bluuebunny/crossref_metadata_2025_split huggingface dataset:
TAMI-04. TUMOR TREATING FIELDS (TTFIELDS) HINDER GLIOMA CELL MOTILITY THROUGH REGULATION OF MICROTUBULE AND ACTIN DYNAMICS
Authors: Voloshin Tali , Schneiderman Rosa , Volodin Alexandra , Shamir Reuben , Kaynan Noa , Zeevi Einav , Koren Lilach , Klein-Goldberg Anat , Paz Rom , Giladi Moshe , Bomzon Zeev , Weinberg Uri , Palti Yoram 
Published: December 2020

Abstract
The ability of glioma cells to invade adjacent brain tissue remains a major obstacle to therapeutic disease management. Therefore, the development of novel treatment modalities that disrupt glioma cell motility could facilitate greater disease control. Tumor Treating Fields (TTFields), encompassing alternating electric fields within the intermediate frequency range, is an anticancer treatment delivered to the tumor region through transducer arrays placed non-invasively on the skin. This novel loco-regional treatment has demonstrated efficacy and safety and is FDA-approved in patients with glioblastoma and malignant pleural mesothelioma. TTFields are currently being investigated in other solid tumors in ongoing trials, including the phase 3 METIS trial (brain metastases from NSCLC; NCT02831959). Although established as an anti-mitotic treatment, the anti-metastatic potential of TTFields and its effects on cytoskeleton rapid dynamics during cellular motility warrant further investigation. Previous studies have demonstrated that TTFields inhibits metastatic properties of cancer cells. Identification of a unifying mechanism connecting the versatile TTFields-induced molecular responses is required to optimize the therapeutic potential of TTFields. In this study, confocal microscopy, computational tools, and biochemical analyses were utilized to show that TTFields disrupt glioma cellular polarity by interfering with microtubule assembly and directionality. Under TTFields application, changes in microtubule organization resulted in activation of GEF-H1, which led to an increase in active RhoA levels and consequent focal adhesion formation with actin cytoskeleton architectural changes. Furthermore, the optimal TTFields frequency for inhibition of invasion in glioma cells was 300 kHz, which differed from the optimal anti-mitotic frequency leading to glioma cell death of 200 kHz. The inhibitory effect of TTFields on migration was observed at fields intensities of 0.6 V/cm RMS (below the threshold of 1 V/cm RMS previously reported for cytotoxic effects). Together, these data identify discrete TTFields effects that disrupt processes crucial for glioma cell motility.
TAMI-08. A TALE OF TWO TELOMERE MAINTENANCE MECHANISMS: TERT EXPRESSION AND THE ALT PATHWAY INDUCE UNIQUE MRS-DETECTABLE METABOLIC REPROGRAMMING IN LOW-GRADE GLIOMAS
Authors: Viswanath Pavithra , Batsios Georgios , Gillespie Anne Marie , Luchman Hema Artee , Costello Joseph , Pieper Russell , Ronen Sabrina 
Published: December 2020

Abstract
Telomeres are nucleoprotein structures at chromosomal ends that shorten with cell division and constitute a natural barrier to proliferation. In order to proliferate indefinitely, all tumors require a telomere maintenance mechanism (TMM). Telomerase reverse transcriptase (TERT) expression is the TMM in most tumors, including low-grade oligodendrogliomas (LGOGs). In contrast, low-grade astrocytomas (LGAs) use the alternative lengthening of telomeres (ALT) pathway as their TMM. As molecular hallmarks of tumor proliferation, TMMs are attractive tumor biomarkers and therapeutic targets. Non-invasive imaging of TMM status will, therefore, allow assessment of tumor proliferation and treatment response. However, translational methods of imaging TMM status are lacking. Here, we show that TERT expression and the ALT pathway are associated with unique magnetic resonance spectroscopy (MRS)-detectable metabolic reprogramming in LGOGs and LGAs respectively. In genetically-engineered and patient-derived LGOG models, TERT expression is linked to elevated 1H-MRS-detectable NAD(P)/H, glutathione, aspartate and AXP. In contrast, the ALT pathway in LGAs is associated with higher α-ketoglutarate, glutamate, alanine and AXP. Importantly, elevated flux of hyperpolarized [1-13C]-alanine to pyruvate, which depends on α-ketoglutarate, is a non-invasive in vivo imaging biomarker of the ALT pathway in LGAs while elevated flux of hyperpolarized [1-13C]-alanine to lactate, which depends on NADH, is an imaging biomarker of TERT expression in LGOGs. Mechanistically, the ALT pathway in LGAs is linked to higher glutaminase (GLS), a key enzyme for α-ketoglutarate biosynthesis while TERT expression in LGOGs is associated with elevated nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme for NADH biosynthesis. Notably, TERT expression and the ALT pathway are linked to MRS-detectable metabolic reprogramming in LGOG and LGA patient biopsies, emphasizing the clinical validity of our observations. Collectively, we have identified unique metabolic signatures of TMM status that integrate critical oncogenic information with noninvasive imaging modalities that can improve diagnosis and treatment response monitoring for LGOG and LGA patients.
TAMI-09. ENERGY METABOLISM AND THERAPEUTIC T CELL EFFICACY IN THE GLIOBLASTOMA MICROENVIRONMENT
Authors: Logun Meghan , Colonna Maxwell , Edison Arthur , Karumbaiah Lohitash 
Published: December 2020

Abstract
Glioblastoma (GBM), like most cancers, undergo metabolic alterations to primarily utilize aerobic glycolysis in the hypoxic tumor microenvironment (TME). Similarly, activated T cells switch to glycolysis upon antigen recognition to cope with proliferation needs but are not equally equipped to survive in the hypoxic TME. Metabolic reprogramming within GBM TME contributes to therapeutic resistance and tumor progression, but the effects of metabolic alterations on therapeutic T cell survival and efficacy have not been fully elucidated. We hypothesized that hypoxia in GBM/T-cell co-cultures will significantly impair T cell proliferation and function. We conducted in vitro co-culture assays and nuclear magnetic resonance (NMR)–based assessments in hypoxic (1%O2) or normoxic conditions to detect metabolic changes in real-time. Imaging cytometry for cell cycle assessment demonstrated that GSCs were unaffected by hypoxia, but roughly 90% of healthy T cells arrested in G0/G1 along with significant reduction in glycan precursor UDP-GlcNAc presence. Media samples over 96h in normal and hypoxic oxygen conditions from cells in solitary or co-cultures were analyzed using a Bruker Avance III HD spectrometer at 600 MHz for comparison over time using PCA analysis of metabolic intermediate differences. After 16h, there was observable differences in produced metabolites between the T cells cultured alone or co-culture with GSCs, compared to the GSCs alone or media alone controls. Quantifiable changes in glucose, lactate, fumarate, acetate and pyruvate, among others, indicated a large shift in T cell metabolism dependent on oxygen conditions and co-culture interactions, while GSCs are less metabolically responsive to culture conditions. Ongoing experiments will examine precise changes in UDP-GlcNAc and glycosylation precursors in T cells and CAR-T cells via targeted NMR analysis, which we expect will help us understand energy dependent mechanisms of T cell exhaustion and lead to development of novel strategies to sustain T cell function in the hostile TME.
TAMI-18. SINGLE-CELL SEQUENCING AND GENETIC LABELING TO CHARACTERIZE HYPOXIC GBM CELLS IN THEIR MICROENVIRONMENT
Authors: Sattiraju Anirudh , Marallano Valerie , Ramakrishnan Aarthi , Brusco Concetta , Shen Li , Wojcinski Alexandre , Kesari Santosh , Friedel Roland H , Zou Hongyan 
Published: December 2020

Abstract
Glioblastoma (GBM) recurrence, arising from treatment-resistant hypoxic cells, is a major contributor to patient mortality. We have engineered GBM cell lines with a novel genetic reporter system (“HRE-UnaG”), which sensitively labels hypoxic cells with green fluorescence. Using murine GL261 HRE-UnaG GBM cells in intracranial transplants in immunocompetent hosts, we detected hypoxic cells primarily around pseudopalisading structures. Here, we report our new results of single cell RNA-seq sequencing of intracranial GL261-HRE-dUnaG GBM tumors, which revealed in hypoxic UnaG+ GBM cells upregulation of more than 60 genes, including canonical hypoxia response genes such as Slc2a1, Bnip3, Ldha and Vegfa, and downregulation of a smaller number of genes, such as Rgcc, Cdc20 and Hist1h2ap, which regulate cell cycle progression. This novel GBM hypoxia signature was in TCGA patient samples mostly upregulated in recurrent GBM and associated with worse prognosis, especially within proneural and mesenchymal subtypes. Gene set enrichment analysis revealed that the GBM hypoxia signature was positively correlated with hypoxia, glycolysis, TNFα and MTORC1 signaling, and negatively correlated with oxidative phosphorylation, Myc and DNA repair pathways. Subclustering revealed four distinct subpopulations of hypoxic GBM cells, which were distinguished by differential expression of pathway genes for proliferation, angiogenesis, stress response, and type I/II interferon signaling. Intercellular communication between hypoxic GBM cells and the stromal cells were predicted using NicheNET package. To spatio-temporally label in future studies hypoxic GBM cells and their progeny, we have engineered GBM cells with an inducible, hypoxia-sensitive lineage-tracing reporter (HRE-Cre; floxed-tdTomato). In combination with the HRE-UnaG hypoxia reporter, we will be able to reveal the role of “hypoxia memory” in GBM expansion and recurrence after treatment. Identifying key markers of hypoxic cells in respect to treatment resistance and relapse will lead to improvements in GBM detection and new options for therapeutic treatments.
TAMI-22. VARIATION AMONG INTACT TISSUE SAMPLES REVEALS THE CORE TRANSCRIPTIONAL IDENTITIES OF CELL TYPES IN THE GLIOMA MICROENVIRONMENT
Authors: Schupp Patrick , Oldham Michael 
Published: December 2020

Abstract
Adult low-grade gliomas generally progress to glioblastoma, a more aggressive CNS tumor with an extremely poor prognosis. Despite intensive efforts, numerous promising glioma therapies have failed to provide survival benefits. These failures reflect many factors, including intertumoral heterogeneity and immunosuppression by the tumor microenvironment (TME). We propose a novel approach to addresses these challenges through integrative deconvolution of bulk gene expression data generated from more than 5000 human gliomas and 7000 normal human brain samples. Inherent variation in the cellular composition and cellular activities of these samples allowed us to identify highly correlated modules of genes that represent specific cell types and cell states. By comparing gene coexpression modules in glioma vs. normal human brain, we have identified cell type-specific gene expression changes in the glioma TME that are highly reproducible. In contrast to single-cell methods, which sample only a fraction of the tumor tissue and fail to capture major nonmalignant cell-types, our results derive from billions of cells and thousands of individuals and are therefore highly robust. We find that a number of genes encoding cell-surface proteins are specifically up-regulated in immune and vascular cells of the glioma TME. Surprisingly, among those genes up-regulated in glioma vasculature are multiple members of the angiotensin pathway, suggesting non-canonical roles for these proteins in the glioma setting. We propose that these proteins may form a specific ‘zip code’ for glioma within the brain’s vasculature that can be targeted directly or by conjugation with existing drugs. More generally, our analytical approach has revealed reproducible gene expression changes in specific cell types of the glioma TME that provide more stable therapeutic targets than those that are expressed by genetically mutable malignant cells. We have also discovered novel, aberrantly coexpressed genes in microglia, oligodendrocytes, and astrocytes which we are testing in state-of-the-art human brain assembloid systems.
TAMI-24. BEHAVIOR OF GLIOBLASTOMA STEM-LIKE CELLS WITH KNOWN IDH1 STATUS IN CEREBRAL ORGANOIDS
Authors: Singh Sanjay , Munyeshyaka Maxime , Gumin Joy , Yang Jing , Ledbetter Daniel , Hossain Anwar , Kerrigan Brittany Parker , Lang Frederick 
Published: December 2020

Abstract
Glioblastomas (GBM) exhibit high proliferative index, areas of necrosis, high vascularization, and are highly invasive to normal brain tissues. The most common and lethal form of GBMs are primary GBMs, with no prior clinical history. Whereas, secondary GBMs arise from low-grade gliomas and are associated with IDH1 mutation. Pre-clinical studies of GBM largely depend on patient-derived GBM stem-like cells (GSCs) in vitro and in vivo as orthotopic xenografts. Cerebral organoids (COs) derived from induced pluripotent stem cells can serve as allogenic in vitro model systems to study interactions between normal brain and GSCs. COs have been shown to harbor neural stem cells and their differentiated progenies as well as microglia within distinct niches. Here, we co-cultured 45 day-old COs and MDA-GSCs lines representing mesenchymal sub-group (M-MDA-GSC), classical sub-group (C-MDA-GSC), and IDH1 mutant (IDH1R132H-MDA-GSC). MDA-GSCs stably express fluorescent proteins and is used to track GSCs within COs. These GSC bearing COs were fixed, embedded, sectioned, immuno-stained, and imaged by confocal microscope. There was a positive correlation between GSC numbers in allografted niche and invasion into COs as measured from the edge of organoid, M-MDA-GSC (R2=0.99; 0.89μm/cell), C-MDA-GSC (R2=0.92; 0.66μm/cell), and IDH1R132H-MDA-GSC (R2=0.89; 0.5μm/cell). Additionally, M-MDA-GSCs had significantly high percentage of Ki67+ve invasive cells (24%) in comparison to C-MDA-GSCs (5.1%; p=0.0057). As a measure of interaction of MDA-GSC with normal cells, we assessed proximity of IBA1+ve microglia in GSC niche within organoids and show that M-MDA-GSC and IDH1R132H-MDA-GSC highly co-localized with IBA1+ve microglia on day12 of co-culture. In conclusion, our cerebral organoid-based allograft study shows that mesenchymal GSCs (M-MDA-GSC) are most invasive whereas IDH1 mutant GSCs (IDH1R132H-MDA-GSC) are least invasive. C-MDA-GSCs are least proliferative while invading into normal COs. Uniqueness of CO based allograft system is highlighted by observed similarity between M-MDA-GSC and IDH1R132H-MDA-GSC for their potential to attract IBA1+ve microglia.
TAMI-27. RADIATION THERAPY REPROGRAMS THE TUMOR METABOLISM TO PROMOTE SURVIVAL AND IMMUNOSUPPRESSION IN GLIOBLASTOMA
Authors: De Martino Mara , Daviaud Camille , Vanpouille-Box Claire 
Published: December 2020

Abstract
Immune checkpoint blockade has re-stimulated the interest of immunotherapeutic strategies for glioblastoma (GBM). However, PD-1 blockade in recurrent GBM patients failed to meet the primary endpoint of superior survival (NCT02017717) highlighting the need to identify mechanisms of GBM immune evasion. One possibility is the metabolic landscape that cancer cells generate to promote survival and prevent immune responses. Radiation therapy (RT), a standard of care for GBM, is well-documented to reprogram the metabolism of the tumor microenvironment (TME). Therefore, we hypothesize that RT generates a metabolic shift in the TME to support GBM survival and immunosuppression. To address this question, we first defined RT-induced metabolic changes by Seahorse assay. We observed that mitochondrial respiration and ATP production were significantly increased in 10 gray (Gy)-irradiated GL261 cells after 24hr. Metabolomic profiling of 10Gy-irradiated GL261 cells showed a strong increase in metabolic pathways related to nucleotide, amino acids and lipid metabolism. We next ask whether RT was modulating the fatty acid synthase (FASN), a key lipogenic enzyme. Western blot analysis indicated upregulation of FASN expression in irradiated GL261 cells. Interestingly, flow cytometry of orthotopically implanted GL261 tumors not only confirmed FASN upregulation in cancer cells in vivo, but also revealed a marked FASN expression in tissue-resident microglia after 10Gy focal irradiation. Moreover, flow cytometry analysis showed increased expression of the immunosuppressive markers PD-L1 and CSF-1R in macrophages co-cultured with irradiated GL261. Finally, targeting FASN using shRNA or pharmacological inhibition, significantly reduced the proliferation of GL261 cells. Altogether, our data indicate that RT is rewiring the energy supply of GBM to presumably foster its growth and immunosuppression of the TME by promoting lipid biosynthesis. While much work remains to be done to confirm this hypothesis, our data propose FASN as a novel therapeutic target to overcome RT resistance and reduce immunosuppression in GBM.
TAMI-32. TEMPOROSPATIAL INVASION AND GENETIC EVOLUTION FROM INFRATENTORIAL TO SUPRATENTORIAL COMPARTMENT IN DIFFUSE MIDLINE GLIOMA
Authors: Georgescu Maria-Magdalena 
Published: December 2020

Abstract
Diffuse midline gliomas (DMGs) are very aggressive pediatric brain tumors with dismal prognosis due to therapy-resistant tumor growth and invasion. We performed the first integrated histologic/genomic/proteomic analysis of 21 tumor foci from three pontine DMG cases with supratentorial dissemination. Histone H3.3 K27M was the driver mutation, usually at high variant allele fraction due to recurrent chromosome 1q copy number gain, in combination with germline variants in ATM, FANCM and MYCN genes. Both previously reported and novel recurrent copy number variations and somatic pathogenic mutations in chromatin remodeling, DNA damage response and PI3K/MAPK growth pathways were variably detected, either in multiple or isolated foci. Proteomic analysis showed global upregulation of histone H3, lack of K27 tri-methylation, and further impairment of polycomb repressive complex 2 by ASXL1 downregulation. Activation of oncogenic pathways resulted from combined upregulation of N-Myc, SOX2, p65/p50 NF-kB and STAT3 transcription factors, EGFR, FGFR2, PDGFRa/b and MET receptor tyrosine kinases, and downregulation of PHLPP1/2, PTEN and p16/INK4A tumor suppressors. Upregulation of SMAD4, PAI-1, CD44, and c-Src in multiple foci most likely contributed to invasiveness. This integrated comprehensive analysis allowed spatiotemporal modeling of tumor progression and identified two general pathways of supratentorial invasion, and a multitude of migratory subpopulations within the infratentorial compartment. It also delineated common signaling pathways and potential therapeutic targets, revealing an unsuspected activation of a multitude of oncogenic pathways that may explain the resistance of DMG to current therapies.
TAMI-37. SEX DIFFERENCES IN REDOX STATE UNDERLIE GLUTAMINE DEPENDENCY IN MALE GLIOBLASTOMA
Authors: Sponagel Jasmin , Zhang Shanshan , Jones Jill , Chinnaiyan Prakash , Rubin Joshua , Ippolito Joseph 
Published: December 2020

Abstract
Glioblastoma (GBM) is the most aggressive primary brain tumor in adults. GBM occurs more commonly in males but female patients survive significantly longer. Understanding the molecular mechanisms underlying this clinical sex disparity could support novel treatment strategies to improve outcomes for GBM patients. In this regard, we found that male and female GBM patient tissues differ in their metabolite profiles and that male GBM exhibit a higher abundance of amino acid metabolites. We confirmed these findings in a murine model of GBM. Furthermore, we found that male GBM cells are more sensitive to amino acid deprivation. This male-specific dependency on amino acids is almost entirely driven by amino acids involved in the synthesis of the antioxidant glutathione. Glutaminase 1 (GLS1) mediates the conversion from glutamine to glutamate, a crucial component of glutathione. We found that male GBM cells are more sensitive to GLS1 inhibition with the clinical inhibitor CB-839. This correlated with significantly increased reactive oxygen species (ROS) in male GBM. We further confirmed sex differences in redox state through pharmacological depletion of glutathione, which resulted in a significant increase in ROS and cell death in male GBM cells. Moreover, assays of glutathione oxidation demonstrated that male GBM cells exist in a chronically oxidized state. Finally, we found that mitochondrial structure and function, including TCA cycle flux, NADH levels, and antioxidant activity, differ between male and female GBM cells. Together, these data suggest that (1) male and female GBM differ in their amino acid requirements, (2) male GBM are more dependent on glutamine to regulate ROS levels, and (3) sex differences in mitochondrial physiology may result in ROS accumulation and increased susceptibility to drugs targeting the redox state in male GBM. Our data reveal novel metabolic targets for GBM and underline the importance of considering sex in metabolic targeting approaches.
TAMI-49. THE ALTERNATIVE SPLICING FACTOR MBNL1 INHIBITS GLIOBLASTOMA TUMOR INITIATION AND PROGRESSION BY REDUCING HYPOXIA-INDUCED STEMNESS
Authors: Voss Dillon , Sloan Anthony , Bar Eli , Bar Eli 
Published: December 2020

Abstract
Muscleblind-like-proteins (MBNL) belong to a family of tissue-specific RNA metabolism-regulators that control pre-messenger RNA-splicing (AS). Inactivation of MBNL causes an adult-to-fetal AS transition, resulting in the development of myotonic dystrophy. We have previously shown that the aggressive brain cancer, glioblastoma (GBM), maintains stem-like features (GSC) through hypoxia-induced responses. Accordingly, we hypothesized that the hypoxia-induced responses in GBM might also include MBNL-based AS to promote tumor progression. When cultured in hypoxia, GSCs rapidly export MBNL1 out of the nucleus resulting in significant inhibition of MBNL1 activity. Notably, the hypoxia-regulated inhibition of MBNL1 also resulted in evidence of adult-to-fetal alternative splicing transitions. Forced expression of a constitutively active isoform of MBNL1 inhibited GSC self-renewal and tumor initiation in orthotopic transplantation models. Using a tetracycline-inducible system, induced expression of MBNL1 in established orthotopic tumors dramatically inhibited tumor progression resulting in a significant prolongation of survival. This study reveals that MBNL1 plays an essential role in GBM stemness and tumor progression, whereby hypoxic responses within the tumor inhibit MBNL1 activity, promoting stem-like phenotypes and tumor growth. Reversing these effects on MBNL1 may, therefore, yield potent tumor-suppressor activities, uncovering new therapeutic opportunities to counter this devastating disease.