Caspase signalling controls microglia activation and neurotoxicity.

Activation of microglia and inflammation-mediated neurotoxicity are suggested to play a decisive role in the pathogenesis of several neurodegenerative disorders. Activated microglia release pro-inflammatory factors that may be neurotoxic. Here we show that the orderly activation of caspase-8 and caspase-3/7, known executioners of apoptotic cell death, regulate microglia activation through a protein kinase C (PKC)-δ-dependent pathway. We find that stimulation of microglia with various inflammogens activates caspase-8 and caspase-3/7 in microglia without triggering cell death in vitro and in vivo. Knockdown or chemical inhibition of each of these caspases hindered microglia activation and consequently reduced neurotoxicity. We observe that these caspases are activated in microglia in the ventral mesencephalon of Parkinson's disease (PD) and the frontal cortex of individuals with Alzheimer's disease (AD). Taken together, we show that caspase-8 and caspase-3/7 are involved in regulating microglia activation. We conclude that inhibition of these caspases could be neuroprotective by targeting the microglia rather than the neurons themselves.

Aberrant immunostaining pattern of the CD24 glycoprotein in clinical samples and experimental models of pediatric medulloblastomas.

The CD24 glycoprotein is a mediator of neuronal proliferation, differentiation and immune suppression in the normal CNS, and a proposed cancer biomarker in multiple peripheral tumor types. We performed a comparative analysis of CD24 gene expression in a large cohort of pediatric and adult brain tumors (n = 813), and further characterized protein expression in tissue sections (n = 39), primary brain tumor cultures (n = 12) and a novel orthotopic group 3 medulloblastoma xenograft model. Increased CD24 gene expression was demonstrated in ependymomas, medulloblastomas, anaplastic astrocytomas and glioblastomas, although medulloblastomas displayed higher expression than all other tumor entities. Preferential expression of CD24 in medulloblastomas was confirmed at protein level by immunostaining and computerized image analysis of cryosections. Morphologies and immunophenotyping of CD24(+) cells in tissue sections tentatively suggested disparate functions in different tumor subsets. Notably, protein staining of medulloblastoma cells was associated with prominent cytoplasmic and membranous granules, enabling rapid and robust identification of medulloblastoma cells in clinical tissue samples, as well as in experimental model systems. In conclusion, our results implicate CD24 as a clinically and experimentally useful medulloblastoma immunomarker. Although our results encourage further functional studies of CD24 as a potential molecular target in subsets of brain tumors, the promiscuous expression of CD24 in vivo highlights the importance of specificity in the future design of such targeted treatment.

Phagocytic properties in tumor astrocytes.

In glioblastoma multiforme (GBM), the pathophysiological events preceding and promoting an uncontrolled and remarkable growth is largely unknown. Studies on gliomas and macrophage expression have shown high levels of phagocytic cells, that is, microglial cells. It has also been demonstrated that human astrocytic cells and rat glioma cells are capable of phagocytosis. The purpose of this study was to investigate a potential phagocytic property in human GBM cells in tumor biopsies from surgery. With an immunhistochemical double staining using macrophage markers (CD68 and CD163) and human telomerase reverse transcriptase (hTERT) as a marker for neoplastic cells, we found high levels of double positive cells in human GBM. In hematoxylin-erythrosin stained sections, we also identified fragmented cell components in the cytoplasm of tumor cells. In our judgement, many neoplastic cells in GBM are also positive for macrophage markers. We suggest that human astroglial tumor cells may have phagocytic properties or phagocyte-like properties. This may represent a latent capacity of self-defence, evoked under certain circumstances. It is likely that these properties substantially help the tumors thrive and expand.

Standardizing evaluation of sarcoma proliferation- higher Ki-67 expression in the tumor periphery than the center.

Soft tissue sarcomas often present as large and histopathologically heterogenous tumors. Proliferation has repeatedly been identified as a prognostic factor and immunostaining for Ki-67 represents the most commonly used proliferation marker. There is, however, a lack of consensus on how to evaluate Ki-67 staining regarding optimal cut-off levels, selection of tumor areas, and the number of tumor cells to evaluate. We assessed the impact of targeting peripheral versus central tumor areas using tissue microarray-based staining for Ki-67 throughout the tumor diameter in 25 leiomyosarcomas. In 18/25 tumors, Ki-67 expression was higher in the tumor periphery. If 10% staining tumor nuclei was used as cut-off and the maximal Ki-67 staining section in the tumor periphery was considered, 21/25 tumors would have been classified as highly proliferative compared to 14/25 if the tumor center had been analyzed. Similar results were obtained also when higher cut-off levels were used and if the mean expression rather than the maximal expression was considered and the differences were neither caused by necrosis nor by hypoxia (assessed as HIF-1alpha expression). Our findings suggest that the determination of proliferation in soft tissue sarcomas should be standardized for clinical application of Ki-67 as a prognostic marker.

Spatio-temporal activation of caspase-8 in myeloid cells upon ischemic stroke.

Ischemic stroke (caused by thrombosis, embolism or vasoconstriction) lead to the recruitment and activation of immune cells including resident microglia and infiltrating peripheral macrophages, which contribute to an inflammatory response involved in regulation of the neuronal damage. We showed earlier that upon pro-inflammatory stimuli, the orderly activation of caspase-8 and caspase-3/7 regulates microglia activation through a protein kinase C-δ dependent pathway. Here, we present in vivo evidence for the activation of caspase-8 and caspase-3 in microglia/macrophages in post-mortem tissue from human ischemic stroke subjects. Indeed, CD68-positive microglia/macrophages in the ischemic peri-infarct area exhibited significant expression of the cleaved and active form of caspase-8 and caspase-3. The temporal and spatial activation of caspase-8 was further investigated in a permanent middle cerebral artery occlusion mouse model of ischemic stroke. Increasing levels of active caspase-8 was found in Iba1-positive cells over time in the peri-infarct area, at 6, 24 and 48 h after artery occlusion. Analysis of post-mortem brain tissue from human subject who suffered two stroke events, referred as recent and old stroke, revealed that expression of cleaved caspase-8 and -3 in CD68-positive cells could only be found in the recent stroke area. Analysis of cleaved caspase-8 and -3 expressions in a panel of human stroke cases arranged upon days-after stroke and age-matched controls suggested that the expression of these caspases correlated with the time of onset of stroke. Collectively, these data illustrate the temporal and spatial activation of caspase-8 and -3 in microglia/macrophages occurring upon ischemic stroke and suggest that the expression of these caspases could be used in neuropathological diagnostic work.

Improved prognostication in soft tissue sarcoma: independent information from vascular invasion, necrosis, growth pattern, and immunostaining using whole-tumor sections and tissue microarrays.

In 140 mixed primary soft tissue sarcomas with a median follow-up of 6 years, the prognostic importance of tumor size, tumor depth, grade, necrosis, vascular invasion, and peripheral growth pattern (pushing versus infiltrating) was evaluated on whole-tumor sections. Immunohistochemical expression of Ki-67, p53, cyclin A, bcl-2, beta-catenin, CD44, and P-glycoprotein was determined using tissue microarray from the peripheral growth zone. Local recurrences developed in 17% of the patients and correlated with necrosis, vascular invasion, and cyclin A expression. No local recurrence developed in tumors with a pushing growth pattern, regardless of tumor grade and depth. Metastasis developed in 39% of the patients. Vascular invasion was identified in 36% of the tumors and was the strongest prognostic factor for metastasis with a hazard ratio of 3.5. Growth pattern and tumor necrosis were also strong prognostic factors for metastasis, whereas malignancy grade, tumor size, and tumor depth did not have any independent prognostic value. Immunostaining showed independent prognostic information for Ki-67, beta-catenin, CD44, and P-glycoprotein. The results indicate that whole-tumor sections could facilitate identification of vascular invasion, necrosis, and peripheral growth pattern and that immunohistochemical profiling from the growth zone also provides independent prognostic information for metastasis in soft tissue sarcoma.

Microglia-Secreted Galectin-3 Acts as a Toll-like Receptor 4 Ligand and Contributes to Microglial Activation.

Inflammatory response induced by microglia plays a critical role in the demise of neuronal populations in neuroinflammatory diseases. Although the role of toll-like receptor 4 (TLR4) in microglia's inflammatory response is fully acknowledged, little is known about endogenous ligands that trigger TLR4 activation. Here, we report that galectin-3 (Gal3) released by microglia acts as an endogenous paracrine TLR4 ligand. Gal3-TLR4 interaction was further confirmed in a murine neuroinflammatory model (intranigral lipopolysaccharide [LPS] injection) and in human stroke subjects. Depletion of Gal3 exerted neuroprotective and anti-inflammatory effects following global brain ischemia and in the neuroinflammatory LPS model. These results suggest that Gal3-dependent-TLR4 activation could contribute to sustained microglia activation, prolonging the inflammatory response in the brain.

Expression profiling using tissue microarray in 211 malignant fibrous histiocytomas confirms the prognostic value of Ki-67.

The tissue microarray technology is a high-throughput technique that allows studies of multiple markers in large tumor materials. We performed immunohistochemical profiling using tissue microarray and immunostaining for Ki-67, p53, bcl-2, CD44, cyclin A and Pgp in a series of 211 malignant fibrous histiocytomas (MFHs) with correlation to prognosis. Tissue from 50 local recurrences and 20 metastases was available for comparison with the primary tumors. In univariate analysis, Ki-67 was the only immunohistochemical marker significantly correlated with metastasis with a hazard ratio of 1.9. Multivariate analysis, with tumor size, depth, necrosis, vascular invasion, mitotic rate and Ki-67 expression, revealed an independent prognostic value of tumor size and Ki-67. Local recurrences did not differ from the corresponding primary tumors, whereas metastases showed a trend for upregulation of cyclin A and Pgp. In this large series of MFHs, a tumor size greater than 8 cm and a Ki-67 index of more than 20% were strong and independent prognostic factors for metastasis. In contrast, p53, bcl-2, CD44, cyclin A and Pgp, which have previously been suggested as prognostic factors in soft tissue sarcomas, did not show such correlations. Hence, we suggest that proliferation, as measured by Ki-67 index, should be considered as a prognostic marker in clinical management of pleomorphic soft tissue sarcomas.

Glioma cell proliferation controlled by ERK activity-dependent surface expression of PDGFRA.

Increased PDGFRA signaling is an essential pathogenic factor in many subtypes of gliomas. In this context the cell surface expression of PDGFRA is an important determinant of ligand sensing in the glioma microenvironment. However, the regulation of spatial distribution of PDGFRA in glioma cells remains poorly characterized. Here, we report that cell surface PDGFRA expression in gliomas is negatively regulated by an ERK-dependent mechanism, resulting in reduced proliferation of glioma cells. Glioma tumor tissues and their corresponding cell lines were isolated from 14 patients and analyzed by single-cell imaging and flow cytometry. In both cell lines and their corresponding tumor samples, glioma cell proliferation correlated with the extent of surface expression of PDGFRA. High levels of surface PDGFRA also correlated to high tubulin expression in glioma tumor tissue in vivo. In glioma cell lines, surface PDGFRA declined following treatment with inhibitors of tubulin, actin and dynamin. Screening of a panel of small molecule compounds identified the MEK inhibitor U0126 as a potent inhibitor of surface PDGFRA expression. Importantly, U0126 inhibited surface expression in a reversible, dose- and time-dependent manner, without affecting general PDGFRA expression. Treatment with U0126 resulted in reduced co-localization between PDGFRA and intracellular trafficking molecules e.g. clathrin, RAB11 and early endosomal antigen-1, in parallel with enhanced co-localization between PDGFRA and the Golgi cisternae maker, Giantin, suggesting a deviation of PDGFRA from the endosomal trafficking and recycling compartment, to the Golgi network. Furthermore, U0126 treatment in glioma cells induced an initial inhibition of ERK1/2 phosphorylation, followed by up-regulated ERK1/2 phosphorylation concomitant with diminished surface expression of PDGFRA. Finally, down-regulation of surface PDGFRA expression by U0126 is concordant with reduced glioma cell proliferation. These findings suggest that manipulation of spatial expression of PDGFRA can potentially be used to combat gliomas.

Better prognosis of patients with glioma expressing FGF2-dependent PDGFRA irrespective of morphological diagnosis.

Signaling of platelet derived growth factor receptor alpha (PDGFRA) is critically involved in the development of gliomas. However, the clinical relevance of PDGFRA expression in glioma subtypes and the mechanisms of PDGFRA expression in gliomas have been controversial. Under the supervision of morphological diagnosis, analysis of the GSE16011 and the Repository of Molecular Brain Neoplasia Data (Rembrandt) set revealed enriched PDGFRA expression in low-grade gliomas. However, gliomas with the top 25% of PDGFRA expression levels contained nearly all morphological subtypes, which was associated with frequent IDH1 mutation, 1p LOH, 19q LOH, less EGFR amplification, younger age at disease onset and better survival compared to those gliomas with lower levels of PDGFRA expression. SNP analysis in Rembrandt data set and FISH analysis in eleven low passage glioma cell lines showed infrequent amplification of PDGFRA. Using in vitro culture of these low passage glioma cells, we tested the hypothesis of gliogenic factor dependent expression of PDGFRA in glioma cells. Fibroblast growth factor 2 (FGF2) was able to maintain PDGFRA expression in glioma cells. FGF2 also induced PDGFRA expression in glioma cells with low or non-detectable PDGFRA expression. FGF2-dependent maintenance of PDGFRA expression was concordant with the maintenance of a subset of gliogenic genes and higher rates of cell proliferation. Further, concordant expression patterns of FGF2 and PDGFRA were detected in glioma samples by immunohistochemical staining. Our findings suggest a role of FGF2 in regulating PDGFRA expression in the subset of gliomas with younger age at disease onset and longer patient survival regardless of their morphological diagnosis.

The glioma cell edge--winning by engulfing the enemy?

Malignant glioma and glioblastoma multiforme form the largest group of highly malignant brain tumours, for which there is yet no definitive cure. Different approaches to treatment have been tried, in vain or with minimal benefit for the patient. In addition to surgery, radiation and chemotherapy, immunotherapy aiming at evoking an inflammatory reaction against the tumour itself has been tried. Immunotherapy has shown good results in an experimental mouse model, but no convincing efficacy/success in patients. Why are the gliomas always winning, how do they take the lead? The following phenomena lead us to propose an hypothesis about the reason for the glioma lead: the reported findings of phagocytic activity in reactive and neoplastic astrocytes in animal models and humans; the frequently observed ingested "non-self material"/debris in glioma cells; the markedly high contents of tumour cells with phagocytic phenotype in gliomas and the signs of only limited and temporary inflammatory reactions in different immunotherapy attempts. Whether it being a true phagocytosis, an engulfing or comparable activity by the glioma cells, contributing to the tumour's self defense against e.g. antitumoural therapies, it should be beneficial to attempt hampering these self defense properties e.g. by blocking their engulfing capacity.

Glial progenitor-like phenotype in low-grade glioma and enhanced CD133-expression and neuronal lineage differentiation potential in high-grade glioma.

BACKGROUND: While neurosphere- as well as xenograft tumor-initiating cells have been identified in gliomas, the resemblance between glioma cells and neural stem/progenitor cells as well as the prognostic value of stem/progenitor cell marker expression in glioma are poorly clarified. METHODOLOGY/PRINCIPAL FINDINGS: Viable glioma cells were characterized for surface marker expression along the glial genesis hierarchy. Six low-grade and 17 high-grade glioma specimens were flow-cytometrically analyzed for markers characteristics of stem cells (CD133); glial progenitors (PDGFRalpha, A2B5, O4, and CD44); and late oligodendrocyte progenitors (O1). In parallel, the expression of glial fibrillary acidic protein (GFAP), synaptophysin and neuron-specific enolase (NSE) was immunohistochemically analyzed in fixed tissue specimens. Irrespective of the grade and morphological diagnosis of gliomas, glioma cells concomitantly expressed PDGFRalpha, A2B5, O4, CD44 and GFAP. In contrast, O1 was weakly expressed in all low-grade and the majority of high-grade glioma specimens analyzed. Co-expression of neuronal markers was observed in all high-grade, but not low-grade, glioma specimens analyzed. The rare CD133 expressing cells in low-grade glioma specimens typically co-expressed vessel endothelial marker CD31. In contrast, distinct CD133 expression profiles in up to 90% of CD45-negative glioma cells were observed in 12 of the 17 high-grade glioma specimens and the majority of these CD133 expressing cells were CD31 negative. The CD133 expression correlates inversely with length of patient survival. Surprisingly, cytogenetic analysis showed that gliomas contained normal and abnormal cell karyotypes with hitherto indistinguishable phenotype. CONCLUSIONS/SIGNIFICANCE: This study constitutes an important step towards clarification of lineage commitment and differentiation blockage of glioma cells. Our data suggest that glioma cells may resemble expansion of glial lineage progenitor cells with compromised differentiation capacity downstream of A2B5 and O4 expression. The concurrent expression of neuronal markers demonstrates that high-grade glioma cells are endowed with multi-lineage differentiation potential in vivo. Importantly, enhanced CD133 expression marks a poor prognosis in gliomas.

Concurrent gain of 17q and the MYC oncogene in a medullomyoblastoma.

Medullomyoblastoma (MMB) is a rare cerebellar childhood tumor containing both myoblastic and primitive neuroectodermal components. Similar to the scenario in classical medulloblastoma, which contains the primitive neuroectodermal component only, gain of sequences from the long arm of chromosome 17 (17q) and gain of the MYC gene in 8q have been implicated in the pathogenesis of MMB. Because karyotypic analysis has not previously been performed for MMB, the mechanisms behind genomic imbalances in this tumor have remained unknown. Several other central aspects of this tumor, such as histocytogenetic origin, clinical characteristics, tumor behavior and prognosis, also remain unknown. We here report neuropathological and cytogenetic features of an MMB in a 3-year-old boy. Chromosome banding analysis and multicolor karyotyping revealed a hyperdiploid karyotype including an unbalanced 1; 17 translocation and isochromosome 17q formation, both leading to gain of 17q. There were also two extra copies of chromosome 8, leading to gain of the MYC oncogene, trisomies 5 and 13, and monosomy 9. Clonal chromosome changes were present in both the myoblastic and neuroectodermal components. Our findings support the notion that MMB and classical medulloblastoma arise through similar genetic mechanisms and that the two main tissue components in MMB are clonally related.

Neuroblastomas and medulloblastomas exhibit more Coxsackie adenovirus receptor expression than gliomas and other brain tumors.

Adenoviral vector-mediated treatment is a potential therapy for tumors of the central nervous system. To obtain a significant therapeutic effect by adenoviral vectors, a sufficient infection is required, the power of which depends predominantly on the level of Coxsackie adenovirus receptors. We stained surgical biopsies of central nervous system tumors and neuroblastomas for Coxsackie adenovirus receptors. For gliomas, the level of the receptor was low and markedly variable among individual tumors. By contrast, neuroblastomas and medulloblastomas exhibited a higher degree of Coxsackie adenovirus receptor expression than gliomas and other brain tumors. We conclude that neuroblastomas and medulloblastomas could be suitable for adenovirus-mediated gene therapy. Adverse effects of the treatment, however, must be considered because neurons and reactive astrocytes also express a significant amount of the receptor.

Cell type- and region-dependent coxsackie adenovirus receptor expression in the central nervous system.

Model systems have shown that adenoviral vector mediated transient gene expression can potentially be applied for the treatment of brain tumours, neurodegenerative diseases and brain injuries. Most studies utilized adenovirus serotype 5 (Ad5) based vectors, which as adhesion molecules require the coxsackie adenovirus receptor (CAR) as a critical determinant for cellular infection. In this report, we have systematically characterized CAR expression in the adult human central nervous system (CNS) by using immunohistochemistry. A total of 85 specimens from various CNS regions were investigated for CAR expression in a cell type-dependent context. The most marked staining positivity was found in the choroid plexus and the pituitary gland. The neocortex had scattered positive neurons, while the white matter was mainly negative. We need to consider the possible adverse effects and the possible damage caused by adenoviral gene therapy if the virus-vector also binds to normal brain cells.

Brain tissue microarrays in dementia research: white matter microvascular pathology in Alzheimer's disease.

Tissue microarrays (TMA) consist of up to 1000 cylindrical tissue cores from different donor paraffin blocks relocated into one recipient block, allowing for efficient histopathological studies by fluorescence in situ hybridization, RNA in situ hybridization or immunohistochemistry. On the background of the increasing interest of the TMA technique in cancer research and the suggestion of its application also in studies of non-neoplastic intracranial disorders, the technique was applied to pathologic white matter in AD brains. Eight cases with AD and concomitant white matter pathology were neuropathologically diagnosed on whole brain coronal slides. The TMA technique was used to grade severity of white matter pathology and to quantify small vessels with traditional staining and immunohistochemical markers. These measurements were compared with the whole brain neuropathological assessment. The technique produced good results with preserved tissue structures as confirmed by the whole brain evaluation. Severity of white matter pathology evaluated on the TMA cores correlated negatively with small vessel quantities, and statistically significant differences in vessel quantities paralleled different grades of white matter pathology. It is concluded that the TMA technique could be further utilized in studies of dementing disorders, and may have its advantages in large, clinically well-characterized materials (e.g. in quantitative mapping of white matter changes).

Immunohistochemical patterns in rectal cancer: application of tissue microarray with prognostic correlations.

We utilized the high-throughput tissue microarray method to characterize immunohistochemical expression patterns with correlations to prognosis in rectal cancer. Immunostaining for the markers Ki-67, Bcl-2, p53, EGFR, E-cadherin, beta-catenin, MLH1 and MSH2 was performed in 269 rectal cancers. Expression profiles were correlated to metastasis-free survival. Immunostaining revealed frequent upregulation and/or aberrant staining patterns for several of the markers, but Ki-67, p53, Bcl-2 and EGFR did not show any correlation to prognosis. However, reduced membranous staining for beta-catenin (p = 0.04), lack of cytoplasmic staining for beta-catenin (p = 0.04), reduced membranous staining for E-cadherin (p = 0.02) and lack of cytoplasmic staining for E-cadherin (p = 0.02) correlated with metastatic disease. Multivariate analysis including the factors Dukes' stage and tumor differentiation grade demonstrated increased risk of metastatic disease in tumors with lack of cytoplasmic staining for beta-catenin (HR = 3.1, p = 0.02), reduced membranous staining for beta-catenin (HR = 1.7, p = 0.06) and reduced membranous staining for E-cadherin (HR = 2.1, p = 0.06). Loss of MMR protein expression was confirmed to be a rare event in rectal cancer with loss of MLH1 staining in 3% and MSH2 in 1% of the tumors. The lack of prognostic information contributed by most of these markers suggests that single markers for prognosis may be of limited value in rectal cancer. However, altered expression of beta-catenin and E-cadherin correlated with metastatic disease, and these markers may have prognostic importance in rectal cancer.

Immunohistochemical Loss of the DNA Mismatch Repair Proteins MSH2 and MSH6 in Malignant Fibrous Histiocytomas.

PURPOSE: Soft tissue sarcomas (STS) account for less than 1% of all malignancies and constitute a heterogeneous tumor entity in which malignant fibrous histiocytomas (MFH) represent one-third and are characterized by a lack of type-specific differentiation. A defective mismatch repair (MMR) system cause the familial cancer syndrome hereditary non-polyposis colorectal cancer (HNPCC), and since occasional MFH have been described in HNPCC patients we assessed the contribution of defective MMR to the development of MFH. METHODS: MMR status was characterized in a series of 209 histopathologically reviewed MFH. Tissue microarray sections from the tumors were immunohistochemically stained for the MMR proteins MLH1, MSH2 and MSH6, and cases with aberrant staining were further characterized for microsatellite instability. RESULTS AND DISCUSSION: Two of the 209 STS-a storiform-pleomorphic MFH and a myxofibrosarcoma-showed concomitant loss of MSH2 and MSH6, but retained staining for MLH1 on both cases. The myxoid tumor also had a microsatellite unstable phenotype. These findings, together with previous observations of defective MMR in pleomorphic STS, indicate that these tumors may be part of the HNPCC-associated tumor spectrum and demonstrate that MMR defects occur in a small subset of STS.