Angiostatic immune reaction in colorectal carcinoma: Impact on survival and perspectives for antiangiogenic therapy Elisabeth Naschberger 1 , Roland S. Croner 2 , Susanne Merkel 2 , Arno Dimmler 3 , Philipp Tripal 1 , Kerstin U. Amann 3 , Elisabeth Kremmer 4 , Wolfgang M. Brueckl 5 , Thomas Papadopoulos 3 , Christine Hohenadl 6 , Werner Hohenberger 2 and Michael St€ urzl 1 * 1 Division of Molecular and Experimental Surgery, Department of Surgery, University of Erlangen-Nuremberg, Schwabachanlage 10, Erlangen, Germany 2 Department of Surgery, University of Erlangen-Nuremberg, Krankenhausstr. 12, Erlangen, Germany 3 Institute of Pathology, University of Erlangen-Nuremberg, Krankenhausstr. 8-10, Erlangen, Germany 4 Institute of Molecular Immunology, Helmholtz Center Munich, Marchioninistr. 25, Munich, Germany 5 Department of Medicine I, University of Erlangen-Nuremberg, Ulmenweg 18, Erlangen, Germany 6 Research Institute of Virology and Biomedicine, University of Veterinary Medicine Vienna, Veterin € arplatz 1, Vienna, Austria Angiogenesis and inflammation are the 2 major stroma reactions in colorectal carcinoma (CRC). Guanylate binding protein-1 (GBP-1) is a key mediator of angiostatic effects of inflammation. Therefore, we hypothesized that GBP-1 may be a biomarker of intrinsic angiostasis associated with an improved outcome in CRC patients. GBP-1 was strongly expressed in endothelial cells and immune cells in the desmoplastic stroma of 32% of CRC as deter- mined by immunohistochemical investigation of 388 sporadic CRC. Cancer-related 5-year survival was highly significant (p < 0.001) increased (16.2%) in patients with GBP-1-positive CRC. Multivariate analysis showed that GBP-1 is an independent prognostic factor indicating a reduction of the relative risk of cancer-related death by the half (p 5 0.032). A comparative transcriptome analysis (22,215 probe sets) of GBP-1-positive (n 5 12) and -negative (n 5 12) tumors showed that particularly IFN-c-induced genes including the major antiangiogenic chemo- kines CXCL9, CXCL10 and CXCL11 were coexpressed with GBP- 1. Altogether our findings indicated that GBP-1 may be a novel bio- marker and an active component of a Th-1-like angiostatic immune reaction in CRC. This reaction may affect patient’s response to antiangiogenic therapy and the identification of such tumors may provide a novel criterion for patient selection. Moreover, the induc- tion of a Th-1-like angiostatic immune reaction may be a promising approach for the clinical treatment of CRC. ' 2008 Wiley-Liss, Inc. Key words: guanylate binding protein-1; interferon-c; colorectal carcinoma; angiogenesis; inflammation Colorectal carcinoma (CRC) is the second most common malig- nant tumor, both in men and women, in the western world. 1 Angiogenesis and inflammation are the 2 primary stroma reactions involved in CRC pathogenesis. 2–4 The high impact of angiogenesis on the growth of cancer has been convincingly demonstrated in numerous animal studies. 5,6 Recently, the inhibition of blood vessel growth has also been suc- cessfully applied in human cancer therapy. In a phase III clinical trial the survival of patients with metastatic CRC was significantly prolonged from 15.6 to 20.3 months when VEGF was neutralized by the antibody bevacizumab. 7 However, it is still an enigma why some patients responded to antiangiogenic therapy and others did not. Biomarkers predicting the response to antiangiogenic therapy are urgently needed to select patients that will most likely benefit from this treatment. 8 It is consistently documented that chronic inflammation predis- poses for CRC 9,10 whereas acute inflammation is regarded as an antitumor defense mechanism. 11 The mechanisms how an acute immune reaction translates into beneficial prognosis are subjects of ongoing research. 12–14 Immune cells can release many different mediators, which can either activate or inhibit angiogenesis, tumor cell proliferation and metastasis and may positively or negatively contribute to tumor cell survival. 3,15–18 In CRC with high T cell density a T-helper 1 (Th-1)-like micromilieu with expression of interferon-g (IFN-g) and IFN-g-induced factors such as IFN regu- latory factor-1 (IRF-1), granulysin and granzyme B has been observed and was found to be associated with a positive progno- sis. 19,20 This suggested that an IFN-g-triggered immune response may be beneficial for the patients. It is in agreement with this hy- pothesis that gene therapeutic approaches with the IFN-g-induced CXCL9 chemokine suppressed growth and dissemination of colon carcinoma cells in combination with the Th-1-cytokine IL-2 in an in vivo model. 21 However, in other reports the expression of indole- amine 2,3-dioxygenase (IDO), a strictly IFN-g-dependent protein, did not have a significant impact on overall survival of CRC patients 22 and the tumorigenic properties of metastatic CRC cells were described to be fostered by IFN-g-induced CXCL10 in vitro. 23 This demonstrates that further studies are required to determine the impact of IFN-g-induced genes in the pathogenesis of CRC. Among the most abundantly induced proteins by IFN-g is gua- nylate binding protein-1 (GBP-1). 24–26 GBP-1 belongs to the fam- ily of large GTPases which consists of 7 homologous members. 27 We showed that GBP-1 characterizes endothelial cells exposed to IFN-g, IL-1b and TNF-a, both in vitro and in vivo 28 and mediates the potent antiangiogenic effects of these cytokines. 29,30 Two dif- ferent antiangiogenic functions were found to be exerted by GBP- 1, first of all the inhibition of endothelial cell proliferation 29 and second, the inhibition of endothelial cell invasiveness. 29 The latter Additional Supporting Information may be found in the online version of this article. Abbreviations: CRC, colorectal carcinoma; CXCL9, chemokine (C-X-C motif) ligand 9; CXCL10, chemokine (C-X-C motif) ligand 10; CXCL11, chemokine (C-X-C motif) ligand 11; CXCR3, chemokine (C-X-C motif) receptor 3; EVI, extramural venous invasion; GBP-1, guanylate binding protein-1; IAR, intrinsic angiostatic immune reaction; IC, inflammatory cytokines; IDO, IFN--inducible indoleamine 2,3-dioxygenase; LDAL2, galectin-2; MCP-2, monocyte chemotactic protein-2; OAS2, 2 0 -5 0 -oligoa- denylate synthetase-2; pM, pathological examined distant metastasis; pN, pathological examined spread into regional lymph nodes; pT, pathological examined size of primary tumor; SLAM, signaling lymphocyte activating molecule; UICC, International Union against Cancer. Conflict of Interest: E.N., R.S.C. and M.S. declare that parts of the work are subject of a US provisional patent application entitled ‘‘Method for the detection of Interferon-associated angiostatic tumorstages in colorectal carcinoma’’. M.S. has received royalties from a patent for GBP-1 as a target in therapy. Grant sponsor: ELAN Program of the University of Erlangen-Nurem- berg; Grant numbers: AZ 05.06.05.1, AZ 03.09.22.1; Grant sponsor: Deut- sche Forschungsgemeinschaft; Grant numbers: STU 317/2-1, DFG-GK 1071; Grant sponsor: German Cancer Aid, Interdisciplinary Center for Clinical Research (IZKF) of the University of Erlangen-Nuremberg. The first two authors contributed equally to this work. *Correspondence to: Division of Molecular and Experimental Sur- gery, Department of Surgery, University of Erlangen-Nuremberg, Schwa- bachanlage 10, Erlangen 91054, Germany. Fax: 149-9131-85-32077. E-mail: michael.stuerzl@uk-erlangen.de Received 30 November 2007; Accepted after revision 20 May 2008 DOI 10.1002/ijc.23764 Published online 11 August 2008 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 123, 2120–2129 (2008) ' 2008 Wiley-Liss, Inc. Publication of the International Union Against Cancer