Targeting sphingolipid metabolism with the sphingosine kinase inhibitor SKI-II overcomes hypoxia-induced chemotherapy resistance in glioblastoma cells: effects on cell death, self-renewal, and invasion.

BACKGROUND: Glioblastoma patients commonly develop resistance to temozolomide chemotherapy. Hypoxia, which supports chemotherapy resistance, favors the expansion of glioblastoma stem cells (GSC), contributing to tumor relapse. Because of a deregulated sphingolipid metabolism, glioblastoma tissues contain high levels of the pro-survival sphingosine-1-phosphate and low levels of the pro-apoptotic ceramide. The latter can be metabolized to sphingosine-1-phosphate by sphingosine kinase (SK) 1 that is overexpressed in glioblastoma. The small molecule SKI-II inhibits SK and dihydroceramide desaturase 1, which converts dihydroceramide to ceramide. We previously reported that SKI-II combined with temozolomide induces caspase-dependent cell death, preceded by dihydrosphingolipids accumulation and autophagy in normoxia. In the present study, we investigated the effects of a low-dose combination of temozolomide and SKI-II under normoxia and hypoxia in glioblastoma cells and patient-derived GCSs. METHODS: Drug synergism was analyzed with the Chou-Talalay Combination Index method. Dose-effect curves of each drug were determined with the Sulforhodamine B colorimetric assay. Cell death mechanisms and autophagy were analyzed by immunofluorescence, flow cytometry and western blot; sphingolipid metabolism alterations by mass spectrometry and gene expression analysis. GSCs self-renewal capacity was determined using extreme limiting dilution assays and invasion of glioblastoma cells using a 3D spheroid model. RESULTS: Temozolomide resistance of glioblastoma cells was increased under hypoxia. However, combination of temozolomide (48 µM) with SKI-II (2.66 µM) synergistically inhibited glioblastoma cell growth and potentiated glioblastoma cell death relative to single treatments under hypoxia. This low-dose combination did not induce dihydrosphingolipids accumulation, but a decrease in ceramide and its metabolites. It induced oxidative and endoplasmic reticulum stress and triggered caspase-independent cell death. It impaired the self-renewal capacity of temozolomide-resistant GSCs, especially under hypoxia. Furthermore, it decreased invasion of glioblastoma cell spheroids. CONCLUSIONS: This in vitro study provides novel insights on the links between sphingolipid metabolism and invasion, a hallmark of cancer, and cancer stem cells, key drivers of cancer. It demonstrates the therapeutic potential of approaches that combine modulation of sphingolipid metabolism with first-line agent temozolomide in overcoming tumor growth and relapse by reducing hypoxia-induced resistance to chemotherapy and by targeting both differentiated and stem glioblastoma cells.

Assessment of technical and clinical utility of a bead-based flow cytometry platform for multiparametric phenotyping of CNS-derived extracellular vesicles.

BACKGROUND: Extracellular vesicles (EVs) originating from the central nervous system (CNS) can enter the blood stream and carry molecules characteristic of disease states. Therefore, circulating CNS-derived EVs have the potential to serve as liquid-biopsy markers for early diagnosis and follow-up of neurodegenerative diseases and brain tumors. Monitoring and profiling of CNS-derived EVs using multiparametric analysis would be a major advance for biomarker as well as basic research. Here, we explored the performance of a multiplex bead-based flow-cytometry assay (EV Neuro) for semi-quantitative detection of CNS-derived EVs in body fluids. METHODS: EVs were separated from culture of glioblastoma cell lines (LN18, LN229, NCH82) and primary human astrocytes and measured at different input amounts in the MACSPlex EV Kit Neuro, human. In addition, EVs were separated from blood samples of small cohorts of glioblastoma (GB), multiple sclerosis (MS) and Alzheimer's disease patients as well as healthy controls (HC) and subjected to the EV Neuro assay. To determine statistically significant differences between relative marker signal intensities, an unpaired samples t-test or Wilcoxon rank sum test were computed. Data were subjected to tSNE, heatmap clustering, and correlation analysis to further explore the relationships between disease state and EV Neuro data. RESULTS: Glioblastoma cell lines and primary human astrocytes showed distinct EV profiles. Signal intensities were increasing with higher EV input. Data normalization improved identification of markers that deviate from a common profile. Overall, patient blood-derived EV marker profiles were constant, but individual EV populations were significantly increased in disease compared to healthy controls, e.g. CD36+EVs in glioblastoma and GALC+EVs in multiple sclerosis. tSNE and heatmap clustering analysis separated GB patients from HC, but not MS patients from HC. Correlation analysis revealed a potential association of CD107a+EVs with neurofilament levels in blood of MS patients and HC. CONCLUSIONS: The semi-quantitative EV Neuro assay demonstrated its utility for EV profiling in complex samples. However, reliable statistical results in biomarker studies require large sample cohorts and high effect sizes. Nonetheless, this exploratory trial confirmed the feasibility of discovering EV-associated biomarkers and monitoring circulating EV profiles in CNS diseases using the EV Neuro assay. Video Abstract.

Extracellular vesicles (EVs) are tiny particles released by cells, carrying unique biomolecules specific to their cell of origin. EVs from the central nervous system (CNS) can reach the blood, where they could serve as liquid-biopsy markers for diagnosing brain diseases like neurodegenerative disorders and tumors. This study evaluated a flow cytometry platform (here termed EV Neuro assay), which can detect multiple EV-associated markers simultaneously, to assess its potential for identifying CNS-derived EVs and disease-specific markers in complex samples including the blood. The study compared different sample materials and methods for isolating EVs. We found distinct EV profiles in EVs derived from glioblastoma and human astrocytes, with signal intensities increasing as more EVs were present. Analyzing serum or plasma from patients with brain diseases and healthy individuals, we observed that EV marker intensities were varying between individuals. Importantly, data normalization improved the identification of disease-specific markers, such as CD36⁺EVs in glioblastoma and GALC⁺EVs in multiple sclerosis, which were significantly higher in disease compared to healthy controls. Advanced clustering analysis techniques effectively distinguished glioblastoma patients from controls. Furthermore, a potential correlation between CD107a⁺EVs and neurofilament levels in multiple sclerosis patients was discovered. Overall, the semi-quantitative EV Neuro assay proved useful for profiling EVs in complex samples. However, for more reliable results in biomarker studies, larger sample cohorts and higher effect sizes are necessary. Nonetheless, this initial trial confirmed the potential of the EV Neuro assay for discovering disease-associated EV markers and monitoring circulating EV profiles in CNS diseases.

Metabolic and inflammatory reprogramming of macrophages by ONC201 translates in a pro-inflammatory environment even in presence of glioblastoma cells.

Tumor-associated macrophages facilitate tumor progression and resistance to therapy. Their capacity for metabolic and inflammatory reprogramming represents an attractive therapeutic target. ONC201/TIC10 is an anticancer molecule that antagonizes the dopamine receptor D2 and affects mitochondria integrity in tumor cells. We examined whether ONC201 induces a metabolic and pro-inflammatory switch in primary human monocyte-derived macrophages that reactivates their antitumor activities, thus enhancing the onco-toxicity of ONC201. Contrary to glioblastoma cells, macrophages exhibited a low ratio of dopamine receptors D2/D5 gene expression and were resistant to ONC201 cytotoxicity. Macrophages responded to ONC201 with a severe loss of mitochondria integrity, a switch to glycolytic ATP production, alterations in glutamate transport, and a shift towards a pro-inflammatory profile. Treatment of macrophages-glioblastoma cells co-cultures with ONC201 induced similar alterations in glutamatergic and inflammatory gene expression profiles of macrophages. It induced as well metabolic changes and a pro-inflammatory switch of the co-culture milieu. However, these changes did not translate into increased onco-toxicity. This study provides the first evidence that ONC201 affects macrophage immunometabolism and leads to a pro-inflammatory tumor environment. This speaks in favor of implementing ONC201 in combinatorial therapies and warrants further investigation of the mechanisms of action of ONC201 in macrophages and other immune cells.

MiR-144 overexpression as a promising therapeutic strategy to overcome glioblastoma cell invasiveness and resistance to chemotherapy.

Glioblastoma (GB) is the most aggressive and common form of primary brain tumor, characterized by fast proliferation, high invasion, and resistance to current standard treatment. The average survival rate post-diagnosis is only of 14.6 months, despite the aggressive standard post-surgery treatment approaches of radiotherapy concomitant with chemotherapy with temozolomide. Altered cell metabolism has been identified as an emerging cancer hallmark, including in GB, thus offering a new target for cancer therapies. On the other hand, abnormal expression levels of miRNAs, key regulators of multiple molecular pathways, have been correlated with pathological manifestations of cancer, such as chemoresistance, proliferation, and resistance to apoptosis. In this work, we hypothesized that gene therapy based on modulation of a miRNA with aberrant expression in GB and predicted to target crucial metabolic enzymes might impair tumor cell metabolism. We found that the increase of miR-144 levels, shown to be downregulated in U87 and DBTRG human GB cell lines, as well as in GB tumor samples, promoted the downregulation of mRNA of enzymes involved in bioenergetic pathways, with consequent alterations in cell metabolism, impairment of migratory capacity, and sensitization of DBTRG cells to a chemotherapeutic drug, the dichloroacetate (DCA). Taken together, our findings provide evidence that the miR-144 plus DCA combined therapy holds promise to overcome GB-acquired chemoresistance, therefore deserving to be explored toward its potential application as a complementary therapeutic approach to the current treatment options for this type of brain tumor.

Paclitaxel Treatment and Proprotein Convertase 1/3 (PC1/3) Knockdown in Macrophages is a Promising Antiglioma Strategy as Revealed by Proteomics and Cytotoxicity Studies.

High grade gliomas are the most common brain tumors in adult. These tumors are characterized by a high infiltration in microglial cells and macrophages. The immunosuppressive tumor environment is known to orient immune cells toward a pro-tumoral and anti-inflammatory phenotype. Therefore, the current challenge for cancer therapy is to find a way to reorient macrophages toward an antitumoral phenotype. Previously, we demonstrated that macrophages secreted antitumoral factors when they were invalidated for the proprotein converstase 1/3 (PC1/3) and treated with LPS. However, achieving an activation of macrophages via LPS/TLR4/Myd88-dependent pathway appears yet unfeasible in cancer patients. On the contrary, the antitumor drug Paclitaxel is also known to activate the TLR4 MyD88-dependent signaling pathway and mimics LPS action. Therefore, we evaluated if PC1/3 knock-down (KD) macrophages could be activated by Paclitaxel and efficient against glioma. We report here that such a treatment of PC1/3 KD macrophages drove to the overexpression of proteins mainly involved in cytoskeleton rearrangement. In support of this finding, we found that these cells exhibited a Ca2+ increase after Paclitaxel treatment. This is indicative of a possible depolymerization of microtubules and may therefore reflect an activation of inflammatory pathways in macrophages. In such a way, we found that PC1/3 KD macrophages displayed a repression of the anti-inflammatory pathway STAT3 and secreted more pro-inflammatory cytokines. Extracellular vesicles isolated from these PC1/3 KD cells inhibited glioma growth. Finally, the supernatant collected from the coculture between glioma cells and PC1/3 KD macrophages contained more antitumoral factors. These findings unravel the potential value of a new therapeutic strategy combining Paclitaxel and PC1/3 inhibition to switch macrophages toward an antitumoral immunophenotype.

Functional studies with IgM and IgA immunoglobulins: binding to pIgR, FcαµR, FcµR, and CDC activities.

IgMs are the first antibodies produced by the immune system upon encounter of a possible pathogen and are one of five antibody subclasses in humans. For IgG, the most intensively studied antibody class, the N-linked glycosylation site located in the Fc-domain is directly involved in high affinity binding to the respective receptors and initiation of corresponding immune response. IgM molecules have five N-glycosylation sites and one N-glycosylation site in the J-chain, which can be incorporated in IgM or IgA molecules. There is only limited knowledge available concerning the function of these N-glycosylations in IgMs. To address this question, we produced IgM molecules lacking a particular N-glycosylation site and tested these variants as well as IgA molecules for binding to the known receptors: the polymeric immunoglobulin receptor (pIgR), the dual receptor for IgA and IgM, FcαµR, and the specific receptor for IgM, FcµR. The single glycosylation sites did not show an impact on expression and multimerization, except for variant N402Q, which could not be expressed. In SPR measurements, no major impact on the binding to the receptors by particular glycosylation sites could be detected. In cellular assays, deglycosylated variants showed some alterations in induction of CDC activity. Most strikingly, we observed also binding of IgA to the FcµR in the same affinity range as IgM, suggesting that this might have a physiological role. To further substantiate the binding of IgA to FcµR we used IgA from different origins and were able to confirm binding of IgA preparations to the FcµR.

Glutathione peroxidase 1 activity dictates the sensitivity of glioblastoma cells to oxidative stress.

The high intratumoral and intertumoral heterogeneity of glioblastoma (GBM) leads to resistance to different therapies, and hence, selecting an effective therapy is very challenging. We hypothesized that the antioxidant enzyme status is a significant feature of GBM heterogeneity. The most important reactive oxygen/nitrogen species (ROS/RNS) detoxification mechanisms include superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx). Expression and activity of these enzymes and the cellular response to induced oxidative stress were systematically analyzed and compared between GBM cells and nontransformed glial cells of both human and murine origin. Regardless of cell type or species, all tested cells expressed similar amount of catalase and MnSOD. All except one, GBM cell lines exhibited a deficiency in GPx1 expression and activity. Analysis of GBM tissue sections indicated a heterogeneous profile of weak to moderate expression of GPx1 in tumor cells. GPx1 deficiency led to an accumulation of ROS/RNS and subsequent death of GBM cells after induction of oxidative stress. Astrocytes, microglia/macrophages, and glioma stem cell lines expressed active GPx1 and resisted ROS/RNS-mediated cell death. Pharmacological inhibition or siRNA silencing of GPx1 partially reverted this resistance in astrocytes, indicating the contribution of various antioxidant molecules besides GPx1. The GPx1-expressing GBM cell line on the contrary, became extremely sensitive to oxidative stress after GPx1 inhibition. Altogether, these results highlight GPx1 as a crucial element over other antioxidant enzymes for oxidative stress regulation in GBM cells. Mapping the antioxidant enzyme status of GBM may prove to be a useful tool for personalized ROS/RNS inducing therapies.

Multistability in Macrophage Activation Pathways and Metabolic Implications.

Macrophages are innate immune cells with a dynamic range of reversible activation states including the classical pro-inflammatory (M1) and alternative anti-inflammatory (M2) states. Deciphering how macrophages regulate their transition from one state to the other is key for a deeper understanding of inflammatory diseases and relevant therapies. Common regulatory motifs reported for macrophage transitions, such as positive or double-negative feedback loops, exhibit a switchlike behavior, suggesting the bistability of the system. In this review, we explore the evidence for multistability (including bistability) in macrophage activation pathways at four molecular levels. First, a decision-making module in signal transduction includes mutual inhibitory interactions between M1 (STAT1, NF-KB/p50-p65) and M2 (STAT3, NF-KB/p50-p50) signaling pathways. Second, a switchlike behavior at the gene expression level includes complex network motifs of transcription factors and miRNAs. Third, these changes impact metabolic gene expression, leading to switches in energy production, NADPH and ROS production, TCA cycle functionality, biosynthesis, and nitrogen metabolism. Fourth, metabolic changes are monitored by metabolic sensors coupled to AMPK and mTOR activity to provide stability by maintaining signals promoting M1 or M2 activation. In conclusion, we identify bistability hubs as promising therapeutic targets for reverting or blocking macrophage transitions through modulation of the metabolic environment.

MYC dosage compensation is mediated by miRNA-transcription factor interactions in aneuploid cancer.

We hypothesize that dosage compensation of critical genes arises from systems-level properties for cancer cells to withstand the negative effects of aneuploidy. We identified several candidate genes in cancer multiomics data and developed a biocomputational platform to construct a mathematical model of their interaction network with micro-RNAs and transcription factors, where the property of dosage compensation emerged for MYC and was dependent on the kinetic parameters of its feedback interactions with three micro-RNAs. These circuits were experimentally validated using a genetic tug-of-war technique to overexpress an exogenous MYC, leading to overexpression of the three microRNAs involved and downregulation of endogenous MYC. In addition, MYC overexpression or inhibition of its compensating miRNAs led to dosage-dependent cytotoxicity in MYC-amplified colon cancer cells. Finally, we identified negative correlation of MYC dosage compensation with patient survival in TCGA breast cancer patients, highlighting the potential of this mechanism to prevent aneuploid cancer progression.

Sphingolipid rheostat alterations related to transformation can be exploited for specific induction of lysosomal cell death in murine and human glioma.

The search for cancer cell-specific targets suffers from a lack of integrative approaches that take into account the relative contributions of several mechanisms or pathways involved in cell death. A systematic experimental and computational comparison of murine glioma cells with astrocytes, their nontransformed counterparts, identified differences in the sphingolipid (SL) rheostat linked to an increased lysosomal instability in glioma cells. In vitro and in silico analyses indicate that sphingosine metabolized in lysosomes was preferentially recycled into ceramide, the prodeath member of the rheostat, in astrocytes. In glioma cells, it preferentially was used for production of the prosurvival sphingosine-1-phosphate (S1P). A combination of tumor necrosis factor alpha (TNF-alpha), lipopolysaccharide (LPS), and interferon gamma (IFN-gamma) strongly decreased S1P production that resulted in abnormal lysosome enlargement and cell death associated with mitochondrial dysfunction of glioma cells only. Lack of intracellular S1P in glioma cells was concomitant with protein and lipid accumulation in enlarged lysosomes, indicating a blockade in lysosome recycling, and hence a role for S1P in membrane trafficking. A pharmacological sphingosine kinase inhibitor efficiently replaced the TNF-alpha, LPS, and IFN-gamma combination and killed murine and human glioma cells without affecting astrocytes. Our study provides evidence for a novel mechanism of lysosomal death dependent upon the SL rheostat that can be specifically triggered in glioma cells. It further strengthens the potential of cancer therapies based on specific ceramide pathway alterations.

TNF-alpha- and TRAIL-resistant glioma cells undergo autophagy-dependent cell death induced by activated microglia.

The role of microglia, the brain resident macrophages, in glioma biology is still ill-defined. Despite their cytotoxic potential, these cells that significantly infiltrate the tumor mass seem to support tumor growth rather than tumor eradication. A proper activation of microglia anti-tumor activities within the tumor may provide a valuable additional arm of defense to immunotherapies against brain tumors. We herewith report a detailed characterization of (lipopolysaccharide and interferon-gamma)-induced anti-tumor activities of mouse primary microglia towards two TNF-alpha and TRAIL resistant glioma cell lines, in cell monolayer or spheroid cultures and in collagen-embedded tumor explants. Irrespective of the mouse strain, stimulated microglia secreted proteic factors that decreased proliferation and migration of these glioma cells and efficiently killed them. Death occurred specifically in glioma cells as demonstrated by the lack of toxicity of microglia supernatant towards primary cultures of astrocytes or neurons. Cell death was characterized by the early accumulation of acidic vesicles, phosphatidylserine exposure, appearance of double-membrane cytoplasmic vesicles, extensive zeiosis and a very late loss of DNA in cells that had lost membrane integrity. Inhibition of autophagosome formation efficiently protected glioma cells from death whereas caspase inhibition could only prevent DNA loss but not cytotoxicity. Death however, resulted from a blockade by microglia supernatant of the basal autophagic flux present in the glioma cells. These observations demonstrate that glioma cells resistant to apoptotic death ligands could be successfully and specifically killed through autophagy-dependent death induced by appropriately activated microglia.

Beyond vessels: occurrence and regional clustering of vascular endothelial (VE-)cadherin-containing junctions in non-endothelial cells.

The genes encoding transmembrane glycoproteins of the cadherin family, i.e., the Ca(2+)-dependent cell-cell adhesion molecules, are typically expressed in cell-type- or cell-lineage-specific patterns. One of them, vascular endothelial (VE)-cadherin, is widely considered to be specific for vascular endothelia in which it is either the sole or the predominant cadherin, often co-existing with N-cadherin. This specificity of VE-cadherin for vascular endothelial cells is important not only in blood and lymph vessel biology and medicine, but also for cell-type-based diagnoses, notably those of metastatic tumors. Surprisingly, however, we have recently noted the frequent synthesis, surface exposure, and junction assembly of VE-cadherin in certain other cells, in which this glycoprotein is clustered into adherens junctions (AJs), either alone or in combination with N-cadherin and/or cadherin-11. Such cells include mammalian astrocytes and glioma, probably mostly astrocytoma cells growing in culture, and a specific subtype of astrocytoma in situ. Moreover, VE-cadherin synthesis and AJ assembly, plus the regional clustering of such AJs in certain domains, are not clonally fixed but can appear again and again in cells of the progeny of cloned homogeneous-appearing individual cells, thus resulting in clonal cell colonies that are often heterogeneous in their cadherin junction patterns. We discuss the constitutive presence of VE-cadherin in some non-endothelial cells with respect to certain architectural features and possible physiological and pathogenic functions of the cells, and in comparison with recent reports of VE-cadherin-positive melanomas.

Schwann cells express the macrophage mannose receptor and MHC class II. Do they have a role in antigen presentation?

The mannose receptor (MR) is a transmembrane glycoprotein, postulated to be a link between innate and adaptive immunity. MR is expressed in several cell types but no information is available on that for Schwann cells (SC). We show that rodent SC in primary cultures take up the MR ligand mannosyl/bovine serum albumin-fluorescein isothiocyanate (man/BSA-FITC) in a highly specific manner and bind an antibody against the C-terminus of the murine macrophage MR (anti-cMR). After incubation with man/BSA-FITC, flow cytometry demonstrates 90% positive SC, a dose-dependent increase in tagged cellular components and near total inhibition of the neoglycoprotein uptake by D-mannose or by the mannosylated protein horseradish peroxidase (HRP). Western blot for MR shows that SC share a unique protein of about 180 kDa with peritoneal resident macrophages. Treatment of cultured SC with interferon-gamma (IFN-gamma) or dexamethasone (DM) followed by the addition of man/BSA-FITC and analysis by flow cytometry shows down- or upregulation, respectively, of man/BSA-FITC uptake. Our results show that SC express the MR in a prospectively functional state and suggest an antigen-presenting function of SC, compatible with a role in infectious/inflammatory states of the peripheral nervous system.

Assessing the reliability of gene expression measurements in very-low-numbers of human monocyte-derived macrophages.

Tumor-derived primary cells are essential for in vitro and in vivo studies of tumor biology. The scarcity of this cellular material limits the feasibility of experiments or analyses and hence hinders basic and clinical research progress. We set out to determine the minimum number of cells that can be analyzed with standard laboratory equipment and that leads to reliable results, unbiased by cell number. A proof-of-principle study was conducted with primary human monocyte-derived macrophages, seeded in decreasing number and constant cell density. Gene expression of cells stimulated to acquire opposite inflammatory states was analyzed by quantitative PCR. Statistical analysis indicated the lack of significant difference in the expression profile of cells cultured at the highest (100,000 cells) and lowest numbers (3,610 cells) tested. Gene Ontology, pathway enrichment and network analysis confirmed the reliability of the data obtained with the lowest cell number. This statistical and computational analysis of gene expression profiles indicates that low cell number analysis is as dependable and informative as the analysis of a larger cell number. Our work demonstrates that it is possible to employ samples with a scarce number of cells in experimental studies and encourages the application of this approach on other cell types.

End-stage dying glioma cells are engulfed by mouse microglia with a strain-dependent efficacy.

Microglia phagocytic activity for apoptotic glioma cells is hardly analysed inspite of its relevance to tissue damage prevention. We provide evidence for a phosphatidylserine-independent clearance of mouse glioma cells at an advanced stage of death, suggesting microglia recognition of late apoptotic markers. Dying cells were immediately cleared or stayed for hours in that stage before engulfment occurred. This phagocytic activity was restricted to a microglia subset representing 30 to 70% of the population according to the used strain. Expression of receptors involved in late apoptotic markers recognition therefore seems confined to a subpopulation of microglia and to be strain-dependent.

Comparative analysis of the effects of a sphingosine kinase inhibitor to temozolomide and radiation treatment on glioblastoma cell lines.

Glioblastoma multiforme (GBM) exhibits high resistance to the standard treatment of temozolomide (TMZ) combined with radiotherapy, due to its remarkable cell heterogeneity. Accordingly, there is a need to target alternative molecules enhancing specific GBM autocrine or paracrine mechanisms and amplifying the effect of standard treatment. Sphingosine 1-phosphate (S1P) is such a lipid target molecule with an important role in cell invasion and proliferation. Sphingosine kinase inhibitors (SKI) prevent S1P formation and induce increased production of reactive oxygen species (ROS), which may potentiate radiation cytotoxicity. We analyzed the effect of SKI singular versus combined treatments with TMZ and radiation on 2 human GBM cell lines characterized by a lack of MGMT expression and low or high expression of the anti-oxidant enzyme, glutathione peroxidase 1 (GPx1). Effects were drug concentration-, cell line-dependent and partly ROS-mediated. Clonogenic survival assay demonstrates that SKI was more effective than TMZ in increasing the sensitivity of U87 cells, which express low GPx1 amount, to a 2 Gy X-ray dose. Addition of both SKI and TMZ drastically decreased U87 cells survival compared with the combination temozolomide/radiation. SKI less effectively than TMZ sensitized LN229 cells to the 2 Gy X-ray dose. Its combination to TMZ in absence of irradiation was as efficient as TMZ combination with X-ray. We provide first evidence for SKI as an alternative or complementary treatment to TMZ, and for efficient combinations of low doses of drugs and X-ray. These may help as novel bi-modal and tri-modal therapies to contend with GBM heterogeneity.

Evaluation of Consistency in Spheroid Invasion Assays.

Multicellular tumor spheroids embedded in a matrix represent invaluable tools to analyze cell invasion. Spheroid sizes and invasiveness are the main observables easily measurable to evaluate effects of biological or pharmaceutical manipulations on invasion. They largely account for these 3-D platforms variability, leading to flaws in data interpretation. No method has been established yet that characterizes this variability and guarantees a reliable use of 3-D platforms. Spheroid initial/end sizes and invasiveness were systematically analyzed and compared in spheroids of U87MG cells generated by three different methods and embedded at different times in a collagen matrix. A normality test was used to characterize size distribution. We introduced the linearity-over-yield analysis as a novel mathematical tool to assess end sizes and invasion reproducibility. We further provide a proof of concept by applying these tools to the analysis of a treatment known to be effective beforehand. We demonstrate that implementation of these statistical and mathematical tools warranted a confident quantification and interpretation of in 3-D conducted assays. We propose these tools could be incorporated in a guideline for generation and use of 3-D platforms.

A Novel Computer-Assisted Approach to evaluate Multicellular Tumor Spheroid Invasion Assay.

Multicellular tumor spheroids (MCTSs) embedded in a matrix are re-emerging as a powerful alternative to monolayer-based cultures. The primary information gained from a three-dimensional model is the invasiveness of treatment-exposed MCTSs through the acquisition of light microscopy images. The amount and complexity of the acquired data and the bias arisen by their manual analysis are disadvantages calling for an automated, high-throughput analysis. We present a universal algorithm we developed with the scope of being robust enough to handle images of various qualities and various invasion profiles. The novelty and strength of our algorithm lie in: the introduction of a multi-step segmentation flow, where each step is optimized for each specific MCTS area (core, halo, and periphery); the quantification through the density of the two-dimensional representation of a three-dimensional object. This latter offers a fine-granular differentiation of invasive profiles, facilitating a quantification independent of cell lines and experimental setups. Progression of density from the core towards the edges influences the resulting density map thus providing a measure no longer dependent on the sole area size of MCTS, but also on its invasiveness. In sum, we propose a new method in which the concept of quantification of MCTS invasion is completely re-thought.

The mannose receptor in the brain.

The mannose receptor is a transmembrane glycoprotein mainly expressed by macrophages, that specifically binds to mannosylated molecules and mediates their endocytosis. Known ligands of the receptor are lysosomases and various pathogens. Ligand specificity and cellular distribution provide the mannose receptor with a very important role in homeostasis and in the immune response. Expression of the mannose receptor has recently been demonstrated in the brain. Astrocytes and microglia, two types of glial cells that can be turned into immune-competent cells, are the main site of expression in vivo and in vitro. The mannose receptor mediates in vitro pinocytosis by astrocytes and microglia and phagocytosis by microglia. Expression and endocytic activities of the mannose receptor in these cells are regulated by various cytokines. Based on our current knowledge on mannose receptor activities in brain cells, on its regional and temporal expression in that organ, and on its putative ligands therein, the possible involvement of the mannose receptor in brain homeostasis, neuronal functions, and brain defense is discussed.

Glioblastoma cells induce differential glutamatergic gene expressions in human tumor-associated microglia/macrophages and monocyte-derived macrophages.

Glioblastoma cells produce and release high amounts of glutamate into the extracellular milieu and subsequently can trigger seizure in patients. Tumor-associated microglia/macrophages (TAMs), consisting of both parenchymal microglia and monocytes-derived macrophages (MDMs) recruited from the blood, are known to populate up to 1/3 of the glioblastoma tumor environment and exhibit an alternative, tumor-promoting and supporting phenotype. However, it is unknown how TAMs respond to the excess extracellular glutamate in the glioblastoma microenvironment. We investigated the expressions of genes related to glutamate transport and metabolism in human TAMs freshly isolated from glioblastoma resections. Quantitative real-time PCR analysis showed (i) significant increases in the expressions of GRIA2 (GluA2 or AMPA receptor 2), SLC1A2 (EAAT2), SLC1A3 (EAAT1), (ii) a near-significant decrease in the expression of SLC7A11 (cystine-glutamate antiporter xCT) and (iii) a remarkable increase in GLUL expression (glutamine synthetase) in these cells compared to adult primary human microglia. TAMs co-cultured with glioblastoma cells also exhibited a similar glutamatergic profile as freshly isolated TAMs except for a slight increase in SLC7A11 expression. We next analyzed these genes expressions in cultured human MDMs derived from peripheral blood monocytes for comparison. In contrast, MDMs co-cultured with glioblastoma cells compared to MDMs co-cultured with normal astrocytes exhibited decreased expressions in the tested genes except for GLUL. This is the first study to demonstrate transcriptional changes in glutamatergic signaling of TAMs in a glioblastoma microenvironment, and the findings here suggest that TAMs and MDMs might potentially elicit different cellular responses in the presence of excess extracellular glutamate.