Systemic Administration of a Conditionally Replicating Adenovirus, Targeted to Angiogenesis, Reduced Lung Metastases Burden in Cotton Rats

Michael Peled, Aviv Shaish, Avi Katav, Shoshana Greenberger, Iris Barshack, Reshef Tal, Livnat Bangio, Eyal Breitbart and Dror Harats

Purpose: Angiogenesis is an essential process for solid tumor development. To interfere with angiogenesis, AdPPE3x-E1, an adenovirus that is transcriptionally targeted to replicate in angiogenic endothelial cells, was constructed, by replacing the E1 promoter with the modified preproendothelin-1 promoter, PPE-1-3x, previously shown to induce specific transcription in angiogenic endothelial cells.

Experimental Design: The specificity of AdPPE3x-E1 to endothelial cells was shown by quantitative PCR and immunostaining, and its antiangiogenic effect was evaluated in Matrigel models. The in vivo efficacy of AdPPE3x-E1 was also tested in a cotton rat lung metastases model.

Results: The replication rate of AdPPE3x-E1 in endothelial cells was similar to that of AdCMV-E1, a nonselective replicating adenovector, but the replication rate was reduced up to 60-fold in nonendothelial cells. Moreover, AdPPE3x-E1 reduced endothelial cell viability by 90% whereas nonendothelial cells were not affected. In in vitro and in vivo Matrigel models, endothelial cells infected with AdPPE3x-E1 did not develop capillary-like structures. The systemic administration of AdPPE3x-E1 reduced the lung metastases burden in a cotton rat model by 55%, compared with saline-treated rats, without significant evidence of toxicity. Quantitative PCR analysis showed that the viral copy number of AdPPE3x-E1 was increased 3-fold in the lung metastases but not in the liver, compared with a nonreplicating adenovector control.

Conclusions: We have shown here for the first time an antimetastatic effect induced by an angiogenesis-transcriptionally targeted adenovirus following systemic administration. Because adenovirus replication is more efficient in humans than in cotton rats, we assume a significant effect for AdPPE3x-E1 treatment in fighting human solid tumors and metastases.

Angiopoietin-2 Levels Are Associated with Disease Progression in Metastatic Malignant Melanoma

Iris Helfrich, Lutz Edler, Antje Sucker, Markus Thomas, Sven Christian, Dirk Schadendorf and Hellmut G. Augustin

Purpose: The blood vessel-destabilizing Tie2 ligand angiopoietin-2 (Ang-2) acts in concert with the vascular endothelial growth factor/vascular endothelial growth factor receptor system to control vessel assembly during tumor progression. We hypothesized that circulating soluble Ang-2 (sAng-2) may be involved in melanoma progression.

Experimental Design: Serum samples (n = 98) from melanoma patients (American Joint Committee on Cancer stages I-IV), biopsies of corresponding patients, and human melanoma cell lines were analyzed for expression of Ang-2 and S100β. Multiple sera of a subcohort of 33 patients were tested during progression from stage III to IV. Small interfering RNA-based loss-of-function experiments were done to assess effects of Ang-2 on melanoma cells.

Results: Circulating levels of sAng-2 correlate with tumor progression in melanoma patients (P < 0.0001) and patient survival (P = 0.007). Analysis of serum samples during the transition from stage III to IV identified an increase of sAng-2 up to 400%. Comparative analyses revealed a 56% superiority of sAng-2 as predictive marker over the established marker S100β. Immunohistochemistry and reverse transcription-PCR confirmed the prominent expression of Ang-2 by tumor-associated endothelial cells but identified Ang-2 also as a secreted product of melanoma cells themselves. Corresponding cellular experiments revealed that human melanoma-isolated tumor cells were Tie2 positive and that Ang-2 acted as an autocrine regulator of melanoma cell migration and invasion.

Conclusions: The experiments establish sAng-2 as a biomarker of melanoma progression and metastasis correlating with tumor load and overall survival. The identification of an autocrine angiopoietin/Tie loop controlling melanoma migration and invasion warrants further functional experiments and validate the angiopoietin/Tie system as a promising therapeutic target for human melanomas.

Differential effects of the vascular endothelial growth factor receptor inhibitor PTK787/ZK222584 on tumor angiogenesis and tumor lymphangiogenesis

Tibor Schomber, Adrian Zumsteg, Karin Strittmatter, Ivana Crnic, Helena Antoniadis, Amanda Littlewood-Evans, Jeanette Wood and Gerhard Christofori

Halting tumor growth by interfering with tumor-induced angiogenesis is an attractive therapeutic approach. Such treatments include humanized antibodies blocking the activity of vascular endothelial growth factor (VEGF)-A (bevacizumab), soluble VEGF receptor (VEGFR) constructs (VEGF-Trap), or small-molecule inhibitors of VEGFR signaling, including PTK787/ZK222584 (PTK/ZK), sorafenib, and sunitinib. PTK/ZK has been shown previously to specifically block VEGF-induced phosphorylation of VEGFR-1, -2 and -3 and thereby to inhibit endothelial cell proliferation, differentiation, and tumor angiogenesis. We have investigated the effect of PTK/ZK on tumor angiogenesis and tumor lymphangiogenesis using the Rip1Tag2 transgenic mouse model of pancreatic β cell carcinogenesis. In Rip1Tag2 mice, tumor angiogenesis is predominantly mediated by VEGF-A, and as expected, PTK/ZK efficiently impaired tumor blood vessel angiogenesis and tumor growth. Double-transgenic Rip1Tag2;Rip1VEGF-C and Rip1Tag2;Rip1VEGF-D mice not only exhibit VEGF-A-dependent blood vessel angiogenesis but also tumor lymphangiogenesis induced by the transgenic expression of VEGF-C or -D. In these mouse models, PTK/ZK also repressed tumor blood vessel angiogenesis and tumor growth yet failed to affect tumor lymphangiogenesis and lymphogenic metastasis. Adenoviral delivery of soluble VEGFR-3 also did not prevent tumor lymphangiogenesis in these mice. In contrast, spontaneous tumor lymphangiogenesis, as observed by the stochastic expression of VEGF-C and -D in tumors of neural cell adhesion molecule-deficient Rip1Tag2 mice, was repressed by PTK/ZK and soluble VEGFR-3. The results indicate that the time of onset and the levels of VEGF-C/D expression may be critical variables in efficiently repressing tumor lymphangiogenesis and that pathways other than VEGFR signaling may be involved in tumor lymphangiogenesis.

Angiogenic Switch of Angiopietins-Tie2 System and Its Prognostic Value in Bladder Cancer

Tibor Szarvas, Tobias Jäger, Martin Tötsch, Frank Vom Dorp, Carsten Kempkensteffen, Ilona Kovalszky, Imre Romics, Süleyman Ergün and Herbert Rübben

Purpose: Vascular endothelial growth factor (VEGF), angiopoietins (Ang-1 and Ang-2), and their receptor Tie2 are critically involved in both normal and pathologic angiogenesis. The aim of this study was to explore the role of Ang-1, Ang-2, VEGF, and Tie2 in the development and progression of bladder cancer as well as to examine their prognostic value in this tumor type.

Experimental Design: Tumor samples of 113 bladder cancer patients, normal bladder epithelium of 5 noncancer patients, and two low-grade (UMUC3 and RT4) and two high-grade (J82 and T24) bladder cancer cell lines were analyzed by quantitative real-time PCR. The expression data were analyzed performing Wilcoxon rank-sum and Kaplan-Meier log-rank tests as well as univariate Cox analyses and Cox proportional hazards regression model.

Results: In tissues of noninvasive bladder tumors, Ang-1 expression was significantly lower (P < 0.001), whereas VEGF expression was significantly higher (P = 0.031) than in normal bladder tissue. These findings were also confirmed at the protein level by immunohistochemistry. In contrast, Tie2 and Ang-2 abundance in tumor did not differ significantly from that in normal bladder tissue. Multivariate analysis identified Ang-2 as a strong and independent predictor of tumor recurrence [hazard ratio (HR), 10.18; 95% confidence interval (95% CI), 2.69-38.49; P < 0.001] and Tie2 expression as an independent favorable prognostic factor for both metastasis (HR, 0.31; 95% CI, 0.11-0.89; P = 0.029) and disease-specific survival (HR, 0.25; 95% CI, 0.10-0.62; P = 0.003).

Conclusions: These data show the strongest change in expression of VEGF and Ang-1 in superficial bladder cancer in comparison with normal bladder epithelium and the invasive tumor stages. The prognostic significance of Ang-2 and Tie2 underlines the essential role of angiopoietins-Tie2 system in progression of bladder cancer.

Enhanced Antitumor Efficacy of Clinical-Grade Vasculature-Targeted Liposomal Doxorubicin

Fabio Pastorino, Daniela Di Paolo, Federica Piccardi, Beatrice Nico, Domenico Ribatti, Antonio Daga, Gabriella Baio, Carlo E. Neumaier, Chiara Brignole, Monica Loi, Danilo Marimpietri, Gabriella Pagnan, Michele Cilli, Eugene A. Lepekhin, Seema V. Garde, Renato Longhi, Angelo Corti, Theresa M. Allen, Jinzi J. Wu and Mirco Ponzoni

Purpose: In vivo evaluation of good manufacturing practice-grade targeted liposomal doxorubicin (TVT-DOX), bound to a CD13 isoform expressed on the vasculature of solid tumors, in human tumor xenografts of neuroblastoma, ovarian cancer, and lung cancer.

Experimental Design: Mice were implanted with lung, ovarian, or neuroblastoma tumor cells via the pulmonary, peritoneal, or orthotopic (adrenal gland) routes, respectively, and treated, at different days post inoculation, with multiple doses of doxorubicin, administered either free or encapsulated in untargeted liposomes (Caelyx) or in TVT-DOX. The effect of TVT-DOX treatment on tumor cell proliferation, viability, apoptosis, and angiogenesis was studied by immunohistochemical analyses of neoplastic tissues and using the chick embryo chorioallantoic membrane assay.

Results: Compared with the three control groups (no doxorubicin, free doxorubicin, or Caelyx), statistically significant improvements in survival was seen in all three animal models following treatment with 5 mg/kg (maximum tolerated dose) of TVT-DOX, with long-term survivors occurring in the neuroblastoma group; increased survival was also seen at a dose of 1.7 mg/kg in mice bearing neuroblastoma or ovarian cancer. Minimal residual disease after surgical removal of neuroblastoma primary mass, and the enhanced response to TVT-DOX, was visualized and quantified by bioluminescence imaging and with magnetic resonance imaging. When treated with TVT-DOX, compared with Caelyx, all three tumor models, as assayed by immunohistochemistry and chorioallantoic membrane, showed statistically significant reductions in cell proliferation, blood vessel density, and microvessel area, showing increased cell apoptosis.

Conclusion: TVT-DOX should be evaluated as a novel angiostatic strategy for adjuvant therapy of solid tumors.

Dual targeting of Raf and VEGF receptor 2 reduces growth and metastasis of pancreatic cancer through direct effects on tumor cells, endothelial cells, and pericytes

Sven A. Lang, Philipp Schachtschneider, Christian Moser, Akira Mori, Christina Hackl, Andreas Gaumann, David Batt, Hans J. Schlitt, Edward K. Geissler and Oliver Stoeltzing

The Ras/Raf/MEK pathway represents an important oncogenic signaling pathway in gastrointestinal malignancies, including pancreatic cancer. Although activating B-Raf mutations are infrequent in pancreatic cancer, we hypothesized that targeting Raf could be valuable for therapy of this cancer entity. Moreover, as vascular endothelial growth factor receptor 2 (VEGFR2) is involved in tumor angiogenesis, we sought to investigate the effects of dual inhibition of Raf and VEGFR2 on pancreatic tumor growth, vascularization, and metastasis. Effects of a Raf/VEGFR2 inhibitor (NVP-AAL881) on pancreatic cancer cells, endothelial cells, and vascular smooth muscle cells were determined by Western blotting, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis, and migration assays, respectively. Changes in the expression of VEGF-A or survivin were investigated by ELISA and/or real-time PCR. The growth-inhibitory effects of Raf/VEGFR2 inhibition were additionally evaluated in orthotopic tumor models. Results showed that various Raf isoforms were activated in pancreatic cancer cells and NVP-AAL881 diminished the activation of MEK, Akt, Erk, and also STAT3. Moreover, dual inhibition of Raf/VEGFR2 significantly reduced VEGF expression and impaired cancer cell migration. Importantly, besides blocking VEGF-induced Erk and SAPK phosphorylation in endothelial cells, the Raf inhibitor diminished STAT3 phosphorylation, independent of a VEGFR2 blockade, and reduced the expression of survivin. In addition, cell proliferation and migration of both endothelial cells and vascular smooth muscle cells were significantly reduced. In vivo, blocking Raf/VEGFR2 significantly inhibited orthotopic tumor growth and vascularization and reduced cancer metastasis. In conclusion, blocking Raf exerts growth-inhibitory effects on pancreatic tumor cells, endothelial cells, and pericytes and elicits antiangiogenic properties. Dual targeting of Raf and VEGFR2 appears to be a valid strategy for therapy of pancreatic cancer.