[CANCER RESEARCH 61, 1786 –1790, March 1, 2001] Advances in Brief Vascular Endothelial Growth Factor C Promotes Tumor Lymphangiogenesis and Intralymphatic Tumor Growth 1 Terhi Karpanen, 2 Mikala Egeblad, 2 Marika J. Karkkainen, Hajime Kubo, Seppo Yla ¨-Herttuala, Marja Ja ¨a ¨ttela ¨, and Kari Alitalo 3 Molecular/Cancer Biology Laboratory, Haartman Institute and Ludwig Institute for Cancer Research, FIN-00014 University of Helsinki, Finland [T. K., M. J. K., H. K., K. A.]; Apoptosis Laboratory, Danish Cancer Society, DK-2100 Copenhagen, Denmark [M. E., M. J.]; and A. I. Virtanen Institute, University of Kuopio, FIN-70211 Kuopio, Finland [S. Y-H.] Abstract Many solid tumors produce vascular endothelial growth factor C (VEGF-C), and its receptor, VEGFR-3, is expressed in tumor blood vessels. To study the role of VEGF-C in tumorigenesis, we implanted MCF-7 human breast carcinoma cells overexpressing recombinant VEGF-C orthotopically into severe combined immunodeficient mice. VEGF-C increased tumor growth, but unlike VEGF, it had little effect on tumor angiogenesis. Instead, VEGF-C strongly promoted the growth of tumor-associated lymphatic vessels, which in the tumor periphery were commonly infiltrated with the tumor cells. These effects of VEGF-C were inhibited by a soluble VEGFR-3 fusion protein. Our data suggest that VEGF-C facilitates tumor metastasis via the lymphatic vessels and that tumor spread can be inhibited by blocking the interaction between VEGF-C and its receptor. Introduction VEGF-C 4 is a ligand for the lymphatic endothelial receptor VEGFR-3, but it binds also to VEGFR-2, which is the major mito- genic signal transducer for VEGF in blood vascular endothelial cells (1–3). VEGF-C stimulates almost exclusively lymphangiogenesis when applied to differentiated chick chorioallantoic membrane (4) or when overexpressed in the skin of transgenic mice (5). However, more recent studies report that VEGF-C also stimulates angiogenesis in mouse cornea (6), in developing chorioallantoic membrane of chick embryos (6), and in ischemic hind limbs of rabbits (7). Many tumors express VEGF-C, and the expression level has been suggested to correlate with tumor angiogenesis and metastasis via the lymphatic system (8 –10). VEGFR-3 is normally expressed predominantly in the lymphatic vessels in adults (11–13), but this receptor is also induced in the angiogenic blood vascular endothelium of many tumors (9, 14, 15). To study the possible effects of VEGF-C on tumor growth, angiogenesis, and lymphangiogenesis, we overexpressed VEGF-C in human MCF-7 breast carcinoma cells, which otherwise produce min- imal levels of this growth factor (16). The VEGF-C-overexpressing or vector-transfected cells were then implanted orthotopically and grown as tumors in the mammary fad pads of SCID mice. Materials and Methods Plasmid Expression Vectors. The cDNAs coding for the human VEGF-C or VEGF 165 were introduced into the pEBS7 plasmid (17). The same vector was used for the expression of the soluble receptor chimeras VEGFR-3-Ig, containing the first three immunoglobulin homology domains of VEGFR-3 fused to the Fc-domain of human immunoglobulin  chain and VEGFR-1-Ig, containing the first five immunoglobulin homology domains of VEGFR-1 in a similar construct (18). Production and Analysis of Transfected Cells. The MCF-7S1 subclone of the human MCF-7 breast carcinoma cell line was transfected with plasmid DNA by electroporation, and stable cell pools were selected and cultured as described previously (19). The cells were metabolically labeled in methionine and cysteine free MEM (Life Technologies, Inc.) supplemented with 100 Ci/ml [ 35 S]methionine and [ 35 S]cysteine (Redivue Pro-Mix; Amersham Pharmacia Biotech). The labeled growth factors were immunoprecipitated from the conditioned medium using antibodies against VEGF-C (1) or VEGF (MAB293; R & D Systems). The immunocomplexes and the VEGFR-Ig fusion proteins were precipitated using protein A-Sepharose (Amersham Pharmacia Biotech), washed twice in 0.5% BSA, 0.02% Tween 20 in PBS, and once in PBS and analyzed in SDS-PAGE under reducing conditions. Cell Proliferation and Tumorigenesis Assays. Cells (20,000/well) were plated in quadruplicate in 24-well plates, trypsinized on replicate plates after 1, 4, 6, or 8 days, and counted using a hemocytometer. Fresh medium was provided after 4 and 6 days. For the tumorigenesis assay, subconfluent cultures were harvested by trypsinization and washed twice, and 10 7 cells in PBS were inocu- lated into the fat pads of the second (axillar) mammary gland of ovarectomized SCID mice, carrying s.c. 60-day slow-release pellets containing 0.72 mg of 17-estradiol (Innovative Research of America). The ovarectomy and implanta- tion of the pellets were done 4 – 8 days before tumor cell inoculation. Tumor length and width were measured twice weekly in a blinded manner, and the tumor volume was calculated as the length  width  depth  0.5, assuming that the tumor is a hemi-ellipsoid and the depth is the same as the width (20). Histology and Quantitation of the Blood Vessels. The tumors were excised, fixed in 4% paraformaldehyde (pH 7.0) for 24 h, and embedded in paraffin. Sections (7 m) were immunostained with monoclonal antibodies against PECAM-1 (PharMingen), VEGFR-3 (21), PCNA (Zymed Laborato- ries), or polyclonal antibodies against LYVE-1 (a kind gift from Dr. David G. Jackson, University of Oxford, Oxford, United Kingdom; Ref. 22), VEGF-C (1) or laminin (a kind gift from Dr. Karl Tryggvason, Karolinska Institute, Stockholm, Sweden) according to published protocols (14). The average of the number of the PECAM-1-positive vessels was determined from three areas (60) of the highest vascular density (vascular hot spots) in a section. All histological analysis was done using blinded tumor samples. Adenoviral Expression of Soluble VEGFR-3 and Evan’s Blue Draining Assay. The cDNA coding for the VEGFR-3-Ig fusion protein was subcloned into the pAdCMV plasmid, constructed by subcloning the human cytomega- lovirus immediate-early promoter, the multiple cloning site, and the bovine growth hormone gene polyadenylation signal from the pcDNA3 (Invitrogen) into the pAdBglII vector, and the adenoviruses were produced as described previously (23). The VEGFR-3-Ig or LacZ control (23) adenoviruses, 10 9 Received 12/29/00; accepted 1/18/01. 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 the Finnish Academy, the Sigrid Juselius Foundation, the University of Helsinki Hospital, the State Technology Development Center, the European Union, the Finnish Cancer Organization, Finnish Cultural Foundation, Ida Montini Foundation, Emil Aaltonen Foundation, Research and Science Foundation of Farmos, the Danish Cancer Society, the Danish Medical Research Council, and the Jens Aage and Edith Ingeborg Sørensen Memorial Foundation. 2 These authors contributed equally to this work. 3 To whom requests for reprints should be addressed, at Molecular/Cancer Biology Laboratory, Haartman Institute, P. O. Box 21 (Haarmaninkatu 3), FIN-00014 University of Helsinki, Finland. Phone: 358-9-191-26434; Fax: 358-9-191-26448; E-mail: Kari.Alitalo@Helsinki.fi. 4 The abbreviations used are: VEGF-C, vascular endothelial growth factor C; VEGFR, VEGF receptor; SCID, severe combined immunodeficient; PCNA, proliferating cell nuclear antigen; Ig, immunoglobulin. 1786 Research. on December 8, 2021. © 2001 American Association for Cancer cancerres.aacrjournals.org Downloaded from