bluuebunny/crossref_metadata_2025_split huggingface dataset:
CNSC-27. AN IDO-PROTAC HIGHLIGHTS A NOVEL TRYPTOPHAN METABOLISM- INDEPENDENT ROLE FOR IMMUNOSUPPRESSIVE IDO IN HUMAN GLIOBLASTOMA
Authors: Bommi Prashant , Monsen Paige , Bommi Lakshmi , Zhai Lijie , Bell April , Lauing Kristen , Mi Xinlei , Kocherginski Masha , Horbinski Craig , Lukas Rimas , Schiltz Gary , Wainwright Derek 
Published: November 2022

Abstract

INTRODUCTION
Indoleamine 2,3-dioxygenase 1 (IDO) is an immunosuppressive enzyme that catabolizes the essential amino acid, tryptophan (Trp), into the metabolite, kynurenine. IDO is expressed in &gt;90% of patient resected GBM. IDO suppresses the anti-brain tumor immune response, in-part, through non-metabolic activities. To determine how IDO non-metabolically suppresses the anti-GBM immune response, IDO-protein degrader (IDO-proteolysis targeting chimera; IDO-PROTAC) effects were studied in multiple human models of GBM.

METHODS
The IDO-expressing GBM cell lines, U87, U138, as well as the patient derived xenograft (PDX) line, GBM43, were treated with either IDO-PROTAC, mutant PROTAC, IDO enzyme inhibitor, or IDO siRNA, followed by RNA-seq analysis and/or mass spectrometry with quantitative proteomics using tandem mass tag (TMT) labelling.

RESULTS
Transcriptomic analysis revealed differentially expressed genes that were commonly regulated after treatment with the IDO-PROTAC as compared to treatment with the mutant PROTAC or IDO enzyme inhibitor groups in U87, U138, and GBM43 cells. Mass spectrometry analysis found 34 unique proteins that were differentially expressed inside of human GBM cells, with an additional 20 unique proteins that were identified in the supernatant of cultured human GBM cells after IDO-PROTAC treatment. Meta-analysis of the transcriptomic and proteomic analyses identified a novel factor that was unique to IDO-PROTAC treatment. GO terms that were enriched after IDO-PROTAC treatment identified nucleoside kinase activity as well as metallocarboxypeptidase activity.

CONCLUSIONS
This study discovered multiple new targets and pathways that immunosuppressive IDO regulates through a non-metabolic function. Functional analyses that validate the newly-discovered IDO-dependent, IDO-enzyme independent factors, are ongoing.
TMIC-37. SINGLE-CELL CHARACTERIZATION OF THE IMMUNE LANDSCAPE OF EXTREME LONG-TERM SURVIVORS WITH MALIGNANT GLIOMA
Authors: Decraene Brecht , Antoranz Asier , Verbeke Tatjana , Vanmechelen Maxime , Nazari Pouya , Solie Lien , Dubroja Nikolina , Derweduwe Marleen , Spans Lien , Bempt Isabelle Vanden , Sciot Raf , De Smet Frederik , De Vleeschouwer Steven 
Published: November 2022

Abstract
Glioblastoma Multiforme (GBM) remains the most common malignant primary brain tumor with a dismal prognosis that rarely exceeds beyond two years despite extensive therapy, which consists of maximal safe surgical resection, radiotherapy and/or chemotherapy. Recently, it has become clear that GBM is not one homogeneous entity and that both intra-and intertumoral heterogeneity contribute significantly to differences in tumoral behavior which may consequently be responsible for differences in survival. Strikingly and despite its dismal prognosis, small fractions of GBM patients seem to display extreme extended survival compared to the large majority of patients. The underlying mechanisms for this peculiarity remain largely unknown however, even though emerging data suggest that both cancer cell-autonomous and microenvironmental factors and their interplay probably play an important role. We used high-dimensional, multiplexed immunohistochemistry to spatially, and cytometry by time-of-flight to quantitively, characterize the cell constitution and interactions within the tumor microenvironment (TME) in 21 extreme long-term survivors (living over 10 year) and 42 deeply matched controls and therefore short-term survivors (living under 1.5 year) on a single cell level. For all tumors (epi)genetic data was also collected. We identified a high level of both inter-and intrapatient heterogeneity defined by several distinct tumoral niches, as well as described interactions within these niches and with the surrounding infiltrating immune cells of the TME in GBM. Finally, by linking patient characteristics with the heterogeneous immune composition we are able to create an immune stratification that can be linked to patient survival in GBM. Therefore, this study is an essential initial step towards strategies to alter the TME in a favorable way with a personalized modulation strategy.
TMIC-40. TRANSCRIPTOMIC CHARACTERIZATION OF PATIENT GLIOMAS AND DERIVED MODEL SYSTEMS REVEALS ENVIRONMENTAL INFLUENCE ON NEURODEVELOPMENTAL CELLULAR STATES
Authors: Bayley Nicholas , Tse Christopher , Minami Jenna , Zhu Henan , Yan Weihong , Baufeld Lynn , Gosa Laura , Ta Lisa , Yong William , Prins Robert , Cloughesy Timothy , Liau Linda M , Graeber Thomas G , Nathanson David 
Published: November 2022

Abstract
Bulk tumor and single-cell RNA sequencing have revealed the remarkable molecular heterogeneity and plasticity of gliomas, leading to the definition of tumor subtypes and cellular states describing inter- and intra-tumoral levels of heterogeneity respectively. While there has been great interest in creating and revising these classifications, the combination of selective pressures from molecular alterations within the tumor cell as well as extrinsic factors within the tumor microenvironment (TME) driving these levels of heterogeneity remain to be fully described. The brain TME is a complex, regionally heterogeneous ecosystem of communicating non-malignant and malignant cell types and scavenge-able nutrients and metabolites. We hypothesized that distinct environmental contexts within and across tumors may drive the intra- and inter-tumoral heterogeneity of gliomas. To identify tumorigenic programs impacted by environmental context, we performed bulk RNA sequencing of over 40 triplets of patient glioma samples and their matched derivative models established in direct orthotopic mouse xenografts and conventional gliomasphere cultures. Comparative analyses revealed environment-associated programs including in vivo immune and neuroglial signaling as well as altered in vitro lipid metabolism and upregulated cell migration. Next, to investigate associations between tumor cellular state and environment-associated programs we performed single-cell RNA sequencing of 4 patient and model system triplets. By annotating single cell profiles with previously defined single cell atlases of the developing and adult brain, 9 major states reflecting neurodevelopmental cell types were identified. Single cell profiling further revealed cell state-specific expression of environment-associated programs. We simultaneously observed the divergence of model cellular state compositions towards states expressing programs associated with their environment. We conclude that the transcriptional evolution of cellular states is connected to their ecological role within the TME. Consequently, we find grade- and diagnosis-associated composition differences between patient tumors predictive of their ability to establish a model in vitro.
TMIC-44. TARGETING BRACHYURY REPROGRAMS ENDOTHELIAL TRANSDIFFERENTIATION OF GLIOBLASTOMA
Authors: Hsu Wesley , Zehri Aqib , Hu Yunping 
Published: November 2022

Abstract
Glioblastoma (GBM) is the most common and aggressive primary brain tumor with a median survival of only 15 months and a 5-year survival of less than 5% despite the best available treatments. Tumor recurrence/progression and therapy resistance are major obstacles for GBM treatment. GBM is characterized by intensive vascular proliferation that is associated with tumor cell growth, invasion, resistance to chemo/radiotherapy, and decreased disease-free survival. Increasing data have shown the existence of cells with endothelial characteristics called tumor derived endothelial cells, which come from the transdifferentiation of GBM cells and, more specifically, from GBM stem cells (GSCs). However, the molecular mechanisms underlying this process remain largely unknown. GSCs are tightly regulated by distinct transcriptional programs that are driven by nuclear transcription factors. Brachyury is a transcription factor expressed in normal, undifferentiated embryonic notochord in the axial skeleton and plays an important role in stem cell development and differentiation during normal embryonic development. Here, we show that brachyury is highly expressed in patient-derived GBM cells. Functional studies demonstrate that brachyury is regulated by fibroblast growth factor receptor 1 (FGFR1)/mitogen-activated protein kinase (MAPK). Our studies further disclose that FGFR1/MAPK-directed brachyury activation promotes GSC survival and endothelial formation via vascular endothelial growth factor receptor 2 (VEGFR2), whereas pharmacological inhibition of FGFR1 and MAPK or shRNA-mediated downregulation of brachyury decreases GSC transdifferentiation into endothelial cells and suppresses GBM cell growth and stemness via VEGFR2. Our findings highlight the importance of FGFR1/MAPK-regulated brachyury activation in GSC transdifferentiation into endothelial cells via VEGFR2, and targeting the FGFR1-MAPK-brachyury-VEGFR2 signal pathway may represent a novel therapeutic strategy by reprogramming GSCs driving vascular transdifferentiation and tumor progression.
TMIC-46. DISRUPTING TRICELLULAR TIGHT JUNCTIONS TO ENHANCE BLOOD-BRAIN-BARRIER PERMEABILITY IN GLIOMA MODELS
Authors: Dalmage Mahalia , Kundu Nabanita , Kwak Minhye , Lim Sanghee , Conrad Christina , Jackson Sadhana 
Published: November 2022

Abstract

BACKGROUND
The blood-brain-barrier (BBB) is predominantly regulated by brain endothelial cells held together by junctional proteins. The BBB poses a significant hinderance to CNS drug entry of effective chemotherapy agents. Previous BBB studies have shown that disruption of tricellular and/or bicellular tight junctional proteins transiently increased CNS drug permeability.

OBJECTIVE
To evaluate the effect of drug proteins angubindin-1, C-CPE, and C-CPEmt on tight junction proteins angulin-1, claudin-3, and claudin 5, respectively, aimed at transiently enhancing BBB permeability and decreasing glioma growth.

METHODS
We evaluated junctional disruption with drug proteins, angubindin-1 (600µg/mL), C-CPE (200µg/mL), and C-CPEmt (200µg/mL) on rat brain endothelium. Endothelial junctional integrity studies were assessed by immunoblotting and cell-cell electrical impedance assays. Treatment effects on rat malignant glioma (S635) was measured via migration and rat glioma models.

RESULTS
Overall, we observed a time-dependent effect of drug proteins on junctional expression and function. Immunoblotting demonstrated a significant decrease (p&lt; 0.05) in angulin-1 expression 5-hours after angubindin-1 treatment, while claudin-3 and claudin-5 expression barely decreased between 2 and 24-hours after C-CPE and C-CPEmt treatment, respectively. Cell-cell integrity was disrupted by 73%, 52%, and 69% compared with control, 3-hours after angubindin-1, C-CPE, and C-CPEmt treatment, respectively. Studies assessing qualitative junctional expression changes after drug proteins are ongoing. Gliomas cells expressed high angulin-1, and interestingly, migration was 50% decreased with angubindin-1 treatment, yet no migration changes were evident with C-CPE or C-CPEmt treatment. Rat glioma model studies evaluating liposomal doxorubicin combined with angubindin-1 are currently being explored to display effects on CNS drug concentrations, cytotoxicity, junctional expression, and survival.

DISCUSSION
Transient disruption of BBB tricellular junctions was seen with angubindin-1 treatment. Additionally, cell migration was hindered in angulin-1 overexpressing gliomas. These findings are promising and demonstrate the need for more combinational therapies aimed at increasing BBB permeability while also impairing glioma progression.
TMIC-53. DEVELOPMENT OF A GEO-TAGGED TUMOR SAMPLE REGISTRY; LINKAGE OF TUMOR SAMPLE LOCATION TO IMAGING CHARACTERISTICS
Authors: Lynes John , Rubino Sebastian , Etame Arnold , Liu James , Beer-Furlan Andre , Tran Nam , Ruiz Alexia , Macaulay Rob , Vogelbaum Michael 
Published: November 2022

Abstract

Background
Significant intra- and inter-heterogeneity exists in gliomas. This provides clinical, radiological, diagnostic, and treatment challenges. To date, there have been few efforts to comprehensively catalog information obtained in the operating room that spatially links neuro-navigation localization to imaging characteristics, gross intraoperative visual findings, and pathological/molecular features. The value of this spatial localization is probably greatest in high grade gliomas, which have been demonstrated to have intra-tumoral histological and genetic/epigenetic heterogeneity.

METHODS
An IRB-approved institutional registry of patients undergoing clinically-indicated surgery with use of an image-guidance system (IGS) was launched in November 2019 and as of June 1, 2022 includes nearly 500 patients, of which 243 were diagnosed with gliomas. Intraoperatively, locations within the gross tumor or tumor-infiltrated brain were sampled at each surgeon’s discretion, and each sample was linked to their precise location with the IGS system (“geo-tagged”). The registry includes information regarding surgeon; anesthesia technique; use of intraoperative tumor fluorescence; tumor location and volume; pathologic diagnosis and molecular features, and sample number.

RESULTS
Of 243 gliomas, 26 were low grade and 217 were high grade with 174 being glioblastoma. For enhancing tumors, volume of enhancement ranged from 0.31 to 127.0 cm3 with an average of 22.9 cm3. Tumors were widely distributed throughout the cerebrum with 133 left-sided tumors, 110 right-sided and 32 spanning multiple lobes or deep subcortical structures including the brainstem. 51% of surgeries were under awake anesthesia, and 40% were performed using fluorescence guidance. The average number of navigation image-linked samples collected per tumor was 3.67; 3.48 in low grade gliomas, and 3.69 in high grade gliomas. Samples are archived in frozen and/or formalin-fixed, paraffin-embedded formats for future research.

CONCLUSION
This registry provides the foundation for correlation of imaging, intraoperative findings, and pathology in brain tumors, and it will support detailed laboratory/translational investigations addressing tumor heterogeneity.
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

Abstract
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

Abstract
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&lt; 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

Abstract
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

Abstract
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.