Durmus, Rolf W. Sparidans, Els Wagenaar, Jos H. Beijnen Alfred H. Schinkel Pharmaceutical Research (2015) Hanna Sallinen, Tommi Heikura, Jonna Koponen, Veli-Matti Kosma, Seppo Heinonen, Seppo Yl盲-Herttuala Maarit Anttila BMC Cancer (2014)