Glioblastoma Samples Research Articles

Abnormal Vessels Potentially Accelerate Glioblastoma Proliferation by Inducing the Protumor Activation of Macrophages.

Glioblastoma (GBM) involves disruptions in the blood-brain barrier (BBB) and alterations in the immune microenvironment, including the activation of glioma-associated macrophages (GAMs). Vascular endothelial growth factor inhibitors, commonly used in recurrent GBM treatment, can influence these processes. This study investigates the relationship between BBB disruption and GAM activation, focusing on plasmalemma vesicle-associated protein (PLVAP), a marker of BBB disruption, and α1-acid glycoprotein (AGP), an inflammatory protein implicated in tumor progression. PLVAP expression was analyzed by immunohistochemistry (IHC) in human GBM samples to determine correlations with tumor grade, proliferation, and GAM activation. Pre- and post-bevacizumab treatment GBM samples were compared to assess changes in BBB integrity and macrophage activity. AGP's role in GAM activation was studied through invitro assays and glioma implantation in AGP knockout mice, with assessments of tumor growth and angiogenesis. Results showed elevated PLVAP expression in higher-grade gliomas, correlating with increased tumor proliferation and GAM activation, particularly around PLVAP-positive vessels. Bevacizumab treatment reduced PLVAP expression and macrophage activity. AGP localized to regions of BBB disruption, promoting macrophage-mediated tumor growth invitro. AGP knockout mice demonstrated reduced angiogenesis and prolonged survival. Spatial analysis revealed increased expression of macrophage-inducing molecules near PLVAP-positive vessels. These findings suggest PLVAP as a marker of BBB disruption and glioma malignancy. AGP, associated with BBB leakage, contributes to GAM activation and tumor progression, highlighting its potential as a therapeutic target for GBM.

Genes related to neural tube defects and glioblastoma.

There are many similarities between early embryonic development and tumorigenesis. The occurrence of neural tube defects (NTDs) and glioblastoma (GBM) are both related to the abnormal development of neuroectodermal cells. To obtain genes related to both NTDs and GBM, as well as small molecule drugs with potential clinical application value. We performed bioinformatics analysis on transcriptome sequencing data of retinoic acid (RA)-induced NTDs mice, human NTDs samples and GBM samples. RT-qPCR, Western blot, and immunohistochemistry were used to validate the expression of candidate genes. Our results indicated that two genes at mRNA and protein levels have been well verified in both NTDs mouse and GBM human samples, namely, Poli and Fgf1. Molecular docking and validating in vitro were performed for FGF1 against pazopanib by using Autodock and Biacore. Cytological experiments showed that pazopanib significantly inhibited the proliferation of GBM tumor cells and mouse neural cells, promoted apoptosis, and had no effect on GBM tumor cells migration. Overall, our results demonstrated that Fgf1 abnormally expressed at different developmental stages, it may be a potentially prenatal biomarker for NTDs and potential therapeutic target for GBM. Pazopanib may be a new drug for the treatment of GBM tumors.

Investigation of metabolic features of glioblastoma tissue and the peritumoral environment using targeted metabolomics screening by LC-MS/MS and gene network analysis

The metabolomic profiles of glioblastoma and surrounding brain tissue, comprising 17 glioblastoma samples and 15 peritumoral tissue samples, were thoroughly analyzed in this investigation. The LC-MS/MS method was used to analyze over 400 metabolites, revealing significant variations in metabolite content between tumor and peritumoral tissues. Statistical analyses, including the Mann–Whitney and Cucconi tests, identified several metabolites, particularly ceramides, that showed significant differences between glioblastoma and peritumoral tissues. Pathway analysis using the KEGG database, conducted with MetaboAnalyst 6.0, revealed a statistically sig­nificant overrepresentation of sphingolipid metabolism, suggesting a critical role of these lipid molecules in glio­blastoma pathogenesis. Using computational systems biology and artificial intelligence methods implemented in a cognitive platform, ANDSystem, molecular genetic regulatory pathways were reconstructed to describe potential mechanisms underlying the dysfunction of sphingolipid metabolism enzymes. These reconstructed pathways were integrated into a regulatory gene network comprising 15 genes, 329 proteins, and 389 interactions. Notably, 119 out of the 294 proteins regulating the key enzymes of sphingolipid metabolism were associated with glioblastoma. Analysis of the overrepresentation of Gene Ontology biological processes revealed the statistical significance of 184 processes, including apoptosis, the NF-kB signaling pathway, proliferation, migration, angiogenesis, and py­roptosis, many of which play an important role in oncogenesis. The findings of this study emphasize the pivotal role of sphingolipid metabolism in glioblastoma development and open new prospects for therapeutic approaches modulating this metabolism.

Genetically Engineered Brain Organoids Recapitulate Spatial and Developmental States of Glioblastoma Progression.

Glioblastoma (GBM) is an aggressive form of brain cancer that is highly resistant to therapy due to significant intra-tumoral heterogeneity. The lack of robust in vitro models to study early tumor progression has hindered the development of effective therapies. Here, this study develops engineered GBM organoids (eGBOs) harboring GBM subtype-specific oncogenic mutations to investigate the underlying transcriptional regulation of tumor progression. Single-cell and spatial transcriptomic analyses revealed that these mutations disrupt normal neurodevelopment gene regulatory networks resulting in changes in cellular composition and spatial organization. Upon xenotransplantation into immunodeficient mice, eGBOs form tumors that recapitulate the transcriptional and spatial landscape of human GBM samples. Integrative single-cell trajectory analysis of both eGBO-derived tumor cells and patient GBM samples reveal the dynamic gene expression changes in developmental cell states underlying tumor progression. This analysis of eGBOs provides an important validation of engineered cancer organoid models and demonstrates their utility as a model of GBM tumorigenesis for future preclinical development of therapeutics.

Disease stage-specific role of the mitochondrial pyruvate carrier suppresses differentiation in temozolomide and radiation-treated glioblastoma.

The mitochondrial pyruvate carrier (MPC), a central metabolic conduit linking glycolysis and mitochondrial metabolism, is instrumental in energy production. However, the role of the MPC in cancer is controversial. In particular, the importance of the MPC in glioblastoma (GBM) disease progression following standard temozolomide (TMZ) and radiation therapy (RT) remains unexplored. Leveraging in vitro and in vivo patient-derived models of TMZ-RT treatment in GBM, we characterize the temporal dynamics of MPC abundance and downstream metabolic consequences using state-of-the-art molecular, metabolic, and functional assays. Our findings unveil a disease stage-specific role for the MPC, where in post-treatment GBM, but not therapy-naïve tumors, the MPC acts as a central metabolic regulator that suppresses differentiation. Temporal profiling reveals a dynamic metabolic rewiring where a steady increase in MPC abundance favors a shift towards enhanced mitochondrial metabolic activity across patient GBM samples. Intriguingly, while overall mitochondrial metabolism is increased, acetyl-CoA production is reduced in post-treatment GBM cells, hindering histone acetylation and silencing neural differentiation genes in an MPC-dependent manner. Finally, the therapeutic translations of these findings are highlighted by the successful pre-clinical patient-derived orthotopic xenograft (PDOX) trials utilizing a blood-brain-barrier (BBB) permeable MPC inhibitor, MSDC-0160, which augments standard TMZ-RT therapy to mitigate disease relapse and prolong animal survival. Our findings demonstrate the critical role of the MPC in mediating GBM aggressiveness and molecular evolution following standard TMZ-RT treatment, illuminating a therapeutically-relevant metabolic vulnerability to potentially improve survival outcomes for GBM patients.

Detecting copy-number alterations from single-cell chromatin sequencing data by AtaCNA.

Single-cell assay of transposase-accessible chromatin sequencing (scATAC-seq) unbiasedly profiles genome-wide chromatin accessibility in single cells. In single-cell tumor studies, identification of normal cells or tumor clonal structures often relies on copy-number alterations (CNAs). However, CNA detection from scATAC-seq is difficult due to the high noise, sparsity, and confounding factors. Here, we describe AtaCNA, a computational algorithm that accurately detects high-resolution CNAs from scATAC-seq data. We benchmark AtaCNA using simulation and real data and find AtaCNA's superior performance. Analyses of 10 scATAC-seq datasets show that AtaCNA could effectively distinguish malignant from non-malignant cells. In glioblastoma, endometrial, and ovarian cancer samples, AtaCNA identifies subclones at distinct cellular states, suggesting an important interplay between genetic and epigenetic plasticity. Some tumor subclones only differ in small-scale (10-20 Mb) CNAs, demonstrating the importance of high-resolution CNA detection. These data show that AtaCNA can aid in integrative analysis to understand the complex heterogeneity in cancer.

Cell type transcriptional identities are maintained in cultured ex vivo human brain tissue.

It is becoming more broadly accepted that human-based models are needed to better understand the complexities of the human nervous system and its diseases. The recently developed human brain organotypic culture model is one highly promising model that requires the involvement of neurosurgeons and neurosurgical patients. Studies have investigated the electrophysiological properties of neurons in such ex vivo human tissues, but the maintenance of other cell types within explanted brain remains largely unknown. Here, using single-nucleus RNA sequencing, we systematically evaluate the transcriptional identities of the various cell types found in six patient samples after fourteen days in culture (83,501 nuclei from day 0 samples and 45,738 nuclei from day 14 samples). We used two pediatric temporal lobectomy samples, an adult frontal cortex sample, two IDH wild-type glioblastoma samples, and one medulloblastoma sample. We found remarkably high correlations of day 14 transcriptional identities to day 0 tissue, especially in tumor cells (r = 0.90 to 0.93), though microglia (r = 0.86), oligodendrocytes (r = 0.80), pericytes (r = 0.77), endothelial cells (r = 0.78), and fibroblasts (r = 0.76) showed strong preservation of their transcriptional profiles as well. Astrocytes and excitatory neurons showed more moderate preservation (r = 0.66 and 0.47, respectively). Because the main difficulty with organotypic brain cultures is the acquisition of human tissue, which is readily available to neurosurgeons, this model is easily accessible to neurosurgeon-scientists and neurosurgeons affiliated with research laboratories. Broad uptake of this more representative model should prompt advances in our understanding of many uniquely human diseases, lead to more reliable clinical trial performance, and ultimately yield better therapies for our patients.

Daily glucocorticoids promote glioblastoma growth and circadian synchrony to the host.

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults with a poor prognosis despite aggressive therapy. Here, we hypothesized that daily host signaling regulates tumor growth and synchronizes circadian rhythms in GBM. We find daily glucocorticoids promote or suppress GBM growth through glucocorticoid receptor (GR) signaling depending on time of day and the clock genes, Bmal1 and Cry. Blocking circadian signals, like vasoactive intestinal peptide or glucocorticoids, dramatically slows GBM growth and disease progression. Analysis of human GBM samples from The Cancer Genome Atlas (TCGA) shows that high GR expression significantly increases hazard of mortality. Finally, mouse and human GBM models have intrinsic circadian rhythms in clock gene expression invitro and invivo that entrain to the host through glucocorticoid signaling, regardless of tumor type or host immune status. We conclude that GBM entrains to the circadian circuit of the brain, modulating its growth through clock-controlled cues, like glucocorticoids.

Integrated analysis of disulfidptosis-related genes SLC7A11, SLC3A2, RPN1 and NCKAP1 across cancers

Disulfidptosis, a newly identified form of regulated cell death associated with disruption of disulfide bond formation in the endoplasmic reticulum, involves the dysregulation of disulfidptosis-related genes (DRGs) that may contribute to cancer development and progression. However, the molecular mechanisms and clinical implications of DRGs in different cancer types remain poorly characterized. Therefore, in this comprehensive study, we investigated the expression, prognostic value, and functional roles of four recently identified DRGs (SLC7A11, SLC3A2, RPN1, and NCKAP1) across various cancers. Especially, in clinical samples of glioblastoma, we found that RPN1 was significantly correlated with patient survival. Through mutation landscape analysis, we identified diverse missense mutations in these DRGs, with NCKAP1 exhibiting the highest mutation frequency (5.9% in skin cutaneous melanoma). Additionally, we observed positive correlations between these DRGs and tumor stemness (DNAss DNA stemness score and RNAss RNA stemness score) as well as RNA modifications, particularly m6A modification, in several cancer types. Furthermore, high expression of SLC7A11, RPN1, and NCKAP1 was positively associated with infiltration of T-helper type 2 (Th2) cells in various cancers, while high expression of SLC7A11, SLC3A2, and RPN1 correlated with tumor mutational burden (TMB) in 10, 4, and 8 tumor types, respectively. Utilizing a protein–protein interaction network, we identified the RHO GTPases Activate WASPs and WAVEs pathway as significantly enriched, suggesting the involvement of these DRGs in cancer-related signaling pathways. Collectively, our findings provide novel insights into the molecular mechanisms and clinical implications of DRGs in pan-cancer, highlighting their potential as biomarkers and therapeutic targets for cancer treatment.

EPCO-44. SPATIAL TRANSCRIPTOMICS ENABLES STATISTICAL MODELING OF SEX DIFFERENCE IN GENE CO-EXPRESSION NETWORK AND IMMUNE LANDSCAPE OF GLIOBLASTOMA

Abstract Glioblastoma (GBM) is characterized by inter- and intra-tumor molecular heterogeneity and demonstrated cell plasticity. Sex differences have been observed in various aspects: a male:female GBM incidence ratio is 1.6:1; standard of care treatments (debulking, radiation, and temozolomide) benefit females more than males. 14 human GBM samples from two study sites were subject to spatial profiling on the Vizgen Merscope platform to examine whether distinct biological and therapeutic niches in GBM were evident, to understand how the tumor microenvironments (TME) contributed to the heterogeneity of GBM, and to interrogate sex difference in the immune landscape of GBM. Multiple sets of spatial probes were designed to capture the cell identities (cancer stem cells, astrocytes, oligodendrocytes, microglia, macrophages, etc.) and the spatially-resolved expression patterns of genes with different functions. Two of the 14 GBM samples were also profiled on another FISH-based spatial platform (Veranome). The data from the two platforms exhibited a linear correlation. We explored the gene-gene co-expression network, and calculated the interaction probabilities between genes which were then compared for sex difference. We found that female samples showed significantly lower (two-fold) CD4+/CD8A+ T-cell count ratios than male samples. We also observed sample-specific spatial niches of different GBM cell subtypes and immune cell populations. Our large-scale spatial transcriptomics study revealed intrinsic sex-based differences in immune response to tumor and illuminated microenvironmental influences on GBM.

EPCO-19. MULTIOMIC PROFILING OF EPIGENETIC HETEROGENEITY IN GLIOBLASTOMA AT SINGLE-CELL RESOLUTION USING MULTIOMIC SINGLE-CELL CUT&TAG

Abstract INTRODUCTION Recent evidence suggests epigenetic heterogeneity may be the primary driver of cell phenotype and treatment resistance in glioblastoma (GBM). Epigenetic heterogeneity is defined by alterations of chromatin histone modifications with activating marks such as H3K27 acetylation and repressive marks such as H3K27 trimethylation. Combinations of these histone marks govern the activity of functional genomic elements such as enhancers or promoters. Single-cell (“sc-”) technologies have only recently been adapted to the study of epigenetic cell states and significant technical challenges have limited their widespread adoption, especially in complex tissues. Here, we report the development of an optimized method for performing tissue processing and simultaneous single-cell epigenetic and transcriptomic analyses in flash frozen GBM samples. METHODS Building on our prior work, we developed a new protocol, multiomic single-cell cleavage-under-targets and tagmentation, or “mscCUT&TAG”. We optimized nuclei isolation, cleanup and permeabilization from flash frozen GBM and reaction conditions for the scCUT&TAG portion of the assay. Critically, these changes maintained high-quality, intact nuclei in suspension while limiting well-described technical issues such as nuclei clumping and sample loss. Furthermore, we incorporated the ability to multiplex both patient samples and histone marks. RESULTS We applied 10X scRNA-seq, 10X Multiome (RNA+ATAC-seq), and mscCUT&TAG for five histone marks to the same GBM sample, enabling an integrated evaluation of the tumor’s transcriptome, chromatin accessibility and epigenetic regulation at single-cell resolution. mscCUT&TAG enabled the first assessment of single-cell genome segmentation in GBM. We integrated across modalities to identify specific tumor cell populations, cell-type specific coordinated changes in epigenetics and transcriptome, and define functional regulatory elements. CONCLUSIONS We developed a novel multiomic protocol, mscCUT&TAG, that allows for a comprehensive analysis of epigenetic heterogeneity in GBM. We anticipate this protocol will enhance our understanding of fundamental biology and potential therapeutic avenues in GBM.

BIOM-18. MAPPING TUMOR HABITATS IN IDH-WILD TYPE GLIOBLASTOMA: INTEGRATING MR IMAGING, PATHOLOGIC, AND RNA DATA FROM IVY GLIOBLASTOMA ATLAS PROJECT

Abstract BACKGROUND To identify biologic correlates and spatially validate intratumoral subregions (tumor habitat) using diffusion-weighted and dynamic susceptibility contrast-imaging on a comprehensive pathology map of the isocitrate dehydrogenase (IDH)-wild type whole glioblastoma sample using the Ivy Glioblastoma Atlas Project (Ivy GAP). METHODS Complete data of 20 patients with 168 slides of IDH-wild type glioblastoma were obtained from the IvyGAP (http://glioblastoma.alleninstitute.org/). On MRI, tumor habitats were defined using voxel-wise clustering of apparent diffusion coefficient (ADC) and cerebral blood volume (CBV) maps for contrast-enhancing lesion (CEL) and peritumoral non-enhancing lesion (NEL). On pathology slides, normalized areas of leading edge (LE), infiltrating tumor (IT), cellular tumor (CT), hypervascular lesion (CThypervascular), and perinecrotic lesion (CTperinecrotic) were obtained. Gross specimen pictures were co-registered on MRI images for co-localization and correlation between MRI tumor habitats. Pathologic areas were calculated using Pearson’s correlation coefficient. RNA sequencing of 67 RNA-seq samples was assessed using 4 Neftel subtypes and further correlated with pathology. RESULTS Six tumor habitats were correlated: hypervascular, hypovascular cellular, and hypovascular hypocellular habitats for CEL and NEL. CT was strongly correlated with hypovascular cellular habitat in CEL (C2, r = 0.238, p =.005). IT was strongly correlated with hypovascular cellular habitat in NEL (C5, r = 0.294, p =.017). CThypervascular was correlated with hypervascular habitat in NEL (r = 0.195, p =.023). CTperinecrotic was correlated with imaging necrosis (r = 0.199, p =.005). Astrocyte-like subtypes were positively correlated with IT (r = 0.256, p <.001), while mesenchymal-like subtypes were positively correlated with CTperinecrotic area (r = 0.246, p <.001). CONCLUSION Pathologically matched tumor subregions were cellular tumor with hypovascular cellular habitat in CEL and infiltrative tumor with hypovascular cellular habitat in NEL. Identification of the most aggressive tumor portion, and infiltrative tumor portion using non-invasive imaging can be achieved using MRI tumor habitats.

PATH-20. CLINICAL FINDINGS AND PROGNOSIS OF ADULT GLIOBLASTOMA HARBORING ABRAF V600E MUTATION

Abstract BACKGROUND AND AIM Although the BRAF V600E mutation is commonly associated with pediatric low-grade gliomas, glioblastomas (GB) with this mutation (BRAF-GB) have also been reported. The recent emergence of RAF and MEK inhibitors for BRAF V600E-positive tumors highlights the potential for targeted therapy in a subset of GB patients. This study aimed to characterize the clinical features and prognosis of BRAF-mutant glioblastomas. MATERIALS AND METHODS A retrospective analysis was conducted on a cohort of patients diagnosed with GB (confirmed by 2021 WHO criteria) above 16 years of age. Targeted genetic analysis for mutations in BRAF V600E, TERT promoter, and CDKN2A/B was performed on the GB samples. Clinical features of BRAF-GB were analyzed, and patients were further categorized into three groups: BRAF-GB, typical GB, and molecular GB. Subsequent analysis compared the prognosis among these groups. RESULTS Our study identified 11 cases (5.1%) harboring the BRAF V600E mutation among 215 adult GB patients. Patients with BRAF-GB exhibited a significantly younger median age of onset (38-year-old) compared to the typical GB (N = 167, 64-year-old) and molecular GB (N = 37, 61-year-old) groups. Further analysis of the BRAF-GB cohort (n=11) revealed a high prevalence (82%) of homozygous CDKN2A/B deletions, and only one case (9%) harbored a TERT promoter mutation. Interestingly, three BRAF-GB patients presented with hemorrhagic onset. Four cases showed evidence of prior lesions, with one case diagnosed histologically as a low-grade glioma 14 years earlier. The BRAF-GB group exhibited significantly longer median progression-free survival (32.8 months) and overall survival (84.2 months) compared to both the typical GB group (9.6 months and 18.9 months, respectively) and the molecular GB group (14.1 months and 32.1 months, respectively). CONCLUSIONS Early-age onset, with hemorrhagic onset or presence of prior lesions are factors that could lead to recommendation of BRAF analysis for adult GB patients.

CNSC-03. HIGH EXPRESSION OF PICCOLO AND SYNTAXIN-1A IN PAEDIATRIC MEDULLOBLASTOMA DUE TO BOTH: HIGH NEURONAL FRACTION AND ENDOGENOUS EXPRESSION

Abstract Emerging evidence indicates that the “healthy” nervous system contributes to the pathogenesis of cancer. Tumor cells were found to form ultra-long protrusions with distinct synapses, mainly in the infiltrative zone, resulting in a neuron-to-brain tumor synaptic communication. Located at the presynapse, proteins of the cytomatrix of the active zone have been identified to maintain highly specialized functions in neurons. Further, the proteins were shown to be involved in progression in various tumors, but little is known about their role in brain tumors. This study examined the expression of CAZ proteins in glioma and medulloblastoma. Tumor samples of Astrocytoma, glioblastoma, pilocytic astrocytoma and medulloblastoma were obtained during neurosurgical resection. Transcription rate of following CAZ proteins was measured via qPCR: SNAP25, VAMP1, VAMP2, SYT1, PCLO, BSN, STX1A. To examine amount of neuronal tissue TUBB3 and for glial fraction GFAP were quantified. A medulloblastoma cell line (DAOY) was furthermore analyzed to evaluate origin of high expressed proteins. Medulloblastoma showed high expression of PCLO (pilocytic astrocytoma vs. medulloblastoma p=0.0126) and STX1A (pilocytic astrocytoma vs. medulloblastoma p=0.0006) compared to. Likewise a high transcriptional level of TUBB3 could be seen with significance to pilocytic astrocytoma (2.2 ± 1.4 vs. 0.2 ± 0.17; p = 0.0018), which could indicate a high amount of neuronal tissue in given medulloblastoma samples. To rule out a causal link between high proportion of neuronal tissue and high CAZ expression, a paediatric medulloblastoma cell line was analysed. High expression of PCLO and STX1A in absence of TUBB3 was shown (3.8 and 2.01 vs. 0.059), indicating endogenous expression of CAZ proteins in medulloblastoma.

Discovery of key molecular signatures for diagnosis and therapies of glioblastoma by combining supervised and unsupervised learning approaches

Glioblastoma (GBM) is the most malignant brain cancer and one of the leading causes of cancer-related death globally. So, identifying potential molecular signatures and associated drug molecules are crucial for diagnosis and therapies of GBM. This study suggested GBM-causing ten key genes (ASPM, CCNB2, CDK1, AURKA, TOP2A, CHEK1, CDCA8, SMC4, MCM10, and RAD51AP1) from nine transcriptomics datasets by combining supervised and unsupervised learning results. Differential expression patterns of key genes (KGs) between GBM and control samples were verified by different independent databases. Gene regulatory network (GRN) detected some important transcriptional and post-transcriptional regulators for KGs. The KGs-set enrichment analysis unveiled some crucial GBM-causing molecular functions, biological processes, cellular components, and pathways. The DNA methylation analysis detected some hypo-methylated CpG sites that might stimulate the GBM development. From the immune infiltration analysis, we found that almost all KGs are associated with different immune cell infiltration levels. Finally, we recommended KGs-guided four repurposable drug molecules (Fluoxetine, Vatalanib, TGX221 and RO3306) against GBM through molecular docking, drug likeness, ADMET analyses and molecular dynamics simulation studies. Thus, the discoveries of this study could serve as valuable resources for wet-lab experiments in order to take a proper treatment plan against GBM.

TMIC-78. CHEMOTHERAPY INDUCED IMMUNOGENIC RESPONSE IN GLIOMA AND IMPLICATIONS FOR THERAPEUTIC STRATEGIES

Abstract INTRODUCTION Many glioblastoma (GBM) treatments fail because they treat tumor cells in isolation and ignore the surrounding microenvironment. In our phase 1b clinical trial of delivery of Topotecan (TPT) via Convection Enhanced Delivery (CED) in glioma, we demonstrated effective tumor cell elimination accompanied by a robust inflammatory response. We hypothesized that TPT induces an immunogenic response resulting in an unfavorable inflammatory landscape. METHODS To characterize chemotherapy’s effect on myeloid cells, we analyzed pre- and post-treatment biopsies from our trial using bulk RNA sequencing to measure canonical myeloid markers. We validated survival benefit of CED in vivo in our tumor model and characterized post-treatment tissue using single cell RNA sequencing (scRNA seq) after 7 days of treatment. We treated slice cultures generated from surgical samples of GBM with TPT and performed scRNA seq and histologic analysis to assess microenvironment changes after 24 hours of treatment. We used several in vitro assays to characterize the direct and indirect mechanisms of myeloid activation. RESULTS Bulk RNA sequencing of post treatment biopsies demonstrates upregulation of markers associated with activated and exhausted immune cell populations such as IBA1, CD68, MSR1, MARCO and loss of markers associated with homeostatic microglia such as P2RY12 and TMEM119. Similar results are found within 7 days of treatment in vivo, however, are not found in human derived samples treated acutely. Treatment of in vitro mono and co-culture systems demonstrate a tumor cell death dependent activation of myeloid cell populations via immunogenic molecules. CONCLUSIONS Chemotherapies eliminate proliferating glioma cells but also induce inflammation. While many investigators have tried to leverage this activation, our results suggest that chronic inflammation contributes to poor prognosis and recurrence. The identification of molecules responsible for this activation point to new therapeutic targets aimed at controlling treatment-induced inflammation, as part of a combinatorial approach to treat GBM.

IMMU-72. TYPE I INTERFERON SIGNALING WITHIN GLIOBLASTOMA CELLS PROMOTES T-CELL EXHAUSTION AND RESISTANCE TO CHECKPOINT INHIBITORS

Abstract Glioblastoma (GBM) remains among the most fatal cancers with few treatment modalities and resistance to modern immunotherapeutic treatment paradigms including immune checkpoint inhibitors (ICI). ICIs neutralize the regulatory signals that disable T cells and have demonstrated remarkable efficacy against a variety of cancers, though not in GBM. The impotence of ICI in GBM has been linked to the tumor’s remarkable capacity to elicit T cell exhaustion, though the mechanism remains poorly understood. Chronic interferon (IFN) cytokine signaling is known to promote immunosuppression and Tex in several cancer types as well as resistance to ICI. However, the role of IFN in GBM has not been investigated despite evidence that IFN signatures exist within clinical samples of GBM. Syngeneic murine GBM cell lines CT2A and SB28 were utilized for in vivo and in vitro studies. Anti-mouse PD-1 and CD8 antibodies or PBS control were injected intraperitoneally. CRISPR editing was used to create validated knockouts (KO) of the type I IFN receptor (IFNAR1) in CT2A. Mice bearing IFNAR1 KO intracranial tumors have improved median survival (p < 0.05) and are exquisitely sensitive to checkpoint anti-PD-1 blockade (p < 0.05) with the majority of mice surviving. Furthermore, surviving mice reject the subsequent intracranial re-challenge with parental CT2A tumors compared to controls (p <0.05) suggesting central memory responses mediating rejection. We conclude that abrogation of Type I IFN signaling is sufficient to extend survival which is further enhanced by ICI. Work is ongoing to further elucidate the precise mechanism that mediates this response and to identify a therapeutic intervention that enables tumor-specific inhibition of the IFN pathway with preliminary work suggesting a CD8 T cell mediated response and reversal of the T cell exhaustion phenotype.

EPCO-07. INTEGRATING SPATIALLY RESOLVED MULTI-OMICS DATA TO UNCOVER DYSFUNCTIONAL METABOLISM DRIVEN NETWORKS THAT ENHANCE INFILTRATION OF DIFFUSE GLIOMAS

Abstract BACKGROUND Diffuse infiltration is an aggressive feature of high-grade gliomas with survival implications. The contribution of crosstalk between non-neoplastic and neoplastic cells to tumor infiltration remains largely understudied due to the lack of profiling techniques that retain spatial information. Spatial multi-omic profiling is a promising approach to comprehensively analyze transcript-omics, prote-omics and metabol-omics on the same tissue section while preserving information about the spatial organization of cells. Integration of these spatial studies allows for inferring the consequences of complex cell-cell communication underlying tumor infiltration. We hypothesize that the glioma edge is enriched with pro-infiltration ligands-receptors driving tumor infiltration. Strategies that disrupt these ligand-receptor networks may suppress glioma infiltration and improve clinical outcomes. METHODS We utilized a glioma tissue micro-array (TMA) to establish the neoplastic and non-neoplastic heterogeneity in the GBM infiltration edge. Each TMA slide consists of pathologist annotated tumor core and edge samples of Glioblastoma (IDH Wildtype, WHO Grade 4; n=10), Diffuse Astrocytoma (IDH Mutant, WHO Grade 3; n=3), Oligodendroglioma (IDH mutant, WHO Grade 2; n=5) and 2 non-brain control samples. We performed spatial multi-omics profiling on adjacent sections of the TMA using 10x Xenium spatial transcriptomics, imaging mass cytometry (IMC) and mass spectrometry imaging (MSI). Integration, visualization, and quantification of the spatial data was done on VisioPharm. RESULTS In GBM, we identified candidate mRNA transcripts and proteins for ligands enriched at the infiltrating edge (compared to tumor core; p<0.0001) that correlated with a poor progression free survival (PFS; r2=0.22). These candidate ligands were also significantly enriched at the edge of oligodendroglioma WHO Grade 2 and astrocytoma WHO Grade 3. CONCLUSIONS Spatial multi-omics profiling on a TMA consisting of Glioma WHO grades 2-4 identified differentially expressed and targetable pro-tumor infiltration ligands associated with lower PFS in GBM. Functional studies to uncover the role of metabolites enriched at the glioma edge for the expression of the identified pro-infiltration ligands are ongoing.

BIOM-17. EVALUATION OF CARBONIC ANHYDRASE TYPE IX (CA IX) EXPRESSION AS TARGET FOR RADIOLIGAND THERAPY IN GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma represents an unmet need in neuro-oncology. Radioligand therapies are effective and approved in extracranial malignancies such as prostate cancer and neuroendocrine tumors, but data on their use is limited in brain tumors. In addition, rational target selection in gliomas is complicated by tumor heterogeneity. In extracranial tumors, clinical trials of radionuclide treatments targeting carbonic anhydrase IX (CA IX) are ongoing. METHODS Tumor samples of glioblastoma (isocitrate dehydrogenase [IDH]-wildtype) were retrieved from the Neuro-Biobank of the Medical University of Vienna. Formalin-fixed, paraffin-embedded tissue specimen were stained using rabbit anti-CA IX antibody (clone EP161, Cell Marque) on a Ventana Benchmark Ultra platform (Roche Diagnostics). CA IX expression was assessed semiquantitatively. RESULTS Overall, 29 patient samples were included in this study. Median age at surgery was 65 years (range: 55 – 74), and 22/29 (75.9%) were male. Median overall survival (OS) reached 11.6 months (95% confidence interval: 7.3 – 16.3). In total, 24/29 (82.8%) of specimen were positive for CA IX, of whom 20/29 (69.0%) exhibited moderate to high membranous CA IX expression and 4/29 (13.8%) showed weak immunoreactivity. CA IX staining displayed pronounced spatial heterogeneity, as positive areas were primarily seen in pseudo-palisading tumor cells around necrotic foci. Patients with stronger CA IX staining tended to have longer OS (median: 14.2 months) compared to those with absent or weak CA IX expression (median OS: 6.2 months; p = 0.075). Inclusion of further samples of different glioma subtypes is ongoing. CONCLUSIONS CA IX could represent a promising target for radioligand therapy in glioblastoma given the strong CA IX immunoreactivity in most samples. Intratumoral heterogeneity in CA IX expression should be considered in the design of appropriate compounds and the choice of optimal radionuclides to maximize efficacy.

CNSC-26. INVESTIGATING THE ROLE OF QUAKING IN REGULATING MHC II ANTIGEN PRESENTATION IN TUMOR-ASSOCIATED MACROPHAGES OF GBM

Abstract Glioblastoma (GBM) is the most common and malignant primary brain tumor with a median survival rate of 14 months. Despite the success of immunotherapy in treating various cancer types, its efficacy remains poor in GBM. Such limitation can be attributed to the profound immunosuppressive tumor microenvironment of GBM enriched with the tumor-associated macrophages (TAMs). Quaking (Qki) is an RNA-binding protein that is mutated or deleted in more than 35% of GBM patients. Decreased levels of Qki in TAMs associates with worsened survival rates and responsiveness to immunotherapy in GBM mouse and patients. Moreover, Qki is a tumor suppressor gene in GBM and a major regulator in phagocytosis, a fundamental biological process in delivering antigens to major histocompatibility complex class II (MHC II). MHC II-restricted antigen presentation in macrophages of GBM is important in maintaining the fitness of cytotoxic and helper T cells required in anti-tumor response. However, the mechanism through which Qki regulates MHCII expression and presentation has yet to be elucidated. I hypothesize that Qki promotes MHC II expression in TAMs, thereby constraining the development and progression of GBM. In vitro assays like immunofluorescent staining, qRT-PCR, cell co-culture system, and flow cytometry will be performed involving QPP (Qk-/-Trp53-/-; Pten-/-) mouse brains, QPP cells, and mouse macrophage cell lines. A positive correlation between transcriptomic and protein expression levels of Qki, MHC II, and CIITA (a master regulator of MHC II) was confirmed in both mouse QPP and human GBM samples. Moreover, bone-marrow-derived macrophages activated by QPP cell supernatant shows upregulated levels of Qki and thus MHC II genes. A preliminary conclusion is that Qki plays a permissive role in MHCII expression in TAMs of GBM. Further downstream impact of Qki in TAMs will be evaluated in T cell activity and in transcription of MHC II in TAMs.