[CANCER RESEARCH 64, 8507– 8511, December 1, 2004] Advances in Brief Identification of a Binding Partner for the Endothelial Cell Surface Proteins TEM7 and TEM7R Akash Nanda, 1 Phillip Buckhaults, 2 Steven Seaman, 5 Nishant Agrawal, 1 Paula Boutin, 3 Srinivas Shankara, 3 Mariana Nacht, 3 Beverly Teicher, 3 Jason Stampfl, 4 Sujay Singh, 4 Bert Vogelstein, 1 Kenneth W. Kinzler, 1 and Brad St. Croix 5 1 The Howard Hughes Medical Institute, Sidney Kimmel Comprehensive Cancer Center and Program in Human Genetics and Molecular Biology, Johns Hopkins Medical Institutions, Baltimore, Maryland; 2 Department of Pathology, University of South Carolina School of Medicine, South Carolina Cancer Center, Division of Basic Research, Columbia, South Carolina; 3 Genzyme Molecular Oncology, Framingham, Massachusetts; 4 Imgenex Corporation, San Diego, California; and 5 Tumor Angiogenesis Section, Mouse Cancer Genetics Program, National Cancer Institute at Frederick, Frederick, Maryland Abstract Tumor endothelial marker 7 (TEM7) was recently identified as an mRNA transcript overexpressed in the blood vessels of human solid tumors. Here, we identify several new variants of TEM7, derived by alternative splicing, that are predicted to be intracellular (TEM7-I), se- creted (TEM7-S), or on the cell surface membrane (TEM7-M) of tumor endothelium. Using new antibodies against the TEM7 protein, we con- firmed the predicted expression of TEM7 on the cell surface and demon- strated that TEM7-M protein, like its mRNA, is overexpressed on the endothelium of various tumor types. We then used an affinity purification strategy to search for TEM7-binding proteins and identified cortactin as a protein capable of binding to the extracellular region of both TEM7 and its closest homologue, TEM7-related (TEM7R), which is also expressed in tumor endothelium. The binding domain of cortactin was mapped to a unique nine-amino acid region in its plexin-like domain. These studies establish the overexpression of TEM7 protein in tumor endothelium and provide new opportunities for the delivery of therapeutic and imaging agents to the vessels of solid tumors. Introduction Targeting the endothelial cells that line tumor blood vessels is a promising new strategy for the treatment of cancer (1). However, realization of the full potential of a vascular-directed approach will require the exploitation of new targets that are expressed predomi- nantly on tumor endothelium (2). In a systematic attempt to uncover such targets, we recently used serial analysis of gene expression on endothelial cells isolated from human normal or malignant colorectal tissues. These studies led to the identification of several novel tumor endothelial markers (TEMs), the most abundant of which was called TEM7 (3). In situ hybridization studies validated the expression of TEM7 in the endothelium of colorectal cancer and demonstrated that TEM7 mRNA was also abundantly expressed in the endothelium of a variety of other human cancer types including, breast, lung, and brain tumors. Based on the full-length nucleotide sequence, TEM7 seems to be a typical type I transmembrane protein containing a signal peptide followed by a nidogen-like domain and a single hydrophobic domain (Fig. 1A). The only other protein that seems to share significant homology to TEM7 is TEM7-related (TEM7R), another putative cell surface protein that also contains a nidogen-like domain. In situ hybridization revealed that TEM7R, like TEM7, was abundantly expressed in the endothelium of malignant colorectal cancer but was absent or rare in normal colonic mucosa (4). By developing antibodies against human TEM7, we now demonstrate that TEM7 protein, like its mRNA, is elevated in tumor tissues and is expressed predominantly by the endothelial cells of tumor-infiltrating blood vessels. To provide potential insights into the structure and function of this endothelial protein, we then attempted to identify TEM7-binding partners using affinity chromatography. Materials and Methods Tumor Endothelial Marker 7 Antibodies. An anti-TEM7 polyclonal antibody was made by immunizing rabbits with a DNA vector designed to express the extracellular coding sequence of the TEM7 protein (amino acids 23– 412; Genovac, Freiburg, Germany). Two anti-TEM7 monoclonal antibod- ies, clone IM193 [an immunoglobulin (Ig) G] and clone IM568 (an IgM), were raised against the peptide sequence NNLSPKTKGTPVHLGTI that resides in the extracellular region of TEM7-M, proximal to the transmembrane domain (Imgenex, San Diego, CA). Immunostaining. Immunohistochemistry on paraffin sections using IM193 antibody and immunofluorescence on fresh-frozen sections using IM568 antibody were performed as described previously (5). Immunoelectron Microscopy. Fresh colorectal cancer specimens were fixed in 4% paraformaldehyde and 0.1% glutaraldeyde in PBS (pH 7.2) overnight at 4°C. Samples were rinsed in PBS, incubated with 0.25% tannic acid (Mallinckrodt, St. Louis, MO) in PBS for 1 hour and rinsed again, and un– cross-linked glutaraldeyde was reduced with 50 mmol/L NH 4 Cl in PBS. Samples were washed in 0.1 mol/L maleate buffer before en bloc staining with 2% uranyl acetate in 0.1 mol/L maleate buffer. After a graded ethanol series, dehydrated samples were infiltrated and embedded with L.R. white resin. Samples were polymerized in tightly sealed gelatin capsules for 2 days at 40°C. Seventy- to 80-nm-thick sections were cut and picked up on Formvar-coated nickel grids. After incubation with IM568 monoclonal antibody and 6-nm gold-conjugated antimouse secondary antibody (The Jackson Laboratory, Bar Harbor, ME), sections were post-fixed with 2% glutaraldehyde and stained with uranyl acetate followed by lead citrate. All grids were viewed and photographed on a Philips CM 120 TEM (Philips, Eindhoven, The Nether- lands) operating at 80 Kv. Immunoblotting. Normal human colonic mucosa, colorectal tumors, normal rabbit hepatic tissue, and VX2 liver tumors were snap frozen immediately after surgical resection and stored at -80°C before use. Tissues were homogenized in TNT buffer [50 mmol/L Tris (pH 7.5), 75 mmol/L NaCl, and 1% Triton X-100 Received 7/30/04; revised 9/27/04; accepted 10/11/04. Grant support: National Cancer Institute, Department of Health and Human Services (B. St. Croix), the Miracle Foundation (B. Vogelstein), Genzyme Molecular Oncology (Genzyme; K. Kinzler), and National Institutes of Health grant CA57345 (K. Kinzler). 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. Note: A. Nanda and P. Buckhaults contributed equally to this work. A. Nanda is a student in the Medical Scientist Training Program and the Program in Human Genetics. Under a licensing agreement between the Johns Hopkins University and Genzyme, technologies related to serial analysis of gene expression and the TEMs were licensed to Genzyme for commercial purposes, and B. Vogelstein, K. Kinzler, and B. St. Croix are entitled to a share of the royalties received by the university from the sales of the licensed technologies. The serial analysis of gene expression technology is freely available to academia for research purposes. K. Kinzler is a consultant to Genzyme. The university and researchers (B. Vogelstein and K. Kinzler) own Genzyme stock, which is subject to certain restrictions under university policy. The terms of these arrangements are being managed by the university in accordance with its conflict of interest policies. Requests for reprints: Brad St. Croix, Tumor Angiogenesis Section, Mouse Cancer Genetics Program, National Cancer Institute at Frederick, Frederick, MD 21702. E-mail: stcroix@ncifcrf.gov. ©2004 American Association for Cancer Research. 8507 Research. on February 19, 2016. © 2004 American Association for Cancer cancerres.aacrjournals.org Downloaded from