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Lymphotoxin- Receptor Immune Interaction Promotes Tumor Growth

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Lymphotoxin- Receptor Immune Interaction Promotes Tumor Growth [CANCER RESEARCH 62, 4034–4040, July 15, 2002] Lymphotoxin-␤ Receptor Immune Interaction Promotes Tumor Growth by Inducing Angiogenesis1 Thomas Hehlgans, Benjamin Stoelcker, Peter Stopfer, Peter Mu¨ller, Grigore Cernaianu, Markus Guba, Markus Steinbauer, Sergei A. Nedospasov, Klaus Pfeffer, and Daniela N. Ma¨nnel2 Departments of Pathology/Tumor Immunology [T. H., B. S., P. S., P. M., D. N. M.] and Surgery [G. C., M. G., M. S.], University of Regensburg, D-93042 Regensburg, Germany; Institute of Medical Microbiology, Immunology, and Hygiene, Technical University of Munich, D-81675 Munich, Germany [K. P.]; and Laboratory of Molecular Immunology, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, and Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119991 Moscow, Russia [S. A. N.] ABSTRACT activated when the balance of angiogenic inhibitors to stimulators is shifted toward a proangiogenic milieu (9). Potent inducers of angio- Growth of solid fibrosarcoma tumors in mice was inhibited by the genesis have been identified, e.g., bFGF, VEGF, and angiopoietins release of a soluble lymphotoxin-␤ receptor inhibitor (LT␤R-immuno- (reviewed in Ref. 10). There is great interest in identifying and globulin fusion protein) from the tumor cells. Tumor growth arrest in ␣ ␤ modulating antiangiogenic pathways and in antiangiogenic drug de- mice deficient in the ligand LT 1 2 demonstrated the requirement for ␤ ␣ ␤ velopment for therapeutic purposes. activation of the LT R on the tumor cells by host cell-derived LT 1 2. Activation of the LT␤R resulted in enhanced release of macrophage Here we provide evidence that activation of LT␤R on fibrosarcoma inflammatory protein-2. Blocked angiogenesis was revealed in LT␤R tumor cells is necessary for angiogenesis and solid tumor growth. ␣ ␤ ␤ inhibitor-producing tumor nodules by immunohistochemistry and in vivo Prevention of LT 1 2-LT R signaling inhibited tumor angiogenesis microscopy. The growth arrest of LT␤R inhibitor-producing fibrosarco- and neovascularization, and resulted in tumor growth arrest. In addi- mas was overcome by forced MIP-2 expression in the tumor cells. Thus, tion, we show that LT␤R activation on the tumor cells induced LT␤R activation on tumor cells by activated host lymphocytes can initiate enhanced release of MIP-2, an angiogenic CXC chemokine (11). a novel proangiogenic pathway leading to organized tumor tissue devel- Thus, our studies identify the interaction of activated LT␣ ␤ -carying opment. 1 2 lymphocytes with LT␤R-expressing tumor cells as initiating event for a previously unknown proangiogenic pathway. INTRODUCTION ␣ ␤ 3 The membrane-bound heterotrimer consisting of LT 1 2 binds as MATERIALS AND METHODS a functional ligand specifically to the LT␤R, a member of the TNF receptor family. Interestingly, LT␤R is expressed on many cell types Animals. Male 20–25 g C57BL/6 mice were purchased from Charles River with the exception of lymphocytes, whereas expression of the ligand (Sulzfeld, Germany). Mice deficient for the LT␤R (6) and LT␣/LT␤-deficient is apparently restricted to activated lymphocytes (1). Currently the mice4 had been backcrossed more than six times to the C57BL/6 background. biological functions of this ligand-receptor system are not completely Experiments were performed with age- and sex-matched mice. understood. It has been shown that signaling through the LT␤R Tumor Cells. The methylcholanthrene-induced fibrosarcoma (BFS-1) was generated in a female C57BL/6 mice as described (12). The tumor cells were induced death in some human adenocarcinoma tumor lines (HT-29 ␥ maintained in vitro in DMEM high glucose medium (Life Technologies, Inc., and WiDr) in the presence of IFN- (2). Combined in vivo treatment Karlsruhe, Germany) supplemented with 5% heat inactivated FCS (Life Tech- of human adenocarcinoma cells (WiDr), which form solid tumors in nologies, Inc.) and 0.05 ng/ml gentamicin (PAA Laboratories, Linz, Austria). immunocompromised mice, with an agonistic anti-LT␤R antibody Tumor cells (1.5 ϫ 106 in 50 ␮l) were inoculated i.d. on the back of mice, and and human IFN-␥ resulted in tumor growth arrest. In addition, sig- tumor growth was measured as described recently (13). naling through LT␤R has been reported to induce NF␬B activation DNA Constructs and Transfectants. The mp55TNFR-Fc and mLT␤R-Fc and chemokine production in a cell type restricted manner (3, 4). expression constructs were generated by insertion of the extracellular domains Additional results have established a constitutive requirement for of the mp55TNFR or the mLT␤R into the Signal pIG plus vector (R&D LT␣ ␤ signaling in maintaining normal levels of secondary lymph- Systems, Wiesbaden, Germany). Stable transfectants expressing the 1 2 ␤ oid tissue chemokine and B lymphocyte chemoattractant in lymphoid p55TNFR-Fc or LT R-Fc fusion protein or MIP-2 were prepared by transfec- tion of BFS-1 cells with the corresponding expression construct using N-[1- tissue (5). Recent studies using genetically modified mice indicated ␤ (2,3-dioleoyloxyl)propyl]-N,N,N-trimethylammoniummethyl sulfate (Roche that LT R is critically involved in lymphoid organogenesis and in the Diagnostics, Mannheim, Germany), following the manufacturer’s instructions. generation of adaptive humoral immune responses (6–8). Transfected cells were selected and maintained in G418 (0.8 mg/ml) or It is generally accepted today that tumor growth is angiogenesis- hygromycin (0.02 mg/ml; PAA Laboratories). dependent and that every increment of tumor growth requires an Flow Cytometry. Expression of mLT␤R on BFS-1 cells was detected by increment of vascular growth. Tumors lacking angiogenesis remain flow cytometry on a FACStar Plus (Becton Dickinson, San Jose, CA) using a dormant indefinitely and rapid logarithmic growth follows the acqui- specific rat anti-mLT␤R monoclonal antibody (mLTR1C5, IgG2a) followed sition of blood supply. The tumor angiogenic switch seems to be by a FITC-conjugated mouse antirat IgG at concentrations of 10 ␮g/ml or an irrelevant isotype-matched rat IgG as control. The mLTR1C5 had been gen- erated by immunizing a rat with mLT␤R-Fc fusion protein, fusing the spleen Received 12/17/01; accepted 5/7/02. The costs of publication of this article were defrayed in part by the payment of page cells with SP2 myeloma cells (14), and screening the resulting hybridoma charges. This article must therefore be hereby marked advertisement in accordance with supernatants for positive staining of LT␤R-transfected Chinese hamster ovary 18 U.S.C. Section 1734 solely to indicate this fact. cells. 1 S. A. N. is International Research Scholar of Howard Hughes Medical Institute. 2 Western Blot Analysis. Supernatants of BFS-1 cells stably transfected To whom requests for reprints should be addressed, at Department of Pathology/Tumor ␤ Immunology, University of Regensburg, F.-J.-Strauss-Allee 11, D-93042 Regensburg, Ger- with the p55TNFR-Fc or the mLT R-Fc expression construct were incubated many. Phone: 49-941-944-6626; Fax: 49-941-944-6602; E-mail: daniela.maennel@klinik. with 10 mg protein G-Sepharose and incubated at 4°C for 1 h. Precipitated uni-regensburg.de. proteins were resolved on 12% SDS-PAGE and blotted on polyvinylidene 3 ␣ ␤ ␣ The abbreviations used are: LT 1 2, one molecule lymphotoxin- and two molecules lymphotoxin-␤;LT␤R, lymphotoxin-␤ receptor; TNF, tumor necrosis factor; NF␬B, nuclear factor ␬B; bFGF, basic fibroblast growth factor; VEGF, vascular endothelial 4 T. Plo¨tzel, M. B. Alimzhanov, D. Kuprash, S. A. Nedospasov, and K. Pfeffer. growth factor; MIP, macrophage inflammatory protein; MVD, microvascular density; Generation and characterization of mice with simultaneous inactivation of LT␣ and LT␤ ROI, region of interest; IL, interleukin; TNFR, tumor necrosis factor receptor. genes, manuscript in preparation. 4034 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2002 American Association for Cancer Research. TUMOR ANGIOGENESIS INDUCED BY LT␤ RECEPTOR ACTIVATION difluoride membrane (Immobilon-P, 0.45 ␮m; Millipore, Eschborn, Germany). shown by flow cytometric analysis using a monoclonal anti-mLT␤R The membrane was blocked with 5% dry milk in Tris-buffered saline for 1 h antibody (mLTR1C5; Fig. 1a). The expression of LT␤R was also at ambient temperature followed by incubation with antihuman IgG1 (1 mg/ confirmed at mRNA level by reverse transcription-PCR using specific ml) in incubation buffer (1% dry milk in Tris-buffered saline) for 1 h. After primers for mLT␤R (data not shown). washing, bound antihuman IgG1 was reacted with goat antimouse-conjugated BFS-1 cells stably transfected with cDNA of LT␤R-Fc or horseradish peroxidase (Sigma Biochemicals, Deisenhofen, Germany) in in- p55TNFR-Fc expressed comparable amounts of the corresponding cubation buffer for1hatambient temperature. The membrane was washed again and developed with the enhanced chemiluminescence kit (Energene, fusion proteins, whereas in the supernatants of nontransfected tumor Regensburg, Germany) following the manufacturer’s instructions. cells no fusion protein was detectable by Western blot analysis (Fig. Determination of MIP-2 and VEGF. MIP-2 and VEGF production was 1b). Binding of both secreted Fc fusion proteins to their corresponding determined using the corresponding ELISA kits (R&D Systems) according to ligands was verified by flow cytometric analysis in staining activated ␣ ␤ the manufacturer’s instructions. PMN1 cells, which express LT 1 2 on their surface (1) with super- Immunohistochemical
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