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

Tumour-derived CSF2/granulocyte macrophage colony stimulating factor controls myeloid cell accumulation and progression of gliomas

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

Crosstalk between Chemokine Receptor CXCR4 and Cannabinoid Receptor CB2 in Modulating Breast Cancer Growth and Invasion

Cannabinoids bind to cannabinoid receptors CB(1) and CB(2) and have been reported to possess anti-tumorigenic activity in various cancers. However, the mechanisms through which cannabinoids modulate tumor growth are not well known. In this study, we report that a synthetic non-psychoactive cannabinoid that specifically binds to cannabinoid receptor CB(2) may modulate breast tumor growth and metastasis by inhibiting signaling of the chemokine receptor CXCR4 and its ligand CXCL12. This signaling pathway has been shown to play an important role in regulating breast cancer progression and metastasis.We observed high expression of both CB(2) and CXCR4 receptors in breast cancer patient tissues by immunohistochemical analysis. We further found that CB(2)-specific agonist JWH-015 inhibits the CXCL12-induced chemotaxis and wound healing of MCF7 overexpressing CXCR4 (MCF7/CXCR4), highly metastatic clone of MDA-MB-231 (SCP2) and NT 2.5 cells (derived from MMTV-neu) by using chemotactic and wound healing assays. Elucidation of the molecular mechanisms using various biochemical techniques and confocal microscopy revealed that JWH-015 treatment inhibited CXCL12-induced P44/P42 ERK activation, cytoskeletal focal adhesion and stress fiber formation, which play a critical role in breast cancer invasion and metastasis. In addition, we have shown that JWH-015 significantly inhibits orthotopic tumor growth in syngenic mice in vivo using NT 2.5 cells. Furthermore, our studies have revealed that JWH-015 significantly inhibits phosphorylation of CXCR4 and its downstream signaling in vivo in orthotopic and spontaneous breast cancer MMTV-PyMT mouse model systems.This study provides novel insights into the crosstalk between CB(2) and CXCR4/CXCL12-signaling pathways in the modulation of breast tumor growth and metastasis. Furthermore, these studies indicate that CB(2) receptors could be used for developing innovative therapeutic strategies against breast cancer

Cannabinoid pharmacology in cancer research: A new hope for cancer patients?

Cannabinoids have been used for many centuries to ease pain and in the past decade, the endocannabinoid system has been implicated in a number of pathophysiological conditions, such as mood and anxiety disorders, movement disorders such as Parkinson's and Huntington's disease, neuropathic pain, multiple sclerosis, spinal cord injury, atherosclerosis, myocardial infarction, stroke, hypertension, glaucoma, obesity, and osteoporosis. Several studies have demonstrated that cannabinoids also have anti-cancer activity and as cannabinoids are usually well tolerated and do not produce the typical toxic effects of conventional chemotherapies, there is considerable merit in the development of cannabinoids as potential anticancer therapies. Whilst the presence of psychoactive effects of cannabinoids could prevent any progress in this field, recent studies have shown the value of the non-psychoactive components of cannabinoids in activating apoptotic pathways, inducing anti-proliferative and anti-angiogenic effects. The aforementioned effects are suggested to be through pathways such as ERK, Akt, mitogen-activated protein kinase (MAPK) pathways, phosphoinositide 3-kinase (PI3K) pathways and hypoxia inducible factor 1 (HIF1), all of which are important contributors to the hallmarks of cancer. Many important questions still remain unanswered or are poorly addressed thus necessitating further research at basic pre-clinical and clinical levels. In this review, we address these issues with a view to identifying the key challenges that future research needs to address

Integration of P2Y receptor-activated signal transduction pathways in G protein-dependent signalling networks

The role of nucleotides in intracellular energy provision and nucleic acid synthesis has been known for a long time. In the past decade, evidence has been presented that, in addition to these functions, nucleotides are also autocrine and paracrine messenger molecules that initiate and regulate a large number of biological processes. The actions of extracellular nucleotides are mediated by ionotropic P2X and metabotropic P2Y receptors, while hydrolysis by ecto-enzymes modulates the initial signal. An increasing number of studies have been performed to obtain information on the signal transduction pathways activated by nucleotide receptors. The development of specific and stable purinergic receptor agonists and antagonists with therapeutical potential largely contributed to the identification of receptors responsible for nucleotide-activated pathways. This article reviews the signal transduction pathways activated by P2Y receptors, the involved second messenger systems, GTPases and protein kinases, as well as recent findings concerning P2Y receptor signalling in C6 glioma cells. Besides vertical signal transduction, lateral cross-talks with pathways activated by other G protein-coupled receptors and growth factor receptors are discussed

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Malignant gliomas are fast-growing, heterogeneous and invasive brain tumors strongly infiltrated by non-tumor cells. Glioma attracts variety of immune cells, in particular microglia/macrophages and re-program these cells into immunosuppressive, tumor-supporting cells. Factors responsible for pro-invasive macrophage polarization and shaping tumor microenvironment in tumor-supporting manner are poorly known. We analyzed glioma secretome using proteomical approach and identified lactadherin (Mfge8) and osteopontin (Spp1) in microglia-activating fractions. Both osteopontin and lactadherin are αvβ3/αvβ5 integrin ligands able to interact with receptors present on microglia and macrophages and thus could be involved in pro-invasive polarization of microglia/macrophages. Moreover, both Spp1 and Mfge8 are overexpressed in glioma cells, but not in non-transformed astrocytes. C6 glioma cells stably expressing shRNA specific to lactadherin (shMfge8), osteopontin (shSpp1) and negative shRNA (shNeg) were implanted into striatum of Wistar rats. There was no difference in proliferation and viability of C6 glioma cells, cells stably expressing shRNA specific to lactadherin, ostopontin and negative shRNA in vitro, that demonstrates the negligible effect of autocrine production of both protein on tumor cell growth. Knockdown of Spp1 and Mfge8 resulted in significant reduction of tumor volume in rat model of glioma. Immunochemical analysis of brain sections revealed similar numbers of infiltrating microglia/macrophages (Iba1 staining), but the reduced number of ameboid, arginase 1 expressing cells in Mfge8 – depleted tumor. Treatment of endothelial cells with rhMFGE8 revealed significant effect of that protein on angiogenesis in vitro, however lactadherin-depleted tumors do not exhibit reduced blood vessel density in rat glioma model. FACS analysis showed that silencing of Spp1 does not affect total number of CD11b-positive cells, but strongly modulates microenvironment by leading to significant changes in percentage of Tc and Treg cells infiltrating tumor-bearing hemisphere. Our results suggest that glioma-derived integrin ligands are important factor in polarization of glioma infiltrating microglia/macrophages into the pro-invasive phenotype and its targeting could be a new therapeutic strategy

Identification of new scaffolds with anti-tumor action toward human glioblastoma cells

Glioblastoma (GBM), a malignant brain tumor, is one of the most aggressive human neoplasms due to its molecular heterogeneity, diffusive growth and multiple mechanisms of resistance to chemotherapeutics. Frequent dysfunctions of tumor suppressors and oncogenes, which lead to overactivation of prosurvival and antiapoptotic signals, are driven by genetics as well as epigenetic abnormalities. The epigenetic mechanisms are de-regulated in gliomas as a result of an aberrant activation or inactivation of enzymes maintaining and modifying the epigenome, such as histone deacetyltranferases (HDAC). Herein, we demonstrate the effects of new compounds from three structurally and functionally unrelated groups on two human glioblastoma cell lines, which are highly resistant to radiation and a majority of anticancer drugs. The first group was comprised of several potential HDAC inhibitors bearing a 4-vinylbiphenyl skeleton incorporating both hydroxyamic and non-hydroxyamic head groups, and the other two groups of compounds contain a 3-arylidene-oxindole scaffold and an isothiazolonaphthoquinone core, respectively. The results demonstrate the promising antitumor efficacy of some of the compounds and provides insight into molecular mechanisms of their cytotoxic action. The compounds containing an isothiazolonaphthoquinone core and HDAC inhibitors with an indolyl-substituted biphenyl-4-yl-acrylohydroxamic acid showed the highest efficacy against tumor cells. The observed proapoptotic effect of certain potential HDAC inhibitors on glioma cells provides a new therapeutic strategy based on the direct modulation of glioma growth using a specific HDAC inhibitor. These new and effective scaffolds can be considered as promising candidates for further investigation