Simultaneous blockade of VEGFR-1 and VEGFR-2 activation is necessary to efficiently inhibit experimental melanoma growth and metastasis formation Jens Gille 1,2 * , Regina Heidenreich 2,3 , Andreas Pinter 1 , Judith Schmitz 2 , Beatrice Boehme 1 , Daniel J. Hicklin 4 , Reinhard Henschler 5 and Georg Breier 2,6 1 Department of Dermatology, J.W. Goethe-University, Frankfurt am Main, Germany 2 Department of Molecular Cell Biology, Max-Planck-Institute, Bad Nauheim, Germany 3 Department of Dermatology, Eberhard-Karls-University, T € ubingen, Germany 4 Department of Immunology, ImClone Systems Incorporated, New York, NY 5 Institute of Transfusion Medicine, German Red Cross Blood Center, Frankfurt am Main, Germany 6 Department of Pathology, University of Technology, Dresden, Germany Metastasis continues to be the major cause of morbidity and mor- tality in malignant melanoma. In our study, we explored whether inhibition of VEGFR-1 or VEGFR-2 signaling conveys distinct suppressive effects on B16 melanoma subcutaneous growth and metastasis formation. The inhibition of VEGFR-1 or -2 alone had no significant influence on both melanoma growth and metastasis formation. In contrast, simultaneous blockade of VEGFR-1 and -2 signaling strongly suppressed progression in both B16 tumor mod- els. There was no expression of VEGFR-1 or -2 detectable on the B16 cells used, excluding the melanoma cells as direct therapeutic targets. Analyzing the contribution of progenitor-like cells during melanoma metastasis formation, we observed an enhanced prolif- eration and mobilization of VEGFR-11 myeloid and VEGFR-21 endothelial cells with progenitor potential by the induction of mel- anoma lung metastasis, which was not influenced by interference with VEGFR signaling. These results indicate that the antimeta- static effects exerted by combined inhibition of VEGFR-1 and -2 signaling were mediated via targeting cell populations other than progenitors only. Sole inhibition of VEGFR-1 signaling led to a strong reduction of the CD45-positive inflammatory infiltrate in the tumor tissue. However, the formation of lung metastasis was not affected, indicating that inhibition of the inflammatory response was not sufficient to efficiently block B16 melanoma me- tastasis development. Taken together, our data suggest that in the utilized B16 tumor models the blockade of both the inflammatory and the VEGFR-2-dependent angiogenic response are necessary to effectively inhibit solid tumor growth and formation of lung metastasis by B16 melanoma cells. ' 2007 Wiley-Liss, Inc. Key words: experimental melanoma; neoplasm metastasis; pathologic neovascularization; vascular endothelial growth factor receptors The incidence of melanoma has strongly increased over the last 40 years. 1 Melanoma metastasis shows a high rate of mortality due to the strong resistance to conventional therapy, including chemotherapy and radiation therapy. Up to now, no therapeutic regimen has yet been proven to significantly impact long-term out- come of metastatic melanoma patients in multicenter randomized studies. The propensity of tumor cells to colonize remote organs is greatly affected by active tumor-host interactions as well as by factors that are inherent both to the tumor cells and to the locally invaded host microenvironment. 2,3 One characteristic feature of cancer is the induction of an inflammatory response in the tumor tissue. The inflammatory infiltrate, composed of a diverse leuko- cyte population (neutrophils, macrophages, mast cells and lym- phocytes), may promote tumor progression by the production of cytokines, growth factors and proteases. 4 Secreted cytokines can directly influence tumor cell proliferation, whereas the release of proangiogenic factors like vascular endothelial growth factor (VEGF), IL-8 and matrix metalloproteinases (MMPs) may enable micrometastasis to acquire the ability to eventually become vascu- larized. In addition, remodeling of extracellular matrix compo- nents might further alter the balance towards proangiogenic stim- uli, enhancing neovascularization. 5,6 There is accumulating evidence suggesting that bone marrow- derived endothelial progenitor cells (EPCs) contribute to adult neovascularization. Moreover, subsets of hematopoietic progenitor cells (HPCs) may be recruited to sites of cancer cell prolifera- tion. 7,8 Plasma elevation of angiogenic factors, including VEGF and placenta growth factor (PlGF), has been shown to induce mo- bilization and recruitment of both vascular endothelial growth fac- tor receptor-2 (VEGFR-2)1EPCs and VEGFR-11HPCs, 9,10 con- tributing to tumor cell survival and neovessel-dependent tumor growth. However, the extent to which postnatal vasculogenesis as opposed to angiogenesis contributes to neovessel-dependent tumor development is still a matter of controversy. 11 VEGF and its receptor tyrosine kinases VEGFR-1 and -2 have received great attention as important therapeutic targets in patho- logic angiogenesis. 12,13 Inhibition of VEGFR-2 signaling was dem- onstrated previously to suppress tumor angiogenesis and growth in different tumor models. 14–18 More recently, anti-VEGFR-1 anti- body (Ab) strategies have been shown to block angiogenesis in both malignant and inflammatory diseases. 19 While the degree of anti-VEGFR-1-mediated inhibition alone may vary, 19 simultane- ous inhibition of both VEGFR-1 and -2 tends to augment the anti- vascular effects of anti-VEGFR-2 in a local tumor model, which was attributed in part to blocking recruitment of VEGFR1 bone marrow-derived progenitors. 7 In our study we analyzed the influence of VEGFR signaling on subcutaneous B16 melanoma growth and lung metastasis forma- tion. The growth of solid B16 melanoma was not significantly influenced by the inhibition of VEGFR-1 or -2 alone through the retroviral gene transfer of VEGFR mutants. Blockade of VEGFR-1 by a neutralizing Ab strongly reduced the inflammatory infiltrate of B16 melanoma metastasis without a significant decrease in metas- tasis formation. Also, the inhibition of endothelial cell (EC) func- tion by the inhibition of VEGFR-2 signaling had no significant impact on the formation of B16 melanoma lung metastasis. In con- trast, simultaneous blockade of both, VEGFR-1 and -2 signaling strongly suppressed the growth of subcutaneous melanoma and metastasis formation. However, the enhanced mobilization and proliferation of VEGFR-11 myeloid cells and VEGFR-21 endo- thelial progenitors induced by the formation of lung metastasis was not altered by the blockade of VEGFR signaling. Therefore, the This article contains supplementary material available via the Internet at http://www.interscience.wiley.com/jpages/0020-7136/suppmat. Jens Gille and Regina Heidenreich contributed equally to this work. Grant sponsor: Deutsche Forschungsgemeinschaft; Grant numbers: Gi 229/5-1, SFB/TR23; Grant sponsor: BMBF; Grant sponsor: EU. *Correspondence to: Zentrum der Dermatologie, Klinikum der J.W. Goethe-Universit€ at, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany. Fax: 149-69-6301-7948. E-mail: gille@em.uni-frankfurt.de Received 10 May 2006; Accepted after revision 6 November 2006 DOI 10.1002/ijc.22531 Published online 17 January 2007 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 120, 1899–1908 (2007) ' 2007 Wiley-Liss, Inc. Publication of the International Union Against Cancer