bluuebunny/crossref_metadata_2025 huggingface dataset:
TMIC-57. USING SINGLE-CELL SEQUENCING AND SPATIAL TRANSCRIPTOMICS TO IDENTIFY CANCER-ASSOCIATED FIBROBLASTS IN GLIOBLASTOMA AND DEFINING THEIR PRO-TUMORAL EFFECTS
Authors: Jain Saket , Rick Jonathan , Joshi Rushikesh , Beniwal Angad , Spatz Jordan , Gill Sabraj , Chang Alexander , Chalif Eric , Haddad Alexander F , Costello Joseph F , Diaz Aaron , Heiland Dieter Henrik , Aghi Manish
Published: November 2022

Cancer-associated fibroblasts (CAFs) constitute a key component of the tumor microenvironment. Pro-tumoral cancer-associated fibroblasts were presumed absent in glioblastoma given the lack of brain fibroblasts. Using single-cell RNA sequencing we identified CAFs in patient GBMs. CAFs were identified using a negative selection strategy to filter endothelial, epithelial, immune cells and pericytes and for the positive expression of previously defined CAF markers. Copy number variation (CNV) analysis was performed to distinguish CAFs from malignant cells. Single-cell spatial transcriptomics from 16 GBM patients confirmed the proximity of CAFs to mesenchymal GBM stem cells (GSCs), endothelial cells, and M2-macrophages. Serial trypsinization of glioblastoma specimens yielded cells with CAF morphology and transcriptomic profile. CAFs were chemotactically attracted to GSCs and CAFs induced GSC proliferation. To identify CAF and GSC interaction mediators, we created a resource of inferred crosstalk by mapping expression of receptors to their cognate ligands, identifying PDGF and TGFB as mediators of GSC effects on CAFs, and osteopontin and HGF as mediators of CAF-induced GSC enrichment. Furthermore, CAFs were found to induce M2-macrophage polarization by producing the EDA fibronectin variant which binds macrophage toll-like receptor 4 (TLR4) in a targetable manner. Glioblastoma CAFs were enriched in the subventricular zone which houses the neural stem cells that houses GSCs. Including CAFs in GSC-derived xenografts induced in vivo tumor growth and reduced survival in two different xenograft models. These findings are among the first to identify glioblastoma CAFs and their GSC interactions, making them an intriguing target for Glioblastomas.
TMIC-59. GLIOBLASTOMA INDUCES THE DIFFERENTIATION AND RECRUITMENT OF NON-CANONICAL ANTI-TUMORAL NEUTROPHILS FROM SKULL BONE MARROW
Authors: Lad Meeki , Beniwal Angad , Jain Saket , Gill Sabraj , Aghi Manish
Published: November 2022

The effects of tumor-associated neutrophils (TANs) on glioblastoma biology remain poorly understood. Flow cytometric analysis of 5 newly diagnosed glioblastoma fresh tissue specimens surprisingly revealed a high fraction (21.0-34.1%) of TANs expressed MHCII, a marker of antigen-presenting cells not classically associated with neutrophils and not expressed in matched peripheral blood (PBNs). Transcriptomic profiling confirmed that patient TANs upregulated expression of MHCII subunits (HLA-DR), chaperones (HLA-DM), and costimulatory ligands (CD86/83). Ex vivo cocultures further demonstrated that TANs activated patient-matched naïve T cells in an MHCII-dependent manner, while PBNs did not (CD25 MFI fold-change: 1.1 vs 3.2, p< 0.001). The antitumoral relevance of this property was confirmed in a syngeneic mouse glioma model, wherein αLy6G-mediated neutrophil depletion in T-cell-competent mice (Balb/c, n= 13) yielded endpoint tumors that had reduced CD8+ T cell infiltration (p= 0.0024) and were 2.4-fold larger by BLI (p= 0.0383) than controls, but had no bearing on tumor burden in T-cell-deficient (athymic) mice. Given the absence of MHCII+ neutrophils in circulation, we interrogated the inducibility of this phenotype by tumor-conditioned media in murine marrow/blood, finding that only immature Ly6Glow bone marrow neutrophils had sufficient plasticity to express MHCII and process foreign antigen as measured by DQ-ovalbumin uptake/proteolysis. Because of the non-inducibility of PBNs, we investigated the hypothesis that MHCII+ TANs were recruited to the glioblastoma microenvironment from adjacent skull bone marrow by labeling this space with CFMDA in tumor-implanted mice; at 72h, skull marrow-derived neutrophils contributed disproportionately to MHCII+ TANs compared to all TANs (41.7% vs 9.3%). As confirmation, in the first-ever scRNA-seq of human TANs, we demonstrated via pseudotime analysis that MHCII+ neutrophils represent a developmental lineage seen in TANs but not matched PBNs. Given the immunostimulatory effects of this population, mechanisms to promote neutrophil egress and tumor infiltration from the skull marrow may have therapeutic value.
TMIC-62. INHIBITION OF TUMOR-ASSOCIATED COL1A1 MATRIX ARRESTS GLIOMA MESENCHYMAL TRANSFORMATION AND REPROGRAMS THE TUMOR MICROENVIRONMENT
Authors: Comba Andrea , Faisal Syed M , Varela Maria Luisa , Argento Anna , Dunn Patrick , Abel Clifford , Hollon Todd , Al-Holou Wajd , Zamler Daniel , Quass Gunnar , Apostolides Pierre , Kish Phillip , Perelman Jacqueline , Jacobs Nicole , Kahana Alon , Brown Christine , Kleer Celina , Motsch Sebastien , Castro Maria , Lowenstein Pedro
Published: November 2022

Tumor mesenchymal transformation (MT) is a hallmark of high-grade gliomas. The mesenchymal state is associated with specific changes related to cell adhesion, migration, and the extracellular matrix. Collagen 1a1 (COL1A1) is a main component of the extracellular matrix in gliomas, and its expression correlates inversely with patient survival. However, the cellular and molecular mechanisms of the tumor-associated COL1A1 matrix in gliomas remains elusive. Our study integrates histopathological features, spatially resolved transcriptomics, cellular dynamics and microenvironment alterations associated with MT in high-grade gliomas. Using deep learning analysis of mouse and human glioma histological samples we identified that the density of areas of MT, named oncostreams, correlates with tumor aggressiveness. Spatial transcriptomics analysis, using laser capture microdissection, identified a signature enriched in extracellular matrix related proteins, in which COL1A1 appeared as a key determinant of mesenchymal organization. Correspondingly, human and mouse high-grade gliomas showed prominent alignment of collagen fibers along these mesenchymal fascicles and higher COL1A1 expression compared to low-grade gliomas. Moreover, RNA fluorescent multiplex assays identified at single cell level that different cells within glioma tumors contribute to COL1A1 expression, including neoplastic cells and perivascular non-neoplastic cells such as ACTA2+, CYR61+ and FAP+. Inhibition of COL1A1 using genetically engineered mouse models decreased areas of mesenchymal transformation and increased survival. COL1A1 downregulation impaired tumor cell proliferation and remodeled the tumor microenvironment by reducing CD68+ macrophages/microglia cells, CD31+ endothelial cells, ACTA2+, CYR61+ and FAP+ perivascular cells, and increased GFAP+ astrocytes infiltration withing the tumor mass. Further studies, using ex-vivo glioma explants demonstrated that CO1A1 downregulation decreased collective invasion of the normal brain, supporting its importance in tumor progression. We propose that COL1A1 expression is a valuable marker for diagnosis, and COL1A1 depletion within glioma tumors is a promising direct or complementary therapeutic approach to reprogram mesenchymal transformation, and halt tumor growth.
CNSC-30. IN VIVO DYNAMICS OF ASTROCYTE-GLIOBLASTOMA TUMOR NETWORKS
Authors: Yang Yvonne , Reyhan Ekin , Schubert Marc , Denninger Robert , Wißmann Niklas , Pramatarov Rangel , Wick Wolfgang , Kuner Thomas , Winkler Frank , Venkataramani Varun
Published: November 2022

As one of the most aggressive incurable primary brain tumors, glioblastomas show a high invasivity and therapeutic resistance caused by their cellular and molecular heterogeneity. In previous studies, we showed that glioma cells interconnect using membrane protrusions called tumor microtubes to form a therapy-resistant malignant network. Here, we extend the concept of tumor networks to heterogeneous connectivity with the glial microenvironment. Using high-resolution light microscopy, ultrastructural tissue imaging, patch-clamp electrophysiology, intravital structural and functional microscopy, we characterize tumor-astrocyte connectivity. First, we used high-resolution light microscopy with immunohistochemistry and ultrastructural imaging to discover and characterize gap junctional connections between tumor cells and astrocytes. Next, we probed functional, heterotypic connections using dye filling with patch-clamp electrophysiology. Employing calcium imaging, we could demonstrate bidirectional communication patterns between tumor cells and astrocytes. We further characterized the stability of these astrocyte-glioma networks by using longitudinal imaging of single cells over several weeks in patient-derived xenograft models. For this, we used sulforhodamine 101 (SR101) as a marker for astrocyte-glioma connectivity to simultaneously visualize tumor cells and their glial microenvironment. The percentage of SR101 uptake in glioma cells increases over time, showing an increasing integration of tumor cells into the tumor-and astrocyte network during tumor evolution. Tumor cells, which are unconnected to other tumor cells or astrocytes, are the main drivers of invasion while a subgroup of glioma cells with stable astrocyte connectivity stay in place over several weeks. Lastly, we showed how these functional, heterotypic connections contribute to therapeutic resistance in the context of radiotherapy. In conclusion, we investigated multicellular networks between glioblastoma cells and astrocytes, their plasticity and role for therapeutic resistance.
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

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

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

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

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

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 > 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

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

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

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

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

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

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.
MODL-25. ANALYSIS OF TUMOR CELL HETEROGENEITY IN A HUMAN NEURAL PROGENITOR-BASED MODEL OF GLIOBLASTOMA REVEALS NEURODEVELOPMENTAL PROGRAMS AND CYTOSKELETAL DYNAMICS
Authors: DeSouza Patrick , Qu Xuan , Ishahak Matthew , McCornack Colin , Annamalai Devi , Batista Luis , Wang Ting , Maher Christopher , Millman Jeffrey , Kim Albert
Published: November 2022

Glioblastoma (GBM) is the most common primary malignant neoplasm with poor survival despite treatment. Developing effective therapies remains challenging due to intratumoral heterogeneity, which drives therapeutic resistance and recurrence. To understand how genetic events alter the epigenome to enhance clonal fitness, we developed an isogenic human neural progenitor cell (NPC)-based model of the proneural (PRO) GBM subtype. We introduced TERT promoter (TERTp) C228T and gain-of-function TP53 R248Q mutations in H1 human embryonic stem cells. Wildtype (WT) cells, single TERTp, and double TERTp/TP53 mutants underwent differentiation to NPCs. Lentiviral transduction of double mutants with PDGFRA D842V resulted in triple mutant PRO NPCs. Bulk and single cell transcriptomics of our model system revealed hundreds of gene expression changes with increased mesoderm and human GBM mesenchymal (MES) subtype signatures in single TERTp and double TERTp/TP53 mutants, versus WT cells. TERTp mutation increased telomerase expression and activity, conferring proliferative advantage and immortalization in NPCs and astrocytes. Additionally, TERT expression was further increased in TERTp/TP53 mutants. Surprisingly, triple mutant PRO NPCs, versus WT, displayed < 100 differentially expressed genes, associated with neurodevelopmental and dynamic cytoskeletal processes. Evolution analyses using gene counts signature and splicing dynamics revealed a developmental trajectory model from WT to additive mutants to PRO NPCs. Only triple mutant PRO NPCs formed tumors after intracranial injection in athymic nude mice, with mean survival of 100 days. Tumors presented histopathological features of GBM, and single cell transcriptomic analyses revealed evolution from immune-interacting to both neural progenitor- and neuronal-like subpopulations, with similar cell cycling signatures. Transcription factor genes related to WNT signaling and lineage commitment as well as glial and neuronal cytoskeletal genes exhibited epigenetic selection in vivo, signatures also observed in PRO NPCs in vitro. Our model thus provides opportunity for dissection of epigenetic and functional mechanisms underlying serial mutations during PRO tumor evolution.
MODL-32. IMPACT OF PRDM6 ON CHROMATIN ACCESSIBILITY, GENE EXPRESSION, AND MEDULLOBLASTOMA FORMATION
Authors: Schmidt Christin , Husain Sarah , Carlson Annika , Cohen Sarah , Westelman Samantha , Wang Linyu , Weiss William , Schwer Bjoern
Published: November 2022

Group 4 medulloblastoma (MB) is the most common medulloblastoma subgroup and shows high incidence of metastasis and late-onset relapse. Group 4 MBs lack a unifying oncogenic driver and treatment targets, despite extensive genomic characterization. Group 4 MBs are characterized by recurrent genetic alterations in chromatin modifiers, amplification of stemness genes, and putative enhancer "hijacking" events. A substantial fraction of Group 4 MBs are characterized by enhancer hijacking through tandem duplication of SNCAIP, resulting in high expression of PRDM6, a putative transcriptional repressor and histone methyltransferase. Some PRDM6-overexpressing MBs show additional mutations in chromatin regulators and high MYCN expression. We set out to elucidate the impact and oncogenic potential of sustained PRDM6 expression in early neural stem cell populations and patient-derived medulloblastoma cells. We find that PRDM6 expression in human iPSC-derived neuroepithelial stem cells (NESCs) results in tumor growth in mice, albeit at low penetrance. Moreover, we find that PRDM6 expression in MYCN-overexpressing NESCs does not further alter tumor aggressiveness or survival in vivo. Notably, PRDM6 overexpression in the patient-derived Group 3 MYC-amplified D283-Med cell line causes significantly increased aggressiveness of tumor growth and shorter survival in mice. At the cellular level, PRDM6 localizes to the nucleus, suggesting a role in gene expression regulation. Consistent with this notion, ATAC-seq and RNA-seq analysis of PRDM6-NESCs and PRDM6-D283-Med cells reveals major changes in chromatin accessibility and gene expression, including upregulation of integrin family members and genes related to focal adhesion and extracellular matrix-receptor interaction pathways. We conclude that PRDM6 promotes tumor growth in NESCs and may play a role in tumor maintenance through interactions with the extracellular matrix and tumor microenvironment.
MODL-34. DECELLULARIZATION OF HUMAN AUTOPSY BRAIN TISSUE TO GENERATE A 3D EXTRACELLULAR MATRIX FOR MEDULLOBLASTOMA AND ATYPICAL TERATOID/RHABDOID TUMOUR MODELLING
Authors: McCrorie Phoebe , Porter Harry , Vinohar Jeffy , Diksin Mohammed , Scurr David , Rahman Ruman
Published: November 2022

INTRODUCTION

Childhood medulloblastoma (MB) and atypical teratoid/rhabdoid tumours (AT/RT) are malignant brain tumours occurring in the posterior fossa, for which prognoses remains particularly poor for the MB Group 3 subtype characterised by amplification of the Myc oncogene and for AT/RT in general. Current in vitro models for these neoplasms rely on non-coated plastic, various hydrogels, or animal-derived extracellular matrix (ECM), which fail to recapitulate the physiological environment from which the cells are derived from.

METHODS

We have developed a method to decellularize ex vivo human brain tissue from different anatomical locations for the use in 3D in vitro models. Human cerebellar brain tissue was harvested from autopsy brain and sectioned into small cubes before bathing in a sodium dodecyl sulfate/phosphate-buffered saline mixture for several days, before washing and lyophilising.

RESULTS

The optimised method for generation of decellularized human brain ECM successfully removes nuclei as confirmed by histological staining and DNA quantification (DNA reduction of ≥ 60%). Orbitrap-Secondary Ion Mass Spectrometry analysis confirmed the retention of the ECM components laminin (C9H11N3O2Na+), fibronectin (C9H14N4O2Na+ and C20H33N7O5Na+) and collagen (C4H5N2O2+), with a reduction in cell membrane lipid components (glycerophosphocholine, C9H19NPO4+; phosphocholine, C5H15NPO4+; and choline, C5H14NO+) relative to control tissue, with a < 2 ppm accuracy, which was further corroborated by glycosaminoglycan and collagen assays. Multiple molecular subtype-specific AT/RT and MB cell lines have been successfully grown on decellularized cerebellar-ECM/PEGDA hydrogel, showing no reduction in metabolic viability using PrestoBlue and Cell Titer Glo assays.

CONCLUSIONS

This methodology offers an innovative human-only high-throughput 3D drug screening model, whereby patient-derived MB or AT/RT cells are co-cultured with healthy human cerebellar astrocytes upon decellularized cerebellar ECM, which we term ‘Tumoursphere Matrices’.
MODL-35. A NOVEL PATIENT-DERIVED TUMOR ORGANOID MODEL IDENTIFIES HDACI PANOBINOSTAT AS HIGHLY EFFECTIVE DRUG IN MENINGIOMAS
Authors: Jungwirth Gerhard , Cao Junguo , Warta Rolf , Moustafa Mahmoud , Abdollahi Amir , Andreas Unterberg , Herold-Mende Christel
Published: November 2022

Tumor-organoids (TOs) are novel, complex three-dimensional ex vivo tissue cultures that under optimal conditions accurately reflect the genotype and phenotype of the original tissue with preserved cellular heterogeneity and morphology. They offer a new and exciting platform for studying cancer biology and directing personalized therapies. Our study aimed to establish TOs from meningioma (MGM) and explore drug responses of selected drugs.TOs were established by controlled reaggregation of freshly prepared single-cell suspension of MGM tissue samples in 384-well plates. We were capable of forming several hundred TO equal in size with a success rate of 98%. TOs consisted of largely viable cells, whereas dead cells were predominantly found outside of the organoid. H&E and IF stainings confirmed the successful establishment of dense tissue-like structures. On the molecular level, transcriptional and mutational profiles were conserved in TOs. To query the suitability and reliability of TOs for robust large-scale drug testing by employing nine highly potent compounds, derived from a drug screening performed on MGM cell lines. In total, standardized TOs from 60 patients were formed, including eight grade II and three grade III MGMs. The FDA-approved epigenetic drug panobinostat demonstrated high anti-meningioma effects in the majority of patients. The efficacy was mediated through inhibition of HDAC1/2 validated by siRNA-mediated knockdown experiments. In an orthotopic in vivo model, treatment with panobinostat extended the median survival of mice, however, the therapy was not curative. To further investigate HDACi resistance, transcriptomes of sensitive and resistant TOs were compared. In resistant tumors, a significant induction of the TGFβ pathway was observed.Taken together, we developed a protocol to generate standardized TO from MGM and preserved histological and molecular features of parental tumor. Drug screening of selected drugs in TOs from 60 patients identified HDACi panobinostat as effective drug through inhibition of HDAC1/2.
MODL-37. DISCERNIBLE INTERINDIVIDUAL PATTERNS OF GLOBAL EFFICIENCY DECLINE DURING THEORETICAL BRAIN SURGERY
Authors: Yueh-Hsin Lin , Dadario Nicholas , Crawford Lewis , Tanglay Onur , Dow Hsu-Kang , Young Isabella , Ahsan Syed Ali , Doyen Stephane , Sughrue Michael
Published: November 2022

Resection of infiltrating brain tumors, such as diffuse gliomas, generally involves resecting a portion of a lobe of the brain. While the concept of localized functions, and the risk to removing these areas, is well established in neurosurgical thinking, the potential that the overall global efficiency of the connectome could be disproportionately disturbed by an intervention in ways which are not immediately obvious have not been formally studied. The current article provides evidence that structural patterns exist in the impact resection of various lobes of the brain has, which also differs between subjects. We utilized diffusion tractography to create structural connectivity graphs from the brains of 80 healthy adults, and then performed every plausible brain surgery in every gross anatomic region of the cerebrum by deleting every possible combination of nodes in the graph which were adjacent to each other, and measured the drop in global efficiency (GE) at each nodal deletion. Not surprisingly, the deletion of some nodes was worse than others, such that in every lobe we studied in every subject, there were combinations of deletions which were worse for GE than removing a greater number of nodes in a different part of the brain. Interestingly, while the worst nodes differed between subjects, there were specific nodes which typically showed up as particularly detrimental regardless of which node was the worst in that person, but that there were patterns of so-called ―connectotype, which could determine which nodes were the worst. Progressive removal of a lobe of the brain leads to patterns of global efficiency decline which are reasonably predictable, but which are not the same between subjects. Given evidence that global efficiency relates to specific neuro-cognitive abilities, this provides a path towards reducing the cognitive footprint of brain surgery.
MODL-38. DEVELOPMENTAL EFFECTS OF MYBL1 ACTIVATION ON MURINE BRAIN AND GLIAL DEVELOPMENT
Authors: Azazmeh Narmen , Rouleau Cecile , Schoolcraft Kate , Jacob Etai , Malinowski Seth , Busanovich John P , Ramkissoon Lori , Kang Yun Jee , Ramkissoon Shakti , Pelton Kristine , Meng Alice , Jones Victor , Bronson Rodderick , Piccioni Federica , Kleinman Claudia , Bandopadhayay Pratiti , Ligon Keith , Beroukhim Rameen
Published: November 2022

Low-grade gliomas (LGG) represent 30% of pediatric brain tumors. Their cellular origins are unknown but are presumed to arise from subtle alterations of progenitor cell cycle regulation during brain development. Rearrangements activating MYB and MYBL1 have been identified as drivers of LGG in angiocentric glioma and diffuse astrocytomas, respectively, but the roles of these genes in the normal brain and the development of LGG are poorly understood. We first performed a developmental analysis of human and mouse Mybl1 expression from bulk and single-cell RNA-sequencing and identified exclusive expression of MYBL1 in neural stem and progenitor cells in the ganglionic eminence. We also found that MYBL1high cell transcriptomes are enriched in genes functionally involved in centromere and mitotic processes, strongly suggesting an association between MYBL1 expression and cellular proliferation states. We next hypothesized that C-terminal truncation may drive tumorigenesis through a direct increase in MYBL1 expression and cell proliferation. We developed a novel Cre-dependent knock-in mouse-model for human truncated MYBL1 expression and tested effects in oligodendroglial(Olig2-cre), astrocytic hGFAP-cre), and somatic(Ubiquitin-cre) cell types. In Ubq-cre:R26-MYBL1-tr mice there was expression and dysplasia in the salivary gland but no significant effects on brain development. In Olig2-Cre+/tg:R26-MYBL1-tr+/fl mice we observed higher susceptibility to motor seizures, early postnatal death without gross or microscopic abnormalities of brain morphology. In contrast, expression in stem cells and astrocytes of hGFAP-Cre+/tg:R26-MYBL1-tr+/flmice drove dramatic abnormalities in brain development and altered proliferation of progenitor and stem cells, but not glioma formation. Single-cell RNA sequencing of MYBL1-tr cells from mNSCs and brains of mice were also used to determine patterns of altered expression driven by MYBL1-tr. These results indicate that aberrant MYBL1 activation affects neural stem/progenitor cell and brain development through altered cell proliferation. Future targeting of the pathways identified may be therapeutically beneficial for patients with LGG driven by MYB-family oncogenes.