Microglia (MG) and macrophages (MPs) represent a significant component of the

Microglia (MG) and macrophages (MPs) represent a significant component of the inflammatory response to gliomas. Among these, myeloid-derived cells are abundant in tumors and have been shown to promote tumorigenesis, angiogenesis and invasion [1]. A class of these cells, designated as myeloid-derived suppressor cells (MDSC), possess immunosuppressive properties that facilitate immune escape based on local microenvironmental factors [2]. MDSCs, however, do not represent a single cell Papain Inhibitor population, but are composed of immature myeloid cells at different stages of cell differentiation. These cells can suppress the immune response by several mechanisms, including the production of arginase 1 (Arg1), which decreases the level of L-arginine that is critical for normal T cell function. Lower levels of arginine are known to reduce T cell receptor chain expression and to promote T cell dysfunction. These cells also secrete nitric oxide and reactive oxygen species which are capable of inducing T cell suppression [3]. In gliomas, myeloid-derived cells are mostly represented by resident microglia (MG) that migrate into the brain during early development, or by infiltrating tumor macrophages (MPs) that arise from circulating monocytes. Although other myeloid cells such as neutrophils and other granulocytes are also present in gliomas, infiltrating MG and MPs (referred to as tumor-associated macrophages or TAMs) have received recent attention due to their involvement in glioma IMPA2 antibody escape from anti-angiogenic agents [4]. As components of the innate immune system, TAMs express a variety of factors that constantly alter tumor microenvironment. These Papain Inhibitor cells can produce proinflammatory molecules such as TNF, IL1, and CXCL10 that can both activate antitumor immune responses and support tumor angiogenesis and invasion [5C10]. TAMs may also secret immunosuppressive cytokines like IL-10 and TGF and matrix-degrading enzymes like MMP2, MMP9, MT1-MMP and cathepsins that promote glioma invasion, immune escape and angiogenesis. So far, most TAM characterization studies have grouped glioma MG and MP as a single cell population, and the contribution of each cell type to glioma microenvironment has been more difficult to evaluate due to overlapping phenotypic and functional similarities. In this study, to evaluate potential variations in MG and MPs function in gliomas, we isolated these cells (and other MDSC) from GL261 murine gliomas based on flow cytometry staining characteristics [11]. A genome-wide microarray expression analysis demonstrated significant upregulation of Arg1 in both tumor MG and MPs as compared to circulating monocytes. These studies also suggested significant similarities in gene expression profiles between tumor MG and MP. In contrast to MPs, however, Arg1 expression in resident MG was delayed and occurred later during tumor growth and was independent of TAM infiltration into gliomas. Evaluation of human tumor specimen also confirmed Arg1 expression in both TAMs and other myeloid-derived cells such as neutrophils. These findings confirm dynamic changes in TAM polarization that is dependent on tumor microenvironmental factors and highlights variations in the contribution of MDSCs to the immunosuppressive glioma milleu. Materials and Methods Reagents and cell lines Luciferase-expressing GL261 glioma cells (GL261-Luc) were obtained from Dr. Karen Aboody’s laboratory in 2006 and were generated as described before [12]. Luciferase-expressing KR158B cells (or K-Luc), an invasive glioma cell line that was derived from spontaneous gliomas in double-mutant mice in Dr. Tyler Jacks Papain Inhibitor laboratory, was a generous gift from Dr. John Sampson in 2011 [13]. Both GL261-Luc and K-Luc cells were cultured in DMEM medium supplemented with 10% FBS (BioWhittaker, Walkersville, MD), 100 U/mL penicillin-G, 100 g/mL streptomycin and 0.01 M Hepes buffer (Life Technologies, Gaithersburg, MD) in a humidified 5% CO2 atmosphere, and their tumorigenicity was authenticated by histological characterization of intracranial gliomas in syngeneic C57BL/6J mice. Tumor implantation Mice were housed and handled in accordance to the guidelines and approval of City of Hope Institutional Animal Care and Use Committee under pathogen-free conditions. All mice were on C57BL/6J background. Knock-in mice that express EGFP under control of the endogenous Cx3cr1 locus were purchased from Jackson Laboratory (Sacramento, CA). CD11b-TKmt-30 mice, a generous gift from Dr. JP Julien, were bred at our institution and PCR genotyped by using Genotyping DNA preparation Kit (Bioland Scientific LLC). Intracranial tumor implantation was performed stereotactically as described before [14]. Briefly, GL261-Luc or K-Luc glioma cells were harvested by trypsinization, counted, and resuspended in culture medium. Female.