Cancer Therapy: Preclinical Nintedanib Is a Highly Effective Therapeutic for Neuroendocrine Carcinoma of the Pancreas (PNET) in the Rip1Tag2 Transgenic Mouse Model Ruben Bill 1 , Ernesta Fagiani 1 , Adrian Zumsteg 1 , Helena Antoniadis 1 , David Johansson 1 , Simon Haefliger 1 , Imke Albrecht 1 , Frank Hilberg 2 , and Gerhard Christofori 1 Abstract Purpose: Pancreatic neuroendocrine tumors (PNET) represent a rare but challenging heterogeneous group of cancers with an increasing incidence over the last number of decades. Herein, we report an in-depth evaluation of the new antiangiogenic small- molecule tyrosine kinase inhibitor (TKI) nintedanib in the pre- clinical Rip1Tag2 transgenic mouse model of neuroendocrine carcinoma of the pancreas (insulinoma). Experimental Design: We have assessed the antiangiogenic and antitumor activity of nintedanib, in comparison with other antiangiogenic TKI, by treating Rip1Tag2 transgenic mice with different treatment schedules complemented with histopatho- logic, cell biologic, and biochemical analyses. Results: Prolonged nintedanib treatment of Rip1Tag2 mice has led to a strong suppression of angiogenesis, accompanied by a reduced tumor burden, which translated into a significant pro- longation of survival. Despite nintedanib's inhibitory action on perivascular cells, the blood vessels remaining after therapy dis- played a considerably mature phenotype with tight perivascular cell coverage and preserved perfusion. Nintedanib treatment did not increase local tumor invasiveness or metastasis to the liver and pancreatic lymph nodes—a phenomenon that has been observed with antiangiogenic treatments of Rip1Tag2 transgenic mice in other laboratories. In contrast with the strong reduction in blood microvessel densities, nintedanib did not have any impact on tumor lymphangiogenesis. Conclusions: Based on our findings, we propose the clinical evaluation of the antiangiogenic drug nintedanib as a new treat- ment modality for PNET patients, notably in a direct comparison with already established therapeutic regimens, such as sunitinib. Clin Cancer Res; 21(21); 4856–67. Ó2015 AACR. Introduction Pancreatic neuroendocrine tumors (PNET), although repre- senting a minority of pancreatic tumors, remain a therapy-chal- lenging heterogeneous group of tumors with increasing incidence over the last decades (1). Whereas 45% to 60% of PNETs are nonfunctional, 40% to 55% produce a variety of hormones leading to different clinical presentations, e.g., hypoglycemic syndrome in the case of insulin-producing PNETs (insulinoma; ref. 2). Aside from cytoreductive surgery, somatostatin analogues, peptide receptor-targeted radiotherapy, and systemic chemother- apy, management of advanced PNETs involves targeted therapies, such as the mTOR inhibitor everolimus or the antiangiogenic small-molecule tyrosine kinase inhibitor (TKI) sunitinib (2). In a phase III clinical trial, sunitinib was shown to be highly effective and significantly prolonged progression-free and overall survival of PNET patients. This study even had to be discontinued early, because of a significantly worse clinical outcome in the placebo group. However, a number of patients experienced treatment- related side effects, such as grade 3 or 4 hypertension and neu- tropenia in 10% and 12% of patients, respectively (3). Reducing adverse events is of particular importance in this cancer entity, since patients usually undergo long-term therapy and usually experience good quality of life even without treatment until late in the course of the disease (4). Despite the encouraging results derived from numerous pre- clinical studies, antiangiogenic therapies targeting mainly the VEGF/VEGF receptor axis have widely failed to substantially increase patient survival in a large number of cancer types (5). In mouse models, the upregulation of alternative proangiogenic factors, such as FGFs, PDGFs, Bv8, and others, has been shown to mediate the resistance to blocking the VEGF-A/VEGFR2 axis. Hence, a simultaneous targeting of the VEGF and the FGF receptor families and other alternative signaling pathways may lead to an improved clinical outcome (6–9). Nintedanib (BIBF1120), a small-molecule kinase inhibitor that targets not only VEGF and PDGF receptors but also FGF receptors and c-Src (10, 11), has recently been shown to yield significant anticancer effects in a variety of preclinical cancer models and in non–small cell lung cancer (NSCLC) patients (10, 12, 13). In a recent phase III clinical trial of nintedanib in NSCLC patients (LUME-Lung 1), the 1 Department of Biomedicine, University of Basel, Basel, Switzerland. 2 Boehringer Ingelheim Austria RCV GmbH & Co KG, Vienna, Austria. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). R. Bill and E. Fagiani contributed equally to this article. Current address for A. Zumsteg: Covagen AG, 8952 Schlieren, Switzerland; and current address for I. Albrecht: Drug Discovery Biology, Actelion Pharmaceutical Ltd, Allschwil, Switzerland. Corresponding Author: Gerhard Christofori, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland. Phone: 41-61-267-3562; Fax: 41-61-267-3566; E-mail: gerhard.christofori@unibas.ch doi: 10.1158/1078-0432.CCR-14-3036 Ó2015 American Association for Cancer Research. 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