bluuebunny/crossref_metadata_2025_split huggingface dataset:
TMIC-08. CLAUDIN-1 UPREGULATION IN GLIOBLASTOMA INDUCED BLOOD-BRAIN-BARRIER DYSFUNCTION VIA A PARACRINE MECHANISM
Authors: Schweiger Brittany , Kievit Forrest 
Published: November 2023

Abstract

BACKGROUND
Blood-brain-barrier (BBB) disruption is a well-known phenomenon in glioblastoma. However, the mechanism driving BBB dysfunction in GBM is still unknown, especially in the previously established vasculature near the invasive edge. In this study, we aimed to determine if GBM paracrine signaling is sufficient to induce BBB dysfunction and identify changes in the tight junctions of the BBB.

METHODS
An in vivo xenograft model was established through an intracranial injection of U-87 MG cells in nude mice. Weekly MRIs were performed to monitor tumor growth. Two weeks after tumor appearance, brains were collected following a 4% PFA perfusion for immunostaining and confocal imaging. An in vitro model of the BBB was created by seeding primary mouse brain microvascular endothelial cells (BMECs) on transwell inserts. Once the BMECs reached confluency with a TEER value of 170-175Ω*cm2, the media was replaced with filtered conditioned media from U-87 MG cell line, an IDH mutant cell line, or BMECs for three days with TEER values monitored each day.

RESULTS
Immunofluorescent staining revealed a significant increase in claudin-1 expression and decrease in claudin-5 expression in the tumor vs normal brain tissue of our in vivo model (p &lt; 0.01). After 24 hours of exposure to conditioned media from both U-87 MG and IDH mutant cells, BMECs had a significant decrease in TEER compared to control and BMEC conditioned media (p &lt; .0001). After 72 hours, TEER values continued to decrease, reaching around 155Ω*cm2, with control BMECs remaining around 187Ω*cm2.

CONCLUSIONS
These results suggest GBM cells secrete factors capable of inducing changes in the tight junction proteins of the BBB and increasing its permeability. Future studies will aim to identify the mechanism in which these changes occur.
TMIC-09. PRIMARY CILIA REGULATE EXTRACELLULAR VESICLE-MEDIATED IMMUNOSUPPRESSION IN HUMAN GLIOBLASTOMA CELLS
Authors: Jung Mi-Yeon , Trichka Josie , Oishi Masahiro , Agyeman-Andoh Afua , Welsh Joshua , George Jubin , Roehrkasse Amanda , Laws Maxwell , Jones Jennifer , Adoro Stanley , Parney Ian , Brown Desmond 
Published: November 2023

Abstract
Glioblastoma is the most common primary malignant brain tumor. Despite maximal safe resection, followed by chemoradiation, median survival is 15 months, and 5-year survival is 6.8%. Efficacy of immunotherapy is limited by glioblastoma-mediated immunosuppression. Extracellular vesicles (EVs) are a heterogeneous group of small membrane-bound particles that are released from all cell types. Glioblastoma-derived EVs regulate the tumor microenvironment and tumor progression in a paracrine manner. They also regulate glioblastoma-mediated systemic immunosuppression in an IL6- and PDL1-dependent manner by inducing myeloid-derived suppressor cells (MDSCs) ultimately resulting in T cell depletion. Primary cilia are ubiquitous microtubule-based organelles that project from the mother centriole and are present on glioblastoma cells. Inhibition of ciliogenesis–through knockdown of KIF3A, IFT88, or ARL13B (structural ciliary proteins required for ciliogenesis)–abolishes IL6 induction in response to chemoradiation and significantly reduces the induction of MDSCs in normal human donor monocytes exposed to glioblastoma-derived EVs. Inhibition of ciliogenesis does not affect the biophysical properties of EVs but our data instead suggest significant alterations in EV packaging and content in response to ciliary loss. These data suggest a novel role for primary cilia in packaging of EV contents and connects these organelles with tumor EV-mediated immunosuppression in glioblastomas. These findings have potential implications for improving the efficacy of immunotherapy in glioblastoma–a universally fatal disease.
TMIC-11. SPATIAL PROFILING OF TUMOR VASCULATURE AT AUTOPSY IN BRAINSTEM GLIOMAS
Authors: Straehla Joelle , Woods Jared , Vogelzang Jayne , Jones Jacquelyn , Ligon Keith , Alexandrescu Sanda 
Published: November 2023

Abstract

BACKGROUND
Diffuse midline glioma (DMG) is a lethal pediatric tumor, and new treatment strategies are urgently needed. To design therapies that can cross the blood-tumor barrier, we designed a pilot study to investigate protein expression on DMG tumor vasculature using spatial profiling, with attention to targetable surface proteins.

METHODS
We identified 5 patients and 4 controls consented to institutional tissue banking protocols with adequate autopsy tissue from multiple anatomical regions within the brainstem. Portions of formalin-fixed, paraffin-embedded tissue were arranged into tissue microarrays profiled using the nanoString GeoMX platform. Briefly, a multiplexed cocktail of 51 primary antibodies tagged with UV-photocleavable oligonucleotide barcodes and fluorescent morphology markers were applied to tissues and 10-14 regions of interest (ROIs) were segmented by vessel area for each patient. ROIs were processed using UV light and barcodes were quantified using ex situ digital counting.

RESULTS
Five patients were included in the pilot study with average age 9.8 years (range 8-13), and overall survival was 9.5 months (range 3-18 months). Three tumors were classified as DMG, H3K27-mutant, WHO grade 4; one as high grade glioma without identified histone alteration but with recurrent DMG drivers (ACVR1 and PIK3CA mutations) and one as infiltrating atypical glioma with high proliferative rate and no DMG driver mutations. Controls ranged from 4-15 years and did not have pathological changes in the brain at death. Protein abundance was compared between cases and control and within cases where tumor-adjacent normal tissue was identified. Several proteins were significantly more abundant on tumor vasculature compared to non-tumor adjacent or control brainstem vessels. Validation studies in primary tissue are ongoing.

CONCLUSIONS
Digital spatial profiling is a promising technology for investigating tumor vasculature and is feasible in autopsy specimens. Work is ongoing to validate candidate biomarkers and expand the cohort to develop a vascular atlas for DMG.
TMIC-13. EXHAUSTED CD8+ T CELLS MAINTAIN LOW-GRADE GLIOMAS IN NF1-MUTANT MICE
Authors: Barakat Rasha , Chatterjee Jit , Mu Rui , Qi Xuanhe , Cobb Olivia , Gao Karen , Gu Xingxing , Smirnov Igor , Kipnis Jonathan , Gutmann David 
Published: November 2023

Abstract
T cells are minor cellular constituents of the brain tumor microenvironment, but have recently been shown to play critical roles in glioma pathobiology and treatment. Previous studies from our laboratory have shown that T cells are required for the formation and growth of low-grade gliomas (LGGs) in murine models of the Neurofibromatosis type 1 (NF1) tumor predisposition syndrome. To better characterize the immune landscape of these pediatric low-grade tumors, we leveraged single cell RNA sequencing of human pediatric pilocytic astrocytomas and genetically engineered mouse LGG (Nf1-optic glioma) models. First, we showed that both human and murine LGGs contain a CD8+ T cell population expressing PD-1 and TIGIT, similar to conventional exhausted T cell populations. Second, these PD-1+/TIGIT+ CD8+ T cells are actively recruited to the tumor site by Ccl2 where they dictate tumorigenesis through the elaboration of Ccl4, a cytokine necessary for microglia Ccl5-mediated support of Nf1-OPG growth. As such, Ccl4 expression was enriched in PD-1+/TIGIT+ CD8+ T cells relative to all other T cell populations within these tumors. Third, this “exhausted” CD8+ T cell population was exclusively present in the optic nerves, but not in the blood, dura, or deep cervical nodes of optic glioma-bearing mice. Fourth, targeting this PD1+/TIGIT+ exhausted CD8+ T cell population with immune checkpoint monoclonal antibody inhibitors reduced CD8+ T cell tumoral recruitment, Ccl4 production, and optic glioma growth. Collectively, these findings establish an obligate role for PD-1+/TIGIT+ CD8+ T cells in Nf1-optic glioma control and suggest that immune checkpoint modulation may be useful for the treatment of LGGs in children.
TMIC-16. THE COLLAGEN RECEPTOR LAIR-1 ALTERS THE GLIOMA MICROENVIRONMENT AND REGULATES TUMOR AGGRESSIVE BEHAVIOR
Authors: Varela Maria , Faisal Syed M , Comba Andrea , Lelling Sayer , Barissi Marcus , Castro Maria , Lowenstein Pedro 
Published: November 2023

Abstract
High-grade gliomas (HGG) are the most common malignant primary brain tumors in adults, with a median survival of 15-18 months. HGG are heterogeneous tumors with a complex extracellular matrix. Our previous work demonstrated the importance of the interaction between tumor cells and Collagen I. Employing genetically engineered mouse models (GEMM) of glioma, we identified Collagen 1α1 (Col1A1) as a regulator of tumor malignancy. Downregulation of Col1A1 in glioma cells extended median survival (MS), and reduced migration and proliferation. Nonetheless, the mechanisms underlining these effects remain unknown. LAIR-1 is a collagen receptor originally described as an inhibitory receptor in immune cells. We now describe the expression of LAIR-1 in glioma cells. Molecular ablation of LAIR-1 from glioma cells in GEMM (NPL: NRAS, shP53, shLAIR-1, and NPAL: NRAS, shP53, shATRX, shLAIR-1) significantly improved MS when compared to controls (NP: NRAS, shP53, and NPAL: NRAS, shP53, shATRX). The NPAL tumors had reduced cell proliferation (PCNA+), higher infiltration of astrocytes (GFAP+) and CD8+ T cells. Cells were obtained from these GEMM and cultured in vitro. LAIR-1 downregulation reduced cell proliferation, in both NPAL and NPL cells. To corroborate LAIR-1’s relationship with cell proliferation, control cells (NPA) were incubated with a LAIR-1 blocking antibody resulting in diminished glioma cell proliferation. When these cells were implanted in the brain LAIR-1 was downregulated resulted in increased MS. In vitro cytokines production assessment showed both NPAL and NPL cell had an enhanced production of RANTES(CCL5), G-CSF and IL-6 production, when compared to their controls. Our data suggest that LAIR-1 expression in glioma cells has a role in glioma malignant behavior both due to tumor cell proliferation and because of tumor composition. We hypothesize that blocking the interaction between LAIR-1 and collagen may improve median survival.
TMIC-23. UNRAVELLING THE HETEROGENEITY OF GLIOBLASTOMA'S VASCULAR LANDSCAPE
Authors: Woolf Zoe , Li Susan , Swanson Molly , Turner Clinton , Schweder Patrick , Correia Jason , Faull Richard , Park Thomas , Dragunow Michael 
Published: November 2023

Abstract

BACKGROUND
Glioblastoma tumours are highly vascular and angiogenic. However, they are also heterogeneous, comprising many different macro- and microenvironments. Reflecting this, tumour-associated blood vessels are variable in their distribution, structure, and function. Despite the significance of vasculature in the development and progression of glioblastoma, its complexity remains poorly defined. Therefore, characterising vascular diversity across the glioblastoma landscape is a crucial step in the development of targeted therapies.

METHODS
40 glioblastoma and 5 epilepsy cases were immunohistochemically labelled with a vascular antibody panel (PDGFRβ, ColIV, αSMA, UEA-1 and GFAP) to identify and subtype vessels. Single-vessel image analysis was performed to analyse vascular coverage and to classify vascular subtypes based on marker co-expression. Labelling with haemoglobin, ZO-1, and claudin-5 allowed for the evaluation of vascular leakage and blood-brain barrier integrity. Tissue segmentation was performed to analyse vascular coverage, vascular subtypes, and vascular integrity across the tumour macro-environment.

RESULTS
No significant difference in gross vascular coverage was found between tumour and epilepsy tissue. However, vascular coverage within glioblastoma varied significantly depending on the tumour macro-environment. Whilst vascular coverage in the tumour periphery was comparable to epilepsy tissue, increased vessel density was observed in the intermediate tumour zone, followed by decreased vessel density towards the tumour core. Reflecting this variation, multiple vessel subtypes, including previously described glomerular microvascular proliferation, mother vessels, vascular malformations and hyperplastic vessels, were identified. These tumour vessels display highly irregular shapes, sizes, and cellular compositions. Finally, glioblastoma tissue displayed a significant increase in haemoglobin leakage and a decrease in vascular integrity compared to epilepsy tissue.

CONCLUSION
Characterising this vascular landscape provides valuable insights for treatment strategies related to hypoxia and angiogenesis, two key factors driving tumour growth. Indeed, targeting specific vessel subtypes to address compromised vascular function and aberrant vascularisation may improve outcomes for glioblastoma patients.
TMIC-24. FERROPTOSIS INDUCES IMMUNOGENIC CELL DEATH AND SYNERGIZES WITH CD47 BLOCKADE TO REPROGRAM THE GLIOMA MICROENVIRONMENT
Authors: Sperring Colin , Argenziano Michael , Banu Matei , Dovas Athanassios , Mela Angeliki , Zhao Wenting , Furnari Julia , Pereira Brianna , Ager Casey , Yeary Mitchell , Shapiro Ben , Kotidis Corina , Adeyuan Oluwaseyi , Higgins Dominique , Upadhyayula Pavan , Mahajan Aayushi , Humala Nelson , Izar Benjamin , Sims Peter , Bruce Jeffrey , Canoll Peter 
Published: November 2023

Abstract
Immunotherapy has thus far proven ineffective in glioblastoma, in part due to the immune cold tumor microenvironment. Here we demonstrate that ferroptosis specifically targets a quiescent, immunosuppressive astrocyte-like glioma population and can therefore be used as a novel inducer of immunogenic cell death to repolarize the immune microenvironment by promoting tumor cell phagocytosis and antigen presentation. First, we characterized the immunogenic effects of ferroptosis by using a glioma cell-myeloid cell co-culture system and patient-derived organotypic slice cultures and performed phagocytosis assays, flow-cytometry, immunohistochemistry, cytokine arrays and single cell RNAseq. GPX4-dependent ferroptosis induced translocation of the pro-phagocytic eat-me signal calreticulin in both murine glioma cells and human tissue samples. Ferroptotic-driven tumor cell death reprogramed local myeloid populations to promote glioma phagocytosis, MHCII-driven antigen presentation, and paracrine cytokine signaling involved in T-cell recruitment. Local delivery of RSL3, a GPX4 inhibitor, in a murine glioma model polarized myeloid cells to a pro-phagocytic phenotype and significantly increased intra-tumoral levels of CD4+ and CD8+ T-cells. Astrocytic-like tumor cells upregulate CD47 as a mechanism to evade phagocytosis. Therefore, we combined ferroptosis with an anti-CD47 monoclonal antibody (aCD47) to enhance reprogramming of the immune cold microenvironment. In an orthotopic murine glioma model, adding aCD47 to RSL3 led to significantly increased tumor phagocytosis, antigen presentation and prolonged survival. Thus, we demonstrate that local delivery of ferroptosis-inducing agents in conjunction with CD47 blockade is an effective multi-modality therapeutic regimen capable of reversing the immunosuppressive glioma microenvironment.
TMIC-25. NEUTROPHIL-INDUCED GLIOBLASTOMA FERROPTOTIC CELL DEATH DEPENDS ON LC3-ASSOCIATED PHAGOCYTOSIS OF NEUTROPHILS BY GLIOBLASTOMA CELLS
Authors: Lu Tong , Yee Patricia , Thorpe Jessica , Wang Hong-Gang , Li Wei 
Published: November 2023

Abstract
Glioblastoma multiforme (GBM) is the most aggressive and common type of primary adult brain cancer. Tumor necrosis is a histological hallmark of GBM and a poor prognostic factor for the disease. Although it is thought to result from chronic ischemia, the molecular mechanisms that induce tumor necrosis remain unclear. Previously, we reported that tumor-associated neutrophils (TANs) contribute to the generation of tumor necrosis through inducing ferroptosis in GBM cells. We showed that accumulation of lipid peroxides within GBM cells depends on neutrophil myeloperoxidase (MPO). The cell killing is associated with transfer of MPO-containing granules to GBM cells. (Yee et al., 2020, Nature Communications, DOI: 10.1038/s41467-020-19193-y). During further study of Neutrophil-induced glioblastoma ferroptotic cell death, we found that neutrophil engulfment by GBM cells is required for intercellular transfer of neutrophil contents and the upregulation of lipid peroxides in GBM cells. The engulfment process can be inhibited by RGDS peptides, which inhibit the function of RGDS-containing integrins. Furthermore, we used immunofluorescence imaging to show that internalized neutrophils colocalize to molecular components of the LC-3 associated phagocytosis (LAP) machinery. Disruption of these components either through pharmacological or genetic means rescues GBM cells from neutrophil-induced cell death. We then used an orthotopic xenograft GBM mouse model to demonstrate that disruption of the LAP machinery in tumor cells reduces tumor necrosis and improves survival. Altogether, this study identified a novel ferroptosis-inducing process, which can help to understand how neutrophils contribute to tumor necrosis in GBM. Additionally, the results suggested that targeting LAP may have therapeutic benefits for GBM treatment.
TMIC-26. TAMS AND HYPOXIA ENHANCE GLIOBLASTOMA STEM-LIKE CELL ENRICHMENT AND MAY IMPACT RADIORESISTANCE
Authors: Karwacinski Victor , Carneiro-Lobo Tatiana , Olson Cheryl , Constante Mayerli , Mesci Pinar , Brat Daniel , Muotri Alysson 
Published: November 2023

Abstract
The dismal survival rates of glioblastoma (GBM) patients are due, at least in part, to therapeutic resistance of GBM stem-like cells (GSCs) and their ability to repopulate recurrent tumors. Our goal was to determine if tumor-associated macrophages (TAMs) or hypoxia enhance the enrichment of GSCs in a manner that could impact radioresistance. First, we established GBM-brain cortical organoids (GBM-BCOs) to model human GBM. We co-cultured GFP-labeled GBM neurospheres with BCOs for 14 days and observed a tumor take rate of 100%. Next, we evaluated whether hypoxia enhanced TAM migration into GBM-BCOs by incorporating THP-1 cells into GFP-GBM-BCOs and incubating them for 24 h under normoxic or hypoxic conditions. We found that hypoxia significantly increased THP-1 cell influx into GBM-BCOs. Further, we investigated whether TAMs influenced the enrichment of GSCs in GBM neurospheres and in GBM-BCOs. We found that co-culture of THP-1 cells with either GBM neurospheres or with GBM-BCOs enhanced the CD133+ population after 72 h hypoxia, correlating with our IF studies of human GBM samples that show increased CD133+ and IBA1+ cells in hypoxic peri-necrotic zones compared to non-necrotic regions. Next, we established Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI), an advanced methodology for monitoring cell cycle phases for investigating the role of hypoxia and radiation on GBM. Flow cytometry analysis of patient-derived GBM cells revealed that the fraction of G1 cells decreased and the fraction of G2/M cells increased 24 h after 3 Gy radiation compared to non-irradiated cells in normoxia. In contrast, GBM cells exposed to hypoxia do not show such cell cycle shifts after radiation and have an enriched population of cells in G0 in both radiated and non-irradiated cells. Our results indicate that hypoxia and TAMs play critical roles in GSC enrichment and may impact radioresistance.
TMIC-27. THE ROLE OF AQUAPORIN-4 IN GLIOBLASTOMA-ASSOCIATED VASOGENIC CEREBRAL EDEMA
Authors: Joshi Tanvi , Hambardzumyan Dolores 
Published: November 2023

Abstract
Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. Vasogenic cerebral edema is a severe complication that occurs in a majority of GBM patients, leading to a significant increase in intracranial pressure, accompanying neurological deficits, and increased mortality rates. Edema in GBM patients is almost exclusively managed by the corticosteroid dexamethasone (DEX), which has recently been found to have several drawbacks, including immunosuppression and interference with radiation therapy. Thus, there is an urgent need to better understand the mechanisms of edema resolution in GBM in order to identify alternative treatment strategies. The water channel aquaporin-4 (AQP4) is a critical player in the brain's water homeostasis, with increased levels in GBM compared to the normal brain. To gain more insight into the role of AQP4 in GBM-associated edema, we used the RCAS/tv-a system to generate de novo GBM with different driver mutations in Aqp4 knockout (KO), heterozygous (HET), and wild-type (WT) mice. Multiplex IF staining combined with RNAScope of tumors showed that AQP4 expression co-localized with GFAP-positive reactive astrocytes in perivascular areas. Our results indicated that tumor-bearing mice in an Aqp4-deficient background had increased edema content and decreased survival, suggesting that AQP4 is important for edema resolution from tumors. Loss of Aqp4 also significantly decreased astrocyte coverage in the tumor core, but not in the tumor periphery. Moreover, anti-VEGFA treatment only improved vessel leakage and reduced edema in WT tumor-bearing mice. Ongoing studies are focused on characterizing the impact of Aqp4 loss on the gene expression profile of tumor core-specific astrocytes using single-cell RNA sequencing and spatial transcriptomics. We are also studying the cellular distribution of AQP4 in GBM and the effect of DEX treatment on AQP4 levels.
TMIC-30. HYPOXIA-INDUCED COMPLEMENT COMPONENT 3 PROMOTES AGGRESSIVE TUMOR GROWTH IN THE GLIOBLASTOMA MICROENVIRONMENT
Authors: Rosberg Rebecca , Smolag Karolina , Sjölund Jonas , Johansson Elinn , Bergelin Christina , Wahldén Julia , Pantazopoulou Vasiliki , Pietras Kristian , Ceberg Crister , Blom Anna , Pietras Alexander 
Published: November 2023

Abstract
Glioblastoma is the most aggressive primary adult brain tumor. Treatment includes surgery, irradiation and chemotherapy. Still, virtually all patients suffer a treatment resistant recurrent tumor. In previous studies from our lab, we have investigated the tumor microenvironment contribution to tumor progression, showing that stromal astrocytes become reactive upon irradiation and hypoxia and generate tumor-supportive conditions for glioblastoma cells. Common features of astrocyte reactivity in several neurological diseases, include upregulation of complement proteins including complement component 3 (C3). However, it remains unexplored if these complement proteins are expressed in stromal astrocytes in glioblastoma. In a genetically engineered glioma mouse model, C3 was expressed in the invasive front, hypoxic, and perivascular tumor areas. TCGA data analysis revealed an overall strong correlation between hypoxia and complement gene expression signatures in glioblastoma patients. Astrocytes and glioma cells cultured in hypoxic conditions upregulated C3, as well as other genes associated with an infiltrative phenotype of GBM. In hypoxia, glioma cell lines, but not astrocytes, upregulated the C3a Receptor. GBM tumor single-cell sequencing data confirmed a subpopulation of highly C3-expressing astrocytes, enriched for cellular pathways comprising epithelial mesenchymal transition (EMT), TNF-alpha, hypoxia, and IFN-signaling. In vitro, C3 enhanced proliferation of glioma cells specifically under hypoxic conditions. Blocking C3a-C3aR signaling in vivo prolonged survival of glioma-bearing mice both alone and in combination with radiotherapy. Overall, our data indicate a strong link between hypoxia and complement expression in the brain tumor microenvironment of glioblastoma, where local expression of complement proteins lead to tumor promoting signaling. Inhibition of C3a-C3aR enhanced survival of glioma-bearing mice and could represent a promising avenue for further investigations.