Active neurons exert a mitogenic effect on normal neural precursor and oligodendroglial precursor cells, the putative cellular origins of high-grade glioma (HGG). By using optogenetic control of cortical neuronal activity in a patient-derived pediatric glioblastoma xenograft model, we demonstrate that active neurons similarly promote HGG proliferation and growth in vivo. Conditioned medium from optogenetically stimulated cortical slices promoted proliferation of pediatric and adult patient-derived HGG cultures, indicating secretion of activity-regulated mitogen(s). The synaptic protein neuroligin-3 (NLGN3) was identified as the leading candidate mitogen, and soluble NLGN3 was sufficient and necessary to promote robust HGG cell proliferation. NLGN3 induced PI3K-mTOR pathway activity and feedforward expression of NLGN3 in glioma cells. NLGN3 expression levels in human HGG negatively correlated with patient overall survival. These findings indicate the important role of active neurons in the brain tumor microenvironment and identify secreted NLGN3 as an unexpected mechanism promoting neuronal activity-regulated cancer growth.

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Purpose To investigate ferumoxytol-enhanced MRI as a noninvasive imaging biomarker of macrophages in adults with high-grade gliomas. Materials and Methods In this prospective study, adults with high-grade gliomas were enrolled between July 2015 and July 2017. Each participant was administered intravenous ferumoxytol (5 mg/kg) and underwent 3.0-T MRI 24 hours later. Two sites in each tumor were selected for intraoperative sampling on the basis of the degree of ferumoxytol-induced signal change. Susceptibility and the relaxation rates R2* (1/T2*) and R2 (1/T2) were obtained by region-of-interest analysis by using the respective postprocessed maps. Each sample was stained with Prussian blue, CD68, CD163, and glial fibrillary acidic protein. Pearson correlation and linear mixed models were performed to assess the relationship between imaging measurements and number of 400× magnification high-power fields with iron-containing macrophages. Results Ten adults (four male participants [mean age, 65 years ± 9 {standard deviation}; age range, 57-74 years] and six female participants [mean age, 53 years ± 12 years; age range, 32-65 years]; mean age of all participants, 58 years ± 12 [age range, 32-74 years]) with high-grade gliomas were included. Significant positive correlations were found between susceptibility, R2*, and R2' and the number of high-power fields with CD163-positive (r range, 0.64-0.71; P < .01) and CD68-positive (r range, 0.55-0.57; P value range, .01-.02) iron-containing macrophages. No significant correlation was found between R2 and CD163-positive (r = 0.33; P = .16) and CD68-positive (r = 0.24; P = .32) iron-containing macrophages. Similar significance results were obtained with linear mixed models. At histopathologic analysis, iron particles were found only in macrophages; none was found in glial fibrillary acidic protein-positive tumor cells. Conclusion MRI measurements of susceptibility, R2*, and R2' (R2* - R2) obtained after ferumoxytol administration correlate with iron-containing macrophage concentration, and this shows their potential as quantitative imaging markers of macrophages in malignant gliomas. © RSNA, 2018 Online supplemental material is available for this article.

Medulloblastoma is the most common malignant brain tumor of childhood. Group 3 medulloblastoma, the most aggressive molecular subtype, frequently disseminates through the leptomeningeal cerebral spinal fluid (CSF) spaces in the brain and spinal cord. The mechanism of dissemination through the CSF remains poorly understood, and the molecular pathways involved in medulloblastoma metastasis and self-renewal are largely unknown. Here we show that NOTCH1 signaling pathway regulates both the initiation of metastasis and the self-renewal of medulloblastoma. We identify a mechanism in which NOTCH1 activates BMI1 through the activation of TWIST1. NOTCH1 expression and activity are directly related to medulloblastoma metastasis and decreased survival rate of tumor-bearing mice. Finally, medulloblastoma-bearing mice intrathecally treated with anti-NRR1, a NOTCH1 blocking antibody, present lower frequency of spinal metastasis and higher survival rate. These findings identify NOTCH1 as a pivotal driver of Group 3 medulloblastoma metastasis and self-renewal, supporting the development of therapies targeting this pathway.

Diffuse intrinsic pontine glioma (DIPG) is a universally fatal malignancy of the childhood central nervous system, with a median overall survival of 9-11 months. We have previously shown that primary DIPG tissue contains numerous tumor-associated macrophages, and substantial work has demonstrated a significant pathological role for adult glioma-associated macrophages. However, work over the past decade has highlighted many molecular and genomic differences between pediatric and adult high-grade gliomas. Thus, we directly compared inflammatory characteristics of DIPG and adult glioblastoma (GBM). We found that the leukocyte (CD45+) compartment in primary DIPG tissue samples is predominantly composed of CD11b + macrophages, with very few CD3+ T-lymphocytes. In contrast, T-lymphocytes are more abundant in adult GBM tissue samples. RNA sequencing of macrophages isolated from primary tumor samples revealed that DIPG- and adult GBM-associated macrophages both express gene programs related to ECM remodeling and angiogenesis, but DIPG-associated macrophages express substantially fewer inflammatory factors than their adult GBM counterparts. Examining the secretome of glioma cells, we found that patient-derived DIPG cell cultures secrete markedly fewer cytokines and chemokines than patient-derived adult GBM cultures. Concordantly, bulk and single-cell RNA sequencing data indicates low to absent expression of chemokines and cytokines in DIPG. Together, these observations suggest that the inflammatory milieu of the DIPG tumor microenvironment is fundamentally different than adult GBM. The low intrinsic inflammatory signature of DIPG cells may contribute to the lack of lymphocytes and non-inflammatory phenotype of DIPG-associated microglia/macrophages. Understanding the glioma subtype-specific inflammatory milieu may inform the design and application of immunotherapy-based treatments.

The p53 transcription factor is a critical barrier to pancreatic cancer progression. To unravel mechanisms of p53-mediated tumor suppression, which have remained elusive, we analyzed pancreatic cancer development in mice expressing p53 transcriptional activation domain (TAD) mutants. Surprisingly, the p5353,54 TAD2 mutant behaves as a "super-tumor suppressor," with an enhanced capacity to both suppress pancreatic cancer and transactivate select p53 target genes, including Ptpn14. Ptpn14 encodes a negative regulator of the Yap oncoprotein and is necessary and sufficient for pancreatic cancer suppression, like p53. We show that p53 deficiency promotes Yap signaling and that PTPN14 and TP53 mutations are mutually exclusive in human cancers. These studies uncover a p53-Ptpn14-Yap pathway that is integral to p53-mediated tumor suppression.

EGF receptor variant III (EGFRvIII) is the most common variant of the EGF receptor observed in human tumors. It results from the in frame deletion of exons 2-7 and the generation of a novel glycine residue at the junction of exons 1 and 8. This novel juxtaposition of amino acids within the extra-cellular domain of the EGF receptor creates a tumor specific and immunogenic epitope. EGFRvIII expression has been seen in many tumor types including glioblastoma multiforme (GBM), breast adenocarcinoma, non-small cell lung carcinoma, ovarian adenocarcinoma and prostate cancer, but has been rarely observed in normal tissue. Because this variant is tumor specific and highly immunogenic, it can be used for both a diagnostic marker as well as a target for immunotherapy. Unfortunately many of the monoclonal and polyclonal antibodies directed against EGFRvIII have cross reactivity to wild type EGFR or other non-specific proteins. Furthermore, a monoclonal antibody to EGFRvIII is not readily available to the scientific community.

Glioblastoma multiforme (GBM) is a highly aggressive form of brain cancer associated with a very poor prognosis. Recently, the initiation and growth of GBM has been linked to brain tumor-initiating cells (BTICs), which are poorly differentiated and share features with neural stem cells (NSCs). Here we describe a kinome-wide RNA interference screen to identify factors that control the tumorigenicity of BTICs. We identified several genes whose silencing induces differentiation of BTICs derived from multiple GBM patients. In particular, knockdown of the adaptor protein TRRAP significantly increased differentiation of cultured BTICs, sensitized the cells to apoptotic stimuli, and negatively affected cell cycle progression. TRRAP knockdown also significantly suppressed tumor formation upon intracranial BTIC implantation into mice. Together, these findings support a critical role for TRRAP in maintaining a tumorigenic, stem cell-like state.

Salla disease and infantile sialic acid storage disease are autosomal recessive lysosomal storage disorders caused by mutations in the gene encoding sialin, a membrane protein that transports free sialic acid out of the lysosome after it is cleaved from sialoglycoconjugates undergoing degradation. Accumulation of sialic acid in lysosomes defines these disorders, and the clinical phenotype is characterized by neurodevelopmental defects, including severe CNS hypomyelination. In this study, we used a sialin-deficient mouse to address how loss of sialin leads to the defect in myelination. Behavioral analysis of the sialin(-/-) mouse demonstrates poor coordination, seizures, and premature death. Analysis by histology, electron microscopy, and Western blotting reveals a decrease in myelination of the CNS but normal neuronal cytoarchitecture and normal myelination of the PNS. To investigate potential mechanisms underlying CNS hypomyelination, we studied myelination and oligodendrocyte development in optic nerves. We found reduced numbers of myelinated axons in optic nerves from sialin(-/-) mice, but the myelin that was present appeared grossly normal. Migration and density of oligodendrocyte precursor cells were normal; however, a marked decrease in the number of postmitotic oligodendrocytes and an associated increase in the number of apoptotic cells during the later stages of myelinogenesis were observed. These findings suggest that a defect in maturation of cells in the oligodendrocyte lineage leads to increased apoptosis and underlies the myelination defect associated with sialin loss.

Bioenergetic failure and oxidative stress are common pathological hallmarks of amyotrophic lateral sclerosis (ALS), but whether these could be targeted effectively for novel therapeutic intervention needs to be determined. One of the reported contributors to ALS pathology is mitochondrial dysfunction associated with excessive mitochondrial fission and fragmentation, which is predominantly mediated by Drp1 hyperactivation. Here, we determined whether inhibition of excessive fission by inhibiting Drp1/Fis1 interaction affects disease progression. We observed mitochondrial excessive fragmentation and dysfunction in several familial forms of ALS patient-derived fibroblasts as well as in cultured motor neurons expressing SOD1 mutant. In both cell models, inhibition of Drp1/Fis1 interaction by a selective peptide inhibitor, P110, led to a significant reduction in reactive oxygen species levels, and to improvement in mitochondrial structure and functions. Sustained treatment of mice expressing G93A SOD1 mutation with P110, beginning at the onset of disease symptoms at day 90, produced an improvement in motor performance and survival, suggesting that Drp1 hyperactivation may be an attractive target in the treatment of ALS patients.

Current in vivo neuroimaging techniques provide limited field of view or spatial resolution and often require exogenous contrast. These limitations prohibit detailed structural imaging across wide fields of view and hinder intraoperative tumor margin detection. Here we present a novel neuroimaging technique, speckle-modulating optical coherence tomography (SM-OCT), which allows us to image the brains of live mice and ex vivo human samples with unprecedented resolution and wide field of view using only endogenous contrast. The increased visibility provided by speckle elimination reveals white matter fascicles and cortical layer architecture in brains of live mice. To our knowledge, the data reported herein represents the highest resolution imaging of murine white matter structure achieved in vivo across a wide field of view of several millimeters. When applied to an orthotopic murine glioblastoma xenograft model, SM-OCT readily identifies brain tumor margins with resolution of approximately 10 μm. SM-OCT of ex vivo human temporal lobe tissue reveals fine structures including cortical layers and myelinated axons. Finally, when applied to an ex vivo sample of a low-grade glioma resection margin, SM-OCT is able to resolve the brain tumor margin. Based on these findings, SM-OCT represents a novel approach for intraoperative tumor margin detection and in vivo neuroimaging.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Since surviving patients experience severe neurocognitive disabilities, better and more effective treatments are needed to enhance their quality of life. Casein kinase 2 (CK2) is known to regulate cell growth and survival in multiple cancers; however, the role of CK2 in MB is currently being studied. In this study, we verified the importance of CK2 in MB tumorigenesis and discovered that inhibition of CK2 using the small molecule inhibitor, CX-4945, can sensitize MB cells to a well-known and tolerated chemotherapeutic, temozolomide (TMZ). To study the role of CK2 in MB we modulated CK2 expression in multiple MB cells. Exogenous expression of CK2 enhanced cell growth and tumor growth in mice, while depletion or inhibition of CK2 expression decreased MB tumorigenesis. Treatment with CX-4945 reduced MB growth and increased apoptosis. We conducted a high-throughput screen where 4000 small molecule compounds were analyzed to identify compounds that increased the anti-tumorigenic properties of CX-4945. TMZ was found to work synergistically with CX-4945 to decrease cell survival and increase apoptosis in MB cells. O-6-methylguanine-DNA methyltransferase (MGMT) activity is directly correlated to TMZ sensitivity. We found that loss of CK2 activity reduced β-catenin expression, a known MGMT regulator, which in turn led to a decrease in MGMT expression and an increased sensitivity to TMZ. Our findings show that CK2 is important for MB maintenance and that treatment with CX-4945 can sensitize MB cells to TMZ treatment.

The mammalian brain contains neurogenic niches that comprise neural stem cells and other cell types. Neurogenic niches become less functional with age, but how they change during ageing remains unclear. Here we perform single-cell RNA sequencing of young and old neurogenic niches in mice. The analysis of 14,685 single-cell transcriptomes reveals a decrease in activated neural stem cells, changes in endothelial cells and microglia, and an infiltration of T cells in old neurogenic niches. T cells in old brains are clonally expanded and are generally distinct from those in old blood, which suggests that they may experience specific antigens. T cells in old brains also express interferon-γ, and the subset of neural stem cells that has a high interferon response shows decreased proliferation in vivo. We find that T cells can inhibit the proliferation of neural stem cells in co-cultures and in vivo, in part by secreting interferon-γ. Our study reveals an interaction between T cells and neural stem cells in old brains, opening potential avenues through which to counteract age-related decline in brain function.

Recent evidence suggests that the accumulation of iron, specifically ferrous Fe2+, may play a role in the development and progression of neurodegeneration in Alzheimer's disease (AD) through the production of oxidative stress.

Clinical imaging performance using a fluorescent antibody was compared across 3 cancers to elucidate physical and biologic factors contributing to differential translation of epidermal growth factor receptor (EGFR) expression to macroscopic fluorescence in tumors. Methods: Thirty-one patients with high-grade glioma (HGG, n = 5), head-and-neck squamous cell carcinoma (HNSCC, n = 23), or lung adenocarcinoma (LAC, n = 3) were systemically infused with 50 mg of panitumumab-IRDye800 1-3 d before surgery. Intraoperative open-field fluorescent images of the surgical field were acquired, with imaging device settings and operating room lighting conditions being tested on tissue-mimicking phantoms. Fluorescence contrast and margin size were measured on resected specimen surfaces. Antibody distribution and EGFR immunoreactivity were characterized in macroscopic and microscopic histologic structures. The integrity of the blood-brain barrier was examined via tight junction protein (Claudin-5) expression with immunohistochemistry. Stepwise multivariate linear regression of biologic variables was performed to identify independent predictors of panitumumab-IRDye800 concentration in tissue. Results: Optimally acquired at the lowest gain for tumor detection with ambient light, intraoperative fluorescence imaging enhanced tissue-size dependent tumor contrast by 5.2-fold, 3.4-fold, and 1.4-fold in HGG, HNSCC, and LAC, respectively. Tissue surface fluorescence target-to-background ratio correlated with margin size and identified 78%-97% of at-risk resection margins ex vivo. In 4-μm-thick tissue sections, fluorescence detected tumor with 0.85-0.89 areas under the receiver-operating-characteristic curves. Preferential breakdown of blood-brain barrier in HGG improved tumor specificity of intratumoral antibody distribution relative to that of EGFR (96% vs. 80%) despite its reduced concentration (3.9 ng/mg of tissue) compared with HNSCC (8.1 ng/mg) and LAC (6.3 ng/mg). Cellular EGFR expression, tumor cell density, plasma antibody concentration, and delivery barrier were independently associated with local intratumoral panitumumab-IRDye800 concentration, with 0.62 goodness of fit of prediction. Conclusion: In multicancer clinical imaging of a receptor-ligand-based molecular probe, plasma antibody concentration, delivery barrier, and intratumoral EGFR expression driven by cellular biomarker expression and tumor cell density led to heterogeneous intratumoral antibody accumulation and spatial distribution whereas tumor size, resection margin, and intraoperative imaging settings substantially influenced macroscopic tumor contrast.

Among high-grade brain tumors, glioblastoma is particularly difficult to treat, in part due to its highly infiltrative nature which contributes to the malignant phenotype and high mortality in patients. In order to better understand the signaling pathways underlying glioblastoma invasion, we performed the first large-scale CRISPR-Cas9 loss of function screen specifically designed to identify genes that facilitate cell invasion. We tested 4,574 genes predicted to be involved in trafficking and motility. Using a transwell invasion assay, we discovered 33 genes essential for invasion. Of the 11 genes we selected for secondary testing using a wound healing assay, 6 demonstrated a significant decrease in migration. The strongest regulator of invasion was mitogen-activated protein kinase 4 (MAP4K4). Targeting of MAP4K4 with single guide RNAs or a MAP4K4 inhibitor reduced migration and invasion in vitro. This effect was consistent across three additional patient derived glioblastoma cell lines. Analysis of epithelial-mesenchymal transition markers in U138 cells with lack or inhibition of MAP4K4 demonstrated protein expression consistent with a non-invasive state. Importantly, MAP4K4 inhibition limited migration in a subset of human glioma organotypic slice cultures. Our results identify MAP4K4 as a novel potential therapeutic target to limit glioblastoma invasion.

In this work, we propose an approach to generate whole-slide image (WSI) tiles by using deep generative models infused with matched gene expression profiles. First, we train a variational autoencoder (VAE) that learns a latent, lower-dimensional representation of multi-tissue gene expression profiles. Then, we use this representation to infuse generative adversarial networks (GANs) that generate lung and brain cortex tissue tiles, resulting in a new model that we call RNA-GAN. Tiles generated by RNA-GAN were preferred by expert pathologists compared with tiles generated using traditional GANs, and in addition, RNA-GAN needs fewer training epochs to generate high-quality tiles. Finally, RNA-GAN was able to generalize to gene expression profiles outside of the training set, showing imputation capabilities. A web-based quiz is available for users to play a game distinguishing real and synthetic tiles: https://rna-gan.stanford.edu/, and the code for RNA-GAN is available here: https://github.com/gevaertlab/RNA-GAN.

E2F transcription factors are central regulators of cell division and cell fate decisions. E2F4 often represents the predominant E2F activity in cells. E2F4 is a transcriptional repressor implicated in cell cycle arrest and whose repressive activity depends on its interaction with members of the RB family. Here we show that E2F4 is important for the proliferation and the survival of mouse embryonic stem cells. In these cells, E2F4 acts in part as a transcriptional activator that promotes the expression of cell cycle genes. This role for E2F4 is independent of the RB family. Furthermore, E2F4 functionally interacts with chromatin regulators associated with gene activation and we observed decreased histone acetylation at the promoters of cell cycle genes and E2F targets upon loss of E2F4 in RB family-mutant cells. Taken together, our findings uncover a non-canonical role for E2F4 that provide insights into the biology of rapidly dividing cells.

The limited entry of anticancer drugs into the central nervous system represents a special therapeutic challenge for patients with brain metastases and is primarily due to the blood brain barrier (BBB). Albumin-bound Evans blue (EB) dye is too large to cross the BBB but can grossly stain tissue blue when the BBB is disrupted. The course of tumor development and the integrity of the BBB were studied in three preclinical breast cancer brain metastasis (BCBM) models. A luciferase-transduced braintropic clone of MDA-231 cell line was used. Nude mice were subjected to stereotactic intracerebral inoculation, mammary fat pad-derived tumor fragment implantation, or carotid artery injections. EB was injected 30 min prior to euthanasia at various timepoints for each of the BCBM model animals. Serial bioluminescent imaging demonstrated exponential tumor growth in all models. Carotid BCBM appeared as diffuse multifocal cell clusters. EB aided the localization of metastases ex vivo. Tumor implants stained blue at 7 days whereas gross staining was not evident until day 14 in the stereotactic model and day 28 for the carotid model. EB assessment of the integrity of the BBB provides useful information relevant to drug testing in preclinical BCBM models.

Routine brain MRI surveillance frequently diagnoses small, asymptomatic brain metastases from non-small cell lung cancer (NSCLC) that are effectively treated with stereotactic radiosurgery (SRS). A subset of patients, however, may die prior to the onset of symptoms. This study identifies clinical features that distinguish neurologically-asymptomatic NSCLC brain metastases patients that die prior to routine 3 month follow-up after SRS.