Tumour tissue is infiltrated by myeloid cells that are reprogrammed into alternatively activated/regenerative (M2) macrophages. The contribution of major signalling pathways and their ...modulators/targets involved in the macrophage reprogramming is poorly known. Glioblastoma (malignant brain tumour) attracts and reprograms brain-resident microglia and peripheral macrophages into cells that increase invasion, angiogenesis and suppress antitumour immunity. Using a 'function-first' approach and glioma secretome proteomics we identified osteopontin and lactadherin as proteins that cooperatively activate amoeboid transformation, phagocytosis and motility of primary microglia cultures via integrins and FAK-Akt (focal adhesion kinase-Akt) signalling. A synthetic peptide interfering with integrin ligands blocks glioma-microglia communication, functional activation and M2 gene expression. We found that osteopontin/secreted phosphoprotein 1 (Spp1) produced by non-transformed cells acts as a proinflammatory factor inducing inflammatory signalling and M1 genes, and counteracts the action of lactadherin. Using constructs encoding functional mutants of osteopontin, we demonstrated sequential processing of Spp1 by thrombin and matrix metalloproteinase-3 and/or -7 (MMP-3 and/or -7) in glioma cells, which generates a microglia-activating form devoid of the inflammatory activity, while retaining the M2 reprogramming potential. A similar form of osteopontin is secreted by human glioma cells but not normal human astrocytes. Knockdown of osteopontin or lactadherin in glioma cells reduces intracranial glioma growth, blocks amoeboid transformation of myeloid cells and affects M2 reprogramming of microglia/macrophages. Our findings demonstrate how glioma cells misuse macrophage-activating signals and redesign primarily proinflammatory signals towards their advantage to induce M2 reprogramming of tumour-infiltrating brain macrophages.
P142 Pszczółkowska, D; Ellert-Miklaszewska, A; Gieryng, A ...
European journal of cancer supplements,
11/2015, Letnik:
13, Številka:
1
Journal Article
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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 αvβ 3/ αvβ 5 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.
Monitoring of bioprocesses and thus observation and identification of such processes is one of the main aims of bioprocess engineering. It is of vital importance in bioprocess development to improve ...the overall productivity by avoiding unintentional limitations to ensure not only optimal process conditions but also the observation of established production processes. Furthermore, reproducibility needs to be improved and final product quality and quantity be guaranteed. Therefore, an advanced monitoring and control system has been developed, which is based on different in‐line, on‐line and at‐line measurements for substrates and products. Observation of cell viability applying in‐line radio frequency impedance measurement and on‐line determination of intracellular recombinant target protein using the reporter protein T‐Sapphire GFP based on in‐line fluorescence measurement show the ability for the detection of critical process states. In this way, the possibility for the on‐line recognition of optimal harvest times arises and disturbances in the scheduled process route can be perceived.
An advanced monitoring and control system was developed which is based on different in‐line, on‐line and at‐line measurements for substrates and products. Using the information achieved in this paper, fully automated processes can be established and process strategies can be developed to obtain a higher productivity.
Cannabinoids were shown to induce apoptosis of glioma cells in vitro and tumor regression in vivo, but mechanisms of their antiproliferative action remain elusive. In the present studies, C6 cells ...were exposed to a synthetic cannabinoid, WIN 55,212-2, which produced down-regulation of the Akt and Erk signalling pathways prior to appearance of any sign of apoptosis. We hypothesized that cannabinoid-induced cell death may be mediated by a Bcl-2 family member—Bad, whose function is hampered by these kinases due to control of its phosphorylation state. Using Western blot analysis, we found that levels of phosphorylated Bad, but not total Bad protein, decreased under exposure to WIN 55,212-2. WIN 55,212-2 treatment further resulted in mitochondrial depolarization and activation of caspase cascade. Thus, we suggest that the increase of proapoptotic Bad activity is an important link between the inhibition of survival pathways and an onset of execution phase of cannabinoid-induced glioma cell death.
A sophisticated measurement system for at‐line determination of the main C‐source glucose, the by‐product acetate, and the N‐source ammonium for high cell density cultivations (HCDC) of Escherichia ...coli K12 TG1 is presented. One flow diffusion technique (FDA) system is used for glucose measurement in the range of 0.5 up to 40 gL–1 in the cultivation broth. Another FDA system detects the amount of the undesired by‐product acetate. The ammonium concentration in the range of 0.2 to 2.5 gL–1 is determined on‐line by a flow injection analysis (FIA) system. For verification purposes, an HPLC system which is also connected to the bioreactor for at‐line measurements is utilized. Several HCDC with cell densities of more than 100 gL–1 have been carried out. The courses of growth‐determining substrates have been detected at‐line. All used systems have shown an excellent compliance with off‐line measurements.
During the cultivation of E. coli, well known for the efficient production of recombinant proteins, like hormones and enzymes, also metabolic by‐products are formed that might cause a decrease in growth and productivity performance. A sophisticated measurement system for at‐line determination of the C source glucose, the by‐product acetate and the N source ammonium for high cell density cultivations of E. coli K12 TG1 is presented. Two different flow diffusion analysis systems and a flow injection analysis system were applied as at‐line measurement devices. For verification purposes, an off‐line HPLC system was used.
Abstract The efficacy of cannabinoids against high-grade glioma in animal models, mediated by two specific receptors, CB1 and CB2, raised promises for targeted treatment of the most frequent and ...malignant primary brain tumors. Unlike the abundantly expressed CB1, the CB2 receptor shows a restricted distribution in normal brain. Although brain tumors constitute the second most common malignancy in children and the prevalence of histological types of brain tumors vary significantly between the adult and pediatric populations, cannabinoid receptor expression in pediatric tumors remains unknown. In the present study, we compared the expression of the CB2 receptor in paraffin-embedded sections from primary brain tumors of adult and pediatric patients. Most glioblastomas expressed very high levels of CB2 receptors and the expression correlated with tumor grade. Interestingly, some benign pediatric astrocytic tumors, such as subependymal giant cell astrocytoma (SEGA), which may occasionally cause mortality owing to progressive growth, also displayed high CB2 immunoreactivity. The high levels of CB2 expression would predestine those tumors to be vulnerable to cannabinoid treatment. In contrast, all examined cases of embryonal tumors (medulloblastoma and S-PNET), the most frequently diagnosed malignant brain tumors in childhood, showed no or trace CB2 immunoreactivity. Our results suggest that the CB2 receptor expression depends primarily on the histopathological origin of the brain tumor cells and differentiation state, reflecting the tumor grade.
An abstract of an article by S. Stark and A. Ellert on aging in place in a naturally occurring retirement community is presented. The study aims to determine if removing environmental barriers in the ...home would result in improved performance and satisfaction with performance of activities of daily living.
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