Preclinical Development Selective Targeting of Interferon g to Stromal Fibroblasts and Pericytes as a Novel Therapeutic Approach to Inhibit Angiogenesis and Tumor Growth Ruchi Bansal 1 , Tushar Tomar 1 , Arne € Ostman 3 , Klaas Poelstra 1,2 , and Jai Prakash 1,2,3,4 Abstract New approaches to block the function of tumor stromal cells such as cancer-associated fibroblasts and pericytes is an emerging field in cancer therapeutics as these cells play a crucial role in promoting angiogenesis and tumor growth via paracrine signals. Because of immunomodulatory and other antitumor activities, IFNg , a pleiotropic cytokine, has been used as an anticancer agent in clinical trials. Unfortunately only modest beneficial effects, but severe side effects, were seen. In this study, we delivered IFNg to stromal fibroblasts and pericytes, considering its direct antifibrotic activity, using our platelet-derived growth factor-beta receptor (PDGFbR)-binding carrier (pPB-HSA), as these cells abundantly express PDGFbR. We chemically conjugated IFNg to pPB-HSA using a heterobifunctional PEG linker. In vitro in NIH3T3 fibroblasts, pPB-HSA-IFNg conjugate activated IFNg -signaling (pSTAT1a) and inhibited their activation and migration. Furthermore, pPB-HSA-IFNg inhibited fibroblasts-induced tube formation of H5V endo- thelial cells. In vivo in B16 tumor-bearing mice, pPB-HSA-IFNg rapidly accumulated in tumor stroma and pericytes and significantly inhibited the tumor growth while untargeted IFNg and pPB-HSA carrier were ineffective. These antitumor effects of pPB-HSA-IFNg were attributed to the inhibition of tumor vascu- larization, as shown with a-SMA and CD-31 staining. Moreover, pPB-HSA-IFNg induced MHC-II expression specifically in tumors compared with untargeted IFNg , indicating the specificity of this approach. This study thus shows the impact of drug targeting to tumor stromal cells in cancer therapy as well as provides new opportunities to use cytokines for therapeutic application. Mol Cancer Ther; 11(11); 2419–28. Ó2012 AACR. Introduction In the past decade, the complexity of the tumor micro- environment has been extensively studied, and this knowledge has contributed to the development of new therapies for cancer (1). Apart from cancer cells, solid tumors contain large amounts of tumor stroma compris- ing a variety of cell types such as cancer-associated fibro- blasts (CAF), pericytes, endothelial cells, infiltrated immune cells, and cancer stem cells. Among them, CAFs are the major cell type that play a crucial role in tumor- igenesis and metastasis (1, 2) by secreting various cyto- kines and growth factors (e.g., VEGF, HGF, SDF-1a), which act in a paracrine/exocrine fashion on other cell types, thereby activating tumor-inducing processes (2–4). In addition to CAFs, pericytes are another important cell type, having phenotypic characteristics of mesenchymal cells and fibroblasts. These pericytes stabilize endotheli- um by surrounding the blood vessels and support angio- genesis by secreting VEGF (1). Both stromal fibroblasts and pericytes, collectively referred here as stromal cells, express high levels of platelet-derived growth factor-beta receptor (PDGFbR) and its expression in tumor stroma has been inversely correlated with the survival rate in patients with different types of cancer (5, 6). Also, studies have shown that inhibition of the functions of these stromal cells using a PDGFbR inhibitor (imatinib) leads to inhibition of angiogenesis and thereby reduction in tumor growth (7, 8). These data indicate the key role of the tumor stromal cells in tumor development; therefore, selective targeting to stromal cells for cancer therapeutics is of great interest and could provide highly attractive strategies to treat cancer. Among potent anticancer agents, IFNg has been shown to possess multiple potent antitumor properties. IFNg is a Authors' Affiliations: 1 Department of Pharmacokinetics, Toxicology and Targeting, Graduate School for Drug Exploration (GUIDE), University of Groningen; 2 BiOrion Technologies BV, MediTech Centre UMCG, L.J. Zielstraweg 1, Groningen, The Netherlands; 3 Cancer Centre Karolinska, Department of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden; and 4 Department of Targeted Therapeutics, MIRA Institute of Biomedical Technology and Technical Medicine, University of Twente, Enschede, The Netherlands Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Corresponding Author: Jai Prakash, Department of Targeted Therapeu- tics, MIRA Institute of Biomedical Technology and Technical Medicine, University of Twente, Enschede, The Netherlands. Phone: 31-53-489- 2412; Email: j.prakash@utwente.nl doi: 10.1158/1535-7163.MCT-11-0758 Ó2012 American Association for Cancer Research. Molecular Cancer Therapeutics www.aacrjournals.org 2419 Downloaded from http://aacrjournals.org/mct/article-pdf/11/11/2419/2321001/2419.pdf by guest on 16 June 2022