M2-like macrophages induce colon cancer cell invasion via matrix metalloproteinases.

The inflammatory milieu plays an important role in colon cancer development and progression. Previously, we have shown that tumor-associated macrophages (TAMs), an important component of the tumor microenvironment, are enriched in tumors compared with normal tissue and confer a poorer prognosis. In the present study, we found that matrix metallopeptidase-9 (MMP-9), which degrades extracellular matrix proteins, was increased in biopsies from colon cancer patients and in mouse xenografts with SW480 cell-derived tumors. SW480 colon cancer cells exposed to M2-like macrophage-conditioned medium (M2-medium) exhibited increased MMP-9 mRNA, protein expression and gelatinase activity. A similar effect was obtained by the addition of tumor necrosis factor-α (TNFα) and leukotriene D4 (LTD4 ). MMP-9 expression and activity were reduced by a TNFα blocking antibody adalimumab and a cysteinyl leukotriene receptor 1 (CysLTR1, the receptor for LTD4 ) antagonist montelukast. M2-medium also induced changes in the epithelial-mesenchymal transition (EMT) markers E-cadherin, ß-catenin, vimentin, and snail in SW480 cells. We also found that both M2-medium and TNFα and LTD4 induced stabilization/nuclear translocation of ß-catenin. Furthermore, we also observed an elongated phenotype that may indicate increased invasiveness, as confirmed in a collagen I invasion assay. M2-medium increased the invasive ability, and a similar effect was also obtained by the addition of TNFα and LTD4 . The specific MMP inhibitor I or adalimumab and montelukast reduced the number of invasive cells. In conclusion, our findings show that M2-medium enriched in TNFα and LTD4 promote colon cancer cell invasion via MMP-9 expression and activation and the induction of EMT.

Membrane-type 1 metalloproteinase is upregulated in microglia/brain macrophages in neurodegenerative and neuroinflammatory diseases.

We previously reported that glioma cells induce the expression of membrane-type 1 metalloproteinase (MT1-MMP or MMP-14) in tumor-associated microglia/macrophages and promote tumor growth, whereas MMP-14 expression in microglia under physiological conditions is very low. Here, we show that the increase in MMP-14 expression is also found in microglia/macrophages associated with neurodegenerative and neuroinflammatory pathologies in mouse models as well as in human biopsies or post-mortem tissue. We found that microglial/macrophage MMP-14 expression was upregulated in Alzheimer's disease tissue, in active lesions of multiple sclerosis, and in tissue from stage II stroke as well as in the corresponding mouse models for the human diseases. In contrast, we observed no upregulation for MMP-14 in microglia/macrophages in the early phase of stroke or in the corresponding mouse model, in human amyotrophic lateral sclerosis (ALS) tissue or in a mouse model of ALS as well as in human cases of acute brain trauma. These data indicate that MMP-14 expression is not a general marker for activated microglia/macrophages but is upregulated in defined stages of neuroinflammatory and neurodegenerative diseases and that there is generally a good match between mouse models and human brain pathologies.

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.

Novel glioblastoma markers with diagnostic and prognostic value identified through transcriptome analysis.

PURPOSE: Current methods of classification of astrocytoma based on histopathologic methods are often subjective and less accurate. Although patients with glioblastoma have grave prognosis, significant variability in patient outcome is observed. Therefore, the aim of this study was to identify glioblastoma diagnostic and prognostic markers through microarray analysis. EXPERIMENTAL DESIGN: We carried out transcriptome analysis of 25 diffusely infiltrating astrocytoma samples [WHO grade II--diffuse astrocytoma, grade III--anaplastic astrocytoma, and grade IV--glioblastoma (GBM)] using cDNA microarrays containing 18,981 genes. Several of the markers identified were also validated by real-time reverse transcription quantitative PCR and immunohistochemical analysis on an independent set of tumor samples (n = 100). Survival analysis was carried out for two markers on another independent set of retrospective cases (n = 51). RESULTS: We identified several differentially regulated grade-specific genes. Independent validation by real-time reverse transcription quantitative PCR analysis found growth arrest and DNA-damage-inducible alpha (GADD45alpha) and follistatin-like 1 (FSTL1) to be up-regulated in most GBMs (both primary and secondary), whereas superoxide dismutase 2 and adipocyte enhancer binding protein 1 were up-regulated in the majority of primary GBM. Further, identification of the grade-specific expression of GADD45alpha and FSTL1 by immunohistochemical staining reinforced our findings. Analysis of retrospective GBM cases with known survival data revealed that cytoplasmic overexpression of GADD45alpha conferred better survival while the coexpression of FSTL1 with p53 was associated with poor survival. CONCLUSIONS: Our study reveals that GADD45alpha and FSTLI are GBM-specific whereas superoxide dismutase 2 and adipocyte enhancer binding protein 1 are primary GBM-specific diagnostic markers. Whereas GADD45alpha overexpression confers a favorable prognosis, FSTL1 overexpression is a hallmark of poor prognosis in GBM patients.

Upregulation of ASCL1 and inhibition of Notch signaling pathway characterize progressive astrocytoma.

Astrocytoma is the most common type of brain cancer constituting more than half of all brain tumors. With an aim to identify markers describing astrocytoma progression, we have carried out microarray analysis of astrocytoma samples of different grades using cDNA microarray containing 1152 cancer-specific genes. Data analysis identified several differentially regulated genes between normal brain tissue and astrocytoma as well as between grades II/III astrocytoma and glioblastoma multiforme (GBM; grade IV). We found several genes known to be involved in malignancy including Achaete-scute complex-like 1 (Drosophila) (ASCL1; Hash 1). As ASCL has been implicated in neuroendocrine, medullary thyroid and small-cell lung cancers, we chose to examine the role of ASCL1 in the astrocytoma development. Our data revealed that ASCL1 is overexpressed in progressive astrocytoma as evidenced by increased levels of ASCL1 transcripts in 85.71% (6/7) of grade II diffuse astrocytoma (DA), 90% (9/10) of grade III anaplastic astrocytoma (AA) and 87.5% (7/8) of secondary GBMs, while the majority of primary de novo GBMs expressed similar to or less than normal brain levels (66.67%; 8/12). ASCL1 upregulation in progressive astrocytoma is accompanied by inhibition of Notch signaling as seen by uninduced levels of HES1, a transcriptional target of Notch1, increased levels of HES6, a dominant-negative inhibitor of HES1-mediated repression of ASCL1, and increased levels of Notch ligand Delta1, which is capable of inhibiting Notch signaling by forming intracellular Notch ligand autonomous complexes. Our results imply that inhibition of Notch signaling may be an important early event in the development of grade II DA and subsequent progression to grade III AA and secondary GBM. Furthermore, ASCL1 appears to be a putative marker to distinguish primary GBM from secondary GBM.

Cysteinyl leukotrienes and their receptors: bridging inflammation and colorectal cancer.

Long-standing inflammation has emerged as a hallmark of neoplastic transformation of epithelial cells and may be a limiting factor of successful conventional tumor therapies. A complex milieu composed of distinct stromal and immune cells, soluble factors and inflammatory mediators plays a crucial role in supporting and promoting various types of cancers. An augmented inflammatory response can predispose a patient to colorectal cancer (CRC). Common risk factors associated with CRC development include diet and lifestyle, altered intestinal microbiota and commensals, and chronic inflammatory bowel diseases. Cysteinyl leukotrienes are potent inflammatory metabolites synthesized from arachidonic acid and have a broad range of functions involved in the etiology of various pathologies. This review discusses the important role of cysteinyl leukotriene signaling in linking inflammation and CRC.

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

BACKGROUND: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: Our results thus show that activation of TLR2 along with TLRs 1 and/or 6 converts microglia into a glioma supportive phenotype.