bluuebunny/crossref_metadata_2025_split huggingface dataset:
TMIC-70. HARMONIZED SINGLE-CELL LANDSCAPE, INTERCELLULAR CROSSTALK AND TUMOR ARCHITECTURE OF GLIOBLASTOMA
Authors: Ruiz-Moreno Cristian , Salas Sergio Marco , Samuelsson Erik , Brandner Sebastian , Kranendonk Mariette , Nilsson Mats , Stunnenberg Hendrik 
Published: November 2022

Abstract
Glioblastoma (GBM) is the most aggressive and deadliest brain tumor in adults. Single-cell RNA sequencing (scRNA-seq) has helped to grasp the complexity of the cell states and dynamic changes within the tumor microenvironment (TME). To better capture the molecular underpinnings of transcriptomic variation across different individuals and reconstruct the intricate TME, it is necessary to gather a significant cohort that can empower our understanding of the complex GBM biology. In addition, little is known about the spatial disposition of the cells within the tumor, neighboring patterns, and intercellular crosstalk. Taking advantage of the recent developments in computational methods, we set out to build the GBM cellular landscape and created the ‘GBM-VERSE’, a curated reference that combines 16 datasets, and 113 patients spanning over 330 thousand cells. Our resource characterizes cells at different levels (coarse and detailed), identifying gene programs that define each cell type/state and providing a harmonized annotation. We demonstrated the applicability of the GBM-VERSE to map for newly generated data, providing a robust annotation tool for other GBM scRNA-seq datasets. We expanded the GBM-VERSE through transfer learning which increased the likelihood of identifying under-represented phenotypic states and incorporating cell types that were not present in the core reference. From the biological point of view, the GBM-VERSE facilitated the construction of an inferred global map of the cell-cell interactome. Our analysis highlighted the crucial role of MES-like cancer cells as the primary source of pro-angiogenic signaling. We employed the GBM-VERSE to chart the spatial architecture of GBM by deconvolving next-generation sequencing-based ST data. We found a well-defined spatiotemporal distribution of the neoplastic cells that were further inspected using an in situ sequencing-based platform. The GBM-VERSE represents a framework that allows the streamlined integration, and interpretation of new data and provides a platform for exploratory analysis, hypothesis generation, and testing.
TMIC-72. ALL-TRANS RETINOIC ACID INDUCES DURABLE TUMOR IMMUNITY IN IDH MUTANT GLIOMAS AND CIRCUMVENTS REPRESSION OF CRBP1-RETINOIC ACID AXIS
Authors: Zhang Xiaoran , Rao Aparna , Cillo Anthony , Mallela Arka , Krueger Katharine , Bruno Tullia , Amankulor Nduka 
Published: November 2022

Abstract
Diffuse gliomas are fatal, immunologically cold tumors characterized by mutations in isocitrate dehydrogenase 1 or 2 (IDH1/2) genes. IDH mutations have been shown to result in global genomic hypermethylation and drastically altered transcriptomic landscape. We and others have shown that IDH mutation is direct responsible for the immunological cold landscape observed in IDH mutated glioma. Using a combination of animal models and patient-derived glioma stem cells (GSCs), we identify retinoic acid signaling as a primary regulator of innate and adaptive immune activation in IDHm gliomas. Using the Sleeping Beauty (SB) orthotopic murine model of IDH glioma, which faithfully recapitulates the TME seen in patients, we show that intracellular accumulation of oncometabolite 2-HG leads to hypermethylation and transcriptional repression of Retinol Binding Protein-1 (RBP1), which encode CRBP1, a key chaperone protein required for intracellular all-trans-retinoic-acid biosynthesis in glial cells. Restoration retinoic acid signaling through the addition of all-trans retinoic acid (ATRA) resulted in spontaneous tumor regression and prolonged survival. ATRA administration is further correlated with increased CD8, NK, M1 macrophages and decreased Treg and MDSC populations within the TME. Single-cell sequencing of the TME shows increased type I interferon signaling, myeloid cell polarization, T cell activation, and TCR diversity. Using knockdown and overexpression techniques in patient-derived GSCs, we show that CRBP1 activity is critical for NK mediated immune surveillance and cytotoxicity through regulation of NKG2D ligand expression and immune cell migration through differential regulation of CXCL12 and CCL2. In summary, epigenetic silencing of RBP1 by 2-HG induced hypermethylation leads to a global immune dysregulation resulting in an immunologically cold landscape. Restoration of retinoic acid signaling is a promising immunotherapeutic approach for the treatment of IDHm gliomas.
TMIC-74. THERAPEUTICALLY REPROGRAMMING MICROGLIA TO ANTI-TUMORAL ACTIVATION STATES IN DIFFUSE MIDLINE GLIOMA, H3K27 MUTANT
Authors: Keane Lily , Perez Mercedes Posada , Friess Lara , Joseph Bertrand 
Published: November 2022

Abstract
Diffuse midline glioma (DMG), H3K27M mutant, is one of the most challenging paediatric cancers to date with poor treatment options and dismal survival outcomes. Therefore, new treatment options are desperately needed. Previously we found that activation of the microglial tumour-supportive phenotype by DIPG is associated with a transient H3K27me3 downregulation suggesting a role for this pathway in inducing this activation state. However, it should be noted, that microglia do not carry the H3-K27M mutation when analysed in DIPG patient tissue, otherwise characteristic of the cancer cells. Ezh2 is a histone methyltransferase and the catalytic subunit of the polycomb repressor complex 2 (PRC2), responsible for tri-methylation of histone H3 at lysine 27. We found that repression of Ezh2 in microglia induced an anti-tumour phenotype resulting in decreased cancer cell invasion capability, increased microglial phagocytosis, and tumour-related cell death. Antisense RNA strategies were used to target Ezh2 gene expression in BV2 microglia and we have now carried out RNA sequencing on scramble BV2 or siEzh2 microglia co-cultured with DIPG tumour cells to further understand how EZH2, PRC2 and H3K27me3 regulates DIPG-induced microglia activation. Further work has found that activation of the microglial tumour supportive phenotype, in this context, is determined by a complex epigenetic mechanism involving both H3K4me3 and H3K27me3 and their associated methyltransferases. These histone marks are often associated with bivalent regulation of distinct gene transcription programs, and we are currently investigating if this is the case for DIPG-induced activation states using chromatin immunoprecipitation techniques. Overall, our results indicate that microglia, can be reprogrammed epigenetically by EZH2 inhibition to exert anti-tumoral responses and that microglia represent a new therapeutic avenue in DIPG. This future work should provide novel mechanisms governing pro-tumoral microglial activation states and new therapeutic targets that can be combined with EZH2 inhibition to enhance anti-tumoral microglia activation states.
TMIC-75. TUMOR ENDOTHELIAL CELL-DERIVED ASPORIN DIFFERENTIALLY REGULATES GLIOBLASTOMA GROWTH
Authors: Muthukrishnan Sree Deepthi , Pham Amy , Alvarado Alvaro , Kawaguchi Riki , Kornblum Harley 
Published: November 2022

Abstract
Gliomas are the most prevalent and malignant brain tumors. A major histopathological feature that distinguishes the high-grade (IV) glioblastoma (GBM) from slow-growing low-grade (II and III) glioma is microvascular hyperproliferation. Although gliomas are highly vascularized tumors, anti-angiogenic therapies have failed to prevent recurrence and significantly improve patient survival. To determine if tumor endothelial cells (TEC) display molecular heterogeneity, we performed bulk RNA-sequencing on cultured CD31+ TEC from low-grade (II/III), and high-grade (IV, primary and recurrent) gliomas. Low- and high-grade TEC exhibited significant molecular heterogeneity, with increased enrichment of extracellular matrix and cell cycle-related gene sets in low-grade and enrichment of cytokine and immune response-related gene sets in high-grade TEC. We also found significant functional differences in proliferation capacity, and resistance to anti-angiogenic therapies between low-and high-grade TEC. Co-transplantation of low-grade TEC with tumor cells significantly inhibited GBM growth and enhanced survival, whereas high-grade TEC promoted growth of tumors in orthotopic xenografts in vivo. Differential gene expression analysis revealed several proteoglycans distinctly expressed by low-grade TEC. Of the highly enriched molecules in low-grade TEC, Asporin (ASPN), a small leucine-rich repeat proteoglycan (SLRP) significantly inhibited growth and proliferation of IDH1-wt GBM cells but did not affect IDH1-mutant tumor cells. Co-transplantation of TEC-lacking ASPN with IDH1-wt GBM partially rescued the growth-inhibitory effect of low-grade TEC on tumor growth and reduced survival. Ongoing experiments are aimed at determining the molecular mechanism underlying the growth-inhibitory effects of TEC-derived Asporin on glioma.
TMIC-77. SINGLE-CELL CHARACTERIZATION OF HUMAN GBM REVEALS REGIONAL DIFFERENCES IN TUMOR-INFILTRATING LEUKOCYTE ACTIVATION
Authors: Schmassmann Philip , Roux Julien , Dettling Steffen , Hogan Sabrina , Shekarian Tala , Martins Tomas A , Ritz Marie-Françoise , Herter Sylvia , Hutter Gregor 
Published: November 2022

Abstract
Clinical trials of systemic T cell checkpoint blockade in GBM patients showed only disappointing results. This may be attributed in part to the immunosuppressive components of the GBM immune tumor microenvironment (iTME). Therefore, major efforts have been undertaken to describe the GBM iTME on a single cell level. However, human data on the composition of the iTME in different tumor regions (contrast enhancing tumor center versus peripheral infiltration zone) remain scarce. Here, we performed high-depth single-cell RNA sequencing (scRNAseq) on patient-matched biopsies from the tumor center and the peripheral infiltration zone of five primary GBM patients. Additionally, peripheral blood mononuclear cells (PBMC) of the same patients were included in the scRNAseq analysis to explore the transcriptional changes occurring during tumor infiltration of circulating immune cells. Through analysis of &gt; 45’000 cells, we revealed a distinct regional transcriptional profile of microglia (MG) and monocyte-derived macrophages (MdM), a non-reactive/exhausted MG subcluster in the GBM iTME and an impaired interferon-response signature in the peripheral cytotoxic cell compartment. Comparing CD8+ T-cells from the tumor periphery to PBMC-derived CD8+ T-cells of the same patient revealed a CX3CR1+ CD8+ T-cell population with effector memory phenotype which is enriched in the PBMC but lacking in the tumor periphery. Peripheral CD8+ T-cells were mainly composed of tissue-resident memory CD8+ T-cells with exhausted effector functions. Our analysis provides a large-scale dissection of GBM-associated cell types complemented by patient-matched PBMCs, serving as a high dimensional reference map of the human GBM iTME.
TMIC-79. MYELOID-DIRECTED TREATMENT USING THE TLR7/8 AGONIST RESIQUIMOD IMPROVES THE SURVIVAL OF MICE WITH MEDULLOBLASTOMA AND ENHANCES EFFICACY OF RADIOTHERAPY
Authors: Pauneto Coral del Mar Alicea , Hwang Duhyeong , Park Christopher , Sokolsky Marina , Gershon Timothy 
Published: November 2022

Abstract
We show the therapeutic potential of myeloid-directed immunomodulatory treatment for medulloblastoma. Patients with medulloblastoma, the most common malignant pediatric brain tumor, need new treatments, as standard therapy produces disabling neurotoxicities and fails 20% of patients. About 30% of medulloblastomas show hyperactivation of the SHH (Sonic Hedgehog) signaling pathway, and these tumors have large myeloid populations in the tumor microenvironment (TME). We analyzed whether activating toll-like receptors on these myeloid cells would slow tumor growth. Our prior single-cell RNA sequencing studies in both patient samples and mouse models showed that 10% of the cells in SHH medulloblastoma are myeloid cells and that these cells uniquely express TLR7 and TLR8. We treated mice genetically engineered to develop SHH medulloblastomas with the TLR7/8 agonist resiquimod, administered systemically either as free drug or in polyoxazoline nanoparticles (POx-resiquimod), and compared POx-resiquimod+radiotherapy to radiotherapy alone. We found that POx-resiquimod extended the survival time of mice with medulloblastoma, while free drug failed to show benefit. PK studies showed that POx-resiquimod increased tumor drug exposure, consistent with increased efficacy. Mechanistically, POx-resiquimod increases tumor myeloid populations and decreased the fraction of myeloid cells that expressed IGF1. POx-resiquimod plus radiation therapy, moreover, was superior to either POx-resiquimod alone, or radiotherapy alone. Together our data show that the TLR7/8 agonist resiquimod, delivered in nanoparticle formulation, produces a significant anti-tumor effect in SHH medulloblastoma, and increased the efficacy of radiotherapy. As radiotherapy is the mainstay of current medulloblastoma treatment, we propose that TLR7/8-agonist therapy such as resiquimod may be added to current regimens to reduce the radiation dose needed for efficacy, and to increase the fraction of successfully treated patients.
MODL-01. TRACTABLE PATIENT-DERIVED MODELS FOR PRECLINICAL THERAPEUTIC STUDIES OF CNS METASTASES
Authors: Vojnic Morana , Odintsov Igor , Offin Michael D , Lui Allan J W , Khodos Inna , Chang Qing , Mattar Marissa S , De Stanchina Elisa , Ladanyi Marc , Somwar Romel 
Published: November 2022

Abstract

INTRODUCTION
Brain metastases are the most common brain tumors and occur in 10-30% of cancer patients, whereas leptomeningeal disease (LMD) occurs in approximately 5% of adults with systemic malignancies. Tractable preclinical disease models that faithfully represent metastasis to the brain and recapitulate LMD are needed to improve our understanding of the biological basis of CNS disease as well developing effective therapeutic strategies. Our goal in this study was to generate representative preclinical disease models using two methods.

METHODS
We isolated tumor cells from CSF of 16 patients with cytologically proven LMD (9 NSCLC, 1 melanoma, 1 ovarian cancer, 1 endometrial cancer, and 4 breast cancer) and implanted the cells subcutaneously into the flank of immunocompromised mice. Cell lines were also generated from PDX tissues. Models were characterized by next-generation sequencing (NGS). We also generated a model of CNS metastasis of kinase-driven sarcoma by intracardiac (IC) injection of human mesenchymal stem cells (HMSC) expressing a SPECC1L::RET fusion (CRISPR-Cas9 gene editing). The cells (HMSC-RET) were also labeled with a luciferase construct to allow non-invasive bioluminescence imaging.

RESULTS
We established three PDX models (2 lung, 1 ovarian) from CSF (19% success rate compared to approximately 33% for solid tumors) and matched cell lines from the resulting PDX tissues. Intracardiac injection of HMSC-RET cells resulted in tumors establishing in several peripheral organs and the brain.

SUMMARY AND CONCLUSIONS
We have established disease models of CNS metastasis and LMD. Translational studies where patients with clinical suspicion of LMD undergo CSF sampling, NGS/ctDNA analysis, and PDX modeling are crucial in improving our understanding of this metastatic compartment and investigating novel treatment paradigms. Future studies will be focused on examining the biochemical and genetic nature of these tumors as well as developing effective therapeutic strategies.
MODL-04. ESTABLISHMENT OF A RELIABLE GLIOBLASTOMA TUMOR-ORGANOID PLATFORM ENABLES AUTOMATED HIGH-THROUGHPUT DRUG SCREENING ADVANCING PERSONALIZED MEDICINE
Authors: Herold-Mende Christel , Paul Adrian , Cao Junguo , Warta Rolf , Andreas Unterberg , Abdollahi Amir , Jungwirth Gerhard 
Published: November 2022

Abstract
Patient-derived tumor organoids (TOs) generated from tumor tissues within a few days are promising new models since under optimal conditions they preserve genotype, phenotype, the tumor microenvironment and the cellular heterogeneity of the parental tumor. This exciting new technology could provide representative avatars of the patient tumor in large numbers and thus can be exploited for drug discovery and drug screening purposes more efficiently than conventional cell lines or xenografted mouse models. We established standardized TOs from fresh glioblastoma (GBM) tissue after enzymatic dissociation into single cell suspensions and seeding cells into anti-adhesive 384-well plates. Over 1000 standardized TOs were generated per patient with a success rate of over 90%. Morphologically, TOs formed after two days. Size was stable over 10 days, with a slow outgrowth in some cases. A steady increase of the ATP signal over time indicated proliferative activity in TOs. Immunofluorescent stainings for tumor cells (GFAP), immune cells (macrophages and T-cells), and extracellular matrix (Tensascin C) confirmed a strong resemblance of TOs to their corresponding parental tumor, which was further substantiated by RNA-seq and DNA-methylation. Next, automated robot-based high-throughput drug screening (aHTS) on TOs was performed using a drug library of 166 FDA-approved antineoplastic drugs. Cell viability was assessed by CellTiter-Glo3D. In total, aHTS on TOs from 11 GBM patients including two recurrent GBMs showed a strong heterogeneity of drug responses. The top ten best-performing drugs consisted of 35 compounds targeting 14 different modes of action. Proteasome and HDAC-inhibitors demonstrated the highest efficacy. Respective targets were validated by knockdown experiments in GBM cells. Altogether, our data corroborate the future use of standardized GBM TOs as patient avatars for personalized drug testing by utilizing an automated high-throughput drug screening platform to better account for inter-patient-individual drug responses.
MODL-06. ASSESSMENT OF ONCOLYTIC VIRUS SPECIFICITY AND CYTOTOXICITY IN A HYBRID GLIOBLASTOMA-CEREBRAL ORGANOID MODEL
Authors: Ferreira Rodolfo Sanches , Araujo Bruno H S , Okamoto Oswaldo 
Published: November 2022

Abstract
Recent studies have demonstrated potent oncolytic effects of wild-type Zika virus (ZIKV) against primary central nervous system (CNS) tumors, including Medulloblastoma, Atypical Teratoid/Rhabdoid Tumor (AT/RT), and Glioblastoma (GBM). However, the neurotropism of ZIKV urges further evaluation of specific tumor-targeting properties and comparative toxicity to non-neoplastic neural cells in order to address its therapeutic potential. We have developed a hybrid organoid model by co-culturing GBM cells with mature human cerebral organoids and assessed cytotoxicity of the Brazilian ZIKV isolate (ZIKVBR) towards normal brain organoid cells and GBM cells. Human induced pluripotent stem cells (hiPSC)-derived cerebral organoids were co-cultured with 105 GFP-expressing cells of three different GBM cell lines. A total of 20.000 PFU of ZIKVBR or mock condition was added per co-cultured model. Viable cells were analyzed by flow cytometry (FC) and fluorescence microscopy at different time points. ZIKVBR presence was assessed by PFU assay. Although ZIKVBR infection did not cause a pronounced reduction of GFP+ cell proportion over time in all GBM cell lines, cell viability analysis showed a greater amount of non-viable GFP+ cells over GFP- cells in all ZIKVBR-treated groups, compared to their corresponding mock groups. PFU assays of cell culture supernatants confirmed the presence of infectious viral particles in treated groups and absence in mock groups. Our data reveal that ZIKVBR has a potential oncolytic effect characterized by preferential killing of GBM tumor cells over normal cerebral cells, in a hybrid organoid model. These findings support the development of an oncolytic virus therapy platform based on ZIKV.
MODL-19. CREATION OF AN IN VITRO MODEL SYSTEM FOR THE STUDY OF H3.1K27M DMG
Authors: Lagas Joseph , Yang Lihua , Becher Oren , Rubin Joshua 
Published: November 2022

Abstract
Diffuse Midline Gliomas (DMG) are a devastating group of pediatric high-grade gliomas that occur in the brainstem with a median survival of less than 1 year. A greater understanding of the early tumorigenic events is essential for the development of effective therapeutics. DMG is characterized by founder mutations in histone H3, either H3.1K27M or H3.3K27M. These mutations cause global hypomethylation, resulting in aberrant gene expression. It is unknown how this mechanism contributes to tumorigenesis. Interestingly, H3.1K27M DMG show an increased incidence in females, whereas H3.3K27M DMG shows no sex difference. This illustrates that the tumorigenic potential of H3.1K27M may be different between the sexes. Few models of DMG incorporate the study of H3.1K27M, even though it represents a unique opportunity to obtain valuable information on the tumorigenesis of DMG through the study of the sex difference. Additionally, an important regulatory element, a stem loop structure, present in mammals is missing from all previous models studying H3.1K27M. Thus, we have created an in vitro model system for H3.1K27M DMG that incorporates the stem loop structure utilizing the RCAS mouse model system. This system utilizes RCAS vectors and a RCAS-ntva transgenic mouse line to deliver specific mutations to nestin expressing cells in the brainstem, including neural and glial progenitor cells, the potential cells of origin. Confirmed through immunofluorescence staining, we can isolate a pure population of NPCs and transduce them with RCAS vectors in vitro to produce stable expression of H3.1K27M. Verified via immunocytochemistry and flow cytometry, delivering H3.1K27M with the stem loop included limits its expression to S phase of the cell cycle, mimicking the human disease biology. These results demonstrate that we have created an in vitro H3.1K27M DMG model system for the study of sex differences and tumorigenesis in DMG.
MODL-21. EFFICACY OF DIFFERENT LAYOUTS IN TREATING INFRATENTORIAL TUMORS WITH TUMOR TREATING FIELDS (TTFIELDS)
Authors: Naveh Ariel , Marciano Tal 
Published: November 2022

Abstract
TTFields are an anti-mitotic technique used to treat solid tumors by delivering low-intensity (1–4 V/cm) alternating electric fields to the tumor, with frequencies between 100–500 kHz. This modality is FDA-approved for treating glioblastoma (GBM). Administration of TTFields is conducted using two pairs of transducer arrays placed on the patient’s scalp. The layouts of the transducer arrays is a key factor in determining the field distribution within the patient’s body. The NovoTAL system is used for determining the array layouts used for supratentorial tumors. In previous works, we suggested layouts for infratentorial layouts, as well as layouts that target the entirety of the brain with field distribution of higher uniformity. However, in some clinical cases, patients may have an infratentorial tumor and a single supratentorial tumor simultaneously. In such a situation, the question of whether we can treat both tumors with an infratentorial layout arises. In this work, we examined all GBM available layouts (NovoTAL, infratentorial and whole brain). We used a realistic healthy patient model (Duke, by ITI’S, Zurich, Switzerland) to assess field distributions resulting for the different layouts. We then created maps of expected TTFields dose for a 10 cm sized tumor placed in each point in the brain, and ranked the different regions by best layout groups. Our results show that for infra-ventricular tumors, the whole brain layouts may provide higher field distributions than any of the NovoTAL or infratentorial layouts. For supra-ventricular tumors, NovoTAL layouts provide stronger fields. A transition zone in which both layout groups provided similar results was observed as well. We conclude that the best option of currently available transducer array layouts for treating the presented clinical case depends on the location of the supratentorial tumor, whether it is above the large ventricles or beneath them.