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Meningiomas are the most common primary intracranial tumors. There are no effective medical therapies for meningioma patients, and new treatments have been encumbered by limited understanding of meningioma biology. Here, we use DNA methylation profiling on 565 meningiomas integrated with genetic, transcriptomic, biochemical, proteomic and single-cell approaches to show meningiomas are composed of three DNA methylation groups with distinct clinical outcomes, biological drivers and therapeutic vulnerabilities. Merlin-intact meningiomas (34%) have the best outcomes and are distinguished by NF2/Merlin regulation of susceptibility to cytotoxic therapy. Immune-enriched meningiomas (38%) have intermediate outcomes and are distinguished by immune infiltration, HLA expression and lymphatic vessels. Hypermitotic meningiomas (28%) have the worst outcomes and are distinguished by convergent genetic and epigenetic mechanisms driving the cell cycle and resistance to cytotoxic therapy. To translate these findings into clinical practice, we show cytostatic cell cycle inhibitors attenuate meningioma growth in cell culture, organoids, xenografts and patients.

Intratumor heterogeneity underlies cancer evolution and treatment resistance1–5, but targetable mechanisms driving intratumor heterogeneity are poorly understood. Meningiomas are the most common primary intracranial tumors and are resistant to all current medical therapies6,7. High-grade meningiomas cause significant neurological morbidity and mortality and are distinguished from low-grade meningiomas by increased intratumor heterogeneity arising from clonal evolution and divergence8. Here we integrate spatial transcriptomic and spatial protein profiling approaches across high-grade meningiomas to identify genomic, biochemical, and cellular mechanisms linking intratumor heterogeneity to the molecular, temporal, and spatial evolution of cancer. We show divergent intratumor gene and protein expression programs distinguish high-grade meningiomas that are otherwise grouped together by current clinical classification systems. Analyses of matched pairs of primary and recurrent meningiomas reveal spatial expansion of sub-clonal copy number variants underlies treatment resistance. Multiplexed sequential immunofluorescence (seqIF) and spatial deconvolution of meningioma single-cell RNA sequencing show decreased immune infiltration, decreased MAPK signaling, increased PI3K-AKT signaling, and increased cell proliferation drive meningioma recurrence. To translate these findings to clinical practice, we use epigenetic editing and lineage tracing approaches in meningioma organoid models to identify new molecular therapy combinations that target intratumor heterogeneity and block tumor growth. Our results establish a foundation for personalized medical therapy to treat patients with high-grade meningiomas and provide a framework for understanding therapeutic vulnerabilities driving intratumor heterogeneity and tumor evolution.

Gliomas synaptically integrate into neural circuits1,2. Previous research has demonstrated bidirectional interactions between neurons and glioma cells, with neuronal activity driving glioma growth1–4 and gliomas increasing neuronal excitability2,5–8. Here we sought to determine how glioma-induced neuronal changes influence neural circuits underlying cognition and whether these interactions influence patient survival. Using intracranial brain recordings during lexical retrieval language tasks in awake humans together with site-specific tumour tissue biopsies and cell biology experiments, we find that gliomas remodel functional neural circuitry such that task-relevant neural responses activate tumour-infiltrated cortex well beyond the cortical regions that are normally recruited in the healthy brain. Site-directed biopsies from regions within the tumour that exhibit high functional connectivity between the tumour and the rest of the brain are enriched for a glioblastoma subpopulation that exhibits a distinct synaptogenic and neuronotrophic phenotype. Tumour cells from functionally connected regions secrete the synaptogenic factor thrombospondin-1, which contributes to the differential neuron–glioma interactions observed in functionally connected tumour regions compared with tumour regions with less functional connectivity. Pharmacological inhibition of thrombospondin-1 using the FDA-approved drug gabapentin decreases glioblastoma proliferation. The degree of functional connectivity between glioblastoma and the normal brain negatively affects both patient survival and performance in language tasks. These data demonstrate that high-grade gliomas functionally remodel neural circuits in the human brain, which both promotes tumour progression and impairs cognition.

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BACKGROUND The Hedgehog pathway directs gene expression programs that are essential for glioblastoma stem cells in vitro, but a clinical trial suggested Hedgehog pathway inhibition does not improve glioblastoma outcomes (ABTC-0904). In pancreatic and bladder cancer, Hedgehog signaling through the tumor microenvironment induces stromal morphogens to inhibit cancer stem cell proliferation. Thus, we hypothesized Hedgehog signaling through the glioblastoma microenvironment may inhibit glioblastoma stem cell proliferation in vivo. METHODS Hedgehog signaling was studied using patient-derived, fluorescently-labeled IDH wild-type glioblastoma cells (GBM6, SF11956, SF11907, GPMP004, GPMP005) in vitro, in vivo after intracranial implantation in mice, or in 3D co-culture with iPSC-derived organoids comprised of human astrocytes or neurons. Genetic loss-of-function experiments were performed using CRISPR interference. Cell proliferation, stem cell marker expression, Sonic Hedgehog (SHH) expression, or primary cilia architecture were assessed using immunofluorescence, confocal microscopy, or live-cell imaging. Hedgehog signaling or morphogen expression were assessed using QPCR or single-cell RNA sequencing. Pharmacologic experiments were performed using vismodegib to inhibit the Hedgehog pathway, or FK506 to induce stromal morphogen expression. RESULTS Vismodegib increased glioblastoma cell proliferation and stem cell marker expression in vivo or in co-culture with astrocyte organoids. Single-cell RNA sequencing demonstrated glioblastoma cell co-culture with astrocyte organoids induced Hedgehog target genes and stem cell markers in glioblastoma cells, or stromal morphogens in astrocytes, which also expressed primary cilia. CRISPRi suppression of SHH in glioblastoma cells increased glioblastoma cell proliferation and stem cell marker expression in co-culture with astrocyte organoids. Neither vismodegib nor SHH suppression induced glioblastoma cell proliferation or stem cell marker expression in vitro or in co-culture with neuron organoids. FK506 induced astrocyte morphogen expression, inhibiting glioblastoma cell proliferation, stem cell marker expression, and the effects of vismodegib in co-culture with astrocyte organoids. CONCLUSIONS SHH from glioblastoma cells signals through astrocytes to inhibit cancer stem cell proliferation.

INTRODUCTION Fractionated radiotherapy is a first-line treatment for glioblastoma, but daily ionizing radiation prevents durable immune infiltration of the tumor microenvironment. Hypofractionated radiotherapy is used to treat glioblastomas at recurrence, but the impact of hypofractionated radiotherapy on the glioblastoma immune microenvironment is incompletely understood. Here we define immune microenvironment changes across multiple immunocompetent intracranial mouse models of glioblastoma after treatment with ionizing radiation mimicking stereotactic radiosurgery (SRS) in humans. METHODS Syngeneic GL261 (3x105 cells/mouse) or SB28 glioblastoma cells (3x104 cells/mouse) were implanted into the frontal lobe of immunocompetent C57BL/6J mice (18 mice/arm x 4 arms). Intracranial bioluminescence was used to assess glioblastoma growth. After tumor engraftment, glioblastomas were treated with conformal SRS (18Gy/1Fx) or sham. Glioblastomas were collected for histologic, single-cell, or molecular analyses 5 days after treatment (6 mice/arm) or at the time of euthanasia after monitoring for survival (12 mice/arm). Glioblastoma immune microenvironment responses were assessed using (1) H&E, (2) single cell mass cytometry (CyTOF) or IHC to define or validate immune cell changes, respectively, or (3) multiplexed cytokine assays to elucidate molecular mechanisms reprograming the glioblastoma immune microenvironment in response to SRS. RESULTS SRS attenuated glioblastoma growth and prolonged survival compared to sham treatment in both immunocompetent intracranial mouse models (GL261: 14 days versus 27 days, p< 0.001, SB28: 19 days versus 22 days, p=0.001). CyTOF showed SRS decreased immunosuppressive macrophage infiltration and increased microglia or CD8+ T cell infiltration of the glioblastoma immune microenvironment. Histologic analyses validated T cell and microglia infiltration after SRS. Glioblastoma cytokine analysis revealed inhibition of pro-tumor/anti-inflammatory cytokines (IL6, LIF) after SRS. CONCLUSIONS Single-fraction SRS durably reprograms glioblastoma macrophage, microglia, and CD8+ T cell populations in preclinical models, suggesting SRS or inhibition of pro-tumor/anti-inflammatory mechanisms underlying the immunosuppressive glioblastoma microenvironment represent immunogenic therapies that may offer a benefit to patients with glioblastoma.

Intratumor heterogeneity drives cancer evolution and resistance to therapy, but unbiased approaches to elucidate mechanisms driving intratumor heterogeneity have been lacking. Here, we integrate spatial gene expression programs and protein signaling mechanisms across 16 meningioma samples to define how intratumor heterogeneity drives molecular classification, temporal evolution, or spatial evolution of the most common primary intracranial tumor. Spatial transcriptomic analysis was performed on 38,718 regions and spatial profiling of 72 proteins comprising proliferation, stress, microenvironment, immune, or signaling modules was performed on 82 regions. DNA methylation, copy number variant, targeted gene expression profiling, targeted DNA sequencing, histologic, or immunohistochemical analyses (Ki67, H3K27me3, p16) were performed on all meningiomas to study intratumor heterogeneity in the context of pre-existing classification schemes. Primary meningioma cells, CRISPR interference, pharmacology, and 3D co-culture models were used for mechanistic and functional validation. Spatial analyses revealed significant intratumor or intertumor heterogeneity irrespective of meningioma histologic subtype or grade, DNA methylation group (Merlin-intact, Immune-enriched, Hypermitotic), copy number variant (1p loss, 1q gain, 6p gain, 9p loss, 14q loss, 22q loss), gene expression risk score, or driver mutation (NF2, CDKN2A/B, TERT promoter, BAP1, SMARCB1, ARID1A). Spatial analyses on paired histologically or molecularly distinct regions from individual meningiomas (n=4), or on paired primary and recurrent meningioma samples (n=9), revealed conserved proliferation, immune, or signaling mechanisms underlying meningioma evolution. Mechanistic and functional studies validated therapeutic vulnerabilities across spatial clusters to combinations of FDA-approved small molecules inhibiting the cell cycle (abemaciclib), epigenetic regulators (vorinostat), the DNA damage response (nariparib), MAPK signaling (erlotinib, selumetinib), or PI3K-AKT signaling (copanlisib). In summary, these data define spatial gene expression programs and protein signaling mechanisms driving meningioma evolution and therapeutic vulnerability, shedding light on past clinical failures and elucidating novel combinations of concurrent or sequential molecular therapies to treat meningiomas that are resistant to standard interventions.

Boron neutron capture therapy (BNCT) is an encouraging therapeutic modality for cancer treatment. Prostate-specific membrane antigen (PSMA) is a cell membrane protein that is abundantly overexpressed in prostate cancer and can be targeted with radioligand therapies to stimulate clinical responses in patients. In principle, a spatially targeted neutron beam together with specifically targeted PSMA ligands could enable prostate cancer-targeted BNCT. Thus, we developed and tested PSMA-targeted poly(lactide

Glioblastoma responses to immune checkpoint inhibition (ICI) are rare, and the molecular mechanisms underlying ICI responses are incompletely understood. Thus, serial glioblastoma samples are valuable resources for identifying biomarkers or therapeutic targets to increase the efficacy of ICI in patients with glioblastoma. We obtained paired glioblastoma samples from 7 patients who underwent sequential surgery, ICI, and eventual salvage surgery for recurrence. Patients were distinguished as ICI responders (n=3) or non-responders (n=4) based on (1) MRI evidence of tumor stability/reduction over 6+ months after ICI, or (2) pathologic evidence of predominant treatment effect at the time of salvage surgery after ICI. FFPE sections from each tumor (n=14) were stained using H&E or IHC/IF for macrophages/microglia (CD68) or T cells (CD3) and analyzed using light or fluorescence microscopy. Six regions-of-interest (ROIs) comprising viable tumor were selected neuropathologist from each sample (n=84 ROIs). ROIs were analyzed using quantitative spatial profiling of 72 proteins on the Nanostring Digital Profiler platform. Glioblastomas responding to ICI were enriched in T-cell proteins (CD3, CD4, CD8) and T-cell activation markers (CD25) at the time of salvage compared to initial surgery. Markers of MAPK signaling were suppressed in pre-ICI samples compared to post-ICI samples in responders. p-ERK was suppressed in post-ICI samples compared to pre-ICI samples in non-responders. Myeloid proteins (CD68, CD163, CD11c) were enriched in post-ICI samples compared to pre-ICI samples in non-responders. Principle components analysis revealed p-ERK and immune proteins (CD3, CD4, CD8, CD20, CD11c, CTLA4, CD68, CD45, CD56, and CD127) accounted for 62% of the variance among pre-ICI and post-ICI samples in responders. In conclusion, temporospatial protein profiling of human glioblastomas reveals molecular mechanisms and biomarkers underlying responses to immune checkpoint inhibition. These data establish a foundation for functional studies to reprogram the immunosuppressive glioblastoma microenvironment and sensitize tumors to immune checkpoint inhibition.

The glioblastoma microenvironment is an immunosuppressive barrier to therapeutic innovation. We hypothesized intratumor convection enhanced delivery (CED) of gene therapy vectors could reprogram the glioblastoma immune microenvironment and elucidate therapeutic vulnerabilities. To test this, SB28 or GL261 glioblastoma allografts were implanted into immunocompetent mice and treated with CED of attenuated adeno-associated virus 9 vectors (AAV9) encoding experimental cytokines (Il1b, Ccl4, or Apoa1) underlying infiltration or activation of anti-tumor immune cells in other intracranial tumors. Serial intracranial bioluminescence was used to assess glioblastoma growth and animals were monitored for survival. The impact of gene therapy perturbations on the glioblastoma immune microenvironment was assessed using histology, immunohistochemistry, single-cell mass cytometry (CyTOF), and multiplexed cytokine assays. Serial body weight and systemic cytokine measurements showed no evidence of treatment toxicity. AAV9-APOA1 or AAV9-IL1B gene therapy CED treatments attenuated SB28 growth and prolonged survival, decreasing immunosuppressive macrophage infiltration and increasing CD8 T cell and microglia infiltration of the glioblastoma microenvironment compared to control AAV9 vectors. Gene therapy CED treatments did not attenuate GL261 growth or prolong survival, but CyTOF of human glioblastomas (n=6) in comparison to preclinical models revealed untreated GL261 glioblastomas were endogenously enriched in CD8 T cells and other lymphoid lineages compared to untreated SB28 or human glioblastomas. Multiplexed cytokine assays demonstrated suppression of intratumor IL6 is a conserved mechanism of action underlying glioblastoma gene therapy responses. Single-cell RNA sequencing analysis of 32,877 cells from human glioblastomas (n=11) showed IL6 is predominantly produced by radial glial like cancer stem cells or endothelial cells in the tumor microenvironment. In support of these findings, survival from intracranial SB28 glioblastomas was prolonged in Il6 knockout C57BL/6J mice compared to wildtype mice. In summary, we report a novel strategy using gene therapy and CED to reprogram the glioblastoma immune microenvironment, revealing IL6 drives glioblastoma immunosuppression and growth.

Prior work demonstrated synaptic integration of malignant gliomas into neural circuits induces local hyperexcitability and tumor proliferation. However, prognostication and therapeutic vulnerabilities are lacking from preclinical models. Here, we integrate in vivo and in vitro neurophysiology spatially matched with gene expression programs and protein signaling mechanisms across 66 IDH WT glioblastoma patients to identify thrombospondin-1 (TSP-1) as a molecular driver of glioma-induced network remodeling. Bulk and single cell RNA-sequencing of 11 intratumoral regions maintaining functional connectivity (13,730 cells analyzed) revealed a distinct neurogenic signature enriched for the synaptogenic factor TSP-1. Mechanistic and functional studies validating therapeutic vulnerabilities to TSP-1 silencing by shRNA knockdown and FDA-approved inhibitors (Gabapentin and LSKL) of excitatory synapse formation through the gabapentin a2d-1 receptor was performed in vitro and in vivo. Glioma-neuron co-culture of TSP-1 overexpressing cells demonstrated increased Ki67 proliferation and tumor microtube (TMT) formation when cultured in the presence of neurons. Pharmacological inhibition of TSP-1 using gabapentin or TSP-1 shRNA inhibited the proliferation and TMT-mediated expansion. Hippocampal xenografted mice with TSP-1 over expressing primary patient cultures demonstrated shorter survival and gabapentin treatment of xenografted mice significantly reduced the proliferation of TSP-1 overexpressing cells in vivo. Electrophysiological properties of glioma-neuron co-cultures analyzed using multi-electrode array (MEA) demonstrated increased neuronal spiking activity and network burst synchrony in the presence of TSP-1 over expressing cells. Strikingly, these increases were eliminated in the presence of gabapentin. We modeled survival risk in patients incorporating the effects of glioma intrinsic neuronal activity, molecular, therapeutic, and clinical factors on overall survival by recursive partitioning. Three risk groups were identified based on tumor intrinsic neuronal activity, pre- and post-operative tumor volume with shortest overall survival in patients with glioma intrinsic neuronal activity. These data identify glioma-induced secretion of TSP-1 as a key contributor of tumor proliferation shedding light on new therapies.

2039 Background: Immune checkpoint inhibition has not improved outcomes for glioblastoma patients, and single- cell approaches reveal the glioblastoma microenvironment is largely comprised of immunosuppressive cells. We hypothesized intratumor convection enhanced delivery (CED) of adenoviral gene therapy could recruit and activate anti-tumor immune cells in the glioblastoma microenvironment. Methods: Syngeneic GL261 (3x105 cells/mouse) or SB28 glioblastoma (3 x 104) cells were implanted into the frontal lobe of immunocompetent C57BL/6J mice (18 mice/arm). Intracranial bioluminescence (BLI) and body weight (BW) measurements were used to assess glioblastoma growth and treatment toxicity, respectively. After tumor engraftment, glioblastomas were treated with conformal ionizing radiation mimicking stereotactic radiosurgery (SRS) in human patients as a positive control for tumor inhibition (18Gy/1Fx). Attenuated adeno-associated virus 9 (AAV9) vectors encoding Gfp as a negative control, or encoding experimental cytokines driving recruitment and activation of anti-tumor immune cells in other intracranial tumors ( Ccl4, Il1b, or Apoa1) were delivered using CED (2x1011 vg/mouse). Glioblastomas were collected for histologic, single-cell, and molecular analyses 5 days after treatment (6 mice/arm) and at endpoints after monitoring for survival (12 mice/arm). CED targeting was validated using AAV9-GFP and confocal microscopy. Treatment responses were assessed using H&E, IHC, multiplexed cytokine assays, and single cell mass cytometry (CyTOF) to define immune cell types in the glioblastoma microenvironment. Results: Histologic analyses revealed AAV9-CCL4, AAV9-IL1B, or SRS induced glioblastoma macrophage infiltration, and AAV9-IL1B, AAV9-APOA1, or SRS induced glioblastoma T cell infiltration. AAV9-APOA1 (18.5 days versus 15 days, p < 0.001) or AAV9-IL1B (16.5 days versus 15 days, p = 0.001) CED treatments prolonged median survival from SB28 allografts. Glioblastoma cytokine analysis revealed inhibition of pro-tumor cytokines (IL6, LIF) after experimental CED treatments. Systemic cytokines were minimally changed by CED treatments. CyTOF showed decreased immunosuppressive macrophage infiltration and increased CD8+ T cell or microglia infiltration of the glioblastoma microenvironment after experimental CED treatments. There was no evidence of systemic or central toxicity in any treatment condition. Conclusions: Convection-enhanced delivered of adenoviral gene therapy reprograms the glioblastoma immune microenvironment and improves survival in an immunologically “cold” syngeneic glioblastoma model.

SummaryGliomas synaptically integrate into neural circuits. Prior work has demonstrated bidirectional interactions between neurons and glioma cells, with neuronal activity driving glioma growth and gliomas increasing neuronal excitability. In this study we wanted to know how glioma induced neuronal changes influence neural circuits underlying cognition and whether these interactions influence patient survival. We use intracranial brain recordings during lexical retrieval language tasks in awake humans in addition to site specific tumor tissue biopsies and cell biology experiments. We find that gliomas remodel functional neural circuitry such that task-relevant neural responses activate tumor-infiltrated cortex, beyond cortical excitation normally recruited in the healthy brain. Site-directed biopsies from functionally connected regions within the tumor are enriched for a glioblastoma subpopulation that exhibits a distinct synaptogenic and neuronotrophic phenotype. Tumor cells from functionally connected regions secrete the synaptogenic factor thrombospondin-1, which contributes to the differential neuron-glioma interactions observed in functionally connected tumor regions compared to tumor regions with less functional connectivity. The degree of functional connectivity between glioblastoma and the normal brain negatively impacts both patient survival and language task performance. These data demonstrate that high-grade gliomas functionally remodel neural circuits in the human brain, which both promotes tumor proliferation and impairs cognition.

Meningiomas arising from the meningothelial central nervous system lining are the most common primary intracranial tumors, and a significant cause of neurologic morbidity and mortality1. There are no effective medical therapies for meningioma patients2,3, and new treatments have been encumbered by limited understanding of meningioma biology. DNA methylation profiling provides robust classification of central nervous system tumors4, and can elucidate targets for molecular therapy5. Here we use DNA methylation profiling on 565 meningiomas integrated with genetic, transcriptomic, biochemical, and single-cell approaches to show meningiomas are comprised of 3 epigenetic groups with distinct clinical outcomes and biological features informing new treatments for meningioma patients. Merlin-intact meningiomas (group A, 34%) have the best outcomes and are distinguished by a novel apoptotic tumor suppressor function of NF2/Merlin. Immune-enriched meningiomas (group B, 38%) have intermediate outcomes and are distinguished by immune cell infiltration, HLA expression, and lymphatic vessels. Hypermitotic meningiomas (group C, 28%) have the worst outcomes and are distinguished by convergent genetic mechanisms misactivating the cell cycle. Consistently, we find cell cycle inhibitors block meningioma growth in cell culture, organoids, xenografts, and patients. Our results establish a framework for understanding meningioma biology, and provide preclinical rationale for new therapies to treat meningioma patients.

SUMMARYMeningiomas arising from the meningothelial central nervous system lining are the most common primary intracranial tumors, and a significant cause of neurologic morbidity and mortality1. There are no effective medical therapies for meningioma patients2,3, and new treatments have been encumbered by limited understanding of meningioma biology. DNA methylation profiling provides robust classification of central nervous system tumors4, and can elucidate targets for molecular therapy5. Here we use DNA methylation profiling on 565 meningiomas integrated with genetic, transcriptomic, biochemical, and single-cell approaches to show meningiomas are comprised of 3 epigenetic groups with distinct clinical outcomes and biological features informing new treatments for meningioma patients. Merlin-intact meningiomas (group A, 34%) have the best outcomes and are distinguished by a novel apoptotic tumor suppressor function ofNF2/Merlin. Immune-enriched meningiomas (group B, 38%) have intermediate outcomes and are distinguished by immune cell infiltration,HLAexpression, and lymphatic vessels. Hypermitotic meningiomas (group C, 28%) have the worst outcomes and are distinguished by convergent genetic mechanisms misactivating the cell cycle. Consistently, we find cell cycle inhibitors block meningioma growth in cell culture, organoids, xenografts, and patients. Our results establish a framework for understanding meningioma biology, and provide preclinical rationale for new therapies to treat meningioma patients.

Background Long non-coding RNAs (lncRNAs) exhibit highly cell type-specific expression and function, making this class of transcript attractive for targeted cancer therapy. However, the vast majority of lncRNAs have not been tested as potential therapeutic targets, particularly in the context of currently used cancer treatments. Malignant glioma is rapidly fatal, and ionizing radiation is part of the current standard-of-care used to slow tumor growth in both adult and pediatric patients. Results We use CRISPR interference (CRISPRi) to screen 5689 lncRNA loci in human glioblastoma (GBM) cells, identifying 467 hits that modify cell growth in the presence of clinically relevant doses of fractionated radiation. Thirty-three of these lncRNA hits sensitize cells to radiation, and based on their expression in adult and pediatric gliomas, nine of these hits are prioritized as lncRNA Glioma Radiation Sensitizers (lncGRS). Knockdown of lncGRS-1, a primate-conserved, nuclear-enriched lncRNA, inhibits the growth and proliferation of primary adult and pediatric glioma cells, but not the viability of normal brain cells. Using human brain organoids comprised of mature neural cell types as a three-dimensional tissue substrate to model the invasive growth of glioma, we find that antisense oligonucleotides targeting lncGRS-1 selectively decrease tumor growth and sensitize glioma cells to radiation therapy. Conclusions These studies identify lncGRS-1 as a glioma-specific therapeutic target and establish a generalizable approach to rapidly identify novel therapeutic targets in the vast non-coding genome to enhance radiation therapy.

BACKGROUND Unlike cancers affecting many solid organs, gliomas exist within the context of complex neural circuitry. It remains unknown whether glioma-neuron interactions play a role in maintaining functional circuits underlying cognition. We test the hypothesis that malignant gliomas remodel functional circuits through glioma-neuron interactions. METHODS Using language processing as a model for functional circuit dynamics, we enrolled 53 patients with dominant hemisphere IDH-wild-type glioblastoma. Task related circuit dynamics were measured using electrocorticography. Magnetoencephalography measures of functional connectivity identified intratumoral connectivity (HFC) and suppressed connectivity (LFC) regions. Primary patient samples and cultures from HFC and LFC-sites were assessed by single-cell RNA sequencing, pre/post-synaptic marker expression, cocultured with murine hippocampal neurons, and induced neuron organoids. Hippocampal tumor xenografts were created. Language/survival statistics were performed to correlate with functional connectivity measures. RESULTS Speech production evokes neuronal population spikes within the entire area of tumor-infiltrated cortex, far beyond the cortical territory normally involved in expressive language. Primary patient samples from HFC-regions are enriched for glioblastoma cells with a synaptogenic profile as characterized by pre-and post-synaptic marker expression at both tissue and cellular levels. RNA-sequencing and proteomic analyses from HFC samples revealed a neurogenic signature including thrombospondin-1 originating from glioma cells in HFC-regions and non-tumor astrocytes in LFC-regions. HFC xenografts demonstrated increased total number of synapses. Importantly, when compared with gliomas without intratumoral functional connectivity, connected gliomas have worse language task performance (r= -0.54,p=0.03) and shorter OS (medianOS-64 weeks compared with 107-weeks,p=0.04). CONCLUSION Glioma infiltrated regions generate task-relevant neural responses, with speech production evoking neuronal activity throughout tumor-involved cortex in the dominant hemisphere. An enriched population of synaptogenic glioma cells are organized within functionally connected intratumoral regions and this confers negative functional and survival outcomes. Together, these findings indicate that malignant gliomas can functionally remodel neural circuitry, thereby impairing neurological function and promoting tumor progression.

Author(s): Vasudevan, H; Magill, S; Seo, K; Villanueva-Meyer, J; Choudhury, A; Liu, SJ; Pekmezci, M; Findakly, S; Hilz, S; LaStella, S; Braunstein, SE; Oberheim, NA; Aghi, MK; Theodosopoulos, PV; Sneed, PK; Berger, MS; McDermott, MW; Lim, D; Ulian, E; Costello, J; Raleigh, D

Additional file 1: Figure S1. (a) Transcriptomic analysis of radiation response. RNA-seq analysis of differentially expressed genes following single dose radiation of U87 cells, along with significant gene ontology terms for upregulated and downregulated genes (n = 2-3 biological replicates per condition). (b) Log2 fold change of lncGRS-1 in U87 cells following radiation, averaged across all replicates from the same condition. Figure S2. Properties of the CRISPRi radiation modifier screen. (a) Proportion of two replicates of the screen population that are expressing sgRNA (BFP positive). Puromycin selection time period highlighted in yellow. Radiation doses indicated by arrows. (b) Z standardized growth (no radiation) and radiation phenotypes for PVT1 in CRISPRi screens. (c) Comparison of z standardized radiation phenotypes (x-axis) and log2 fold change of targeted lncRNA expression from RNA-seq (y-axis) analysis. Z standardized phenotypes were calculated as log2 enrichment normalized by the standard deviation of negative control genes’ phenotypes. lncGRS-1 to lncGRS-9 are labeled by their NCBI gene names. Figure S3. Nanopore direct RNA-seq of spliced reads aligned to the lncGRS-1 through -9 loci in U87 cells, with GENCODE v29 transcript models, Ensembl H3K27Ac layered track, and multiz alignment for conservation (from top to bottom in each subpanel). Figure S4. ASO knockdown of lncGRS-1 demonstrating glioma specific phenotype. (a) Single molecule RNA FISH of lncGRS-1 in DIPG SF8628 cells following transfection of non-targeting ASO (top) or ASO targeting lncGRS-1 (bottom). Scale bar = 5 μm. (b) RT-qPCR of TP53 (p53) transcript levels following ASO knockdown of TP53 in U87 cells. (c) lncGRS-1 locus with locations of sgRNA, ASO, and qPCR primer targets. (d-g) RT-qPCR of lncGRS-1 transcript levels (left) and cell propagation assay (right) following ASO knockdown of lncGRS-1 in SU-DIPG 24 (d), SU-DIPG 25 (e), GBM 43 (f), and HEK293T cells (g). (h) RT-qPCR of POLA1 transcript levels (left) and cell proliferation assay (right) following ASO knockdown of NHA cells (at day 7) or in (i) U87 cells (at day 3). n = 2 - 3 biological replicates per condition in all experiments indicated; error bar = S.D. Figure S5. (a) Cell propagation assay of purified populations of HeLa cells with lncGRS-1 CRISPRi knockdown. (b) Expression values (log2 (TPM + 1)) of lncGRS-1 across cell lines in the CCLE atlas, grouped by disease of origin or tissue type. (c) Top 5 gene ontology terms for upregulated (top) and downregulated (bottom) differentially expressed genes with adj. p val < 0.05, in GBM U87 (left) and DIPG SF8628 (right) 24 hours following lncGRS-1 ASO-mediated knockdown. (d) Scatter plot of genes differentially expressed in either U87 or SF8628 cell lines demonstrating positive correlation in expression changes following lncGRS-1 knockdown. (e) RNA-seq expression values and (f) western blot of protein levels for CDKN1A (p21) with quantification (right) in U87 cells following lncGRS-1 knockdown. (g) Immunohistochemistry of p53BP1 and (h) γH2AX nuclear foci in nuclei of U87 cells following lncGRS-1 knockdown with or without 2 Gy radiation. Scale bar = 5 μm. n = range of 225 to 440 nuclei per replicate across 2 biological replicates per condition. Figure S6. Full size western blot with additional replicate, corresponding to Figure S5f. Figure S7. Radiosensitization of glioma cells in MBO hosts. (a) Quantification of single molecule RNA FISH of lncGRS-1 in iAstrocyte MBO (A-MBO) nuclei following transfection of non-targeting ASO or ASO targeting lncGRS-1. n = 69 and 98 A-MBO nuclei quantified in ASO-Ctrl and ASO #2 conditions, respectively, across 2 independent experiments for each biological condition. (b) Left, fluorescence viability assay of combined (1:1 ratio) iAstrocyte and i3Neuron organoids (AN-MBO) following transfection of non-targeting ASO or ASO targeting lncGRS-1. Right, apoptosis induction assay of AN-MBOs following transfection of non-targeting ASO or ASO targeting lncGRS-1. (n = 3 biological replicates per condition; error bar = S.D.). (c) Fold change in AN-MBO size between day 2 and day 21 of co-culture with growth arrested DIPG SF8628 cells, with negative control or lncGRS-1 ASO, at various doses of fractionated radiation. (n = 5 biological replicates per condition; boxplot represents 1st quartile, median, and 3rd quartile with whiskers = range). (d) Confocal microscopy of AN-MBO 20 days following seeding of RFP+ U87 glioma cells. Nuclei are counterstained with DAPI (blue). Scale bar = 100 μm. (e) Longitudinal fluorescence microscopy of individual AN-MBOs seeded with RFP+ U87 cells. Cultures were treated with non-targeting ASO (Ctrl) or ASO targeting lncGRS-1 combined with 0 Gy, 12 Gy, or 18 Gy of fractionated radiation.

INTRODUCTION Little is known about the mechanisms by which gliomas integrate into functional neural networks and influence complex cognitive processes such as language. Glioma-neuron interactions are bidirectional, with increased neuronal activity promoting tumor growth and the latter in turn influencing neuronal excitability and synaptic connections. It remains unknown whether glioma-neuron interactions play a role in maintaining long-range neural networks subserving cognition in humans. We test the hypothesis that glioma-neuron interactions (“synaptogenic glioma cells”) are enriched within intratumoral high functional connectivity (FC) network hubs, thereby influencing language processing via release of synaptogenic factors into the tumor microenvironment. METHODS We employed magnetoencephalography imaginary coherence measures to identify intratumoral high (HFC) and low (LFC) functional connectivity network hubs in newly diagnosed glioblastoma patients. Primary patient samples and cultures from HFC and LFC sites were assessed for pre and post-synaptic marker expression (IF), cocultured with murine hippocampal neurons, and induced neuron organoids. ECOG Field recordings were performed on HFC/LFC tumors. Secreted proteins were measured from patient serum and LFC/HFC culture supernatant. Language assessments were performed to correlate task performance with FC measures. RESULTS Primary patient samples from HFC regions are enriched for glioblastoma cells with a synaptogenic profile as characterized by pre- and post-synaptic marker expression at both tissue and cellular level (coculture with mouse hippocampal neuron and organoid models). RNA sequencing and proteomic analyses from HFC samples revealed a neurogenic signature including thrombospondin 1 (TSP1). Overexpression of TSP1 in LFC primary patient cultures rescues the synaptogenic and proliferative phenotype. Importantly, we found a linear relationship between intratumoral HFC with patient serum TSP1 (ELISA) with a further correlation with language task performance. CONCLUSION An enriched population of synaptogenic glioma cells are organized within intratumoral high network connectivity regions. Glioma-induced neuronal synaptogenesis contributes to the microenvironment in support of network connectivity through secretion of TSP1.

Long noncoding RNAs (lncRNAs) exhibit highly cell type-specific expression and function, making this class of transcript attractive for targeted cancer therapy. However, the vast majority of lncRNAs have not been tested as potential therapeutic targets, particularly in the context of currently used cancer therapy. Malignant glioma is rapidly fatal, and ionizing radiation is part of the current standard-of-care used to slow tumor growth in both adult and pediatric patients. Here, we used a CRISPR interference (CRISPRi) platform to systematically screen 5689 lncRNA loci in human glioblastoma (GBM) cells, identifying 467 hits that modified cell growth in the presence of clinically relevant doses of fractionated radiation. 34 of these lncRNA hits sensitized cells to radiation, and based on their expression in adult and pediatric glioma, nine of these hits were prioritized as lncRNA Glioma Radiation Sensitizers (lncGRS). Knockdown of lncGRS-1 – a primate-conserved, nuclear-enriched lncRNA – inhibited the growth and proliferation of primary adult and pediatric glioma cells, but not the viability of normal brain cells. Using human brain organoids comprised of mature neural cell types as a 3-dimensional tissue substrate to model the invasive growth of glioma, we found that antisense oligonucleotides (ASOs) targeting lncGRS-1 selectively decreased tumor growth synergistically with radiation therapy. These studies identify lncGRS-1 as a glioma-specific therapeutic target and establish a generalizable approach to rapidly identify novel therapeutic targets in the vast non-coding genome that can enhance the standard-of-care for cancer therapy.

Long non-coding RNAs (lncRNAs) are transcripts >200 nucleotides long with essentially no protein coding potential. While certain lncRNAs play key roles in cancer and therefore represent an important class of therapeutic targets, very few lncRNAs have been studied in glioma. Here, we show that knockdown of a primate-specific lncRNA – referred to as GTT1 – selectively inhibits the growth of both adult and pediatric glioma but does not adversely affect the viability of normal human glia and neurons. In a recent genome-scale CRISPR interference (CRISPRi)-based screen, we identified GTT1 as one of 65 lncRNA genes that modify the growth of U87 glioblastoma cells. To prioritize lncRNAs for further study, we next performed a CRISPRi-based screen to identify lncRNA targets that also sensitize tumor cells to radiation. Both CRISPRi-mediated and antisense oligonucleotide (ASO)-mediated knockdown of GTT1 inhibited the propagation of U87 cells in culture, and this effect synergized with radiation treatment. Furthermore, GTT1 knockdown inhibited the growth of patient-derived glioma cells including adult glioblastoma and pediatric diffuse intrinsic pontine glioma (DIPG) in culture. Although GTT1 is expressed in normal human brain, knockdown of this lncRNA in normal human astrocytes, cortical neurons and fetal forebrain tissues did not reduce cell viability. As GTT1 is primate-specific, knockdown of this lncRNA in mouse xenograft models would not fully assess potential adverse effects. We therefore developed a human brain organoid model comprised of mature astrocytes and functional neurons. Patient-derived glioma cells engrafted into these organoids, and tumors grew in an infiltrative manner. GTT1 knockdown in this brain organoid model strongly impaired tumor growth but did not reduce organoid viability. These studies identify GTT1 as a glioma-specific therapeutic target and illustrate how this human brain organoid tumor model can be used to rapidly evaluate the tumor-specificity of novel therapeutics.

Summary Human astrocytes network with neurons in dynamic ways that are still poorly defined. Our ability to model this relationship is hampered by the lack of relevant and convenient tools to recapitulate this complex interaction. To address this barrier, we have devised efficient coculture systems utilizing 3D organoid-like spheres, termed asteroids, containing pre-differentiated human pluripotent stem cell (hPSC)-derived astrocytes (hAstros) combined with neurons generated from hPSC-derived neural stem cells (hNeurons) or directly induced via Neurogenin 2 overexpression (iNeurons). Our systematic methods rapidly produce structurally complex hAstros and synapses in high-density coculture with iNeurons in precise numbers, allowing for improved studies of neural circuit function, disease modeling, and drug screening. We conclude that these bioengineered neural circuit model systems are reliable and scalable tools to accurately study aspects of human astrocyte-neuron functional properties while being easily accessible for cell-type-specific manipulations and observations., Highlights • Novel three-dimensional “asteroid” coculture system is described • Complex human astrocyte-specific morphology is generated in vitro • This system produces rapid and tight association of astrocytes with synapses, In this article, Krencik and colleagues show that high-density cocultures of pre-differentiated human astrocytes with induced neurons, from pluripotent stem cells, elicit mature characteristics by 3–5 weeks. This provides a faster and more defined alternative method to organoid cultures for investigating human neural circuit function.

BACKGROUND Meningiomas are the most common primary intracranial tumor, and high grade meningiomas are resistant to most cancer therapies. Intratumor heterogeneity is a recognized source of resistance to treatment in numerous malignancies. Thus, we hypothesized that investigating molecular heterogeneity in meningiomas would elucidate biologic drivers and shed light on tumor evolution and mechanisms of resistance. METHODS We collected 86 spatially distinct samples at the time of resection from 13 meningiomas. Seven meningiomas were WHO grade I (46 samples), three were grade II (22 samples), and three were grade III (18 samples). Seven meningiomas were sampled at the time of salvage surgery (48 samples), and 6 were sampled at the time of initial diagnosis (38 samples). We performed multiplatform molecular profiling of these samples to identify drivers of intratumor heterogeneity, and validated our results using meningioma cells co-cultured with human cerebral organoids and RNA sequencing of paired primary and recurrent meningiomas. RESULTS Using bulk RNA sequencing, DNA methylation profiling and phylogenetic analysis of spatially distinct samples, we discovered significant transcriptomic, epigenomic and genomic heterogeneity in meningioma. In particular, we identified chromosomal structural alterations and differences in immune and neuronal signaling that underlie clonal evolution in high grade tumors. Using MRI-stratified bulk RNA sequencing, single nuclear RNA sequencing, RNA sequencing of paired primary and recurrent meningiomas, and live cell microscopy and single cell RNA sequencing of meningioma cells in co-culture with human cerebral organoids, we revealed a rare meningioma cell subpopulation with strong transcriptional concordance to the neural crest, a multipotent embryonic tissue that forms the meninges in development. CONCLUSIONS These data suggest that misactivation of a developmental cell population underlies intratumor heterogeneity in meningioma and that expression of neural crest and immediate early genes are an important step in meningeal oncogenesis.

The technical adequacy of curriculum-based measures in the form of short and simple vocabulary-matching probes to predict students’ performance and progress in science at the secondary level was investigated. Participants were 198 seventh-grade students from 10 science classrooms. Curriculum-based measurements (CBM) were 5-min vocabulary-matching probes administered once weekly over a period of 14 weeks. Criterion measures were knowledge pre- and posttests, the science subtest of the Iowa Test of Basic Skills (ITBS), and course grades. Alternate-form reliability coefficients ranged from r = .64 to .84. Coefficients increased over time and by combining scores across probes. Correlations between scores on the vocabulary-matching and criterion measures ranged from r = .55 to .76. The estimated mean group growth rate on the vocabulary-matching measure was .63 correct matches per week, which was significantly different from zero. The measures produced significant interindividual differences in growth rates, and growth on the measures was related to performance on the ITBS, course grades, and pre–post gains on the knowledge test. Results provide initial support for the technical adequacy of vocabulary-matching as an indicator of performance and progress in science.

This is a pilot study of a multifaceted project to examine current condition of children with mild disabilities and to identify the related issues and possible solutions. The purpose of this stage is to compare and contrast the definition of each category of disabilities from Korea and the United States to identify the differences in prevalence. Using the statistical data and interviews of teachers and parents with children with mild disabilities, issues from each stakeholder were determined and possible solutions were presented. Parent advocacy group, parent education, parent-teacher disability awareness training, and staff development program for preservice teachers and current teachers were suggested as possible options.

Androgen deprivation is currently a standard-of-care, first-line therapy for prostate cancer in the United States. Although this regimen effectively regresses androgen-dependent disease, relapse often occurs in an androgen-independent manner and is associated with poor prognosis. Such castration-resistant prostate cancer represents a major clinical challenge, and the mechanisms underlying castration resistance are not fully understood. Epithelial–mesenchymal transition (EMT) is a key developmental process and has also been implicated in cancer metastasis and therapeutic resistance in recent years. However, the factors contributing to EMT in human cancers remain unclear. Here, we show that both normal mouse prostate tissue and human LuCaP35 prostate tumor explants display an EMT as well as increased stem cell–like features following androgen deprivation. Importantly, we observed similar changes in mesenchymal features in prostate tumors from patients treated with androgen-deprivation therapy. In addition, we have delineated a feedback loop involving the androgen receptor and the Zeb1 transcription factor that seems to mediate this transition. In summary, we show for the first time that androgen deprivation induces EMT in both normal prostate and prostate cancer, revealing a potentially important consequence of a standard-of-care treatment for prostate cancer. This finding could have significant implications for second-line treatment strategies in this clinical setting. Cancer Res; 72(2); 527–36. ©2011 AACR.

Additional file 5: Review history.

Additional file 5: Review history.