for highly malignant gliomas (World Health Organization grade III and IV) there is no successful treatment; patients have an average survival time of approximately 1 y after diagnosis. Glioma cells are highly invasive and infiltrate normal brain tissue, and as a result, surgical resection is always incomplete. Degradation of ECM by membrane-bound and secreted metalloproteases facilitates glioma invasion. In particular, the membrane-bound metalloproteases are pivotal for tumor invasion as they very efficiently digest extracellular matrix proteins and also activate secreted metalloproteases (1) like matrix metalloproteinase-2 (MMP-2, also known as gelatinase A), which is one of the major proteases involved in glioma invasion in mouse models (2) and probably also in humans (3). Hence, membrane-inserted metalloproteases like membrane type 1 matrix metalloproteinase (MT1-MMP) can enable gliomas to invade the brain parenchyma as single cells (4).Microglia are the intrinsic immune cells of the brain; they control the innate and the adaptive immune response in the CNS and are activated by inflammatory or other pathological stimuli (5). Activation of microglial toll-like receptors (TLRs) triggers the innate immune response and can initiate host-defense and tissue repair mechanisms, but also CNS inflammation, neurodegeneration, and trauma (5, 6). As microglial cells are attracted toward glioma in large numbers-glioma tissue consists of as much as 30% microglial cells-and because microglia density in gliomas positively correlates with malignancy, invasiveness, and grading of the tumors (7-9), we investigated if microglia may actively contribute to glioma expansion. Here, we show that soluble factors released from glioma stimulate microglial TLRs, resulting in microglial MT1-MMP expression via the TLR downstream signaling molecules MyD88 and p38 MAPK. In turn, MT1-MMP expression and activity in these immune cells promotes glioma cell invasion and tumor expansion. ResultsGlioma Associated Microglia Over-Express MT1-MMP. We analyzed the expression pattern of the matrix protease MT1-MMP in mouse and human gliomas and found the enzyme to be expressed predominantly in microglial cells closely associated with the tumors. Whereas tumor-free human brain samples showed virtually no MT1-MMP expression, we detected intense MT1-MMP labeling, especially in higher-grade gliomas. Importantly, in human samples, immunolabeling for the microglial marker Iba1 and for MT1-MMP largely overlapped [supporting information (SI) Fig. S1 A-D and Table S1]. Likewise, after injection of a human glioma cell line (U373 cells) into immunodeficient mice, we detected that microglia represent the predominant cell type contributing intratumoral MT1-MMP expression (see Fig. S1E).In our in vivo mouse model, the glioma cells were identified by stable expression of EGFP and microglial cells by immunolabeling for Iba1. In sections obtained from mice 2 weeks after intracerebral injection with isogenic glioma cells (GL261 cells), we found an increased density of mic...

Malignant glioma belong to the most aggressive neoplasms in humans with no successful treatment available. Patients suffering from glioblastoma multiforme (GBM), the highest-grade glioma, have an average survival time of only around one year after diagnosis. Both microglia and peripheral macrophages/monocytes accumulate within and around glioma, but fail to exert effective anti-tumor activity and even support tumor growth. Here we use microarray analysis to compare the expression profiles of glioma-associated microglia/macrophages and naive control cells. Samples were generated from CD11b+ MACS-isolated cells from naïve and GL261-implanted C57BL/6 mouse brains. Around 1000 genes were more than 2-fold up- or downregulated in glioma-associated microglia/macrophages when compared to control cells. A comparison with published data sets of M1, M2a,b,c-polarized macrophages revealed a gene expression pattern that has only partial overlap with any of the M1 or M2 gene expression patterns. Samples for the qRT-PCR validation of selected M1 and M2a,b,c-specific genes were generated from two different glioma mouse models and isolated by flow cytometry to distinguish between resident microglia and invading macrophages. We confirmed in both models the unique glioma-associated microglia/macrophage phenotype including a mixture of M1 and M2a,b,c-specific genes. To validate the expression of these genes in human we MACS-isolated CD11b+ microglia/macrophages from GBM, lower grade brain tumors and control specimens. Apart from the M1/M2 gene analysis, we demonstrate that the expression of Gpnmb and Spp1 is highly upregulated in both murine and human glioma-associated microglia/macrophages. High expression of these genes has been associated with poor prognosis in human GBM, as indicated by patient survival data linked to gene expression data. We also show that microglia/macrophages are the predominant source of these transcripts in murine and human GBM. Our findings provide new potential targets for future anti-glioma therapy.

Gliomas represent the most frequent type of human brain tumor, and their strong invasiveness is a significant clinical problem. Microglia, the immunocompetent cells of the brain, contribute significantly to the tumor and are potential interaction partners of the glioma cells. We studied the impact of the presence of microglia on tumor cell invasion in cultured brain slices. To selectively deplete microglia, the slices were treated with clodronate-filled liposomes. When glioma cells were injected into slices devoid of endogenous microglia, the invasiveness of the tumors was significantly decreased as compared with controls. Inoculation of exogenous microglia together with glioma cells into cultured brain slices increased the infiltrative behavior of the tumor depending on the microglia/glioma cell ratio. Cell culture experiments revealed that soluble factors released from glioma cells strongly stimulate metalloprotease-2 activity in microglia. In the brain slices inoculated with glioma cells, increased activity of metalloprotease-2 was directly correlated with the abundance of microglia. Our data indicate that glioma cells stimulate microglial cells to increase breakdown of extracellular matrix and thereby promote tumor invasiveness.

Primary astrocytomas of World Health Organization grade 3 and grade 4 (HG-astrocytomas) are preponderant among adults and are almost invariably fatal despite multimodal therapy. Here, we show that the juvenile brain has an endogenous defense mechanism against HG-astrocytomas. Neural precursor cells (NPCs) migrate to HG-astrocytomas, reduce glioma expansion and prolong survival by releasing a group of fatty acid ethanolamides that have agonistic activity on the vanilloid receptor (transient receptor potential vanilloid subfamily member-1; TRPV1). TRPV1 expression is higher in HG-astrocytomas than in tumor-free brain and TRPV1 stimulation triggers tumor cell death via the activating transcription factor-3 (ATF3) controlled branch of the ER stress pathway. The anti-tumorigenic response of NPCs is lost with aging. NPC-mediated tumor suppression can be mimicked in the adult brain by systemic administration of the synthetic vanilloid Arvanil, suggesting that TRPV1 agonists hold potential as new HG-astrocytoma therapeutics.