bluuebunny/crossref_metadata_2025_split huggingface dataset:
TMIC-70. A TRANSCRIPTIONALLY ACTIVE EPIGENETIC IMPRINTING OF THE JAK/STAT PATHWAY MEDIATED BY H3.3-G34 MUTATIONS IN PEDIATRIC HIGH-GRADE GLIOMA (PHGG) INDUCES AN IMMUNOREACTIVE TME
Authors: Haase Santiago , Fabiani Maria Belen Garcia , Banerjee Kausjik , Mujeeb Anzar Abdul , Li Yingxiang , Zhu Ziwen , Nunez Felipe , Yu Jennifer , Lowenstein Pedro , Castro Maria 
Published: November 2023

Abstract
Pediatric high-grade gliomas (pHGGs) stand as the primary cause of cancer-associated fatalities among children. Sixteen percent of pHGGs found in children and young adults present Gly34Arg/Val substitutions within the histone H3.3 (H3.3-G34R/V). Using a syngeneic, genetically engineered mouse model (GEMM) and human pHGG cells encoding H3.3-G34R, we showed that this mutation leads to downregulation of the DNA repair pathways and enhanced susceptibility to DNA damage. DNA damage response (DDR) inhibitors in vivo, further increased genetic instability, and activation of the cGAS-STING pathway, which induces the release of immune-stimulatory cytokines. We carried out an epigenetic analysis using ChIP-seq in our GEMM, and our findings indicate that this mutation triggers transcriptionally active chromatin states within genes linked to immune reactivity, particularly JAK-STAT signaling pathway genes. Correspondingly, G34-mutant pHGG cells release immune-activating cytokines that reprogram the immune cells within the tumor microenvironment (TME). Myeloid derived suppressor cells from G34-mutant pHGG are phenotypically less immunosuppressive than the MDCS form histone wild type pHGG. Moreover, G34-mutant pHGG TME exhibit higher frequency of CD8 T cells. Based on these findings, we assessed the efficacy of an adenoviral-mediated immunostimulatory gene therapy (GT). This therapy mediates expression of Flt3L within pHGG TME, which aids dendritic cell recruitment. Thymidine kinase (TK), converts ganciclovir (GSV) into a cytotoxic compound, leading to pHGG cells’ death. GT resulted in 60% of long-term survival in mice harboring G34R pHGG. The animals that survived due to the GT did not develop tumors after a rechallenge with G34R pHGG cells, demonstrating that the treatment induces antitumoral immunological memory. In conclusion, our findings suggest that G34-mutant pHGG patients could benefit from combined therapies that target genetic instability and stimulate the immune system. Funded by NIH/NINDS.
TMIC-73. UNRAVELING THE ALTERED EXTRACELLULAR MATRIX LANDSCAPE IN GLIOBLASTOMA AFTER RADIATION TREATMENT
Authors: Manou Dimitra , Rosberg Rebecca , Pietras Alexander 
Published: November 2023

Abstract

BACKGROUND
Extracellular matrix (ECM) is a complex network of macromolecules, influencing cellular behavior and tissue integrity. Abnormal alterations in the composition and organization of the extracellular matrix have been implicated not only in the pathogenesis of numerous diseases, including cancer but are also related to the state of the disease. Despite advancements in treatment, glioblastoma (GBM) exhibits a high rate of recurrence. Tumor microenvironment (TME) plays a critical role in the regulation of cell behavior, and one important understudied tumor determinant is the ECM. Previous studies of our laboratory revealed that the matrix derived from irradiated astrocytes positively influences the stemness capacity and radio-resistance of GBM cells.

MATERIAL AND METHODS
The first aim of this project is to elucidate the landscape of important extracellular matrix hits in the TME of primary and recurrent GBM, using an in-house single seq database and an ECM-focused proteomic analysis in decellularized tissues. The second aim is to spatially map important neighborhoods with common crucial cell populations including astrocytes and macrophages and specialized regions such as perivascular and hypoxic niches using multiplex analysis.

RESULTS AND CONCLUSIONS
Preliminary data indicate a significant imbalance between fibrous components and glycans in the ECM after irradiation. In light of these considerations, our goal is to understand the TME interactions of cell populations with the matrix and the post-irradiation effect on matrix composition, aiming for an opportunity window for combinational ECM-targeted therapies.
TMIC-74. SPATIAL LANDSCAPE OF MELANOMA BRAIN METASTASES WITH AND WITHOUT PRE-SURGICAL IMMUNE CHECKPOINT BLOCKADE
Authors: Sun Lu , Ding Lizhong , Monteleone Amber , Liau Linda , Hugo Willy , Kim Won , Prins Robert 
Published: November 2023

Abstract
We recently characterized the immune populations of brain metastases samples with or without immune checkpoint blockade (ICB) treatment. In that study, we utilized single cell RNAseq (scRNAseq) and multiplex immunofluorescence (mIF) to demonstrate that ICB induced T cell infiltration and the migration of a unique MRC1+ macrophage subset from the perivascular space into the tumor parenchyma. However, scRNAseq’s tissue dissociation step loses spatial information of cells while mIF has low dimensionality, covering only a few markers for each tissue section. Thus, we applied sequencing-based Visium spatial transcriptomics (ST) to study the spatial architecture of melanoma brain metastases with/without ICB treatment and developed a pipeline that leverages public scRNAseq data as a reference to resolve fine-grained cell types on each Visium spot. ICB was associated with an increased CXCL13+ terminally exhausted T cell subset in close spatial proximity to tumor cells, suggestive of tumor specificity. ICB also reduced multiple MRC1+ macrophage subtypes in the blood vessel regions, supporting our mIF observation. Furthermore, we discovered that MRC1+ macrophages in close proximity to blood vessels expressed elevated TIMP1. TIMP1 is a known inhibitor of MMP9 that prevents ECM degradation and blood vessel damage, thus limiting T cell extravasation. In contrast, MRC1+ macrophages that migrated into the tumor had higher interferon activation and expressed CXCL9 and CXCL12 chemokines, which could recruit T cells via CXCR3 and CXCR4. Such findings could explain the increased T cell infiltration with ICB even in metastatic brain tumors. However, these MRC1+ macrophages also had an M2 signature and expressed multiple immune checkpoint ligands. Such data suggest these cells might also increase immunosuppressive interactions with T cells. In summary, our study reveals how ICB affects the spatial architecture and crosstalk of different cell types within melanoma brain metastases and will provide insights into new therapeutic strategies for brain metastases patients.
TMIC-78. ENDOTHELIAL REGULATION OF THE IMMUNOSUPPRESSIVE PHENOTYPE OF TUMOR-ASSOCIATED MICROGLIA IN GLIOBLASTOMA
Authors: Muthukrishnan Sree Deepthi , Qi Haocheng , Alvarado Alvaro , Kawaguchi Riki , Kornblum Harley 
Published: November 2023

Abstract
Glioblastoma (GBM) is considered a non-immunogenic “cold tumor” with an immunosuppressive microenvironment that blocks T-cell infiltration and function, limiting the efficacy of targeted immunotherapy. Tumor-associated microglia and macrophages (TAM) constitute approximately 30-50% of the non-neoplastic immune cells, and they are closely associated with blood vessels in GBM. However, the precise mechanism by which TAM acquire the immunosuppressive phenotype remains poorly understood. In this study, we show that tumor endothelial cells (TEC) from primary and recurrent GBM express inflammatory cytokines and chemokines that influence the function and polarization of microglia. We demonstrate that conditioned media (CM) of TEC promotes the immunosuppressive phenotype of normal microglia and patient-derived TAM. RNA-sequencing of TAM treated with TEC-CM shows upregulation of immunosuppressive cytokines, as well as T-cell co-inhibitory molecules such as PD-L1. Culturing CD8 T-cells in CM of TAM-polarized by TEC inhibits their cytotoxic function and cytokine production, and induces expression of PD-1 and CTLA-4. Furthermore, gene set enrichment analysis uncovers alterations in calcium signaling-related pathways in TAM polarized by TEC-CM. Blocking calcium signaling with small molecule inhibitors in microglia reverses the immunosuppressive phenotype induced by TEC. To determine whether the interactions between GBM, endothelial cells and TAM promotes immunosuppression in vivo, we utilize the humanized immune system mouse model (huNOG-EXL) that harbor human lymphocytes and monocyte populations. In preliminary studies, we found that co-transplantation of GBM tumor cells with TAM and TEC significantly promotes tumor growth compared with GBM cells alone. Single-cell mass cytometry analysis of peripheral blood mononuclear cells (PBMC) pre- and post-transplant, and scRNA-sequencing and spatial-transcriptomic studies of these co-transplanted mice will potentially reveal key insights into local- and peripheral immune responses regulated by tumor endothelial-microglia interactions. Together, our findings highlight a key role for endothelial-mediated calcium signaling in regulation of the immunosuppressive phenotype of microglia in GBM.
TMIC-80. PERK CONTRIBUTES TO GLIOBLASTOMA PROGRESSION BY REGULATING GLIOBLASTOMA STEM CELL ADAPTATION TO EXTRA CELLULAR MATRIX STIFFENING THROUGH F-ACTIN REMODELING AND FOCAL ADHESION COMPLEX REGULATION
Authors: Khoonkari Mohammad , Liang Dong , Govaerts Chris , Huurman Romy , Kamperman Marleen , van Rijn Patrick , Kruyt Frank 
Published: November 2023

Abstract
The association between a stiffening extracellular matrix (ECM) and increased aggressiveness of gliomas is well established. Originally recognized as a protective mechanism against endoplasmic reticulum (ER) stress, the unfolded protein response (UPR) is primarily responsible for maintaining cellular protein homeostasis and triggering cell death under severe stress conditions. However, emerging evidence indicates that the ER transmembrane UPR sensors, IRE1 and PERK, possess additional functions beyond proteostasis, such as modulating cancer stem cell differentiation, cell adhesion, and migration, which also involve more recently identified protein scaffold functions. In this study, we uncover a novel role of PERK in cellular adaptation to matrix stiffening. Glioblastoma stem cells (GSCs), when cultured on human blood plasma/alginate hydrogels with tuneable stiffness, exhibited stiffness-dependent cellular responses, including cell flattening, differentiation, and enhanced migration. Through genetic depletion and pharmacological inhibition strategies combined with immunofluorescence microscopy, we identified the involvement of a previously found PERK/FLNA signaling mechanism in the adaptive response to matrix stiffness, where F-actin polymerization emerged as a key mechanism. To explore the possible involvement of PERK in sensing ECM stiffness, the expression of various proteins that constitute the focal adhesion complex were studied. Remarkably, PERK also influenced the maturation of focal adhesion complexes components know to be involved in sensing ECM stiffness. Finally, comparative transcriptional profiling of PERK proficient and deficient GSCs uncovered significant alterations in the expression of genes associated with cell adhesion and extracellular matrix (ECM)-related processes. These findings reveal a novel function of PERK in stiffness-dependent cellular adaptations, with ongoing investigations exploring the underlying molecular mechanisms and therapeutic implications for glioblastoma progression
TMIC-85. TGFB2 AND IFNGR2 MRNA EXPRESSION LEVELS IN THE TUMOR MICROENVIRONMENT (TME) PROGNOSITICALLY IMPACT OS IN PEDIATRIC DIPG PATIENTS
Authors: Qazi SanJive , Trieu Vuong 
Published: November 2023

Abstract
DIPG patients expressed significantly higher levels of TGFB2 and IFNGR2 than control pons samples and the expression profile indicated higher levels of TGFB2 than TGFB1 and TGFB3 in DIPG patients suggesting TGFB2 mRNA level is preferentially increased in DIPG patients. TGFB2 is a prognostic indicator (HR for patients in the TGFB2high group of DIPG patients = 2.88 (1.12-7.39); P = 0.028) for poor overall survival (OS) independent of IFNGR2 levels, the age of the patient, and the significant interaction effect observed between IFNGR2 and TGFB2. Predictive modeling using the Cox regression parameters showed that the context of low expression of IFNGR2, TGFB2low resulted in more favorable OS times compared to TGFB2high whereby the median OS time of 11 months for the TGFB2low group of patients was greater than the upper 95% confidence limit for the TGFB2high group of patients (Median = 7 months; Upper 95% Confidence interval = 10 months). At higher levels of IFNGR2 expression, the TGFB2high group of patients exhibited a median OS time of 15 months that was within the (95% confidence interval for the TGFB2low group of patients (Median = 13 months; 95% CI = 10-17 months). Worse survival outcomes comparing DIPG patients with high versus low TGFB2 levels in the context of low levels of IFNGR2 levels suggest that abrogating the TGFB2 mRNA expression in the immunologically cold tumor microenvironment indicated by low levels of IFNGR2 expression can potentially treat pediatric DIPG patients. Furthermore, DIPG patients with low levels of JAK1 or STAT1 mRNA expression in combination with TGFB2high also exhibited poor OS outcomes suggesting that the inclusion of (Interferon-gamma) IFNγ to stimulate and activate JAK1 and STAT1 in anti-tumor immune cells present the brainstem tumor microenvironment can enhance the effect of TGFB2 blockade.
MODL-05. ELUCIDATING THE EPIGENETIC DRIVEN TRANSCRIPTIONAL PROGRAM UNDERLYING THE INVASIVE PHENOTYPE OF THE NOVEL EGFRA289VARIANT OF GBM
Authors: Lin Benjamin , Ziebro Julia , Shelton Abigail , Skinner Kasey , Bash Ryan , Osuka Satoru , Sudarshan Sunil , Binder Zev , O'Rourke Donald M , Nathanson David , Furnari Frank , Miller Christopher 
Published: November 2023

Abstract
Glioblastoma (GBM) is a devastating malignant brain tumor with a dismal 15-month median survival. Current treatments have not been successful at preventing recurrence, thus novel therapeutic modalities are urgently needed. EGFR is a tyrosine kinase receptor that is commonly mutated in ~60% of GBM and is an attractive target for GBM precision oncology. Aberrant EGFR signaling drives tumor proliferation and maintenance, thus further study of common EGFR alterations in GBM, including interstitial deletions (EGFRvIII) and missense mutations (EGFRA289), is needed to develop effective EGFR-targeted therapies. This is especially important in the context of a retrospective study that showed EGFRA289 driven GBM was more clinically aggressive. EGFRA289 positive GBM had a unique, invasive radiographic signature on magnetic resonance imaging (MRI) and patients had a significantly shorter median overall survival of 6-months. We hypothesize that the malignant clinical phenotypes of EGFRA289 GBM are driven by unique epigenomic and transcriptomic, collectively referred to as (epi)genomic, programs linked to EGFRA289 signaling. Prior studies using established cell lines show that EGFRvIII overexpression drives malignant (epi)genomic programs mediated by transcription factor (TF) activation. To better understand variant specific EGFR biology in GBM, we have developed novel genetically engineered mouse astrocyte (mAc) models that overexpress wtEGFR (MA-WT), vIII (MA-v3), and EGFRA289V (MA-A289V). Phospho-immunoblots show that these mAc models have differential EGFR ligand dependance, which match published studies. Despite this difference in EGFR signaling, all mAc models have similar sensitivities to EGFR tyrosine kinase inhibitor (TKI) treatment. However, MA-A289V is more proliferative and migratory compared to the other mAc variants. Preliminary RNA-Seq on our mAc models has identified similar and unique TFs differentially expressed (DE) between MA-v3 and MA-A289V when compared to parental MA-C. Integration of RNA-Seq and CUT&RUN followed by mechanistic genetic interrogation will elucidate the relationship between EGFR variant-specific (epi)genomic mechanisms and their unique malignant phenotype.
MODL-09. SCAFFOLD-BASED (MATRIGELTM) 3D CULTURE TECHNIQUE OF GLIOBLASTOMA RECOVERS A PATIENT-LIKE IMMUNOSUPPRESSIVE PHENOTYPE
Authors: Braun Frank , Rothhammer-Hampl Tanja , Lorenz Julia , Pohl Sandra , Menevse Ayse-Nur , Vollmann-Zwerenz Arabell , Bumes Elisabeth , Büttner Maren , Zoubaa Saida , Proescholdt Martin , Schmidt Nils , Hau Peter , Beckhove Philipp , Winner Beate , Riemenschneider Markus 
Published: November 2023

Abstract
3D (organoid) models in cancer research are considered the novel gold standard for in vitro experiments. Nevertheless, a variety of different protocols exits and the models are often used uncritically in lack of further validation. In this study, we aimed to characterize a scaffold-based (MatrigelTM) 3D glioma cell culture approach. We compared the 3D technique to conventional 2D culturing in its efficacy of generating models and in respect to its exact modeling properties. Models were generated from 62 patients with CNS tumors, most of them with glioblastomas, IDH-wildtype (GBM, n = 43). Pairs of 11 GBM models (2D and 3D) together with corresponding patient material were further characterized using bulk and single-cell RNA sequencing as well as 850k methylation analyses. Functional aspects of 3D tumor organoids were analyzed using a coculture system (Incucyte® SX5 live cell imager) with tumor infiltrating lymphocytes. Compared to conventional 2D culture the MatrigelTM-based 3D protocol was not superior in terms of the successful generation of in vitro models. However, NGS-based bulk and single-cell sequencing of matched pairs of 2D and 3D models showed an altered transcription of key immune regulatory genes in the 3D models, indicating the reoccurrence of an immunosuppressive phenotype. Changes included the presentation of different HLA surface molecules as well as cellular stressors. In the coculturing approach, lymphocyte-mediated cell killing appeared less effective in clearing 3D models than their 2D counterparts. IFN-γ release as well as live cell staining and proliferation analysis pointed towards an elevated resistance of 3D models. In conclusion, we found that the Scaffold-based (MatrigelTM) 3D culture technique depicts clinically highly important aspects of tumor-immune cell interactions, with the potential to explore immunotherapeutic approaches in an easily accessible in vitro system.
MODL-10. SEVERELY IMMUNODEFICIENT NOG-EXL MICE ALLOW FOR HUMANIZATION AND DEVELOPMENT OF A HUMAN GLIOBLASTOMA-DERIVED TUMOR MICROENVIRONMENT
Authors: Binder Zev , Lamrani Lamia , Secreto Anthony , Skuli Nicholas , Jacobson Moriah , Assenmacher Charles-Antoine , Willis Elinor , Radaelli Enrico , O'Rourke Donald M , Alanio Cecile , Nasrallah MacLean P 
Published: November 2023

Abstract
Cellular immunotherapy has resulted in tremendous therapeutic advances across liquid tumors. However, for solid tumors, including glioblastoma (GBM), significant limitations still exist in successfully translating immune-based adoptive cell therapeutics from the bench to the clinic. The majority of preclinical in vivo GBM models either use human cell lines grown in immunodeficient animals or syngeneic tumors grown in immunocompetent animals. Both approaches lack critical immune components of the tumor microenvironment (TME), thus inherently limiting the ability to adequately model immunotherapies. Here, we demonstrate the ability of hematopoietic stem cells (HSCs) from a GBM patient to successfully engraft in the severely immunodeficient NOG-EXL mouse strain and generate a patient-derived immune system. Intraosseous injections of a low number (10,000) of CD34+ HSCs, obtained from a bone marrow draw from the patient at the time of tumor resection, resulted in an average of 20% human CD45+ cells in peripheral blood 17 weeks post-transplant. GBM organoids, established from the same patient, were implanted intracranially at Week 18 and allowed to grow for 5 weeks without significant clinical evidence of graft-vs-host disease or macrophage activation syndrome. Immunohistochemical and flow cytometry analyses of mouse brains, with and without tumors, revealed infiltration of human-derived, CD45+/CD33+ cells, in proximity to the tumor engraftment site. These results demonstrate the ability of patient-obtained CD34+ HSCs to engraft and form a TME in severely immunodeficient mice. Subsequent work will focus on the differences between autologous and allogeneic humanized tumor-bearing mice in the context of the originating tumor. Additional future work will focus on testing cellular therapies directed towards the relevant areas of the autologous model.
MODL-11. EXPLORING THE THERAPEUTIC POTENTIAL OF YAP-TEAD INHIBITORS FOR TARGETING GBM CELL MIGRATION AT THE INFILTRATIVE MARGIN
Authors: Hurtado Thenzing Silva , Pai Balagopal , Ramos Susana Isabel , Hambardzumyan Dolores , Yong Raymund , Tsankova Nadejda 
Published: November 2023

Abstract
Glioblastoma (GBM) is an aggressive brain tumor with a median overall survival of only 12-15 months. Despite remarkable cellular and molecular heterogeneity, all GBM subtypes exhibit an infiltrating growth pattern which leads to incomplete surgical resection and inevitable recurrence, representing a major obstacle for effective treatment and an unmet need for therapy. Our research project aims to investigate the efficacy of YAP-TEAD inhibitors, including Verteporfin (VP), on migratory properties of GBM cells at the interface between the tumor core and the infiltrative tumor margin. Recently, several groups have implicated Hippo pathway YAP-TEAD activation in GBM growth and mesenchymal transition, and our laboratory demonstrated anti-migration and anti-invasion efficacy of the YAP-TEAD inhibitor VP across GBM subtypes, in patient-derived cell and xenograft (PDX) mouse models. Here we set to develop an ex vivo model for high-throughput testing of various YAP-TEAD inhibitors using organotypic slice cultures obtained from PDX and primary glioblastoma resection specimens, focusing on tumor areas around the infiltrative margin. Tumor cells within 250µ-thick organotypic slices were infected with GFP labeled retrovirus and subjected to confocal time-lapse imaging 72-96 hours later. Slices were treated first with vehicle (DMSO) for 4 hours, and subsequently with the YAP-TEAD inhibitor VP for 4 hours. To assess drug impact on tumor cell migration, we gathered imaging data on migration properties such as speed and directionality. Analysis of a PDX model generated from a de-novo GBM surgical resection (G-17969) revealed significantly decreased distance traveled in GFP+ cells treated with VP, compared to DMSO (p-value = 0.0002, n=50 cells). These pilot results corroborate other studies demonstrating anti-invasion properties of VP, contributing towards our goal of establishing a preclinical pipeline for rapid, high-throughput screening of anti-invasion drug inhibitors ex vivo. Key words: Glioblastoma, cell migration, YAP-TEAD inhibitors, time-lapse imaging