Malgorzata Sielska

Characteristics of the alternative phenotype of microglia/macrophages and its modulation in experimental gliomas.

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.

Distinct roles of CSF family cytokines in macrophage infiltration and activation in glioma progression and injury response

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. Copyright

Down-regulation of IKKβ expression in glioma-infiltrating microglia/macrophages is associated with defective inflammatory/immune gene responses in glioblastoma

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.

Characteristics of Phenotype and Pro-Tumorigenic Roles of Glioma Infiltrating Microglia/Macrophages

Recent clinical and experimental studies show an important role of tumor-infiltrating macrophages in tumor growth, metastasis and response to cancer treatments. Tumor-associated macrophages are attracted by tumor-released molecules which induce reprogramming/differentiation of macrophages/myeloid derived cells into anti-inflammatory cells known as alternatively activated, M2-type macrophages, in contrast to inflammatory M1-type. The stromal signals and components of immune microenvironment influencing glioma progression are poorly known and are likely distinct from those implicated in non-nervous system cancers. This review summarizes recent findings on characteristics of immune microenvironment of gliomas. Various functional properties of microglia in the normal and pathological central nervous system are now being revealed because of combinations of bone marrow transplantations and experimental disease models. Here, we describe some of the latest findings on the heterogeneity of glioblastoma-infiltrating brain macrophage, their alternative, immunosuppressive phenotype and contribution to glioma pathology. Recent attempts to determine a profile of cytokine/chemokine production and genes expression profiling on sorted CD11b+ cells from tumor tissues, revealed that many genes induced in M2-type macrophages from other tissues or in the context of helminth infections, are not up-regulated in CD11b+ cells isolated from gliomas. On the other hand, some genes characteristic for the alternative and pro-invasive phenotype: arg-1, mt1-mmp and cxcl14 were consistently activated in those cells. Furthermore, we discuss the potential for targeting interactions between glioma and brain macrophages in therapeutic interventions. Small molecule inhibitors of MAPK signaling with immunosuppressive properties, such as cyclosporine A and minocycline were shown to block infiltration and activation of microglia/macrophages in vitro, in organotypic brain slices and in vivo. Small molecule inhibitors with anti-inflammatory properties significantly reduced infiltration of brain macrophages, angiogenesis and tumor growth in mice, demonstrating that blockade of their proinvasive functions could be a novel therapeutic strategy in malignant gliomas.

Glioblastoma: Anti-tumor Action of Cyclosporin A and Functionally Related Drugs

Human malignant gliomas are highly resistant to current therapeutic approaches. Major signaling pathways that have been identified as playing important roles in glioblastomas are: the PTEN/PI3K/Akt/mTOR and the Ras/Raf/MEK/ERK signaling cascades, which support cell invasion, survival and prevent apoptosis. In the face of tumor resistance to apoptosis, novel agents which can overcome resistance or/and affect cell survival by non-apoptotic mechanisms such as necrosis, senescence, autophagy and mitotic catastrophe, are highly desirable. The present chapter focuses on anti-tumor action of cyclosporin A (CsA) and rapamycin that besides their well known immunosuppressive abilities appear to be multitarget kinase inhibitors and moderately effective anti-tumor agents in glioblastomas in vitro, in vivo and in clinical trials. A compelling evidence shows that cyclosporin A induces growth arrest and programmed cell death in cultured rat and human glioblastoma cells. The molecular mechanism involves accumulation of a cell cycle inhibitor – p21Cip1/Waf1, even in the absence of functional p53 tumor suppressor. In C6 glioma cells with functional TP53 and PTEN tumor suppressors CsA treatment up-regulates fasL expression, activates p53 and intrinsic mitochondrial death pathway, while in human glioblastoma cells with defects in either TP53 or PTEN, none of those effects were observed. Molecular analysis revealed that CsA, trough yet unknown mechanisms, down-regulates PI3K/Akt and mTOR signaling pathways in glioblastoma cells, and interferes with pro-invasive activity of tumor-infiltrating microglia. A systemically applied CsA significantly reduced growth of intracranial gliomas, tumor invasion and angiogenesis. Pharmacological inhibitors of the mTOR pathway: rapamycin, temsirolimus, everolimus and AP23573 were tested as potential targeted drugs in human glioblastoma cultures and in animal models. However, rapamycin and derivatives show moderate efficacy in patients with recurrent glioblastoma multiforme, they deserve further clinical studies, particularly in combination with PI-3 K pathway inhibitors. Defects of innate and adaptive immunity are common in glioblastoma patients contributing to a lack of effective anti-tumor responses. Thus, immunosuppressants such as CsA, rapamycin and its derivatives may be an effective novel strategy to treat drug-resistant gliomas or complement apoptosis based-therapies.

Abstract C230: Osteopontin as a driving force of glioma-related inflammation and a candidate for therapeutic targeting in malignant gliomas?

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.