bluuebunny/crossref_metadata_2025_split huggingface dataset:
CNSC-13. TUMOR INFILTRATION OF GAMMA-DELTA T CELLS IN GLIOBLASTOMA
Authors: Juran Vilem , Piskacek Martin , Tomandlova Marie , Sova Marek , Smrcka Martin , Kazda Tomas , Vybihal Vaclav , Fadrus Pavel , Knight Andrea 
Published: November 2022

Abstract
Gamma-delta (γδ) T cells are innate immunity effector lymphocytes with known prominent anti-tumor reactivity against aggressive glioblastoma (GBM). However, therapeutic approaches have had limited success due to the protective blood-brain-barrier and the immunosuppressive GBM tumor microenvironment. In this study, we determined Vδ1 a Vδ2 γδ T cell populations in peripheral blood and paired tumor tissue samples in patients (n=40) following the resection and throughtout the therapy follow-up. Tumor samples were processed using enzymatic kits and gentleMACSTM Dissociator (Miltenyi Biotec Inc.) and tumor-infiltrating γδ T lymphocytes (TILs) were analyzed by flow cytometry.We found infiltration of both intratumoral CD3+ γδ T cell subsets in 68% tumor samples. We detected Vδ1 γδ T cells in the range 0-0.8% (median 0.26%). Majority of GBM patients presented the Vδ2 subset among TILs in the range 0-13.8% (median 1.5%). Functional studies showed prominent cytotoxicity of magnetically sorted Vδ1 a Vδ2 γδ T cells against GBM cell lines and more importantly against primary tumors. Detailed phenotypic profiling and single-cell sequencing of Vδ2 γδ T cells is currently underway. Next, we identified the EphA2 receptor as one of the targets for tumor-reactive Vδ1 γδ T cells. Specifically, we found that blocking of EphA2 expression resulted in significant inhibition of GBM killing mediated by Vδ1 γδ T cells. Furthermore, Luminex xMAP technology identified significantly elevated levels of stress ligand MICA and check-point inhibitor ligands PD-L1 (B7-H1, CD274) and B7-H3 (CD276) and galectin-9 in patients‘ plasma samples at diagnosis compared to age-matched controls.The patient’s clinical course and therapeutic protocols will be discussed.This study was supported by Ministry of Health, Czech Republic (grant NV19-05-00410 to AK) by Ministry of Health, Czech Republic-conceptual development of research organization (FNBr, 65269705). All rights reserved.
TMET-11. QUINOLINATE PROMOTES ALTERNATIVELY ACTIVATED MACROPHAGE-INDUCED IMMUNE TOLERANCE IN GLIOBLASTOMA THROUGH THE NMDA/PPARG SIGNALING AXIS
Authors: Kesarwani Pravin , Kant Shiva , Zhao Yi , Prabhu Antony , Buelow Katie , Miller C Ryan , Chinnaiyan Prakash 
Published: November 2022

Abstract
There has been considerable interest in understanding the biological consequence and therapeutic implications of aberrant tryptophan metabolism in brain tumors and neurodegenerative diseases. An overwhelming majority of this work has focused on the first-step of tryptophan metabolism (kynurenine); however, this has yet to result in clinical application. Using global metabolomic profiling on &gt;100 patient-derived brain tumors, we identified a 64-fold accumulation of quinolinate (QA), a downstream metabolic intermediate of the tryptophan pathway, in glioblastoma when compared to low-grade glioma. As several metabolites in the tryptophan pathway have been implicated in immune modulation, we sought to determine the impact of QA on the immune microenvironment. We identified the capacity of QA to strongly skew macrophage polarization towards the “pro-tumorigenic” M2-phenotype with suppressive properties, which recent studies suggest play a dominant role in the immune microenvironment in glioblastoma. Intriguingly, QA conferred an “M2-like” phenotype to M1 macrophages and microglia, attenuating their phagocytosis efficiency. We went on to systematically delineate a novel mechanism of macrophage polarization through QA-induced NMDA receptor activation and Foxo1/PPARg signaling. We then determined that tumor cells and host macrophages/microglia work in concert to complete both upstream and downstream metabolism of tryptophan, respectively, resulting in the accumulation of QA. We discovered a very strong positive feedback loop involving the expression of kynureninase (KYNU) in macrophages, an enzyme involved in the downstream metabolism of tryptophan and QA production, making this a lead candidate for targeting this pathway. As this represents a novel target and agents designed to inhibit this enzyme are not commercially available, we generated a Kynu-/- knockout mouse model. Intriguingly, consistent with in vitro data, tumors grown in Kynu-/- mice had a ~50% reduction in M2 macrophages, increased immune activation, decreased growth, and improved overall survival in two orthotopic glioblastoma models, supporting the therapeutic potential of targeting this pathway.
TMET-15. CYTOCHROME C OXIDASE MEDIATES CELLULAR IRON HOMEOSTASIS AND RADIORESISTANCE IN GLIOBLASTOMA
Authors: Ali Md Yousuf , Pullium Casey , Flor Susanne , Goswami Prabhat , Oliva Claudia , Griguer Corinne 
Published: November 2022

Abstract
Defining the molecular mechanism(s) responsible for the adaptive radioresistance in glioblastoma (GBM) is necessary for the development of effective treatment options. The cellular labile iron pool (LIP) is very important for determining the cellular response to radiation, as it contributes to radiation-induced production of reactive oxygen species (ROS). Recently, cytochrome c oxidase (CcO), a mitochondrial heme-containing enzyme also involved in regulating ROS production, was found to be involved in GBM chemoresistance. However, the role of LIP and CcO in GBM radioresistance is not known. Herein, we tested the hypothesis that CcO-mediated alterations in the level of labile iron contribute to adaptive radioresistance. Using an in vitro model of GBM adaptive radioresistance, we found an increase in CcO activity in radioresistant cells that associated with a decrease in the cellular LIP (4.1-fold), decrease in lipid peroxidation (2-fold), and a switch in the CcO subunit 4 (COX4) isoform expression, from COX4-2 to COX4-1. Furthermore, knockdown of COX4-1 in radioresistant GBM cells decreased CcO activity and restored radiosensitivity, whereas overexpression of COX4-1 in radiosensitive cells increased CcO activity and rendered the cells radioresistant. We further observed that radioresistant cells has increased expression of mitoferrin-1, a protein that facilitates the incorporation of iron into mitochondria. Overexpression of mitoferrin-1 in glioma cells increases the activity of mitochondrial protein complexes, decreases the cellular labile iron pool (p&lt; 0.05), and decreases the production of 4-hydroxy nonenal (4-HNE), a lipid peroxidation product. Furthermore, overexpression of mitoferrin-1 significantly increases the anchorage independent colony formation in soft agar assay (p&lt; 0.01). Moreover, mitoferrin-1 overexpressing cells implanted orthotopically in the brain of nude mice significantly decreased survival compared to wild type (p &lt; 0.01). These results indicate that manipulation of cellular and mitochondrial iron homeostasis may provide a strategy to improve therapeutic outcome in patients with GBM.
TMET-35. ONE CARBON AND NUCLEOTIDE METABOLISM ARE NOVEL TARGETS IN PEDIATRIC BRAINSTEM TUMORS
Authors: Mersich Ian , D'Alessandro Angelo , Desai Pankaj , Sallans Larry , Dasgupta Biplab 
Published: November 2022

Abstract
Diffuse intrinsic pontine glioma (DIPG) are inoperable tumors of the brainstem with no cure. The median survival of children with DIPG is less than 2 years and 5-year overall survival is only 1%. Genomic studies have identified recurrent mutations in histone H3, ACVR and TP53 genes in DIPG. However, there are still no breakthrough drug targets for DIPG. To better understand the disease biology and find new drug targets for DIPG, we recently performed joint pathway analysis of DIPG metabolites (untargeted metabolomics) and differentially expressed genes (RNAseq) relative to normal human neural stem cells. We discovered that specific enzymes of the de novo purine biosynthesis pathway (DNPB), serine synthesis and the associated and one-carbon (1C) metabolism are highly active and the most upregulated in DIPG tumors and cell lines. Gain and loss of function experiments as well as pharmacological studies indicated a mechanistic link between DIPG mutations and DNPB pathway and the dependence of DIPG cells on specific DNPB and 1C metabolism enzymes for in vitro and in vivo growth. The 1C pathway is crucial for rapidly proliferating malignant cells as it senses and regulates cellular nutrient status by allocating and cycling 1C-groups between different acceptor compounds. It controls synthesis of nucleotides, amino acids, glycogen and phosphoglyceride precursors, S-adenosylmethionine (SAM), glutathione and NADPH. Thus, besides dispensing carbon atoms it also regulates cellular epigenetic and redox status. An important carbon donor is serine which is synthesized from glucose at 3-phosphoglycerate step in glycolysis. Untargeted as well as carbon and nitrogen tracing experiments indicated large folate pools in DIPG cells that can serve as a buffer to potentially blunt antifolate therapy. Experiments are underway to understand the importance of the folate cycle in DIPG and design novel and translatable combination therapies for DIPG.
TMIC-34. INVESTIGATING THE EFFECT OF IRON IN GLIOBLASTOMA CELL POLARIZATION AND MIGRATION
Authors: Shenoy Ganesh , Palsa Kondaiah , Wade Quinn , Khunsriraksakul Chachrit , Khristov Vladimir , Slagle-Webb Becky , Lathia Justin , Wang Hong-Gang , Connor James 
Published: November 2022

Abstract
Glioblastoma represents one of the most difficult-to-treat malignancies as evidenced by the poor prognosis associated with a diagnosis. The ability of glioblastoma cells to diffusely infiltrate into healthy brain tissue renders complete surgical resection challenging. Consequently, a large majority of glioblastoma patients end up with recurrent disease despite receiving maximally feasible surgical resection and rigorous chemoradiation. This work examined how modulation of cellular iron levels in T98G and LN229 glioblastoma cells impacted migratory capacity. Treatment of T98G or LN229 glioblastoma cells with iron in the form of ferric ammonium citrate (FAC) resulted in significantly reduced migration as assessed by time-lapse phase contrast imaging and wound healing assays. The iron-induced reduction in migration was able to be rescued by the addition of equimolar concentrations of deferoxamine, an iron chelator. Cellular proliferation in response to the iron treatments was quantified using both optical confluence and nucleic-acid-based proliferation assays and it was found that iron treatment at the concentrations used for the migration assays (0 – 300 µM FAC) did not result in reduced proliferation. Mechanistically probing iron’s impact on cell migration revealed that addition of iron resulted in decreased expression of Cdc42, a Rho GTPase that is essential to determining cellular polarity during migration. Functional cellular polarization assays further confirmed that reduced expression of Cdc42 corresponded to reduced cellular polarization. Bioinformatic analysis of CDC42 transcripts revealed the presence of potential iron-responsive-elements that may drive the iron-induced reduction in Cdc42 expression. This work highlights the importance of iron biology in impacting glioblastoma cell phenotype and potentially glioblastoma patient outcomes.
TMIC-35. CROSSTALK BETWEEN TAMS AND GLIOBLASTOMA STEM-LIKE CELLS IN A HYPOXIC MICROENVIRONMENT INDUCES SENESCENCE ARREST AND ENHANCED RADIORESISTANCE
Authors: Carneiro-Lobo Tatiana , Olson Cheryl , Markwell Steven , Mesci Pinar , Muotri Alysson , Brat Daniel 
Published: November 2022

Abstract
Patients with glioblastoma (GBM) have an overall survival of 15 months. These catastrophic survival rates are correlated with systematic relapses that might arise from remaining glioblastoma stem cells (GSCs) left behind after treatments. Our goal was identifying whether radioresistance of GBM is caused by GSCs and how hypoxia and TAMs affect this process. First, we established a glioblastoma-brain cortical organoid (GBM-BCOs) to model human GBM. We co-cultured GFP-labeled GBM neurosphere with BCOs for 14 days and we observed that tumor take rate was 100% for all GBM neurosphere tested. Next, we evaluated whether hypoxia enhanced TAMs migration into GBM-BCOs. We incorporated THP-1 cells into GFP-GBM-BCOS for 14 days and incubated for 24 h under normoxic or hypoxic conditions. Hypoxia increased THP-1 cells influx into GBM-BCOs. Further, we investigated whether TAMs enhanced the enrichment of GSCs in GBM neurosphere and in GBM-BCOs. We found that, co-culture of THP-1 cells with GBM neurosphere or with GBM-BCOs system, enhanced CD133+ population after 72 h hypoxic condition. In addition, IF of GBM human tissue revealed increased of CD133+ and IBA1+ cells in peri-necrotic area compared to non-necrotic area and non-necrotic tumor. To examine whether TAMs enhanced the radioresistant phenotype and the quiescent population, we irradiated GBM neurosphere grown under hypoxic and normoxic conditions with different doses of γ-rays. After irradiation, these cells were co-cultured with TAMs for 72 h under normoxic and hypoxic conditions. MTT assay showed that TAMs enhanced the radioresistant phenotype promoted by hypoxia in GBM neurosphere and an increase in quiescent markers (γ-H2AX and IL-6) were found in our co-culture model after hypoxia exposure. These results indicate, not only hypoxic condition play a key role in the GSCs enrichment and radioresistance, but also crosstalk between TAMs and GSCs promote tumor oncogenesis and contribute to an ineffective anti-tumor response.
TMIC-68. EVALUATING FLASH AND CONVENTIONAL DOSE-RATE RADIATION AND IMMUNE RESPONSE WITH SINGLE-CELL SEQUENCING IN DIFFUSE MIDLINE GLIOMA (DMG)
Authors: Minns Hanna , Padilla Oscar , Wei Hong-Jian , Webster-Carrion Andrea , Tazhibi Masih , McQuillan Nicholas , Zhang Xu , Yeh Rebecca , Zhang Zhiguo , Szalontay Luca , Pavisic Jovana , Garty Guy , Garvin James , Canoll Peter , Vanpouille-Box Claire , Menon Vilas , Olah Marta , Rabadan Raul , Wu Cheng-Chia , Gartrell Robyn 
Published: November 2022

Abstract
Diffuse Midline Glioma – H3K27M mutant (DMG), is a fatal and inoperable pediatric brain tumor with limited treatment options as radiation provides only temporary reprieve, leaving the median survival between 9-15 months. Conventional dose-rate radiation (2Gray/minute, CONV) has been shown in other cancers to recruit an immune component, however, this has not been studied in DMG. Ultra-high dose-rate radiation given at 90 Gray/second (FLASH), is a novel technique associated with decreased toxicity and effective tumor control. Using a syngeneic model of brainstem DMG, we performed single-cell RNA sequencing on CD45+ immune cells isolated from tumors irradiated with 15Gray using FLASH or CONV, and compared to unirradiated tumor and normal brainstem. Isolation of 33,308 immune cells revealed 17 unique clusters, most abundant of which was microglia (73.8%), present in four distinct subtypes representing a spectrum from homeostatic to activated. In the most activated microglia, both FLASH and CONV showed an enrichment in type 1 interferon (IFN1) pathway scores compared to untreated tumors (p&lt; 0.001 and p&lt; 0.001, respectively). The most differential response was found in macrophages (MAC) and dendritic cells (DC) with a robust enrichment of IFN1 pathway scores for CONV compared to FLASH (p&lt; 0.001, MAC and p&lt; 0.001 DC). FLASH showed an increase in anti-inflammatory MAC markers such as Mrc1, Cd163, and Maf and an enrichment of myeloid-derived suppressor cell (MDSC) signature in monocytes, not seen in CONV (p&lt; 0.001). Finally, we correlated our data with publicly available single-cell data taken from the cerebrospinal fluid of DMG patients treated with anti-GD2 CAR T Cell therapy and found similar inflammatory markers characteristic of our unirradiated murine tumors. In summary, our work is the first to study immune alterations comparing different dose-rates of radiation with single-cell resolution in DMG, highlighting the potential for combining radiation and immunotherapy in these tumors.
MODL-26. EVOLUTION OF MULTI-FACETED GLIOBLASTOMA MICROENVIRONMENTS IN 3D
Authors: Senior Jessica , Dalby Amanda , Correia Joao , Pike Jeremy , Jaworska Kayley , Thomas Steve , Smith Alan , Brüning-Richardson Anke 
Published: November 2022

Abstract
Glioblastomas (GBM) account for poor prognosis and dismal survival rates in patients due to their highly aggressive infiltrative nature to rapidly migrate within the brain. Experimental treatments for GBMs using animal models often elicit severe side effects and there are major doubts regarding the usefulness of such in vivo models when undergoing animal to human clinical translation. Here, we describe the development of a series of 3D bioengineered GBM models towards the generation of a more physiologically representative system in which candidate drugs can be tested. Glioma cell lines U87 and U251 derived from human GBM were used along with associated knockdowns of previously described anti-migratory and pro-migratory genes or anti-migratory inhibitors to examine their roles in the actin polymerization pathway in cancer cell migration. GBM models were fabricated by implantation of spheroids within hydrogel microenvironments that were fashioned to exhibit migratory collagen tracts within a non-cell adhesive outer shell. The distribution of collagen was organised to have low, intermediate, and high-density regions within the construct, replicating the complexity of native GBM. 3D models were then cultured under control conditions (media only) or treated with anti-migratory drugs (CCG-1423, rhosin or combination). Using light-sheet and confocal microscopy, migration velocity, cell phenotype and migratory behaviours were analysed in live and fixed tumour mimics. In models where migration is promoted, actin was vastly upregulated and cells assumed a migratory mesenchymal phenotype, whereas under anti-migratory drug treatment, cells were amoeboid in shape with a dramatic reduction in actin expression and consequently limited migration velocity. Here, we have demonstrated that it is possible to develop biologically-relevant GBM models that capture the anisotropic nature of the tumour microenvironment using multi-layer biopolymer engineering. The ultimate goal of this research is to develop technology that can help provide personalised treatments for GBM and subsequently improve patient outcomes.
STEM-02. GENERATION AND CHARACTERISATION OF A LIVING BIOBANK OF POST-SURGICAL RESIDUAL GLIOBLASTOMA TO IDENTIFY NOVEL THERAPEUTIC TARGETS
Authors: McGarrity - Cottrell Connor , Myers Katie , Wang Dennis , Dunning Mark , Bagga Veejay , Al-Tamimi Yahia , Sinha Saurabh , Rominiyi Ola , Collis Spencer 
Published: November 2022

Abstract
Glioblastoma is the most common cancer arising within the brain and contributes to ~190,000 brain tumour related deaths/year globally. Current standard-of-care therapy consists of maximal safe surgical resection, radiotherapy with concomitant then adjuvant chemotherapy (Temozolomide) which triggers the cellular DNA damage response (DDR). However, survival for patients remains poor (median OS ~ 12-15 months). Lack of progress in the development and clinical translation of novel therapeutics is largely attributed to extensive inter- and intra-tumoral heterogeneity and resistant glioma stem cell (GSC) niches. Traditionally, primary ex vivo and in vivo models of glioblastoma have been derived from the resected tumour mass potentially omitting significant genetic, epigenetic and phenotypical characteristics exhibited by post-surgical residual tumour cells that are ultimately responsible for disease recurrence and morbidity. We therefore hypothesise that GSC’s isolated from the invasive edge of the tumour are inherently treatment resistant due to an increased “stemness” and upregulated DDR. Here we display novel methodology for the generation of multiple parallel primary ex vivo GSC cell models from anatomically discrete regions of specific partial lobectomy specimens. Importantly, these samples contain large portions of infiltrated adjacent brain to recapitulate features of resected (tumour core) and typically residual glioblastoma (distal invasive tumour edge). Initial RNA-seq analysis has revealed large divergent transcriptional landscapes of resected vs residual tumour cells and protein expression analyses has identified key differences in DDR proteins and subsequent cellular responses to current standard-of-care radio-chemotherapy regimens. Immunofluorescence analysis of DNA damage markers has also revealed differential capacity & kinetics for repair of DNA damage between resected and residual GSC populations. These data therefore highlight the potential clinical relevance of our novel parallel resected and residual ex vivo glioblastoma models, which will hopefully aid evaluation of novel therapeutics capable of targeting post-surgical GSC subpopulations leading to improved disease outcomes.
STEM-20. ROLE OF CELLULAR PRION PROTEIN IN TEMOZOLOMIDE RESISTANCE OF GLIOBLASTOMA STEM CELLS
Authors: Iglesia Rebeca , Prado Mariana , Alves Rodrigo , Tiek Deanna , Goenka Anshika , Song Xiao , Hu Bo , Hu Bo , Lopes Marilene 
Published: November 2022

Abstract
Glioblastoma (GBM), the most aggressive type of brain tumor, is initiated and maintained by a subpopulation of tumorigenic cells called glioblastoma stem cells (GSCs) that contribute to progression and resistance to therapy. In GBM therapy the alkylating agent temozolomide (TMZ) is the most effective chemotherapist, but resistance is frequent, especially in GSCs located in hypoxia niches. One of the mechanisms of resistance to TMZ involves the modulation of MGMT, which promotes cell survival through the induction of antiapoptotic proteins expression and can be regulated by hypoxia inducible factors (HIFs). Recent data from our group demonstrate that blocking the interaction between the cellular prion protein (PrPC) and one of its main ligands, the heat shock organizing protein (HOP) with a HOP peptide (HOP230-245) can inhibit GSCs self-renewal and glioblastomas growth in vivo. PrPC silencing decreases the expression of stem cells markers, self-renewal, and tumorigenesis of GSCs. Remarkably, a PrPC peptide which mimics HOP binding site (PrPC106-126) can promote HIF1α expression, inducing protection against cell death. Considering these data together, this study aims to identify the participation of PrPC in the TMZ resistance of GSCs mediated by HIF1α. In addition, we will evaluate HOP230-245 peptide potential in sensitizing GSCs to TMZ, since it binds to PrPC at the specific site 106-126 being able to compete with other PrPC ligands by this domain and, consequently, inhibit functions modulated by these interactions. Preliminary trials on stem cell enriched glioblastoma lineage cultures suggest that treatment with TMZ associated HOP230-245 increases cell death compared to TMZ or the peptide alone, sensitizing cells to TMZ treatment and increasing their efficiency. Finally, the search for therapeutic-combined strategies targeting GSCs is imperative to improve the efficacy of treatments against this incurable tumor.