Microglia (brain resident macrophages) accumulate in malignant gliomas and instead of initiating the anti-tumor response, they switch to a pro-invasive phenotype, support tumor growth, invasion, angiogenesis and immunosuppression by release of cytokines/chemokines and extracellular matrix proteases. Using immunofluorescence and flow cytometry, we demonstrate an early accumulation of activated microglia followed by accumulation of macrophages in experimental murine EGFP-GL261 gliomas. Those cells acquire the alternative phenotype, as evidenced by evaluation of the production of ten pro/anti-inflammatory cytokines and expression profiling of 28 genes in magnetically-sorted CD11b+ cells from tumor tissues. Furthermore, we show that infiltration of implanted gliomas by amoeboid, Iba1-positive cells can be reduced by a systematically injected cyclosporine A (CsA) two or eight days after cell inoculation. The up-regulated levels of IL-10 and GM-CSF, increased expression of genes characteristic for the alternative and pro-invasive phenotype (arg-1, mt1-mmp, cxcl14) in glioma-derived CD11b+ cells as well as enhanced angiogenesis and tumor growth were reduced in CsA-treated mice. Our findings define for the first time kinetics and biochemical characteristics of glioma-infiltrating microglia/macrophages. Inhibition of the alternative activation of tumor-infiltrating macrophages significantly reduced tumor growth. Thus, blockade of microglia/macrophage infiltration and their pro-invasive functions could be a novel therapeutic strategy in malignant gliomas.

Gliomas attract brain-resident (microglia) and peripheral macrophages and reprogram these cells into immunosuppressive, pro-invasive cells. M-CSF (macrophage colony-stimulating factor, encoded by the CSF1 gene) has been implicated in the control of recruitment and polarization of macrophages in several cancers. We found that murine GL261 glioma cells overexpress GM-CSF (granulocyte-macrophage colony-stimulating factor encoded by the CSF2 gene) but not M-CSF when compared to normal astrocytes. Knockdown of GM-CSF in GL261 glioma cells strongly reduced microglia-dependent invasion in organotypical brain slices and growth of intracranial gliomas and extended animal survival. The number of infiltrating microglia/macrophages (Iba1(+) cells) and intratumoural angiogenesis were reduced in murine gliomas depleted of GM-CSF. M1/M2 gene profiling in sorted microglia/macrophages suggests impairment of their pro-invasive activation in GM-CSF-depleted gliomas. Deficiency of M-CSF (op/op mice) did not affect glioma growth in vivo and the accumulation of Iba1(+) cells, but impaired accumulation of Iba1(+) cells in response to demyelination. These results suggest that distinct cytokines of the CSF family contribute to macrophage infiltration of tumours and in response to injury. The expression of CSF2 (but not CSF1) was highly up-regulated in glioblastoma patients and we found an inverse correlation between CSF2 expression and patient survival. Therefore we propose that GM-CSF triggers and drives the alternative activation of tumour-infiltrating microglia/macrophages in which these cells support tumour growth and angiogenesis and shape the immune microenvironment of gliomas.

Glioblastoma (GBM) is an aggressive malignancy associated with profound host immunosuppression. Microglia and macrophages infiltrating GBM acquire the pro-tumorigenic, M2 phenotype and support tumor invasion, proliferation, survival, angiogenesis and block immune responses both locally and systematically. Mechanisms responsible for immunological deficits in GBM patients are poorly understood. We analyzed immune/inflammatory gene expression in five datasets of low and high grade gliomas, and performed Gene Ontology and signaling pathway analyses to identify defective transcriptional responses. The expression of many immune/inflammatory response and TLR signaling pathway genes was reduced in high grade gliomas compared to low grade gliomas. In particular, we found the reduced expression of the IKBKB, a gene coding for IKKβ, which phosphorylates IκB proteins and represents a convergence point for most signal transduction pathways leading to NFκB activation. The reduced IKBKB expression and IKKβ levels in GBM tissues were demonstrated by qPCR, Western blotting and immunohistochemistry. The IKKβ expression was down-regulated in microglia/macrophages infiltrating glioblastoma. NFκB activation, prominent in microglia/macrophages infiltrating low grade gliomas, was reduced in microglia/macrophages in glioblastoma tissues. Down-regulation of IKBKB expression and NFκB signaling in microglia/macrophages infiltrating glioblastoma correlates with defective expression of immune/inflammatory genes and M2 polarization that may result in the global impairment of anti-tumor immune responses in glioblastoma.

Background Malignant tumours release factors, which attract myeloid cells and induce their polarisation to pro-invasive, immunosuppressive phenotypes. Brain-resident microglia and peripheral macrophages accumulate in the tumour microenvironment of glioblastoma (GBM) and induce immunosuppression fostering tumour progression. Macrophage colony stimulating factors (CSFs) control the recruitment of myeloid cells during peripheral cancer progression, but it is disputable, which CSFs drive their accumulation in gliomas. Methods The expression of CSF2 (encoding granulocyte-macrophage colony stimulating factor) was determined in TCGA datasets and five human glioma cell lines. Effects of stable CSF2 knockdown in glioma cells or neutralising CSF2 or receptor CSF2Rα antibodies on glioma invasion were tested in vitro and in vivo. Results CSF2 knockdown or blockade of its signalling reduced microglia-dependent glioma invasion in microglia-glioma co-cultures. CSF2-deficient human glioma cells encapsulated in cell-impermeable hollow fibres and transplanted to mouse brains, failed to attract microglia, but stimulated astrocyte recruitment. CSF2-depleted gliomas were smaller, attracted less microglia and macrophages, and provided survival benefit in tumour-bearing mice. Apoptotic microglia/macrophages were detected in CSF2-depleted tumours. Conclusions CSF2 is overexpressed in a subset of mesenchymal GBMs in association with high immune gene expression. Tumour-derived CSF2 attracts, supports survival and induces pro-tumorigenic polarisation of microglia and macrophages.

Novel concepts of anticancer therapies focus on targeting tumor-supportive microenvironment and/or cancer stem-like cells (CSC). We combined two approaches in a quest for novel targets for malignant glioma therapy. Brain macrophages accumulate in malignant gliomas and instead of initiating the anti-tumor response, they switch to a pro-invasive phenotype, support tumor growth, invasion, angiogenesis and cause immunosuppression. We found that one of glioma- derived factors crucial for activation of the pro-invasive phenotype is osteopontin (OPN, SPP1). OPN expression is up-regulated in many cancers, correlating with malignancy grade and poor patient prognosis. We demonstrated hundreds-fold increased expression of OPN in rat C6 glioma and elevated expression in human glioma cells in comparison to non-transformed astrocytes. Thus, we developed C6 glioma cell clones stably expressing control (shNeg) or OPN specific shRNA. Invasion of control and shOPN expressing glioma cells was evaluated in Matrigel assay after culturing cells for 36 h in the presence or absence of brain macrophages (microglia). Quantification of invading glioma cells, stained with a fluorescent dye was performed by laser scanner cytometry. Knockdown of OPN greatly diminished both basal and microglia-induced invasion of glioma cells. To evaluate contribution of OPN to glioma growth in vivo, we injected shOPN or shNeg glioma clones into the striatum of Wistar rats. Silencing of OPN in glioma cells greatly reduced the tumor volume on 15th day after tumor implantation. Staining for Iba-1 positive microglia/macrophages demonstrated no difference in the number of brain macrophages infiltrating gliomas. However, Iba1-positive cells infiltrating tumors with silenced OPN expression displayed ramified morphology, contrary to amoeboid, fully activated cells infiltrating control tumors. As glioma initiating stem-like cells were suggested to induce immunosuppressive macrophages, we hypothesized that OPN expression may differ in CSC. We employed commonly used procedures to enriched in CSC: rhodamine 123 exclusion and sphere-forming assays. Cells negative for rhodamine 123 were considered as a fraction enriched in CSC and sorted. Spheres grown in neural stem cell medium supplemented with epidermal growth factor and basic fibroblast growth factor were collected at 14th day after seeding. Enrichment in CSC was confirmed by increased expression of the markers of cancer stem cells: Nanog and Oct3/4 in selected cell populations. We demonstrate that human glioma cell populations enriched in CSC exhibit higher expression of OPN than bulk cells. To identify signaling pathways regulating OPN expression, glioma cells were exposed to a panel of signal transduction inhibitors, and OPN mRNA and protein levels were determined by qPCR and ELISA. Those studies showed involvement of NF B and ERK signaling pathways in regulation of OPN expression. We conclude that OPN overexpressed in glioma cells, crucial for both tumor invasion and macrophage activation is a potential promising target for glioma therapy. High expression of OPN in CSC points to its crucial role in glioma pathology. The study was supported by grants N N301 290837 and N N301 786240 from the Polish Ministry of Science and Higher Education Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C230.