Characterization of Arginase Expression in Glioma-Associated Microglia and Macrophages

Microglia (MG) and macrophages (MPs) represent a significant component of the inflammatory response to gliomas. When activated, MG/MP release a variety of pro-inflammatory cytokines, however, they also secrete anti-inflammatory factors that limit their cytotoxic function. The balance between pro and anti-inflammatory functions dictates their antitumor activity. To evaluate potential variations in MG and MP function in gliomas, we isolated these cells (and other Gr1+ cells) from intracranial GL261 murine gliomas by FACS and evaluated their gene expression profiles by microarray analysis. As expected, arginase 1 (Arg1, M2 marker) was highly expressed by tumor-associated Gr1+, MG and MP. However, in contrast to MP and Gr1+ cells that expressed Arg1 shortly after tumor trafficking, Arg1 expression in MG was delayed and occurred in larger tumors. Interestingly, depletion of MPs in tumors did not prevent MG polarization, suggesting direct influence of tumor-specific factors on MG Arg1 upregulation. Finally, Arg1 expression was confirmed in human GBM samples, but most Arg1+ cells were neutrophils and not MPs. These findings confirm variations in tumor MG and MP polarization states and its dependency on tumor microenvironmental factors. The Affymetrix GeneChip Mouse Gene 1.0-ST array (Affymetrix, Santa Clara, CA) was used to define gene expression profiles from the samples. Synthesis and labelling of cDNA targets, hybridization and scanning of GeneChips were carried out by the Microarray Core Facility at the City of Hope. Due to the limited starting material, we used a modified two-cycle amplification protocol. Briefly, cRNA was generated using 10ng total RNA according to the manufacturer's protocol by using Affymetrix's GeneChip Whole Transcript Sense Target Labeling Assay in the first cycle of amplification. The cRNA was subjected to RiboMinus kit (Invitrogen, Carlsbad, CA) to remove rRNA. The resulted cRNA was used as the template for another round of amplification using the sense target labeling assay kit. Hybridization cocktails containing 5.5 g of fragmented, end-labeled cDNA were prepared and applied to GeneChip Mouse Gene 1.0 ST arrays. Hybridization was performed for 16 hours, and the arrays were washed and stained with the GeneChip Fluidics Station 450 using FS450_0007 script. Arrays were scanned at 5-m resolution using the Affymetrix GCS 3000 7G and GeneChip Operating Software v. 1.4 to produce .CEL intensity files.