Microenvironment and Immunology Aptamer-Mediated Blockade of IL 4 R a Triggers Apoptosis of MDSCs and Limits Tumor Progression

In addition to promoting tumor progression andmetastasis by enhancing angiogenesis and invasion, myeloidderived suppressor cells (MDSC) and tumor-associated macrophage (TAM) also inhibit antitumor T-cell functions and limit the efficacy of immunotherapeutic interventions. Despite the importance of these leukocyte populations, a simple method for their specific depletion has not been developed. In this study, we generated an RNA aptamer that blocks the murine or human IL-4 receptor-a (IL4Ra or CD124) that is critical for MDSC suppression function. In tumor-bearing mice, this anti–IL4Ra aptamer preferentially targeted MDSCs and TAM and unexpectedly promoted their elimination, an effect that was associated with an increased number of tumorinfiltrating T cells and a reduction in tumor growth.Mechanistic investigations of aptamer-triggered apoptosis in MDSCs confirmed the importance of IL4Ra–STAT6 pathway activation in MDSC survival. Our findings define a straightforward strategy to deplete MDSCs and TAMs in vivo, and they strengthen the concept that IL4Ra signaling is pivotal for MDSC survival. More broadly, these findings suggest therapeutic strategies based on IL4Ra signaling blockades to arrest an important cellular mechanism of tumoral immune escape mediated by MDSCs and TAM in cancer. Cancer Res; 72(6); 1–11. 2012 AACR. Introduction Myeloid-derived suppressor cells (MDSC, CD11bþGr1þ IL4Raþ (1, 2) and tumor-associated macrophages (TAM, CD11bþGr1 F4/80þ; ref. 3) are particularly important in promoting tumor growthmetastasis and angiogenesis (4, 5) and in suppressing the antitumor immune response (6). Although different signaling pathways may be responsible for the MDSC suppressive activity, the IL4Ra–STAT6 pathway seems to be particularly important: it mediates TGF-b production (7), arginase activity (8) and, in conjunction with STAT3 and STAT1 activation, it allows for the production of peroxynitrite and ROS (6) and for the expression ofmatrixmetalloproteinase 9 (9). Although different compounds were shown to inhibit or deplete MDSCs (i.e., STAT3 or PDE5 inhibitors, vitamin D, ATRA, nitroaspirin gemcitabine, and others; ref. 10) and showed reasonable rates of success, their systemic use leads to undesirable side effects and off-target depletion. Selective targeting of MDSCs is therefore necessary to maximize the efficacy of immune therapeutic approaches while minimizing systemic side effects. To this aim, we selected a new RNA aptamer that specifically targets IL4Ra because of the importance of this receptor in MDSC suppressive function. RNA aptamers are nanomolecules that can recognize their targets with extremely high affinity. This class of compounds provides unique advantages as therapeutic agents, such as amenability to chemical modifications, ease of production, and low immunogenicity (11, 12). Here we describe a new RNA aptamer specific for IL4Ra/ CD124. This aptamer selectively targets MDSCs and TAM in vivo, blocks IL4Ra signaling, induces MDSCs apoptosis, and significantly delays tumor progression in 4T1-bearing mice. Materials and Methods Cell lines 4T1, 4T1HAThy1.1luciferase TS/A, and MSC2 cell lines were previously described (13, 14). MSC2 was maintained in Dulbecco's modified Eagle's medium (Invitrogen), supplemented with L-glutamine (2 mmol/L), HEPES (10 mmol/L), 2-mercaptoethanol (20 mmol/L), streptomycin (150 U/mL), penicillin (200 U/mL), and 10% heat-inactivated FBS (Invitrogen). Aptamer selection Aptamers were selected with the optimized magnetic epoxy beads method described in the Supplementary Authors' Affiliations: Department of Microbiology and Immunology, Sylvester Cancer Center, Miller School of Medicine, University of Miami; and Diabetes Research Institute, University of Miami Leonard M. Miller School of Medicine, Miami, Florida Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Current address for F. Roth: Sanford Health South University, Fargo, ND 58103. Corresponding Author: Paolo Serafini, Department of Microbiology and Immunology, University ofMiami, 1600 NW10th Avenue, 3075RMSBBldg (R138), Miami, Fl 33136. Phone: 305-243-7917; Fax: 1-305-243 5522; E-mail: pserafini@med.miami.edu doi: 10.1158/0008-5472.CAN-11-2772 2012 American Association for Cancer Research. Cancer Research www.aacrjournals. org OF1 Research. on January 28, 2018. © 2012 American Association for Cancer cancerres.aacrjournals. org Downloaded from Published OnlineFirst January 26, 2012; DOI: 10.1158/0008-5472.CAN-11-2772