Glioblastomas (GBMs) diffusely infiltrate the brain, making complete removal by surgical resection impossible. The mixture of neoplastic and nonneoplastic cells that remain after surgery form the ...biological context for adjuvant therapeutic intervention and recurrence. We performed RNA-sequencing (RNA-seq) and histological analysis on radiographically guided biopsies taken from different regions of GBM and showed that the tissue contained within the contrast-enhancing (CE) core of tumors have different cellular and molecular compositions compared with tissue from the nonenhancing (NE) margins of tumors. Comparisons with the The Cancer Genome Atlas dataset showed that the samples from CE regions resembled the proneural, classical, or mesenchymal subtypes of GBM, whereas the samples from the NE regions predominantly resembled the neural subtype. Computational deconvolution of the RNA-seq data revealed that contributions from nonneoplastic brain cells significantly influence the expression pattern in the NE samples. Gene ontology analysis showed that the cell type-specific expression patterns were functionally distinct and highly enriched in genes associated with the corresponding cell phenotypes. Comparing the RNA-seq data from the GBM samples to that of nonneoplastic brain revealed that the differentially expressed genes are distributed across multiple cell types. Notably, the patterns of cell type-specific alterations varied between the different GBM subtypes: the NE regions of proneural tumors were enriched in oligodendrocyte progenitor genes, whereas the NE regions of mesenchymal GBM were enriched in astrocytic and microglial genes. These subtypespecific patterns provide new insights into molecular and cellular composition of the infiltrative margins of GBM.
The proliferative and invasive nature of malignant cancers drives lethality. In glioblastoma, these two processes are presumed mutually exclusive and hence termed "go or grow." We identified a ...molecular target that shuttles between these disparate cellular processes-the molecular motor KIF11. Inhibition of KIF11 with a highly specific small-molecule inhibitor stopped the growth of the more treatment-resistant glioblastoma tumor-initiating cells (TICs, or cancer stem cells) as well as non-TICs and impeded tumor initiation and self-renewal of the TIC population. Targeting KIF11 also hit the other arm of the "go or grow" cell fate decision by reducing glioma cell invasion. Administration of a KIF11 inhibitor to mice bearing orthotopic glioblastoma prolonged their survival. In its role as a shared molecular regulator of cell growth and motility across intratumoral heterogeneity, KIF11 is a compelling therapeutic target for glioblastoma.
OBJECTIVE Extent of resection is an important prognostic factor in patients undergoing surgery for glioblastoma (GBM). Recent evidence suggests that intravenously administered fluorescein sodium ...associates with tumor tissue, facilitating safe maximal resection of GBM. In this study, the authors evaluate the safety and utility of intraoperative fluorescein guidance for the prediction of histopathological alteration both in the contrast-enhancing (CE) regions, where this relationship has been established, and into the non-CE (NCE), diffusely infiltrated margins. METHODS Thirty-two patients received fluorescein sodium (3 mg/kg) intravenously prior to resection. Fluorescence was intraoperatively visualized using a Zeiss Pentero surgical microscope equipped with a YELLOW 560 filter. Stereotactically localized biopsy specimens were acquired from CE and NCE regions based on preoperative MRI in conjunction with neuronavigation. The fluorescence intensity of these specimens was subjectively classified in real time with subsequent quantitative image analysis, histopathological evaluation of localized biopsy specimens, and radiological volumetric assessment of the extent of resection. RESULTS Bright fluorescence was observed in all GBMs and localized to the CE regions and portions of the NCE margins of the tumors, thus serving as a visual guide during resection. Gross-total resection (GTR) was achieved in 84% of the patients with an average resected volume of 95%, and this rate was higher among patients for whom GTR was the surgical goal (GTR achieved in 93.1% of patients, average resected volume of 99.7%). Intraoperative fluorescein staining correlated with histopathological alteration in both CE and NCE regions, with positive predictive values by subjective fluorescence evaluation greater than 96% in NCE regions. CONCLUSIONS Intraoperative administration of fluorescein provides an easily visualized marker for glioma pathology in both CE and NCE regions of GBM. These findings support the use of fluorescein as a microsurgical adjunct for guiding GBM resection to facilitate safe maximal removal.
Objective
Somatic variants are a recognized cause of epilepsy‐associated focal malformations of cortical development (MCD). We hypothesized that somatic variants may underlie a wider range of focal ...epilepsy, including nonlesional focal epilepsy (NLFE). Through genetic analysis of brain tissue, we evaluated the role of somatic variation in focal epilepsy with and without MCD.
Methods
We identified somatic variants through high‐depth exome and ultra–high‐depth candidate gene sequencing of DNA from epilepsy surgery specimens and leukocytes from 18 individuals with NLFE and 38 with focal MCD.
Results
We observed somatic variants in 5 cases in SLC35A2, a gene associated with glycosylation defects and rare X‐linked epileptic encephalopathies. Nonsynonymous variants in SLC35A2 were detected in resected brain, and absent from leukocytes, in 3 of 18 individuals (17%) with NLFE, 1 female and 2 males, with variant allele frequencies (VAFs) in brain‐derived DNA of 2 to 14%. Pathologic evaluation revealed focal cortical dysplasia type Ia (FCD1a) in 2 of the 3 NLFE cases. In the MCD cohort, nonsynonymous variants in SCL35A2 were detected in the brains of 2 males with intractable epilepsy, developmental delay, and magnetic resonance imaging suggesting FCD, with VAFs of 19 to 53%; Evidence for FCD was not observed in either brain tissue specimen.
Interpretation
We report somatic variants in SLC35A2 as an explanation for a substantial fraction of NLFE, a largely unexplained condition, as well as focal MCD, previously shown to result from somatic mutation but until now only in PI3K‐AKT‐mTOR pathway genes. Collectively, our findings suggest a larger role than previously recognized for glycosylation defects in the intractable epilepsies. Ann Neurol 2018
Ferroptosis is mediated by lipid peroxidation of phospholipids containing polyunsaturated fatty acyl moieties. Glutathione, the key cellular antioxidant capable of inhibiting lipid peroxidation via ...the activity of the enzyme glutathione peroxidase 4 (GPX-4), is generated directly from the sulfur-containing amino acid cysteine, and indirectly from methionine via the transsulfuration pathway. Herein we show that cysteine and methionine deprivation (CMD) can synergize with the GPX4 inhibitor RSL3 to increase ferroptotic cell death and lipid peroxidation in both murine and human glioma cell lines and in ex vivo organotypic slice cultures. We also show that a cysteine-depleted, methionine-restricted diet can improve therapeutic response to RSL3 and prolong survival in a syngeneic orthotopic murine glioma model. Finally, this CMD diet leads to profound in vivo metabolomic, proteomic and lipidomic alterations, highlighting the potential for improving the efficacy of ferroptotic therapies in glioma treatment with a non-invasive dietary modification.
The ability of gliomas to invade the brain limits the efficacy of standard therapies. In this study, we have examined glioma migration in living brain tissue by using two novel in vivo model systems. ...Within the brain, glioma cells migrate like nontransformed, neural progenitor cells-extending a prominent leading cytoplasmic process followed by a burst of forward movement by the cell body that requires myosin II. In contrast, on a two-dimensional surface, glioma cells migrate more like fibroblasts, and they do not require myosin II to move. To explain this phenomenon, we studied glioma migration through a series of synthetic membranes with defined pore sizes. Our results demonstrate that the A and B isoforms of myosin II are specifically required when a glioma cell has to squeeze through pores smaller than its nuclear diameter. They support a model in which the neural progenitor-like mode of glioma invasion and the requirement for myosin II represent an adaptation needed to move within the brain, which has a submicrometer effective pore size. Furthermore, the absolute requirement for myosin II in brain invasion underscores the importance of this molecular motor as a potential target for new anti-invasive therapies to treat malignant brain tumors.
Ribosome profiling has emerged as a powerful tool for genome-wide measurements of translation, but library construction requires multiple ligation steps and remains cumbersome relative to more ...conventional deep-sequencing experiments. We report a new, ligation-free approach to ribosome profiling that does not require ligation. Library construction for ligation-free ribosome profiling can be completed in one day with as little as 1 ng of purified RNA footprints. We apply ligation-free ribosome profiling to mouse brain tissue to identify new patterns of cell type-specific translation and test its ability to identify translational targets of mTOR signaling in the brain.
Abstract
Aurora kinase A (AURKA) has emerged as a drug target for glioblastoma (GBM). However, resistance to therapy remains a critical issue. By integration of transcriptome, chromatin ...immunoprecipitation sequencing (CHIP-seq), Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq), proteomic and metabolite screening followed by carbon tracing and extracellular flux analyses we show that genetic and pharmacological AURKA inhibition elicits metabolic reprogramming mediated by inhibition of MYC targets and concomitant activation of Peroxisome Proliferator Activated Receptor Alpha (PPARA) signaling. While glycolysis is suppressed by AURKA inhibition, we note an increase in the oxygen consumption rate fueled by enhanced fatty acid oxidation (FAO), which was accompanied by an increase of Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α). Combining AURKA inhibitors with inhibitors of FAO extends overall survival in orthotopic GBM PDX models. Taken together, these data suggest that simultaneous targeting of oxidative metabolism and AURKAi might be a potential novel therapy against recalcitrant malignancies.
Common approaches to gene signature discovery in single‐cell RNA‐sequencing (scRNA‐seq) depend upon predefined structures like clusters or pseudo‐temporal order, require prior normalization, or do ...not account for the sparsity of single‐cell data. We present single‐cell hierarchical Poisson factorization (scHPF), a Bayesian factorization method that adapts hierarchical Poisson factorization (Gopalan et al, , Proceedings of the 31st Conference on Uncertainty in Artificial Intelligence, 326) for de novo discovery of both continuous and discrete expression patterns from scRNA‐seq. scHPF does not require prior normalization and captures statistical properties of single‐cell data better than other methods in benchmark datasets. Applied to scRNA‐seq of the core and margin of a high‐grade glioma, scHPF uncovers marked differences in the abundance of glioma subpopulations across tumor regions and regionally associated expression biases within glioma subpopulations. scHFP revealed an expression signature that was spatially biased toward the glioma‐infiltrated margins and associated with inferior survival in glioblastoma.
Synopsis
Single‐cell Hierarchical Poisson Factorization (scHPF) is a Bayesian factorization method for de novo discovery of both continuously varying and subpopulation‐specific expression patterns in single‐cell RNA‐sequencing data.
scHPF takes genome‐wide molecular counts as input, avoids prior normalization, captures the statistical structure of scRNA‐seq data better than alternative methods, and has fast, memory‐efficient inference on sparse scRNA‐seq data.
Applied to scRNA‐seq of a spatially sampled high‐grade glioma, scHPF reveals regional differences in lineage resemblance within glioma subpopulations.
One regionally biased gene signature enriched in astrocyte‐like glioma cells is associated with poor survival in glioblastoma.
Single‐cell Hierarchical Poisson Factorization (scHPF) is a Bayesian factorization method for de novo discovery of both continuously varying and subpopulation‐specific expression patterns in single‐cell RNA‐sequencing data.
Macrophages are the most common infiltrating immune cells in gliomas and play a wide variety of pro-tumor and anti-tumor roles. However, the different subpopulations of macrophages and their effects ...on the tumor microenvironment remain poorly understood.
We combined new and previously published single-cell RNA-seq data from 98,015 single cells from a total of 66 gliomas to profile 19,331 individual macrophages.
Unsupervised clustering revealed a pro-tumor subpopulation of bone marrow-derived macrophages characterized by the scavenger receptor MARCO, which is almost exclusively found in IDH1-wild-type glioblastomas. Previous studies have implicated MARCO as an unfavorable marker in melanoma and non-small cell lung cancer; here, we find that bulk MARCO expression is associated with worse prognosis and mesenchymal subtype. Furthermore, MARCO expression is significantly altered over the course of treatment with anti-PD1 checkpoint inhibitors in a response-dependent manner, which we validate with immunofluorescence imaging.
These findings illustrate a novel macrophage subpopulation that drives tumor progression in glioblastomas and suggest potential therapeutic targets to prevent their recruitment.
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