ABT-888 enhances cytotoxic effects of temozolomide independent of MGMT status in serum free cultured glioma cells

Background: The current standard of care for Glioblastoma Multiforme (GBM) consists of fractionated focal irradiation with concomitant temozolomide (TMZ) chemotherapy. A promising strategy to increase the efficacy of TMZ is through interference with the DNA damage repair machinery, by poly(ADP-ribose) polymerase protein inhibition(PARPi). The o

A Systematic Comparison Identifies an ATP-Based Viability Assay as Most Suitable Read-Out for Drug Screening in Glioma Stem-Like Cells

Serum-free culture methods for patient-derived primary glioma cultures, selecting for glioma stem-like cells (GSCs), are becoming the gold standard in neurooncology research. These GSCs can be implemented in drug screens to detect patient-specific responses, potentially bridging the translational gap to personalized medicine. Since numerous compounds are available, a rapid and reliable readout for drug efficacies is required. This can be done using approaches that measure viability, confluency, cytotoxicity, or apoptosis. To determine which assay is best suitable for drug screening, 10 different assays were systematically tested on established glioma cell lines and validated on a panel of GSCs. General applicability was assessed using distinct treatment modalities, being temozolomide, radiation, rapamycin, and the oncolytic adenovirus Delta24-RGD. The apoptosis and cytotoxicity assays did not unequivocally detect responses and were excluded from further testing. The NADH- and ATP-based viability assays revealed comparable readout for all treatments; however, the latter had smaller standard deviations and direct readout. Importantly, drugs that interfere with cell metabolism require alternative techniques such as confluency monitoring to accurately measure treatment effects. Taken together, our data suggest that the combination of ATP luminescence assays with confluency monitoring provides the most specific and reproducible readout for drug screening on primary GSCs

Intraobserver and interobserver variability and spatial differences in histologic examination of carotid endarterectomy specimens

IntroductionStudies using histologic examination and protein analysis of atherosclerotic plaques are increasingly being performed, but reproducibility of plaque histology and variation of plaque composition among different parts of the plaque, which are key to reliability of these studies, are relatively unexplored. Therefore, this study investigated the intraobserver and interobserver variability of plaque histology and spatial variability in plaque composition.MethodsAtherosclerotic plaques (n = 100) obtained during carotid endarterectomy were divided into 0.5-cm segments. Paraffin sections were stained and semiquantitatively analyzed (four categories: no, minor, moderate, and heavy) for fat, macrophages, smooth muscle cells, collagen, calcification, thrombus, and overall phenotype. First, to determine the intraobserver and interobserver reproducibility, two independent observers independently analyzed the plaques. Second, to investigate spatial variability in plaque composition, histologic appearances of the culprit lesions (0-segment) were compared with the histologic appearances of adjacent (+5 mm) and more distant (+10 mm) plaque segments of 30 specimens.ResultsThe κ values for intraobserver variability of fat, macrophages, smooth muscle cells, collagen, calcifications, thrombus, and overall phenotype were 0.83, 0.85, 0.71, 0.63, 0.81, 0.80, and 0.86, respectively, and κ values for interobserver variability were 0.68, 0.74, 0.54, 0.59, 0.82, 0.75, and 0.71, respectively. Comparison of the histologic scorings of adjacent segments revealed a mean κ of 0.40 (range, 0.33 to 0.60). When the culprit segment was compared with the more distant segment, the mean κ was 0.24; however, in 91% of cases, the difference between the culprit segment and the distal segment was one category or less.ConclusionSemiquantitative analysis of carotid atherosclerotic plaque histology was well reproducible, both intraobserver and interobserver. Although variation between different plaque segments in histologic appearance was observed, differences were small in almost all cases. Variability in histologic examination needs to be taken into account in studies comparing plaque imaging with histopathology and plaque research studies

The Sequence of Delta24-RGD and TMZ Administration in Malignant Glioma Affects the Role of CD8(+)T Cell Anti-tumor Activity

The conditionally replicating oncolytic adenovirus Delta24-RGD (Ad) is currently under investigation in clinical trials for glioblastoma, including in combination with temozolomide (TMZ), the standard chemotherapy for this tumor. Previously, we showed that the efficacy of Delta24-RGD in a murine model is primarily dependent on the virus-induced anti-tumor immune response. As observed with most chemotherapies, TMZ has pronounced immune-modulating effects. Here, we studied the combined effects of these treatments in a murine glioma model. In vitro, we observed a synergistic activity between Delta24-RGD and TMZ. In vivo, C57BL/6 mice bearing intracranial GL261 tumors were treated with TMZ for 5 days either prior to intratumoral Delta24-RGD injection (TMZ/Ad) or post virus injection (Ad/TMZ). Notably, the Ad/TMZ regimen led to similar tumoral CD8+ T cell influx as the virus-only treatment, but increased the ability of CD8+ T cells to specifically recognize the tumor cells. This was accompanied by improved survival. The TMZ/Ad regimen also improved survival significantly compared to controls, but not compared to virus alone. In this group, the influx of dendritic cells is impaired, followed by a significantly lower number of tumor-infiltrating CD8+ T cells and no recognition of tumor cells. Depletion of either CD4+ T cells or CD8+ T cells impaired the efficacy of Delta24-RGD, underscoring the role of these cells in therapeutic activity of the virus. Overall, we show that the addition of TMZ to Delta24-RGD treatment leads to a significant increase in survival and that the order of sequence of these treatments affects the CD8+T cell anti-tumor activity

The in vivo therapeutic efficacy of the oncolytic adenovirus Delta24-RGD is mediated by tumor-specific immunity

The oncolytic adenovirus Delta24-RGD represents a new promising therapeutic agent for patients with a malignant glioma and is currently under investigation in clinical phase I/II trials. Earlier preclinical studies showed that Delta24-RGD is able to effectively lyse tumor cells, yielding promising results in various immune-deficient glioma models. However, the role of the immune response in oncolytic adenovirus therapy for glioma has never been explored. To this end, we assessed Delta24-RGD treatment in an immune-competent orthotopic mouse model for glioma and evaluated immune responses against tumor and virus. Delta24-RGD treatment led to long-term survival in 50% of mice and this effect was completely lost upon administration of the immunosuppressive agent dexamethasone. Delta24-RGD enhanced intra-tumoral infiltration of F4/80+ macrophages, CD4+ and CD8+ T-cells, and increased the local production of pro-inflammatory cytokines and chemokines. In treated mice, T cell responses were directed to the virus as well a

The ATLAS3D project - XXIV. The intrinsic shape distribution of early-type galaxies

We use the ATLAS3D sample to perform a study of the intrinsic shapes of early-type galaxies, taking advantage of the available combined photometric and kinematic data. Based on our ellipticity measurements from the Sloan Digital Sky Survey Data Release 7, and additional imaging from the Isaac Newton Telescope, we first invert the shape distribution of fast and slow rotators under the assumption of axisymmetry. Theso-obtained intrinsic shape distribution for the fast rotators can be described with a Gaussian with a mean flattening of q=0.25 and standard deviation σq = 0.14, and an additional tail towards rounder shapes.The slow rotators are much rounder, and are well described with a Gaussian with mean q = 0.63 and σq =0.09. We then checked that our results were consistent when applying a different and independent method to obtain intrinsic shape distributions, by fitting the observed ellipticity distributions directly using Gaussian parametrizations for the intrinsic axis ratios. Although both fast and slow rotators are identified as early-type galaxies in morphological studies, and in many previous shape studies are therefore grouped together, their shape distributions are significantly different, hinting at different formation scenarios. The intrinsic shape distribution of the fast rotators shows similarities with the spiral galaxy population. Including the observed kinematic misalignment in our intrinsic shape study shows that the fast rotators are predominantly axisymmetric, with only very little room for triaxiality. For the slow rotators though there are very strong indications that they are (mildly) triaxial.PostprintPeer reviewe

The HDAC inhibitors scriptaid and LBH589 combined with the oncolytic virus Delta24-RGD exert enhanced anti-tumor efficacy in patient-derived glioblastoma cells

Background: A phase I/II trial for glioblastoma with the oncolytic adenovirus Delta24-RGD was recently completed. Delta24-RGD conditionally replicates in cells with a disrupted retinoblastomapathway and enters cells via αvβ3/5 integrins. Glioblastomas are differentially sensitive to Delta24-RGD. HDAC inhibitors (HDACi) affect integrins and share common cell death pathways with Delta24-RGD. We studied the combination treatment effects of HDACi and Delta24-RGD in patient-derived glioblastoma stem-like cells (GSC), and we determined the most effective HDACi. Methods: SAHA, Valproic Acid, Scriptaid, MS275 and LBH589 were combined with Delta24-RGD in fourteen distinct GSCs. Synergy was determined by Chou Talalay method. Viral infection and replication were assessed using luciferase and GFP encoding vectors and hexon-titration assays. Coxsackie adenovirus receptor and αvβ3 integrin levels were determined by flow cytometry. Oncolysis and mechanisms of cell death were studied by viability, caspase-3/7, LDH and LC3B/p62, phospho-p70S6K. Toxicity was studied on normal human astrocytes. MGMT promotor methylation status, TCGA classification, Rb-pathway and integrin gene expression levels were assessed as markers of responsiveness. Results: Scriptaid and LBH589 acted synergistically with Delta24-RGD in approximately 50% of the GSCs. Both dr

Inhibition of PFKFB3 Hampers the Progression of Atherosclerosis and Promotes Plaque Stability

Aims: 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB)3-mediated glycolysis is pivotal in driving macrophage- and endothelial cell activation and thereby inflammation. Once activated, these cells play a crucial role in the progression of atherosclerosis. Here, we analyzed the expression of PFKFB3 in human atherosclerotic lesions and investigated the therapeutic potential of pharmacological inhibition of PFKFB3 in experimental atherosclerosis by using the glycolytic inhibitor PFK158. Methods and Results: PFKFB3 expression was higher in vulnerable human atheromatous carotid plaques when compared to stable fibrous plaques and predominantly expressed in plaque macrophages and endothelial cells. Analysis of advanced plaques of human coronary arteries revealed a positive correlation of PFKFB3 expression with necrotic core area. To further investigate the role of PFKFB3 in atherosclerotic disease progression, we treated 6–8 weeks old male Ldlr–/– mice. These mice were fed a high cholesterol diet for 13 weeks, of which they were treated for 5 weeks with the glycolytic inhibitor PFK158 to block PFKFB3 activity. The incidence of fibrous cap atheroma (advanced plaques) was reduced in PFK158-treated mice. Plaque phenotype altered markedly as both necrotic core area and intraplaque apoptosis decreased. This coincided with thickening of the fibrous cap and increased plaque stability after PFK158 treatment. Concomitantly, we observed a decrease in glycolysis in peripheral blood mononuclear cells compared to the untreated group, which alludes that changes in the intracellular metabolism of monocyte and macrophages is advantageous for plaque stabilization. Conclusion: High PFKFB3 expression is associated with vulnerable atheromatous human carotid and coronary plaques. In mice, high PFKFB3 expression is also associated with a vulnerable plaque phenotype, whereas inhibition of PFKFB3 activity leads to plaque stabilization. This data implies that inhibition of inducible glycolysis may reduce inflammation, which has the ability to subsequently attenuate atherogenesis

The Sequence of Delta24-RGD and TMZ Administration in Malignant Glioma Affects the Role of CD8+T Cell Anti-tumor Activity

The conditionally replicating oncolytic adenovirus Delta24-RGD (Ad) is currently under investigation in clinical trials for glioblastoma, including in combination with temozolomide (TMZ), the standard chemotherapy for this tumor. Previously, we showed that the efficacy of Delta24-RGD in a murine model is primarily dependent on the virus-induced anti-tumor immune response. As observed with most chemotherapies, TMZ has pronounced immune-modulating effects. Here, we studied the combined effects of these treatments in a murine glioma model. In vitro, we observed a synergistic activity between Delta24-RGD and TMZ. In vivo, C57BL/6 mice bearing intracranial GL261 tumors were treated with TMZ for 5 days either prior to intratumoral Delta24-RGD injection (TMZ/Ad) or post virus injection (Ad/TMZ). Notably, the Ad/TMZ regimen led to similar tumoral CD8+ T cell influx as the virus-only treatment, but increased the ability of CD8+ T cells to specifically recognize the tumor cells. This was accompanied by improved survival. The TMZ/Ad regimen also improved survival significantly compared to controls, but not compared to virus alone. In this group, the influx of dendritic cells is impaired, followed by a significantly lower number of tumor-infiltrating CD8+ T cells and no recognition of tumor cells. Depletion of either CD4+ T cells or CD8+ T cells impaired the efficacy of Delta24-RGD, underscoring the role of these cells in therapeutic activity of the virus. Overall, we show that the addition of TMZ to Delta24-RGD treatment leads to a significant increase in survival and that the order of sequence of these treatments affects the CD8+T cell anti-tumor activity