Tyler Steed

In vivo Stimulus-Induced Vasodilation Occurs without IP3 Receptor Activation and May Precede Astrocytic Calcium Increase

Calcium-dependent release of vasoactive gliotransmitters is widely assumed to trigger vasodilation associated with rapid increases in neuronal activity. Inconsistent with this hypothesis, intact stimulus-induced vasodilation was observed in inositol 1,4,5-triphosphate (IP3) type-2 receptor (R2) knock-out (KO) mice, in which the primary mechanism of astrocytic calcium increase—the release of calcium from intracellular stores following activation of an IP3-dependent pathway—is lacking. Further, our results in wild-type (WT) mice indicate that in vivo onset of astrocytic calcium increase in response to sensory stimulus could be considerably delayed relative to the simultaneously measured onset of arteriolar dilation. Delayed calcium increases in WT mice were observed in both astrocytic cell bodies and perivascular endfeet. Thus, astrocytes may not play a role in the initiation of blood flow response, at least not via calcium-dependent mechanisms. Moreover, an increase in astrocytic intracellular calcium was not required for normal vasodilation in the IP3R2-KO animals.

Cell type specificity of neurovascular coupling in cerebral cortex

Identification of the cellular players and molecular messengers that communicate neuronal activity to the vasculature driving cerebral hemodynamics is important for (1) the basic understanding of cerebrovascular regulation and (2) interpretation of functional Magnetic Resonance Imaging (fMRI) signals. Using a combination of optogenetic stimulation and 2-photon imaging in mice, we demonstrate that selective activation of cortical excitation and inhibition elicits distinct vascular responses and identify the vasoconstrictive mechanism as Neuropeptide Y (NPY) acting on Y1 receptors. The latter implies that task-related negative Blood Oxygenation Level Dependent (BOLD) fMRI signals in the cerebral cortex under normal physiological conditions may be mainly driven by the NPY-positive inhibitory neurons. Further, the NPY-Y1 pathway may offer a potential therapeutic target in cerebrovascular disease. DOI: http://dx.doi.org/10.7554/eLife.14315.001

Axinellamines as Broad-Spectrum Antibacterial Agents: Scalable Synthesis and Biology

Antibiotic-resistant bacteria present an ongoing challenge to both chemists and biologists as they seek novel compounds and modes of action to out-maneuver continually evolving resistance pathways, especially against Gram-negative strains. The dimeric pyrrole–imidazole alkaloids represent a unique marine natural product class with diverse primary biological activity and chemical architecture. This full account traces the strategy used to develop a second-generation route to key spirocycle 9, culminating in a practical synthesis of the axinellamines and enabling their discovery as broad-spectrum antibacterial agents, with promising activity against both Gram-positive and Gram-negative bacteria. While their detailed mode of antibacterial action remains unclear, the axinellamines appear to cause secondary membrane destabilization and impart an aberrant cellular morphology consistent with the inhibition of normal septum formation. This study serves as a rare example of a natural product initially reported to be devoid of biological activity surfacing as an active antibacterial agent with an intriguing mode of action.

Iterative Probabilistic Voxel Labeling: Automated Segmentation for Analysis of The Cancer Imaging Archive Glioblastoma Images

BACKGROUND AND PURPOSE: Robust, automated segmentation algorithms are required for quantitative analysis of large imaging datasets. We developed an automated method that identifies and labels brain tumor–associated pathology by using an iterative probabilistic voxel labeling using k-nearest neighbor and Gaussian mixture model classification. Our purpose was to develop a segmentation method which could be applied to a variety of imaging from The Cancer Imaging Archive. MATERIALS AND METHODS: Images from 2 sets of 15 randomly selected subjects with glioblastoma from The Cancer Imaging Archive were processed by using the automated algorithm. The algorithm-defined tumor volumes were compared with those segmented by trained operators by using the Dice similarity coefficient. RESULTS: Compared with operator volumes, algorithm-generated segmentations yielded mean Dice similarities of 0.92 ± 0.03 for contrast-enhancing volumes and 0.84 ± 0.09 for FLAIR hyperintensity volumes. These values compared favorably with the means of Dice similarity coefficients between the operator-defined segmentations: 0.92 ± 0.03 for contrast-enhancing volumes and 0.92 ± 0.05 for FLAIR hyperintensity volumes. Robust segmentations can be achieved when only postcontrast T1WI and FLAIR images are available. CONCLUSIONS: Iterative probabilistic voxel labeling defined tumor volumes that were highly consistent with operator-defined volumes. Application of this algorithm could facilitate quantitative assessment of neuroimaging from patients with glioblastoma for both research and clinical indications.

Characterization and Correction of Geometric Distortions in 814 Diffusion Weighted Images.

Introduction Diffusion Weighted Imaging (DWI), which is based on Echo Planar Imaging (EPI) protocols, is becoming increasingly important for neurosurgical applications. However, its use in this context is limited in part by significant spatial distortion inherent to EPI. Method We evaluated an efficient algorithm for EPI distortion correction (EPIC) across 814 DWI scans from 250 brain tumor patients and quantified the magnitude of geometric distortion for whole brain and multiple brain regions. Results Evaluation of the algorithm’s performance revealed significantly higher mutual information between T1-weighted pre-contrast images and corrected b = 0 images than the uncorrected b = 0 images (p < 0.001). The distortion magnitude across all voxels revealed a median EPI distortion effect of 2.1 mm, ranging from 1.2 mm to 5.9 mm, the 5th and 95th percentile, respectively. Regions adjacent to bone-air interfaces, such as the orbitofrontal cortex, temporal poles, and brain stem, were the regions most severely affected by DWI distortion. Conclusion Using EPIC to estimate the degree of distortion in 814 DWI brain tumor images enabled the creation of a topographic atlas of DWI distortion across the brain. The degree of displacement of tumors boundaries in uncorrected images is severe but can be corrected for using EPIC. Our results support the use of distortion correction to ensure accurate and careful application of DWI to neurosurgical practice.

Immune evasion mediated by PD-L1 on glioblastoma-derived extracellular vesicles

Glioblastoma can suppress immunity by using surface PD-L1 on extracellular vesicles to block T cell receptor–mediated T cell activation. Binding of programmed death ligand-1 (PD-L1) to programmed cell death protein-1 (PD1) leads to cancer immune evasion via inhibition of T cell function. One of the defining characteristics of glioblastoma, a universally fatal brain cancer, is its profound local and systemic immunosuppression. Glioblastoma has also been shown to generate extracellular vesicles (EVs), which may play an important role in tumor progression. We thus hypothesized that glioblastoma EVs may be important mediators of immunosuppression and that PD-L1 could play a role. We show that glioblastoma EVs block T cell activation and proliferation in response to T cell receptor stimulation. PD-L1 was expressed on the surface of some, but not of all, glioblastoma-derived EVs, with the potential to directly bind to PD1. An anti-PD1 receptor blocking antibody significantly reversed the EV-mediated blockade of T cell activation but only when PD-L1 was present on EVs. When glioblastoma PD-L1 was up-regulated by IFN-γ, EVs also showed some PD-L1–dependent inhibition of T cell activation. PD-L1 expression correlated with the mesenchymal transcriptome profile and was anatomically localized in the perinecrotic and pseudopalisading niche of human glioblastoma specimens. PD-L1 DNA was present in circulating EVs from glioblastoma patients where it correlated with tumor volumes of up to 60 cm3. These results suggest that PD-L1 on EVs may be another mechanism for glioblastoma to suppress antitumor immunity and support the potential of EVs as biomarkers in tumor patients.

Differential localization of glioblastoma subtype: implications on glioblastoma pathogenesis

Introduction The subventricular zone (SVZ) has been implicated in the pathogenesis of glioblastoma. Whether molecular subtypes of glioblastoma arise from unique niches of the brain relative to the SVZ remains largely unknown. Here, we tested whether these subtypes of glioblastoma occupy distinct regions of the cerebrum and examined glioblastoma localization in relation to the SVZ. Methods Pre-operative MR images from 217 glioblastoma patients from The Cancer Imaging Archive were segmented automatically into contrast enhancing (CE) tumor volumes using Iterative Probabilistic Voxel Labeling (IPVL). Probabilistic maps of tumor location were generated for each subtype and distances were calculated from the centroid of CE tumor volumes to the SVZ. Glioblastomas that arose in a Genetically Modified Murine Model (GEMM) model were also analyzed with regard to SVZ distance and molecular subtype. Results Classical and mesenchymal glioblastomas were more diffusely distributed and located farther from the SVZ. In contrast, proneural and neural glioblastomas were more likely to be located in closer proximity to the SVZ. Moreover, in a GFAP-CreER; PtenloxP/loxP; Trp53loxP/loxP; Rb1loxP/loxP; Rbl1−/− GEMM model of glioblastoma where tumor can spontaneously arise in different regions of the cerebrum, tumors that arose near the SVZ were more likely to be of proneural subtype (p < 0.0001). Conclusions Glioblastoma subtypes occupy different regions of the brain and vary in proximity to the SVZ. These findings harbor implications pertaining to the pathogenesis of glioblastoma subtypes.

Abstract 2737: Modulation of mitotic DNA damage as a paradigm for glioblastoma therapy

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The clinical efficacies of molecularly targeted glioblastomas therapies have been vastly disappointing. The multitudes of resistance mechanisms suggest that glioblastomas possess highly dynamic molecular circuits grounded in functional redundancy. Emerging data suggests that the expression of functionally redundant oncogenes induced similar forms of cellular stress, requiring hyper-activation of common compensatory pathways to ensure cell viability. Targeting these pathways, therefore, afford potential opportunities for tumor ablation while by-passing the redundancy of oncogenic circuitry. To explore this paradigm, we carried out a siRNA screen to identify synthetic lethal partners of the oncogenic Epidermal Growth Factor Receptor variant III (EGFRvIII) in glioblastoma cells and identified Polo-Like Kinase 1 (PLK1). Treatment using a PLK1 inhibitor, BI2536, or siRNAs induced preferential toxicity to the EGFRvIII expressing glioblastoma cells in multiple in vitro (serum and neurosphere lines) and in vivo models (heterotopic and orthotopic xenograft models). Consistent with the heightened PLK1 requirement, EGFRvIII expressing glioblastomas harbored increased levels of the p-Thr210 PLK1 (an activated form of PLK1). Inhibition of PLK1 by BI2536 treatment induced an increase in the proportion of cells that co-stained for p-Histone H3 and γH2AX foci, suggesting accumulation of mitotic DNA damage. This effect was exacerbated by EGFRvIII expression, implicating induction of mitotic DNA damage as a major contributor to the observed synthetic lethality. Consistent with this observation, EGFRvIII expression induced the formation of aberrant mitosis as well as prolonged mitotic progression. Further supporting an essential role for PLK1 in suppressing DNA damage accumulation, BI2536 treatment significantly enhanced the tumoricidal effect of the DNA damaging chemotherapy, temozolomide. Mechanistically, inhibition of PLK1 suppressed the expression of Rad51, the accumulation of pS14 Rad51 (an active form of Rad51), as well as overall homologous recombination efficiency in vitro. We validated the clinical pertinence of these results using three clinically annotated glioblastoma databases (TCGA, REMEBMRANDT, CGGA). In all three datasets, increased expression of a PLK1 signature consistently associated with increased expression of HR genes and lowered gene expression signature associated with DNA damage accumulation. Supporting our proposed paradigm, the tumoricidal effect of BI2536 was universally observed in a panel of eight murine ink4a/arf (-/-) EGFRvIII expressing glioblastoma clones that developed resistance to EGFR inhibitors by distinct and independent mechanisms. In aggregate, our results support the essential role of PLK1 in suppressing mitotic DNA damage and provide a novel framework for glioblastoma therapy. Citation Format: Ying Shen, Masayuki Nitta, Jie Li, Diahnn Futalan, Tyler Steed, Zack Taich, Jeffrey M. Treiber, Deanna Stevens, Mark A. Schroeder, Jann N. Sarkaria, Hong-Zhuan Chen, Tao Jiang, Bob S. Carter, Fumiko Esashi, Jill Wakosky, Frank Furnari, Webster K. Cavenee, Arshad Desai, Clark C. Chen. Modulation of mitotic DNA damage as a paradigm for glioblastoma therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2737. doi:10.1158/1538-7445.AM2014-2737