Glioma-derived Soluble Factors Research Articles

P52a: Multiplex analysis of 27 cytokines produced by low-grade and high-grade glioma cells

Glioma is the most common type of primary brain tumor. Despite aggressive therapy, glioma virtually always recurs. Soluble factors produced by glioma cells play an important role in progression of tumor growth. Glioma-derived soluble factors can stimulate tumor cell proliferation in autocrine and paracrine manners, affect the tumor microenvironment and promote angiogenesis and metastasis. Besides, glioma-derived cytokines are able to disturb antitumor immune response by inhibiting the functions of effector lymphocytes and inducing immunosuppressive cells. The aim of the present study was to determine the profile of cytokines produced by primary low-grade and high-grade glioma cell cultures. The study was held in 21 patients with brain tumors after receiving a written informed consent. There were 6 patients with histologically verified low-grade glioma (Grade II) and 15 patients – with high-grade glioma (Grade III–IV). Tumor tissues from patients were obtained during surgical resection. Cell suspensions were prepared by mechanical and enzymatic disaggregation followed by culturing in DMEM/F12 medium containing 10% FCS. The levels of 27 cytokines were measured using multiplex analysis (Bio-Rad, USA) in 7-days supernatants collected upon reaching the cellular subconfluence. Low-grade glioma cells produced low level (Me γ and IL-15 (Me 355 pg/ml and Me 57 pg/ml, respectively). The level of growth factors and cytokines, which act as regulators of hemo- and immunopoesis, was low in supernatants of low-grade glioma cultures. Median concentration of G-CSF, GM-CSF, IL-7, FGFb and PDGF was less than 25 pg/ml, but IL-9 concentration was in the middle range (Me 74 pg/ml) . High-grade glioma cultures produced, besides IL-9, also G-GSF (Me 263 pg/ml), FGFb (Me 60 pg/ml) and PDGF (Me 63 pg/ml). The level of FGFb and G-GSF correlated with the glioma grade ( R = 0.87, p = 0.002, and R = 0.66, p = 0.002, respectively). The IL-7 and GM-CSF level in high-grade culture supernatant was low (Me 500 pg/ml) of IL-6 (Me 1933 pg/ml and 3531 pg/ml, respectively) and VEGF (Me 1004 pg/ml and 14887 pg/ml, respectively). VEGF level correlated with glioma grade ( R = 0.86, p = 0.002). Concerning CC-chemokines the level of Eotaxin was low in low-grade glioma cultures (Me 11 pg/ml). The mild-level production was found for MIP-1α (Me 71 pg/ml) and RANTES (231 pg/ml), high-level – for MCP-1 (Me 9468 pg/ml) and MIP-1β (Me 990 pg/ml). High-grade glioma cells produced not only MIP-1α (Me 208 pg/ml) and RANTES (Me 311 pg/ml) in the middle range but also Eotaxin (Me 107 pg/ml). Eotaxin level correlated with the glioma grade ( R = 0.87, p = 0.002). High-grade glioma cells produced highlevel of MCP-1 (Me 9868 pg/ml) and MIP-1β (Me 1923 pg/ml). Both low-grade and high-grade glioma cells produced high level of CXC-chemokines IL-8 and IP-10 (Me >1000 pg/ml). The level of IL-8, which acts as chemoattractant for immune cells as well as for endothelial cells involved in angiogenesis, correlated with glioma grades ( R = 0.72, p = 0.002). Thus, the grade of gliomas associated with broadening the spectrum of cytokines and enhanced level of cytokines production by tumor cells.

P52: Glioma-derived soluble factors pose strong chemoattractants and partially change the cytotoxic activity of IFN-alpha-induced dendritic cells

Dendritic cells (DCs) are the most potent antigen-presenting cells, which play crucial role in initiation and maintenance of immune response. However, DCs of high-grade glioma patients are characterized by some functional impairment including reduced TNF α -mediated antitumor cytotoxic activity and low migratory activity. Tumor-produced soluble molecules can profoundly affect the differentiation and maturation of DCs and influence the functional activity of DCs. The aim of the present study was to determine the effect of supernatants of primary glioma cell lines on the migration and the cytotoxic activity of healthy donor DCs. The study included 11 healthy donors and 7 patients with histologically verified glioma Grade II (2 patients) and glioma Grade IV (5 patients). DCs were generated from peripheral blood monocytes of healthy donors in the presence of GM-CSF and IFN-alpha (IFN-DCs) followed by the addition of lipopolysaccharide (LPS Escherichia coli). Primary glioma cell cultures from tissue samples of studied patients were obtained by mechanical and enzymatic (0.3% collagenase IA) disaggregation followed by culturing in DMEM/F12 medium containing 10% FCS. 7-day supernatants were collected upon reaching the cellular subconfluence. Migratory capacity of donor IFN-DCs in response to supernatants (v/v 25%) of primary glioma cell cultures was measured in trans-well culture chamber. DCs that migrated to the bottom chamber with medium alone as a control or with supernatants were counted by FACS analysis using BD calibration beads. Cytotoxic activity of donor IFN-DCs against tumor cell line HEp-2 was studied using MTT-assay for 24 h at a ratio of DC: HEp-2 1:1. IFN-DCs were preincubated for 2 h with supernatants of primary glioma cell cultures. Migration index of donor IFN-DCs to lymphoid chemokine CCL19 (100 ng/ml) was an average of 6.5 ± 2.1. Migration capacity of IFN-DCs towards concentration gradient of primary glioma culture supernatants was significantly higher. Supernatants of low-grade glioma cultures as well as of high-grade glioma cultures effectively induced chemotaxis of IFN-DCs. Migration index in response to concentration gradient of primary glioma line-derived soluble factors (6 cell lines) ranged from 32.6 ± 9 to 48.4 ± 15.5. To assess the effect of supernatants of primary lines on the cytotoxic activity of IFN-DCs four primary high-grade glioma lines were used. The cytotoxicity of intact LPS-stimulated donor IFN-DCs against HEp-2 cells was an average of 34.8 ± 7.4%. Supernatant pretreating of IFN-DCs reduced the cytotoxic activity of DCs in 8 out of 16 experiments (with an average of 43%). In other 8 cases, supernatants of high-grade glioma lines, in contrast, possessed a mild stimulating activity and enhanced the cytotoxicity of DCs on average about of 34%. Thus, tumor cells in glioma patients produce chemoattractants capable of providing migration of DCs to tumor. At the same time glioma-derived soluble factors change the cytotoxic activity both ways. One can suppose thereupon that impairment of DC cytotoxic activity in glioma patients occurs not only due to tumor cell impact but because of unknown mechanisms.

Toll-like receptor 2 mediates microglia/brain macrophage MT1-MMP expression and glioma expansion

Glioblastomas are the most aggressive primary brain tumors in humans. Microglia/brain macrophage accumulation in and around the tumor correlates with malignancy and poor clinical prognosis of these tumors. We have previously shown that microglia promote glioma expansion through upregulation of membrane type 1 matrix metalloprotease (MT1-MMP). This upregulation depends on signaling via the Toll-like receptor (TLR) adaptor molecule myeloid differentiation primary response gene 88 (MyD88). Using in vitro, ex vivo, and in vivo techniques, we identified TLR2 as the main TLR controlling microglial MT1-MMP expression and promoting microglia-assisted glioma expansion. The implantation of mouse GL261 glioma cells into TLR2 knockout mice resulted in significantly smaller tumors, reduced MT1-MMP expression, and enhanced survival rates compared with wild-type control mice. Tumor expansion studied in organotypic brain slices depended on both parenchymal TLR2 expression and the presence of microglia. Glioma-derived soluble factors and synthetic TLR2 specific ligands induced MT1-MMP expression in microglia from wild-type mice, but no such change in MT1-MMP gene expression was observed in microglia from TLR2 knockout mice. We also found evidence that TLR1 and TLR6 cofunction with TLR2 as heterodimers in regulating MT1-MMP expression in vitro. Our results thus show that activation of TLR2 along with TLRs 1 and/or 6 converts microglia into a glioma supportive phenotype.

Treg Infiltration in Glioma: A Hurdle for Antiglioma Immunotherapy

Tregs play a crucial role in glioma-mediated immunosuppression; hence, tackling the Treg population in patients with malignant glioma could improve the clinical success rate of antiglioma immunotherapy. Therefore, it is of high importance to elucidate the mechanisms responsible for Treg recruitment and retention within the glioma microenvironment. The current paper demonstrates that, in addition to preferential chemoattraction, glioma-derived soluble factors can also induce preferential Treg proliferation and survival. These data identify new targets for Treg modulating strategies.

Induction of prostaglandin E2 synthesis and microsomal prostaglandin E synthase–1 expression in murine microglia by glioma-derived soluble factors

Object Microglia are one of the members of monocyte/macrophage lineage in the central nervous system (CNS) and exist as ramified microglia in a normal resting state, but they are activated by various stimuli, such as tumors. Activated microglia induce immune responses in the CNS, but the precise functions of microglia in glioma microenvironments are not clear. It has been reported that glioma cells produce prostaglandin (PG)E2, which promotes the growth of tumor cells and possesses immunosuppressive activity. The authors previously reported that PGE2 production by peritoneal macrophages was enhanced by glioma-derived soluble factors, which induce an immunosuppressive state. In this study, they investigated PGE2 production by microglia treated with glioma cells and assessed the role of microglia in glioma microenvironments in the mouse. Methods Microglia and peritoneal macrophages were cultured in vitro with or without lipopolysaccharide, and tumor necrosis factor (TNF) and PGE2 in the culture supernatant were measured using L929 bioassay and enzyme immunoassay. The expression of mRNA was measured using reverse transcriptase polymerase chain reaction, and the protein expression was assayed with Western blotting. In some experiments glioma cells and conditioned glioma medium were added to the microglia cultures. Results Glioma cells studied in this report did not produce a significant amount of PGE2. However, the coculture of microglia with glioma cells or conditioned glioma medium led to the production of a large amount of PGE2. The enhancement of PGE2 production by microglia was more significant than that by peritoneal macrophages. The expression of cyclooxygenase (COX)–2 and particularly the expression of microsomal PGE synthase (mPGES)–1 (a terminal enzyme of the arachidonate cascade) in microglia were enhanced by conditioned glioma medium. The enhancement of mPGES-1 expression in microglia was more significant than that in peritoneal macrophages. The production of TNF was suppressed when culturing microglia with conditioned glioma medium, but this suppression was abrogated by the addition of a COX inhibitor (NS-398) and a PGE2 receptor (EP4) antagonist. Furthermore, TNF production was not suppressed in microglia from mPGES-1–deficient mice. Conclusions These results indicate that PGE2 production by microglia is enhanced by conditioned glioma medium, which induces an immunosuppressive state in the CNS. Therefore, the manipulation of microglia, from the standpoint of PGE2, provides investigators with an important strategy to induce an effective antiglioma immune response.

The invasion promoting effect of microglia on glioblastoma cells is inhibited by cyclosporin A

The invasion of tumour cells into brain tissue is a pathologic hallmark of WHO grades II-IV gliomas and contributes significantly to the failure of current therapeutic treatments. Activated microglial cells are abundant in brain tumours and may support tumour invasiveness. We have previously demonstrated that cyclosporin A (CsA) can affect growth of glioma cells in vitro by inhibiting signalling pathways, which are essential for tumour proliferation and invasiveness. In this work, we demonstrate that migration of EGFP-transfected glioblastoma cells in organotypic brain slices was significantly inhibited by treatment with CsA. On average 77% of untreated cells migrated beyond 500 mum, while only 28-33% cells migrated as far in the brain slices treated with CsA (P < 0.001). This inhibitory effect on glioblastoma invasion was lost when glioblastoma cells were injected into microglia-depleted brain slices. Moreover, CsA significantly inhibits intracranial glioma growth in vivo. We demonstrate that microglia-derived factors increase glioma invasiveness in Matrigel assay in vitro and this is associated with activation of the PI-3K/Akt signalling pathway. The invasion promoting effect of microglia is abolished in the presence of CsA. Furthermore, glioma-derived soluble factors induce morphological transformation of microglia and activate MAPK signalling, although production of pro-inflammatory factors was not observed. Our findings that CsA interferes at clinically relevant concentrations with the tumour-promoting role of microglia and impairs invasive growth of glioma cells in vivo may provide a novel therapeutic strategy against gliomas.

Induction of macrophagic prostaglandin E2 synthesis by glioma cells

It has been reported that glioma cells produce prostaglandin (PG)E2, which promotes the growth of tumor cells and possesses immunosuppressive activity, and that cyclooxygenase (COX) inhibitors impede tumor growth and infiltration. Macrophages in tumor-bearing hosts are activated to produce PGE2, which induces an immunosuppressive state. Note, however, that the precise mechanism by which PGE2 induces an immunosuppressive state is still unclear. In this study, the authors investigated the mechanism of PGE2 production in glioma-bearing hosts. The human and murine glioma cells that were studied did not produce a significant amount of PGE2. However, the coculture of human peripheral blood mononuclear cells or murine peritoneal macrophages with glioma cells or conditioned glioma medium led to the production of a large amount of PGE2. In contrast, production of tumor necrosis factor and interleukin (IL)-12p70 by macrophages and cytotoxic T lymphocyte induction were suppressed by culturing with conditioned glioma medium; this suppression was abrogated by the addition of the COX inhibitor indomethacin. The macrophagic expression of COX-2, and particularly the expression of microsomal PGE synthase (mPGES)-1, a terminal enzyme of the arachidonate cascade, was enhanced by the glioma-derived soluble factors. Furthermore, IL-12p70 production was not clearly suppressed in macrophages from mPGES-1-deficient mice. The glioma-derived soluble factors were sensitive to treatment with heat and papain. These results indicated that PGE2 production by macrophages is enhanced by glioma-derived soluble factors, which induce an immunosuppressive state in glioma-bearing hosts. Therefore, the inhibition of PGE2 synthesis, targeting COX-2 and mPGES-1, is an effective treatment for the induction of antiglioma immune responses.

Enhancement of macrophage cytotoxicity against murine gliomas by interferon beta: increase in nitric oxide production in response to glioma-derived soluble factors.

In previous studies interferon-beta (IFNbeta) has been shown to suppress tumor growth. In this report, the antitumor effect of macrophages stimulated with IFNbeta is investigated in murine gliomas in vitro. The authors examined the cytotoxic activity of IFNbeta-stimulated peritoneal macrophages in glioma cells labeled with [3H]thymidine. The addition of IFNbeta enhanced cytotoxic activity in gliomas as well as the nitric oxide (NO) production of macrophages in cocultures. Addition of N(G)-monomethyl-L-arginine (L-NMMA) and L-N6-(1-iminoethyl)-lysine, but not D-NMMA (an inactive analog of L-NMMA), blocked this cytotoxic activity. The addition of IFNbeta had no direct effect on the growth of glioma cells. Because NO was not produced from macrophages treated with IFNbeta alone and IFNbeta-induced cytotoxic activity did not need cell-to-cell contact, the authors suspected that gliomas produce some soluble factors that act as cofactors for IFNbeta-induced cytotoxic activity. Macrophages stimulated with IFNbeta in the presence of glioma culture supernatants showed higher cytotoxicity against glioma cells than macrophages stimulated with IFNbeta alone. Furthermore, NO was markedly produced by IFNbeta-stimulated macrophages in the presence of glial culture supernatants. These data indicate that the antiglioma activity of IFNbeta through macrophages is due to NO produced by macrophages and that glioma-derived soluble factors play a role as an essential cofactor in this activity.

Malignant glioma-derived soluble factors regulate proliferation of normal adult human astrocytes.

Malignant gliomas are characteristically surrounded by marked gliosis. To assess whether glioma-derived products contribute to the proliferation of astrocytes, a feature of the gliosis response, we evaluated the influence of culture supernatants from malignant human glioma lines and tumor cyst fluids collected from two patients with glioblastoma multiforme on the proliferation of non-transformed adult human astrocytes. Both the culture supernatants and cyst fluids significantly increased DNA synthesis in astrocytes as assessed by a double immunofluorescence glial fibrillary acidic protein-bromodeoxyuridine technique. The net proliferative effect mediated by glioma cell line supernatants was tumor growth phase-dependent, being preferentially expressed during the logarithmic phase of glioma cell growth. Specific growth factor molecules and cytokines known to be secreted by gliomas (epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, transforming growth factor-beta, interleukin-6, and tumor necrosis factor-alpha) could not reproduce the mitogenic effects of the glioma-derived soluble factors. Cytokines which can induce DNA synthesis by adult human astrocytes in vitro, gamma-interferon and interleukin-1, were not detected in the culture supernatant of glioma lines used in this study. In conjunction with the documented effects of glioma products on endothelial and lymphoid cells, the current study suggests that soluble glioma products can contribute to the production of surrounding gliosis observed in vivo.