Extracellular Matrix Research Articles

PATHOPHYSIOLOGICAL AND STRUCTURAL PECULIARITIES OF CARDIAC FIBROSIS DEVELOPMENT IN ATRIAL FIBRILLATION

Introduction. In recent years, the study of the pathogenesis of atrial fibrillation (AF) and its complications resulting from this condition has drawn substantial attention from cardiologists. A comprehensive understanding of this issue is essential for investigating the structural and functional disturbances occurring in the heart during AF. These data are also important for developing novel therapies aimed at preventing the onset and progression of cardiac fibrosis. The aim of this study is to identify the primary pathways that activate cardiac connective tissue cells involved in fibrosogenesis, to examine the structural characteristics of this pathological process, and to investigate the role of fibroblasts in initiating and progressing cardiac fibrosis. Materials and Methods. The primary data for this article were collected through an in-depth analysis of recent scientific literature, with a focus on studies published in the last 5–7 years. Results. A detailed review of primary sources reveals that this cardiological issue has only been partially explored. Findings indicate a close link between the pathophysiological and structural mechanisms of AF and numerous resulting complications, with cardiac fibrosis being a primary consequence. Two major forms of fibrous damage to atrial cardiomyocytes play a key role in the development of cardiac fibrosis associated with AF. This includes both reactive and reparative types of cardiac fibrosis, which develop concurrently in atrial fibrillation. These structural changes lead not only to damage and loss of cardiomyocytes but also to pathological remodeling of fibroblasts, the intercellular matrix, and the atria and ventricles. Disruptions in bioelectrical potential conduction are also observed. Activated fibroblasts play a central role in initiating cardiac fibrosis, with key stages of protein synthesis involved in atrial fibrosis now better understood. Specific intracellular signaling pathways, which present potential therapeutic targets for preventing cardiac fibrosis in AF treatment, have been identified. Conclusion. Atrial fibrillation and cardiac fibrosis are interdependent, with each potentially accelerating the progression of the other. These pathological processes are underpinned by significant structural and functional disruptions within cardiac cells and the intercellular matrix. Fibroblasts, myofibroblasts, and intensive fibrous tissue formation within the matrix are pivotal in the development of cardiac fibrosis. A preventive strategy targeting early intervention in cardiac fibrosis shows the greatest therapeutic promise.

TMIC-67. EMERGING ROLE OF EXTRACELLULAR MATRIX STIFFNESS AS A HALLMARK OF GLIOBLASTOMA: INSIGHT TO MECHANICAL MEMORY FORMATION AND PROLONGED INVASION IN THE TUMOR PERIPHERY

Abstract The extracellular matrix (ECM) plays a critical role in cancer progression, with its biophysical properties influencing cancer behavior. ECM stiffness can induce long-lasting adaptations in cancer cells, promoting sustained invasion through a process known as “mechanical memory” (MM). Glioblastoma (GBM) exhibits spatial heterogeneity in stiffness, with stiffer ECM found at the tumor periphery where invasive cells reside and promote recurrence. We hypothesize that increased stiffness at the tumor periphery primes GBM cells for sustained invasion through MM. METHODS Cell lines were established from image-guided human GBM samples from tumor margin and core (n=10 patients, 20 cell lines). Cells from random locations were conditioned in physiologically relevant extracellular matrices for two weeks. Human organotypic assay (n=3), Migration Assays, Atomic Force Microscopy (AFM), Immunocytochemistry, Western Blot, and RNA-sequencing (n=6) were performed. RESULTS GBM cells at higher stiff surfaces displayed increased migration (p<0.01). Magnetic Resonance Elastography (n=9) confirmed GBM heterogeneity, with softer cores (0.2-0.5kPa) transitioning to stiffer margins (>2kPa). Organotypic scaffolds mimicking the tumor margin promoted enhanced migration of GFP-labeled GBM cells. Margin cells and cells conditioned on stiffer matrices demonstrated higher migratory potential compared to their core and soft-conditioned counterparts (p<0.05). These trends persist over time and across multiple passages. ECM stiffness exposure causes nanomechanical, cytoskeletal, and transcriptional adaptations. AFM shows that cells from the margin (n=6) are softer (p<0.01) and more deformable (p<0.01). RNAseq identified upregulation of MM-specific genes in GBM margin cells. The mechanosensitive transcription factor YAP1 emerged as a key driver of MM. Notably, YAP1 is targetable by the FDA-approved drug Verteporfin. YAP1 expression was elevated in margin and stiff-conditioned cells (p<0.05). Stiff-conditioned cells and margin cells displayed increased sensitivity to Verteporfin treatment (p<0.05), with treatment significantly reducing margin migration compared to the core (p<0.001). CONCLUSION Stiffer GBM margins promote the formation of aggressive phenotypes through MM. Targeting YAP1 with Verteporfin, a readily available drug, presents a promising strategy to halt GBM invasion and improve patient outcomes.

IMMU-27. INHIBITION OF THE EXTRACELLULAR MATRIX PROTEIN EFEMP1/FIBULIN-3 REDUCES IMMUNOSUPPRESIVE SIGNALING IN TUMOR STEM CELLS AND INCREASES MACROPHAGE ACTIVATION AGAINST GLIOBLASTOMA

Abstract Glioblastoma tumors remain a formidable challenge for immune-based treatments because of their molecular heterogeneity, poor immunogenicity, and growth in the largely isolated and immunosuppresive neural environment. As the tumor grows, GBM cells change the composition and architecture of the neural extracellular matrix (ECM), affecting the mobility, survival, and function of immune cells such as tumor-associated microglia and infiltrated macrophages (TAMs). We have previously described the unique expression of the ECM protein EFEMP1/fibulin-3 in GBM compared to normal brain and demonstrated that this secreted protein promotes growth and resistance of the GBM stem cell (GSC) population. Here, we describe a novel immunomodulatory role of fibulin-3 and the immuno-boosting effects of targeting this ECM protein. Mice carrying fibulin-3-deficient intracranial tumors showed increased myeloid infiltration and reduced expression of TAM pro-tumoral markers (Arginase, CD206) compared to controls. The opposite was observed in orthotopic tumors overexpressing fibulin-3. In silico dataset analysis revealed positive correlation of fibulin-3 with an immunosuppresive signature in GBM, which was validated in GBM stem cells (GSCs). Accordingly, expression of fibulin-3 by GSCs promoted anti-inflammatory polarization of co-cultured macrophages. We further demonstrated that fibulin-3 regulated the expression of immunosuppresive signals (CSF-1, TGFbeta) and the innate immune checkpoint CD47 in GSCs via autocrine activation of NF-kB signaling. Immunosuppresive signals in GSCs were downregulated by knockdown of fibulin-3 or inhibition of this protein with an anti-fibulin-3 antibody (mAb428.2). Genetic or antibody-mediated targeting of fibulin-3 in GSCs co-cultured with macrophages (U937 and THP1 lines) increased macrophage phagocytosis and reduced the viability of the tumor cells. Furthermore, local delivery of mAb428.2 in mice carrying intracranial GBM resulted in increased infiltration of TAMs expressing pro-inflammatory markers and reducing tumor viability. Our findings show that anti-fibulin-3 approaches, which impact the tumor ECM, can diminish immunosuppression in GBM and boost innate immune responses against the tumor.

Viscosity regulates cell spreading and cell‐extracellular matrix interactions

Fluid viscosity and osmolarity are among some of the underappreciated mechanical stimuli that cells can detect. Abnormal changes of multiple fluidic factors such as viscosity and osmolarity have been linked with diseases such as cystic fibrosis, cancer, and coronary heart disease. Changes in viscosity have been recently suggested as a regulator of cell locomotion. These novel studies focus on cell migration and spreading on glass substrates and through microchannels, and it remains a question whether viscosity impacts the cellular remodeling of extracellular matrices (ECMs). Here, we demonstrate that elevated viscosity induces cellular remodeling of collagen substrates and enhances cell spreading on ECM‐mimetic substrates. Our results expand on recent work showing that viscosity induces increased cellular forces and demonstrates that viscosity can drive local ECM densification. Our data further show that microtubules, Ras‐related C3 botulinum toxin substrate 1 (Rac1), actin‐related protein 2/3 (Arp2/3) complex, Rho‐associated protein kinase 1 (ROCK), and myosin are important regulators of viscosity‐induced ECM remodeling. In the context of viscosity‐induced cell spreading, cells cultured on glass and collagen substrates exhibit markedly different responses to pharmacological treatments, indicating that microtubules, Rac1, and Arp2/3 play distinct roles in regulating cellular spreading depending on the substrate. In addition, our results demonstrate that high osmotic pressures override viscosity‐induced cell spreading by suppressing membrane ruffling. Our results demonstrate viscosity as a regulator of ECM remodeling and cell spreading in a fibrillar microenvironment. We also reveal a complex interplay between viscosity and osmolarity. We anticipate that our research can pave the way for future investigations into the crucial roles played by viscosity in both physiological and pathological conditions.

Role of DDR1 in Regulating MMPs in External Root Resorption

Human periodontal ligament cells (hPDLCs) express matrix metalloproteinases (MMPs), a group of enzymes responsible for the destruction of most extracellular matrix proteins in dental tissues, especially MMP-1, MMP-2, and MMP-13. Exploring the regulatory mechanism of MMPs is crucial for understanding external root resorption (ERR), one of the most severe complications, along with substantial loss of dental tissue, induced by trauma, pulpal infection, tooth bleaching, and orthodontic treatment, etc. Discoidin domain receptor 1 (DDR1), a cell surface receptor binding to collagen, has the potential to regulate the expression of MMP-1, MMP-2, and MMP-13, but the mechanism remains unclear. Thus, the present study aimed to investigate the connection and underlying mechanism between MMP-1, MMP-2, MMP-13, and DDR1 in hPDLCs. Our post-replantation ERR model revealed that Mmp-1, Mmp-2, Mmp-13, and Ddr1 all increased in the sites of ERR. hPDLCs with DDR1 knockdown exhibited a substantial reduction in MMP-1, MMP-2, and MMP-13 expression. To further confirm the underlying mechanism, we conducted further in vitro experiments, including RNA sequencing, RNA interference, RT-qPCR, Western blotting, and ELISA. Based on our results, MMP-1 was positively regulated by the Smad2/3 and MEK-ERK1/2 pathways and negatively regulated by the PI3K-Akt pathway through CCN2. MMP-2 and MMP-13 were positively regulated by the Smad2/3 pathway. MMP-13 was positively regulated by the MEK-ERK1/2 and PI3K/Akt signaling pathways. Collectively, DDR1 is a potent regulator of MMP-1, MMP-2, and MMP-13 expression through the Smad2/3, MEK-ERK1/2, and PI3K/Akt signaling pathways. Clarifying the significance and underlying mechanism by which DDR1 is involved in ERR might bring the chances to hinder the pathogenic process of ERR, hence reducing its incidence rate.

IMMU-25. B-CELL BASED THERAPY (BVAX) PRODUCES ANTIBODIES TO SUPPRESS GLIOBLASTOMA TUMOR MIGRATION AND INVASION

Abstract Glioblastoma (GBM) presents as a highly aggressive and malignant brain tumor with limited treatment options. Despite the availability of existing therapies, the invasive characteristics of GBM frequently result in tumor recurrence and poor prognosis. We previously developed a B-cell-based therapy (BVax) that has shown promising results in its ability to induce therapeutic responses in preclinical models of GBM. Our research revealed potency of BVax can be attributed to its robust antigen-presenting capability, cognate antigen specificity with T cells, and ability to generate tumor-reactive antibodies. The present study aims to characterize the unique antigenic reactivity of BVax-derived antibodies by evaluating their immunological effects and therapeutic potential in both murine models and GBM patient-specific samples. Leveraging CD45.1 and CD45.2 congenic mouse models, we first revealed that BVax preferentially migrated to glioma-bearing brains upon intravenous injection, resulting in a CD38+CD20-CD19+ plasmablast phenotype. To further characterize the reactivity of BVax-derived immunoglobulins (Ig), we utilized proteomic analysis and identified preferential targets of Gelsolin, Fibronectin, Versican, Fibrinogen, and collagen type IV alpha, commonly associated with cell motility and the extracellular matrix. Microdialysis data confirmed the increased presence of many of these BVax-derived Igs-recognized antigens in tumor regions compared to normal brain regions in GBM patients. Utilizing purified BVax-derived antibodies from patient specimen, in vitro cell motility assays demonstrated a significant prevention of the invasion and migration of cell lines from patient derived xenograft (PDX). In this study, we concluded that BVax elicits antitumor activity through targeted migration to tumor-bearing regions, differentiation into plasmablasts, and robust secretion of specific Igs that retain the ability to inhibit functionalities associated with cell invasion and migration. Here, we present the first in-depth study that characterizes GBM patients’ antibody reactivity, specifically a subset of inflammatory B cells (BVax) with therapeutic effects that offer an indication for a new direction of immunotherapies.

TMIC-65. USING BIOINFORMATICS TO INVESTIGATE THE TUMOR MICROENVIRONMENT OF DIFFUSE MIDLINE GLIOMA

Abstract Diffuse midline gliomas, H3K27-altered (DMG) are highly malignant brain tumors with a 2-year survival rate of less than 10 percent. These tumors are primarily localized to the pons and thalamus regions of the brain. The cellular compartment of the tumor microenvironment (TME) which surrounds a tumor is composed of tumor and non-tumor cells, such as immune cells and oligodendrocytes. Communication between tumor cells and non-tumor cells in the TME can promote tumor growth and targeting communication networks could improve patient outcomes. Using a bioinformatics approach and publicly available pediatric DMG single-cell RNA-sequence (scRNA-seq) data, several methods of communication (secreted signaling, cell-cell contact, and extracellular matrix) within the DMG TME were identified. Based on the high communication probability, outgoing and incoming networks from non-tumor cells and tumor cells were inferred, such as the Complement, Galectin, and CD96 pathways. Specific ligand-receptor interactions between non-tumor and tumor cells were also found, including LGALS9-HAVCR2, PDGFB/PDGFRA, and COL4A5/ITGAV. A subsequent literature review was performed to validate these identified pathways in other tumors, including gliomas, and inform future in vitro experiments with primary DMG cell lines.

Epithelial Folding Through Local Degradation of an Elastic Basement Membrane Plate

AbstractEpithelia are polarized layers of cells that line the outer and inner surfaces of organs. At the basal side, the epithelial cell layer is supported by a basement membrane, which is a thin polymeric layer of self‐assembled extracellular matrix (ECM) that tightly adheres to the basal cell surface. Proper shaping of epithelial layers is an important prerequisite for the development of healthy organs during the morphogenesis of an organism. Experimental evidence suggests that local degradation of the basement membrane is one of the mechanisms that can drive epithelial folding. However, how folding emerges in the absence of tissue growth remains elusive. Here, we present a coarse‐grained plate theory model of the basement membrane that assumes force balance between i) cell‐transduced active forces and ii) deformation‐induced elastic forces. We verify key assumptions of this model through experiments in the Drosophila wing disc epithelium and demonstrate that the model can explain the emergence of outward epithelial folds upon local plate degradation. The model accounts for local degradation of the basement membrane as a mechanism for the generation of epithelial folds in the absence of epithelial growth.

BIOM-65. ELEVATED MMP9 EXPRESSION IN ADHERENT AND LARGE VESTIBULAR SCHWANNOMAS: A PROMISING THERAPEUTIC TARGET

Abstract Vestibular schwannoma (VS) is the most common tumor of the cerebellopontine angle affecting up to 1 in 2000 adults and presents significant treatment challenges, especially when the tumor is adherent to the brainstem and cranial nerves. These tumors often require complex surgeries with high risks of complications such as hearing loss, facial paralysis, and incomplete tumor resection. Previous studies have linked immune cell infiltration and protease activation to aggressive VS. MMP9, a protease involved in extracellular matrix (ECM) remodeling, is associated with cancer invasion and progression, suggesting its potential as a biomarker and therapeutic target. This study explores whether MMP9 can classify VS and improve surgical outcomes. METHODS Fresh tumors were collected from patients undergoing surgery to establish primary VS cultures. Transcriptomic profiling was performed to examine expression of genes related to ECM remodeling, protease activation, and immune cell infiltration. MMP9 expression during schwannoma progression was validated using immunofluorescence staining and ELISA. Transwell invasion assays were performed to evaluate the role of MMP9 in schwannoma invasion. The efficacy of small molecule inhibitors against eIF4F (a transcription factor that binds to 5’ UTR of MMP9) and against MMP9 was tested in a mouse model of schwannoma. RESULTS MMP9 was the most abundantly expressed protease in VS. MMP9 expression increased with tumor size, and was higher in adherent than non-adherent VS (49.7 vs. 13.8 ng/mL, p=0.014). MMP9 expression was abundant in perivascular regions in adherent VS. Inhibition of eIF4F reduced schwannoma cell invasion mediated by MMP9.MMP9 inhibition in vivo- reduced tumor burden by 50% in mice. CONCLUSIONS MMP9 is a highly active protease in VS growth and adherent VS. Developing MMP9-targeted therapies could be a breakthrough in managing these challenging tumors.

Connexin 43 hemichannels and related diseases

Abstract Connexin 43 (Cx43) protein forms hemichannels (connexons) and gap junctions, with hemichannels consisting of six Cx43 molecules and gap junctions formed by two hemichannels. While gap junctions are prevalent in organs like the heart and liver, hemichannels are found in specific cell types, such as astrocytes and osteocytes. They allow the passage of small molecules (<1.5 kDa) between the cytoplasm and extracellular matrix. Cx43 hemichannels have emerged as potential therapeutic targets in various diseases, including central nervous system disorders, bone-related diseases, diabetic complications, wound healing, and cancers. Aberrant hemichannel opening can worsen conditions by releasing inflammatory elements, such as causing gliosis in neuronal cells. Conversely, functional hemichannels may inhibit cancer cell growth and metastasis. Recent studies are revealing new mechanisms of Cx43 hemichannels, broadening their therapeutic applications and highlighting the importance of regulating their activity for improved disease outcomes.

CNSC-55. UNCOVERING THE ROLE OF COLLAGEN EXTRACELLULAR MATRIX IN GLIOBLASTOMA BRAIN MICROENVIRONMENT TRANSFORMATION

Abstract Glioblastoma (GBM) is a highly malignant brain tumor characterized by diffuse infiltration and significant heterogeneity, without effective treatment. Mesenchymal-like (MES) glioma cells drive tumor aggression by promoting brain invasion and tumor microenvironment remodeling. Previous spatial transcriptomic analyses identified Collagen, particularly COL1A, as a key extracellular matrix (ECM) component, enriched in MES areas termed oncostreams. Depletion of COL1A1 disrupted oncostreams, influencing the tumor microenvironment to support cell survival, and migration. Reactive astrocytes and macrophages, essential components of the brain microenvironment, impact glioma progression. However, the interplay between glioma-secreted collagen and the brain microenvironment remains poorly understood. To elucidate this, we generated orthotopic mouse glioma models with high-collagen (NPD) and low-collagen (NPDshCol1A1). Our data revealed that downregulation of COL1A1 in glioma cells alters the abundance and spatial distribution of reactive astrocytes and macrophages/microglia. Immunofluorescence and flow cytometry analysis showed that tumors with low-collagen had decreased GFAP+ reactive astrocyte cells at the invasive edge and perivascular niche compared to high-collagen gliomas. Moreover, our results shows that high collagen levels may activate astrocytes through distant communication, as evidenced by the higher presence of reactive astrocytes in the contralateral cortex of NPD tumors. However, low-collagen tumors exhibited an increase in astrocytes (GFAP+) in the tumor core. Furthermore, high-collagen gliomas demonstrated increased macrophage infiltration, promoting an immunosuppressive environment. Using multiplex immunohistochemistry assays, we observed that reactive astrocytes along the invasive tumor edge were associated with glioma cells expressing elevated levels of COL1A1 and TGF-β ligand. In summary, our results show that glioma cell-expressed COL1A1 promotes the activation of peritumoral and perivascular brain-resident astrocytes and macrophages. The interactions of reactive astrocyte with glioma cells enriched in collagen form a feedback loop that enhances tumor invasion and vascular proliferation, suggesting that targeting the ECM-astrocyte interactions could disrupt these processes and offer a therapeutic avenue for GBM.

TMET-08. RADIATION-INDUCED SLC25A22 CONTRIBUTES TO GLIOBLASTOMA RADIORESISTANCE ACQUISITION THROUGH REMODELING GLUTAMATE METABOLISM

Abstract Glioblastoma Multiforme (GBM), a malignant primary brain tumor, is treated by surgical resection followed by chemo- and radiotherapy. Nevertheless, most GBM patients show dismal prognosis due to the radioresistance acquisition of GBM cells via metabolic rewiring. Therefore, it is urgently required to discover how metabolic rewiring attributes to GBM radioresistance, and its targeting strategies. To identify GBM radioresistance driver genes, we previously established radioresistance GBM cells (RGCs) via repetitive in vivo selection, followed by mRNA sequencing, and selected candidate genes that are altered in RGCs (fold change>2 or <0.5, p-value<0.05) and also related with GBM patients’ prognosis. Here, we focused on amino acid transmembrane transporter (Gene Ontology: 0003333) and found that SLC25A22 was the most increased in RGCs. SLC25A22 exhibits uni-directional properties, exporting glutamate to cytosol, resulting in cytosolic glutamate accumulation in RGCs. Using 13C glutamine isotope tracing analysis following mitochondrial fractionation, we quantified the mitochondrial metabolic flux of glutamine. Despite decreased 13C glutamate efflux from RGCs mitochondria after genetic inhibition of SLC25A22, glutamine metabolites (a-ketoglutarate, succinate, malate, fumarate) remained constant. Cytosolic glutamate, as a substrate, is incorporated into various biosynthetic pathways, especially glutathione (GSH) and proline synthesis pathways in radioresistant GBM cells. GSH protects GBM cells by scavenging radiation-induced reactive oxygen species. Whereas, proline, a rate-limiting substrate for collagen biosynthesis, leads GBM cells to show an invasiveness phenotype via extracellular matrix remodeling. Genetic inhibition of SLC25A22 using short hairpin RNA (shRNA) or microRNA-184 mimic suppresses RGCs radioresistance and aggressiveness. Additionally, intranasal administration of microRNA-184 mimic prolonged the survival (median survival: 26 days after irradiation) of GBM-bearing mice, compared with non-target control (median survival: 16 days after irradiation). Together, our study demonstrates that SLC25A22 upregulation confers GBM radioresistance by inducing cytosolic glutamate-driven biosynthesis and suggests SLC25A22 as a therapeutic target to overcome GBM radioresistance.

BIOS-07. GRADUAL METABOLIC ALTERATIONS RESTRUCTURE THE SPATIAL ARCHITECTURE AND IMMUNITY IN GLIOBLASTOMA

Abstract The organized layered structure of the human neocortex contrasts sharply with the chaotic architecture of malignant CNS tumors. The heterogenous nature of glioblastoma (GBM) poses significant challenges to research and treatment, given that effective strategies require consistent biological patterns. Here, we leverage recent advances in spatial biology, integrating spatially resolved transcriptomics and metabolomics to identify drivers of GBM-specific architectural evolution. We performed spatial metabolomics (MALDI) and transcriptomics (Visium n=43 and MERFISH n=25) on glioblastoma patients, with computational analysis conducted using the SPATA2 R package. Our findings reveal that most spatial recurrent patterns in glioblastoma emerge in a gradient-like organization, particularly influenced by proximity to areas of necrosis and hypoxia. Samples without hypoxic regions demonstrated less spatial organization. Metabolomic and transcriptomic profiling indicated enhanced responses to hypoxia, reduced cell proliferation, and elevated expression indicative of the S phase in cell division near necrotic regions. The vicinity to hypoxic areas was associated with increased extracellular matrix remodeling and epithelial-to-mesenchymal transition, suggesting that the invasive capabilities of GBM cells originate from these hypoxic niches. By further integrating spatially resolved TCR-sequencing, we identified that hypoxic gradients drive defined T cell responses with a higher likelihood of T cell dysfunction within a distance of 235 µm (sd: 132 µm) to hypoxia. This suggests that the presence of necrosis, hypoxia, and hypoxia-associated metabolites could serve as potential predictive markers for T-cell behavior and exhaustion in glioblastoma. Our study offers an in-depth analysis of the influence of two prevalent niches in glioblastoma: necrosis and hypoxia. We reveal that the complex architecture of glioblastoma is not solely dictated by genetic randomness and stochastic occurrences but is significantly influenced by spatial proximity to key factors like necrosis and hypoxia. This insight contributes to an evolving model that seeks to unravel the intricate biology underpinning this formidable disease.

BIOM-29. UNMASKING COPPER DEPENDENCE IN GLIOMA: 19-GENE SIGNATURE FOR PATIENT STRATIFICATION AND TARGETED THERAPY

Abstract Adult-type diffuse gliomas exhibit poor prognosis with limited treatment options. Although copper-targeted therapies have been previously explored in these tumors, they have yet to show significant benefits. To understand the role of copper in glioma biology we performed an in silico data analysis on the TCGA glioma cohorts to identify copper-homeostasis related genes (CHRG) with prognostic significance. This was done to enable patient stratification into appropriate treatment arms for future clinical trials involving copper targeted therapies. Furthermore, in vitro experimentation was performed to validate the functional outcomes observed from the in silico results. 19 CHRGs were identified to have prognostic association from the TCGA glioma cohorts. This gene signature regulates a broad range of cellular processes, including metal homeostasis, metabolism, extracellular matrix organization, and immune cell function. The CHRG signature enabled the development of a predictive model that allowed stratification of patients between high and low risk groups. In vitro spheroid growth assays in both U87 cell lines with or without IDH mutation elicited sustained growth inhibition after 6 days post treatment. Furthermore, copper chelation therapy modified the energy potential of these glioma cells in vitro leading to growth inhibition. This study established a predictive model using 19 CHRGs that enable patient stratification between high and low risk glioma patients. Furthermore, our in vitro experiments show the suitability of targeting intracellular copper to inhibit tumor growth. Further research is needed to understand the underlying mechanisms and explore optimal treatment combinations for improved outcomes. We aim to extend this approach to pediatric gliomas for improved risk assessment and personalized medicine.

BIOM-22. SPATIAL, SINGLE-CELL, AND BULK RNA SEQUENCING IDENTIFY MOLECULAR PROGRAMS UNDERLYING MENINGIOMA BRAIN INVASION

Abstract Brain invasion is a negative prognostic factor for meningiomas, but the molecular programs underlying meningioma brain invasion are poorly understood. To address this, we performed transcriptomic analyses of 3 independent datasets: Bulk RNA sequencing from 199 meningiomas (n=33 with brain invasion), single-cell RNA sequencing of 2 regionally distinct samples from the brain tumor interface (BTI) versus tumor core (TC) (n=22,118 single-cell transcriptomes), and spatial RNA sequencing from 6 samples with (n=4) versus without (n=2) brain invasion (n=18,471 spatial transcriptomes). Single-cell and spatial datasets were integrated with Harmony, cell interactions were defined using CellChat, and cross-platform comparisons were performed with the Seurat data integration pipeline. Bulk RNA sequencing revealed enrichment of 1835 genes in meningiomas with brain invasion compared to those without brain invasion, including glial markers (GFAP, NCAN) and markers of extracellular matrix remodeling (PDGFRA, COL9A1). Single-cell RNA sequencing comparison of BTI versus TC meningioma cells identified 2793 genes that were enriched in BTI cells, 268 of which were conserved across bulk and single-cell transcriptomes from brain-invasive meningiomas. Gene ontology enrichment analysis showed molecular programs underlying cell migration and regulation of neurogenesis at the BTI, while glycolysis and antigen presentation programs were enriched in the TC. Cholesterol, CSPG4, and LIF signaling were active in the TC, while SLIT signaling was enriched at the BTI. Spatial RNA sequencing showed enrichment of 439 genes at the BTI, and a conserved set of 12 genes were enriched in BTI meningioma cells across bulk, single-cell, and spatial datasets (TGM2, APOC1, IGFBP6, PLEKHO1, GFPT2, ZYX, IQGAP3, LUM, S100A11, ARPC1B, CDK1, CYCS). Comparison of tumor microenvironment monocytes at the BTI versus TC demonstrated enrichment of 1571 genes that were associated with macrophage migration and activation. These data elucidate molecular programs underlying meningioma brain invasion and provide insights into meningioma intratumor heterogeneity.

MiR-24-3p secreted as extracellular vesicle cargo by cardiomyocytes inhibits fibrosis in human cardiac microtissues

Abstract Background and Aims Cardiac fibrosis in response to injury leads to myocardial stiffness and heart failure. At the cellular level, fibrosis is triggered by the conversion of cardiac fibroblasts (CF) into extracellular matrix–producing myofibroblasts. miR-24-3p regulates this process in animal models. Here, we investigated whether miR-24-3p plays similar roles in human models. Methods and Results Gain– and loss–of–function experiments were performed using human induced pluripotent stem cell–derived cardiomyocytes (hCM) and primary hCF under normoxic or ischaemia–simulating conditions. hCM–derived extracellular vesicles (EVs) were added to hCF. Similar experiments were performed using three-dimensional human cardiac microtissues and ex vivo–cultured human cardiac slices. hCF transfection with miR-24-3p mimic prevented TGFβ1–mediated induction of FURIN, CCND1 and SMAD4—miR-24-3p target genes participating in TGFβ1–dependent fibrinogenesis —, regulating hCF–to–myofibroblast conversion. hCM secreted miR-24-3p as EV cargo. hCM–derived EVs modulated hCF activation. Ischaemia–simulating conditions induced miR-24-3p depletion in hCM-EVs and microtissues. Similarly, hypoxia downregulated miR-24-3p in cardiac slices. Analyses of clinical samples revealed decreased miR-24-3p levels in circulating EVs in acute myocardial infarction (AMI) patients, compared with healthy subjects. Post-mortem RNAScope analysis showed miR-24-3p downregulation in myocardium from AMI patients, compared with patients who died from noncardiac diseases. Berberin, a plant–derived agent with miR-24-3p–stimulatory activity, increased miR-24-3p contents in hCM-EVs, downregulated FURIN, CCND1 and SMAD4, and inhibited fibrosis in cardiac microtissues. Conclusions These findings suggest that hCM may control hCF activation through miR-24-3p secreted as EV cargo. Ischaemia impairs this mechanism, favouring fibrosis.

BIOM-62. CIRCULATING TUMOR CELLS IN MEDULLOBLASTOMA: A NOVEL PERIPHERAL BIOMARKER FOR CNS DISEASE

Abstract BACKGROUND Medulloblastoma is an embryonal tumor of the cerebellum and the most common pediatric malignancy of the central nervous system (CNS). Diagnosis, staging, and disease monitoring require invasive neurosurgical biopsy or MRI. No blood or cerebrospinal fluid (CSF) biomarkers exist. We demonstrate the presence of circulating tumor cell clusters (CTCCs) in patients with medulloblastoma and use a transcriptomic approach to describe their gene expression. Design/Method: We enrolled 34 total patients in a longitudinal study: 24 with medulloblastoma and 10 without malignancy. CTCCs are captured from unprocessed whole blood using a physics-based label-free approach to CTCC isolation. RNA sequencing was performed on 21 samples at time of diagnostic resection from 6 patients with medulloblastoma. Sequencing reads were assessed for quality control, trimmed, and aligned to the human genome. Differential gene expression (DGE) was performed against whole blood from healthy controls. Reactome pathway database of curated, peer-reviewed gene sets are used for gene set enrichment analysis (GSEA). RESULTS The presence of peripherally CTCC patients with medulloblastoma is not previously known and was detected in all patients with medulloblastoma whereas none were observed from patients with non-malignant hematological conditions. RNA-sequencing demonstrate separation on principal component analysis and gene-expression heatmap against whole blood healthy control. DGE shows upregulation of genes indicating stem-like state. GSEA show enrichment of medulloblastoma molecular pathways and gene sets defining integrin, extracellular matrix remodeling, and cell adhesion pathways. Transient elevation of peripheral CTCC followed by return to baseline levels was associated with durable disease response to therapy, whereas persistence or elevation of CTCC in CSF correlated with disease recurrence. CONCLUSION The universal and specific finding of peripherally captured CTCC arising from parent tumor in the CNS within our patient cohort suggests a utility as a tumor biomarker. Future sequencing is planned comparing CTCC transcriptome against parent tumor.

BIOM-26. ROLE OF EXTRA-DOMAIN B FIBRONECTIN IN MODULATING GLIOBLASTOMA CELL INVASIVE PROPERTIES

Abstract Glioblastoma (GBM) is a highly malignant tumor characterized by its resistance to current treatment modalities and high recurrence rates. Extra-domain B Fibronectin (EDB-FN), a glycoprotein not found in normal tissues but selectively expressed in the tumor microenvironment, was previously identified by our team as a promising molecular marker for brain tumors, offering potential for both prognostic prediction and therapeutic targeting. However, the specific role and mechanism of action of EDB-FN in GBM cells remain unclear. In this study, we sought to elucidate the potential role of EDB-FN and the underlying mechanisms contributing to GBM malignancy through in vitro investigations. Our findings reveal that silencing EDB-FN in GBM cells leads to a decrease in cell migration, invasiveness, extracellular matrix adhesion ability, and stem cell-like properties. Furthermore, we discovered that FAK and ERK signaling pathways play a crucial role in GBM cell progression in response to EDB-FN. This study underscores the potential of EDB-FN as a specific biomarker for GBM treatment, paving the way for future research in this area.

TMIC-42. CONQUERING SPP1+ MYELOID-DRIVEN RESISTANCE OF GLIOBLASTOMA TO IL13RA2-TARGETED CAR T CELL THERAPY

Abstract In the evolving landscape of glioblastoma (GBM) therapy, the recent proliferation of clinical trials exploring chimeric antigen receptor (CAR) T cell interventions has garnered attention, although their therapeutic efficacy remains limited. Despite recent progress in elucidating the role of the GBM microenvironment in immunotherapy resistance, particularly regarding intercellular networks and macrophages, significant ambiguities endure, impeding a comprehensive understanding of resistance mechanisms amidst the patient-specific heterogeneity of the tumor microenvironment (TME). In this study, we utilized single-cell RNA sequencing to analyze tumors from 41 glioma patients undergoing IL13Rα2-targeted CAR T cell therapy. Our findings unveiled heightened suppressive extracellular network signatures, particularly SPP-1, predominantly observed within myeloid cells from non-responsive patients. The clinical significance of both SPP1+ myeloid cell abundance and overall expression levels revealed associations with patient outcomes. Subsequently, transcriptomic insights were translated to myeloid cell functions in patients exhibiting high versus low SPP1 expression. SPP1-expressing macrophages displayed diminished antigen presentation, phagocytosis, and cell-mediated cytotoxicity, along with augmented extracellular matrix biogenesis and degradation, iron efflux, and suppressive interleukin pathways. Moreover, SPP1-high macrophages exhibited enhanced ligand-receptor interactions, implying a predisposition towards suppressive activities. Immunometabolism pathways, including lipid and ketone body biogenesis and regeneration, were upregulated in SPP1-expressing macrophages, indicating an increased reliance on non-glycolysis-mediated ATP production. To assess translational implications, the therapeutic efficacy of SPP1 blockade was evaluated in a syngeneic glioma model. Blockade of SPP1 with an anti-SPP1 antibody prior to CAR T cell infusion effectively mitigated the immunosuppressive milieu, reversing resistance in preclinical GBM models to murine IL13Rα2-targeted CAR T cell therapy. Subsequent profiling of tumors post-treatment provided insights into TME alterations following SPP1 blockade therapy. This investigation illuminates the intricate interplay between the TME and CAR T cell therapy resistance in GBM, underscoring SPP1 as a promising therapeutic target to surmount CAR T cell resistance and enhance treatment outcomes.

“Red Flags”: Case Report of Cardiac Amyloidosis with Significant Coronary Artery Disease

BACKGROUND: Cardiac Amyloidosis is a disorder of protein misfolding and metabolism in which insoluble fibrils are deposited in the myocardial extracellular matrix causing organ dysfunction and eventually death. It can exhibit cardiac signs and symptoms, or it can be identified through screening in patients who exhibit extracardiac symptoms of amyloidosis. As there were no clear clinical signs of cardiac amyloidosis and a biopsy is required to show amyloid deposition, the condition has been historically challenging to diagnose. Thus, a high index of suspicion based on the clinical presentation and the outcomes of the preliminary testing arecrucial to determine the approach to diagnosis. CASE SUMMARY: We outline a case of 75-year-old Filipino male who was admitted due to progressive exertional dyspnea. Cardiac Amyloidosis was considered due to evaluation findings of heart failure with preserved ejection fraction with restrictive type of cardiomyopathy. This was subsequently confirmed through extracardiac fat pad biopsy, echocardiographic strain analysis and Technetium (99mTc) Pyrophosphate (PYP) single photon emission computed tomography scan (SPECT). CONCLUSION: This case report discussed the red flags of clinical manifestations of cardiac amyloidosis and highlighted the use of non-invasive diagnostic modalities to diagnose the disease. Cardiac amyloidosis remains a rare entity and with emerging therapies that have the potential to improve patient outcomes, early diagnosis is really important. Having high index of suspicion based on signs and symptoms can lead to early detection and an increased number of patients being referred for treatment.