bluuebunny/crossref_metadata_2025 huggingface dataset:
TMIC-16. THE COLLAGEN RECEPTOR LAIR-1 ALTERS THE GLIOMA MICROENVIRONMENT AND REGULATES TUMOR AGGRESSIVE BEHAVIOR
Authors: Varela Maria , Faisal Syed M , Comba Andrea , Lelling Sayer , Barissi Marcus , Castro Maria , Lowenstein Pedro
Published: November 2023

High-grade gliomas (HGG) are the most common malignant primary brain tumors in adults, with a median survival of 15-18 months. HGG are heterogeneous tumors with a complex extracellular matrix. Our previous work demonstrated the importance of the interaction between tumor cells and Collagen I. Employing genetically engineered mouse models (GEMM) of glioma, we identified Collagen 1α1 (Col1A1) as a regulator of tumor malignancy. Downregulation of Col1A1 in glioma cells extended median survival (MS), and reduced migration and proliferation. Nonetheless, the mechanisms underlining these effects remain unknown. LAIR-1 is a collagen receptor originally described as an inhibitory receptor in immune cells. We now describe the expression of LAIR-1 in glioma cells. Molecular ablation of LAIR-1 from glioma cells in GEMM (NPL: NRAS, shP53, shLAIR-1, and NPAL: NRAS, shP53, shATRX, shLAIR-1) significantly improved MS when compared to controls (NP: NRAS, shP53, and NPAL: NRAS, shP53, shATRX). The NPAL tumors had reduced cell proliferation (PCNA+), higher infiltration of astrocytes (GFAP+) and CD8+ T cells. Cells were obtained from these GEMM and cultured in vitro. LAIR-1 downregulation reduced cell proliferation, in both NPAL and NPL cells. To corroborate LAIR-1’s relationship with cell proliferation, control cells (NPA) were incubated with a LAIR-1 blocking antibody resulting in diminished glioma cell proliferation. When these cells were implanted in the brain LAIR-1 was downregulated resulted in increased MS. In vitro cytokines production assessment showed both NPAL and NPL cell had an enhanced production of RANTES(CCL5), G-CSF and IL-6 production, when compared to their controls. Our data suggest that LAIR-1 expression in glioma cells has a role in glioma malignant behavior both due to tumor cell proliferation and because of tumor composition. We hypothesize that blocking the interaction between LAIR-1 and collagen may improve median survival.
TMIC-23. UNRAVELLING THE HETEROGENEITY OF GLIOBLASTOMA'S VASCULAR LANDSCAPE
Authors: Woolf Zoe , Li Susan , Swanson Molly , Turner Clinton , Schweder Patrick , Correia Jason , Faull Richard , Park Thomas , Dragunow Michael
Published: November 2023

BACKGROUND

Glioblastoma tumours are highly vascular and angiogenic. However, they are also heterogeneous, comprising many different macro- and microenvironments. Reflecting this, tumour-associated blood vessels are variable in their distribution, structure, and function. Despite the significance of vasculature in the development and progression of glioblastoma, its complexity remains poorly defined. Therefore, characterising vascular diversity across the glioblastoma landscape is a crucial step in the development of targeted therapies.

METHODS

40 glioblastoma and 5 epilepsy cases were immunohistochemically labelled with a vascular antibody panel (PDGFRβ, ColIV, αSMA, UEA-1 and GFAP) to identify and subtype vessels. Single-vessel image analysis was performed to analyse vascular coverage and to classify vascular subtypes based on marker co-expression. Labelling with haemoglobin, ZO-1, and claudin-5 allowed for the evaluation of vascular leakage and blood-brain barrier integrity. Tissue segmentation was performed to analyse vascular coverage, vascular subtypes, and vascular integrity across the tumour macro-environment.

RESULTS

No significant difference in gross vascular coverage was found between tumour and epilepsy tissue. However, vascular coverage within glioblastoma varied significantly depending on the tumour macro-environment. Whilst vascular coverage in the tumour periphery was comparable to epilepsy tissue, increased vessel density was observed in the intermediate tumour zone, followed by decreased vessel density towards the tumour core. Reflecting this variation, multiple vessel subtypes, including previously described glomerular microvascular proliferation, mother vessels, vascular malformations and hyperplastic vessels, were identified. These tumour vessels display highly irregular shapes, sizes, and cellular compositions. Finally, glioblastoma tissue displayed a significant increase in haemoglobin leakage and a decrease in vascular integrity compared to epilepsy tissue.

CONCLUSION

Characterising this vascular landscape provides valuable insights for treatment strategies related to hypoxia and angiogenesis, two key factors driving tumour growth. Indeed, targeting specific vessel subtypes to address compromised vascular function and aberrant vascularisation may improve outcomes for glioblastoma patients.
TMIC-24. FERROPTOSIS INDUCES IMMUNOGENIC CELL DEATH AND SYNERGIZES WITH CD47 BLOCKADE TO REPROGRAM THE GLIOMA MICROENVIRONMENT
Authors: Sperring Colin , Argenziano Michael , Banu Matei , Dovas Athanassios , Mela Angeliki , Zhao Wenting , Furnari Julia , Pereira Brianna , Ager Casey , Yeary Mitchell , Shapiro Ben , Kotidis Corina , Adeyuan Oluwaseyi , Higgins Dominique , Upadhyayula Pavan , Mahajan Aayushi , Humala Nelson , Izar Benjamin , Sims Peter , Bruce Jeffrey , Canoll Peter
Published: November 2023

Immunotherapy has thus far proven ineffective in glioblastoma, in part due to the immune cold tumor microenvironment. Here we demonstrate that ferroptosis specifically targets a quiescent, immunosuppressive astrocyte-like glioma population and can therefore be used as a novel inducer of immunogenic cell death to repolarize the immune microenvironment by promoting tumor cell phagocytosis and antigen presentation. First, we characterized the immunogenic effects of ferroptosis by using a glioma cell-myeloid cell co-culture system and patient-derived organotypic slice cultures and performed phagocytosis assays, flow-cytometry, immunohistochemistry, cytokine arrays and single cell RNAseq. GPX4-dependent ferroptosis induced translocation of the pro-phagocytic eat-me signal calreticulin in both murine glioma cells and human tissue samples. Ferroptotic-driven tumor cell death reprogramed local myeloid populations to promote glioma phagocytosis, MHCII-driven antigen presentation, and paracrine cytokine signaling involved in T-cell recruitment. Local delivery of RSL3, a GPX4 inhibitor, in a murine glioma model polarized myeloid cells to a pro-phagocytic phenotype and significantly increased intra-tumoral levels of CD4+ and CD8+ T-cells. Astrocytic-like tumor cells upregulate CD47 as a mechanism to evade phagocytosis. Therefore, we combined ferroptosis with an anti-CD47 monoclonal antibody (aCD47) to enhance reprogramming of the immune cold microenvironment. In an orthotopic murine glioma model, adding aCD47 to RSL3 led to significantly increased tumor phagocytosis, antigen presentation and prolonged survival. Thus, we demonstrate that local delivery of ferroptosis-inducing agents in conjunction with CD47 blockade is an effective multi-modality therapeutic regimen capable of reversing the immunosuppressive glioma microenvironment.
TMIC-25. NEUTROPHIL-INDUCED GLIOBLASTOMA FERROPTOTIC CELL DEATH DEPENDS ON LC3-ASSOCIATED PHAGOCYTOSIS OF NEUTROPHILS BY GLIOBLASTOMA CELLS
Authors: Lu Tong , Yee Patricia , Thorpe Jessica , Wang Hong-Gang , Li Wei
Published: November 2023

Glioblastoma multiforme (GBM) is the most aggressive and common type of primary adult brain cancer. Tumor necrosis is a histological hallmark of GBM and a poor prognostic factor for the disease. Although it is thought to result from chronic ischemia, the molecular mechanisms that induce tumor necrosis remain unclear. Previously, we reported that tumor-associated neutrophils (TANs) contribute to the generation of tumor necrosis through inducing ferroptosis in GBM cells. We showed that accumulation of lipid peroxides within GBM cells depends on neutrophil myeloperoxidase (MPO). The cell killing is associated with transfer of MPO-containing granules to GBM cells. (Yee et al., 2020, Nature Communications, DOI: 10.1038/s41467-020-19193-y). During further study of Neutrophil-induced glioblastoma ferroptotic cell death, we found that neutrophil engulfment by GBM cells is required for intercellular transfer of neutrophil contents and the upregulation of lipid peroxides in GBM cells. The engulfment process can be inhibited by RGDS peptides, which inhibit the function of RGDS-containing integrins. Furthermore, we used immunofluorescence imaging to show that internalized neutrophils colocalize to molecular components of the LC-3 associated phagocytosis (LAP) machinery. Disruption of these components either through pharmacological or genetic means rescues GBM cells from neutrophil-induced cell death. We then used an orthotopic xenograft GBM mouse model to demonstrate that disruption of the LAP machinery in tumor cells reduces tumor necrosis and improves survival. Altogether, this study identified a novel ferroptosis-inducing process, which can help to understand how neutrophils contribute to tumor necrosis in GBM. Additionally, the results suggested that targeting LAP may have therapeutic benefits for GBM treatment.
TMIC-26. TAMS AND HYPOXIA ENHANCE GLIOBLASTOMA STEM-LIKE CELL ENRICHMENT AND MAY IMPACT RADIORESISTANCE
Authors: Karwacinski Victor , Carneiro-Lobo Tatiana , Olson Cheryl , Constante Mayerli , Mesci Pinar , Brat Daniel , Muotri Alysson
Published: November 2023

The dismal survival rates of glioblastoma (GBM) patients are due, at least in part, to therapeutic resistance of GBM stem-like cells (GSCs) and their ability to repopulate recurrent tumors. Our goal was to determine if tumor-associated macrophages (TAMs) or hypoxia enhance the enrichment of GSCs in a manner that could impact radioresistance. First, we established GBM-brain cortical organoids (GBM-BCOs) to model human GBM. We co-cultured GFP-labeled GBM neurospheres with BCOs for 14 days and observed a tumor take rate of 100%. Next, we evaluated whether hypoxia enhanced TAM migration into GBM-BCOs by incorporating THP-1 cells into GFP-GBM-BCOs and incubating them for 24 h under normoxic or hypoxic conditions. We found that hypoxia significantly increased THP-1 cell influx into GBM-BCOs. Further, we investigated whether TAMs influenced the enrichment of GSCs in GBM neurospheres and in GBM-BCOs. We found that co-culture of THP-1 cells with either GBM neurospheres or with GBM-BCOs enhanced the CD133+ population after 72 h hypoxia, correlating with our IF studies of human GBM samples that show increased CD133+ and IBA1+ cells in hypoxic peri-necrotic zones compared to non-necrotic regions. Next, we established Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI), an advanced methodology for monitoring cell cycle phases for investigating the role of hypoxia and radiation on GBM. Flow cytometry analysis of patient-derived GBM cells revealed that the fraction of G1 cells decreased and the fraction of G2/M cells increased 24 h after 3 Gy radiation compared to non-irradiated cells in normoxia. In contrast, GBM cells exposed to hypoxia do not show such cell cycle shifts after radiation and have an enriched population of cells in G0 in both radiated and non-irradiated cells. Our results indicate that hypoxia and TAMs play critical roles in GSC enrichment and may impact radioresistance.
TMIC-27. THE ROLE OF AQUAPORIN-4 IN GLIOBLASTOMA-ASSOCIATED VASOGENIC CEREBRAL EDEMA
Authors: Joshi Tanvi , Hambardzumyan Dolores
Published: November 2023

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. Vasogenic cerebral edema is a severe complication that occurs in a majority of GBM patients, leading to a significant increase in intracranial pressure, accompanying neurological deficits, and increased mortality rates. Edema in GBM patients is almost exclusively managed by the corticosteroid dexamethasone (DEX), which has recently been found to have several drawbacks, including immunosuppression and interference with radiation therapy. Thus, there is an urgent need to better understand the mechanisms of edema resolution in GBM in order to identify alternative treatment strategies. The water channel aquaporin-4 (AQP4) is a critical player in the brain's water homeostasis, with increased levels in GBM compared to the normal brain. To gain more insight into the role of AQP4 in GBM-associated edema, we used the RCAS/tv-a system to generate de novo GBM with different driver mutations in Aqp4 knockout (KO), heterozygous (HET), and wild-type (WT) mice. Multiplex IF staining combined with RNAScope of tumors showed that AQP4 expression co-localized with GFAP-positive reactive astrocytes in perivascular areas. Our results indicated that tumor-bearing mice in an Aqp4-deficient background had increased edema content and decreased survival, suggesting that AQP4 is important for edema resolution from tumors. Loss of Aqp4 also significantly decreased astrocyte coverage in the tumor core, but not in the tumor periphery. Moreover, anti-VEGFA treatment only improved vessel leakage and reduced edema in WT tumor-bearing mice. Ongoing studies are focused on characterizing the impact of Aqp4 loss on the gene expression profile of tumor core-specific astrocytes using single-cell RNA sequencing and spatial transcriptomics. We are also studying the cellular distribution of AQP4 in GBM and the effect of DEX treatment on AQP4 levels.
TMIC-30. HYPOXIA-INDUCED COMPLEMENT COMPONENT 3 PROMOTES AGGRESSIVE TUMOR GROWTH IN THE GLIOBLASTOMA MICROENVIRONMENT
Authors: Rosberg Rebecca , Smolag Karolina , Sjölund Jonas , Johansson Elinn , Bergelin Christina , Wahldén Julia , Pantazopoulou Vasiliki , Pietras Kristian , Ceberg Crister , Blom Anna , Pietras Alexander
Published: November 2023

Glioblastoma is the most aggressive primary adult brain tumor. Treatment includes surgery, irradiation and chemotherapy. Still, virtually all patients suffer a treatment resistant recurrent tumor. In previous studies from our lab, we have investigated the tumor microenvironment contribution to tumor progression, showing that stromal astrocytes become reactive upon irradiation and hypoxia and generate tumor-supportive conditions for glioblastoma cells. Common features of astrocyte reactivity in several neurological diseases, include upregulation of complement proteins including complement component 3 (C3). However, it remains unexplored if these complement proteins are expressed in stromal astrocytes in glioblastoma. In a genetically engineered glioma mouse model, C3 was expressed in the invasive front, hypoxic, and perivascular tumor areas. TCGA data analysis revealed an overall strong correlation between hypoxia and complement gene expression signatures in glioblastoma patients. Astrocytes and glioma cells cultured in hypoxic conditions upregulated C3, as well as other genes associated with an infiltrative phenotype of GBM. In hypoxia, glioma cell lines, but not astrocytes, upregulated the C3a Receptor. GBM tumor single-cell sequencing data confirmed a subpopulation of highly C3-expressing astrocytes, enriched for cellular pathways comprising epithelial mesenchymal transition (EMT), TNF-alpha, hypoxia, and IFN-signaling. In vitro, C3 enhanced proliferation of glioma cells specifically under hypoxic conditions. Blocking C3a-C3aR signaling in vivo prolonged survival of glioma-bearing mice both alone and in combination with radiotherapy. Overall, our data indicate a strong link between hypoxia and complement expression in the brain tumor microenvironment of glioblastoma, where local expression of complement proteins lead to tumor promoting signaling. Inhibition of C3a-C3aR enhanced survival of glioma-bearing mice and could represent a promising avenue for further investigations.
TMIC-32. IMPACT OF LOW-INTENSITY TRANSCRANIAL DIRECT-CURRENT STIMULATION (TDCS) IN GLIOBLASTOMA
Authors: Das Arabinda , McDonald Daniel G , Yazdani Milad , Welsh Cynthia A , Infinger Libby Kosnik , Vandergrift III William A , Varma Abhay K , Patel Sunil J , Cachia David , Zukas Alicia M , Lindhorst Scott M
Published: November 2023

The prognosis of glioblastoma (GBM) remains poor despite multiple attempts over the years to develop better treatment options for patients with this disease. Various challenges include an immunosuppressive tumor microenvironment, poor knowledge of the neuroimmune system, and the presence of the blood−brain barrier amongst others. To try and overcome some of these barriers, we used a low-intensity non-invasive transcranial direct current stimulation (tDCS) to suppress tumor growth and enhance the immune response in preclinical glioblastoma cell culture and animal model. This study reports for the first time that treatment of GBM and rGBM (recurrent GBM) cells with tDCS (50 mV/mm for 30 min), can modulate the expression of human leukocyte antigen (HLA) class II molecules such as HLA-DR alpha, HLA-DR complex, invariant chain (Ii), and HLA-DM (an important catalyst of the class II-peptide loading). Furthermore, we demonstrated in vivo that tDCS [350 μA (1 x 30 min/day) treatment 7 times (within 14 Days) showed a significant (~30%) decrease in GBM tumor growth in an orthotopic allograft GL261 GBM mouse model (at Day 41). Following tDCS treatment, our results demonstrated the downregulation of Ki-67 in treated tumors. Also, we observed the upregulation of CD38 (M1 macrophages) and CD8+ T cells in treated cells in vivo. Based on our preliminary data from in vitro and in vivo studies, we believe that this study suggests that induction of immune components of the HLA class II pathway by tDCS may be a promising therapeutic strategy for the treatment of human GBM.
TMIC-33. UNDERSTAND AND TARGET ASTROCYTE REACTIVITY IN BRAIN TUMORS TO PREVENT RADIORESISTANCE
Authors: Jeannot Pauline , Berg Tracy , Dawson John , Eliott Richard , Carragher Neil , Pietras Alexander
Published: November 2023

Glioblastoma multiforme (GBM) is the most aggressive type of glioma and deadliest brain tumor. Despite aggressive treatments including surgery, chemo-, and radiotherapy, tumors invariably recur as incurable lesions, and the median survival is approximately 15 months after diagnosis. Recurrence is tightly linked to tumor cell resistance to ionizing radiation (IR), a feature that in turn is linked to stem cell characteristics of tumor cells. Previous results from our lab indicate that stromal astrocytes respond to IR with a reactive phenotype that in turn promotes therapeutic resistance of neighboring tumor cells. Our objectives were to i) identify pathways involved in IR-induced astrocyte reactivity; ii) identify compounds able to inhibit IR-induced astrocyte reactivity. We used two complementary approaches to meet our objectives. Using an antibody array we found that JNK and ABL pathways were activated following IR in primary human astrocytes. To validate their involvement in the induction of astrocyte reactive phenotype we used an image-based readout combining the changes in the expression of reactivity markers and in the morphology of astrocytes. For the second approach, we used a similar readout to perform an image-based drug screen which aimed to identify compounds able to inhibit IR-induced astrocyte reactivity. The drug libraries used consisted of approved drugs to get candidates with potential for drug repurposing. We identified 20 compounds inhibiting IR-induced astrocyte reactivity and amongst them, 10 are brain-penetrant compounds that could make them good candidates for repurposing in GBM. An in vivo test is now ongoing with 2 of these compounds in a PDGFA/PDGFB-driven mouse model of GBM to validate their efficiency to increase radiosensitivity of the tumor cell by inhibiting astrocyte reactivity
TMIC-34. SKULL-DERIVED MYELOID CELLS AUGMENT T CELL RESPONSES AND SUPPRESS GLIOBLASTOMA GROWTH
Authors: Lad Meeki , Kalistratova Venina , Young Jacob , Gill Sabraj , Saha Atul , Rios Jennifer , Salha Diana , Lui Austin , Aghi Manish
Published: November 2023

While recent studies have implicated the skull marrow as a functional immune reservoir in CNS pathology, its contribution to malignancy remains unexplored. To that end, we transplanted fluorescently-labeled calvarial flaps into syngeneic glioblastoma-bearing mice and cytometrically characterized tumor-infiltrative immune cells, observing a robust population of skull-derived myeloid cells (SDMCs; 40.3% of all intratumoral myeloid cells) in a Balb/C mouse tumor model (CMFDA-stained flaps, 72h). These SDMCs were robust, remaining viable for 3 weeks in an alternative longitudinal model (C57BL/6 mice, GFP+ flaps). As MHCII+ leukocytes were disproportionately enriched among SDMCs (87.9% vs 67.3%, n=3, p=0.012), we interrogated the immunogenicity of these cells through targeted skull ablation, achieved via 13 Gy irradiation of body-shielded mice prior to orthotopic glioblastoma implantation. Indeed, skull-ablated mice had reduced infiltration by antigen-presenting cells than controls (cDCs: 1.1% vs 4.5%, p=0.002; macrophages: 14.4% vs 36.6%, n=10/group, p< 0.001) and 6-fold greater necrosis (67.7% vs 13.5% apoptotic, p< 0.001), suggesting an impaired ability to mount a controlled, adaptive immune response. To clarify the role of SDMCs in mediating secondary immune function, we administered the CXCR4 antagonist AMD3100 between the inner and outer tables of the skull, inhibiting marrow homing signals and promoting myeloid precursor egress (0.26% vs 0.02% of intratumoral myeloid cells, n=10/group, p< 0.001). This yielded cDC2 expansion (10.4% vs 3.0%, p=0.003) and consequent CD4+ T effector memory differentiation (92.3% vs 60.0%, p=0.007). These differences were clinically meaningful, as skull irradiation accelerated tumor growth and attenuated survival compared to controls, while AMD3100 treatment accomplished the opposite (median OS: 17d vs 22d, p< 0.001; 26.5d vs 22d, p=0.006). Concurrent αCD4/CD8/Thy1.2 administration abrogated the therapeutic effect of AMD3100 entirely, confirming the dependency of SDMC-mediated tumor suppression on T cells. Together, our work clarifies the antitumoral role of skull marrow and represents the first-ever therapeutic modulation of this space.
TMIC-35. TUMOR CELL-DERIVED SPERMIDINE ACCELERATES GLIOBLASTOMA PROGRESSION VIA LYMPHOCYTE DEPLETION
Authors: Kay Kristen , Bayik Defne , Wang Zeneng , Miska Jason , Brown J Mark , Hazen Stanley L , Lathia Justin
Published: November 2023

Therapeutic resistance in glioblastoma (GBM) is partly due to a highly immunosuppressive environment resulting in ineffective anti-tumor immune response. This likely underlies the lack of success of immunotherapies that are successful in other cancers; novel ways to target this environment are needed. Naturally occurring polyamines (PAs) have been identified as a putative therapeutic target in other cancers based on their increased presence and correlation to poorer prognosis. Under normal conditions, spermidine (SPD) – a member of the PA family – is critical for cell growth. While PAs are increased in GBM patients, little is known about their impact on GBM growth. In syngeneic immunocompetent mouse glioma models, mass spectrometry revealed that SPD increases in the tumor microenvironment and circulation as compared to non-tumor-bearing mice. We treated the same models with exogenous SPD, which significantly decreased survival. However, in immunocompromised mice, no survival difference was observed, suggesting SPD drives GBM progression through immune interaction. Immune profiling the mouse models treated with SPD showed peripheral reduction of CD4+ and CD8+ T cells. Due to the observed T cell changes, we repeated the exogenous SPD paradigm in RAG1 knockout mice that do not have circulating B/ T cells. Similar to NSG mice, we see no survival difference, indicating that SPD drives GBM by affecting lymphoid cells. To investigate the origin of SPD in our models, we knocked down the rate limiting enzyme of the PA biosynthesis pathway in glioma cells in immunocompetent mice. Mice implanted with knockdown cells showed a robust survival extension, suggesting that tumor cells produce SPD which affects the tumor microenvironment. Ongoing studies are evaluating uptake from the environment on the progression of GBM using pharmacological inhibitors. Understanding the interactions of PAs and the immune response as a whole will allow us to alter the environment to enhance immunotherapeutic efficacy.
TMIC-37. AN ADVANCED PHAGOCYTOSIS GLIOMA THERAPY BASED ON MICROGLIA: INSIGHT FROM AD THERAPY
Authors: Lu Jie , Mou Yonggao
Published: November 2023

INTRODUCTION

Alzheimer's disease (AD) has an inverse correlation with glioma incidence. AD brain present a suppressive environment for glioma initiation and progression caused by amyloid β42 (Aβ42) activation of microglia. However, the underlying mechanisms remain elusive.

METHODS

Public database of patients with AD and brain cancer were collected and analyzed for the correlation between the two distinct diseases. The level of Aβ42 expression was examined by IHC staining in lower-grade glioma (LGG) and higher-grade glioma (HGG). Glioma-bearing mice were applied to evaluate the tumor suppressive effect induced by oligomer Aβ42 (OAβ42) in vivo. The co-culture system was applied to study the mechanism of OAβ42 activated microglia and enhanced tumor cell phagocytosis. RNA sequencing was performed to identify the underlying molecular mechanism involved in OAβ42 potentiated microglial phagocytosis.

RESULTS

OAβ42 is an innate peptide and a favorable prognostic biomarker of glioma with a prolonged OS in LGG than HGG (P=0.04). Injection of OAβ42 could inhibit the growth of glioma (P=0.0003) through enhancing microglial phagocytosis in the allogeneic mouse model. And the overall survival of the OAβ42 treated mice was longer than the vehicle group (P=.037). The OAβ42 primed microglia phagocytized more glioma cells than vehicle control in a co-culture system (P=0.0007). Autocrine of insulin-like growth factor 1(IGF-1) from OAβ42 activated microglia is essential for engulfing tumor cells.

CONCLUSIONS

Our findings proved OAβ42 modulated microglia could serve as a cellular target for treatment of glioma. Furthermore, our study provides a novel conceptual therapeutic strategy through modulating the function of existing immune cells in the microenvironment.
TMIC-40. BIOPHYSICAL ANALYSIS OF GLIOBLASTOMA CORE AND RIM REVEAL UNIQUE MECHANICAL AND ULTRASTRUCTURAL SIGNATURES
Authors: Mahaffey Bradley , Fowler Zachary , Lung Zoe , Dang Vivien , Frieboes Hermann , Williams Brian , Chen Joseph
Published: November 2023

Glioblastoma (GBM) is a highly aggressive and invasive brain cancer, carrying a median survival of 15 months. This poor prognosis is due, in part, to its resistance to therapy through tumor adaptations mediated by the tumor microenvironment (TME). Efforts to describe the tumor-protective cues in the TME may aid in the development of strategies to combat resistance. Emerging work has indicated that GBM biophysical properties are significant modulators of tumor progression; however, these parameters are poorly described. Here, we investigate the mechanical, extracellular matrix (ECM), and ultrastructural characteristics of tumors in patient matched GBM core and rim tissue. Using atomic force microscopy, immunohistochemistry, and scanning electron microscopy techniques, we find that GBM core is abnormally stiff and dense with large amounts of ECM, while the rim is softer and more porous. The GBM core is uniquely rich in hyaluronic acid and Tenascin-C, ECM proteins known to correlate with tumor stiffness and tumor supportive function. Additionally, dense matrix along with increased levels of hyaluronic acid and Tenascin-C correlate with decreased progression free survival. These data suggest that the stiff core provides increased pro-malignant mechanotransductive cues while the rim presents more permissive routes for GBM invasion. These results reveal a biophysical basis for tumor progression and provides a novel framework for diagnostic strategies and therapeutic intervention.
TMIC-41. LATERAL VENTRICLE PROXIMITY OF GLIOBLASTOMA IS ASSOCIATED WITH LOCALIZED MALIGNANCY-PROMOTING MOLECULAR SIGNATURES IN IMAGE-GUIDED BIOPSIES
Authors: Norton Emily , Ramos-Fresnedo Andres , Cherukuri Yesesri , Whaley Lauren , Schiapparelli Paula , Asmann Yan , Chaichana Kaisorn , Quiñones-Hinojosa Alfredo , Guerrero-Cazares Hugo
Published: November 2023

Glioblastoma (GBM) is the most devastating and common form of primary brain cancer in adults. Tumor location plays a significant role in patient prognosis; in particular, GBM tumors contacting the lateral ventricles (LVs) are more aggressive than LV-distal counterparts. This may be due to interaction of tumors with unique features of the LV microenvironment, including the cerebrospinal fluid (CSF) and neural progenitor cells (NPCs) of the subventricular zone. Despite LV contact having a role on tumor malignancy, studies using bulk tumor samples to identify molecular contributors to LV-contacting prognosis have been only moderately successful, in large part due to intratumoral heterogeneity. Here, we leverage intraoperative surgical navigation to collect matched biopsies of adult LV-contacting primary GBM at locations proximal (< 2 cm) and distal ( > 2 cm) from the LV. Matched samples were collected from ten patients with equal sex distribution and an age range of 46 to 81 years. RNA sequencing of biopsies revealed a localized transcriptional signature of gene expression in LV-proximal locations. These genes include those identified to be associated with GBM malignancy and upregulated in primary GBM cells by CSF exposure or co-culture with NPCs, such as SERPINA3, CD44, and CTSB, as well as many newly identified genes. Implicated biological pathways involved in this transcriptional signature involve inflammation, chemotaxis, Notch signaling, angiogenesis, integrin signaling, and more. The top 50 upregulated genes by LV-proximity of GBM were used to cluster patient transcriptomes from the TCGA GBM database and evaluate the contribution to patient outcome. This analysis revealed a cluster with significantly extended overall survival that displayed a general downregulation of LV-proximity genes. Ultimately, these studies provide evidence that regional tumoral analysis is essential in identifying molecular signatures associated with microenvironmental influence, and that LV-contacting GBM has regional gene expression changes in many genes associated with malignancy.
TMIC-43. THE IMPACT OF MICROGLIA MODULATION ON THE IMMUNE TUMOR MICROENVIRONMENT IN SYNGENEIC GLIOBLASTOMA MOUSE MODELS
Authors: Kaymak Deniz , Shekarian Tala , Ritz Marie-Françoise , Ferrari Nicoletta , Kälin Roland , Glass Rainer , Hutter Gregor
Published: November 2023

BACKGROUND

Glioblastoma is exceptionally resistant to immunotherapies for which the immunosuppressive myeloid compartment is held responsible. As influencer and instructor of T-cell-immunity this compartment mainly consists of Monocyte-derived Cells (MdCs) and tissue-resident (TR) macrophages called microglia (MG). Since the response to immunotherapies depends on antigen presenting cells, we investigate this compartment and its individual role in tumor progression by genetically depleting/activating the TR MG.

METHODS

We made use of two MG-specific and tamoxifen inducible knockout mouse models in which 1) we activate MG by excision of the activation-repressing transcription factor Sall1 and 2) deplete MG by deleting Csf1r on MG. To investigate the effect of MG activation/depletion on the immune tumor microenvironment (iTME), immunocompetent mice were engrafted with syngeneic glioma cells and the iTME subsequently characterized by scRNAseq, spectral flow cytometry and immunofluorescence imaging.

RESULTS

Upon Sall1-KO, MG demonstrate a morphologically reactive phenotype. The deletion of Csf1r depletes the brain parenchyma of MG which rapidly gets repopulated by a morphologically distinct microglia-(like) population. Genetic activation of MG leads to a survival benefit in Gl261-engrafted mice compared to Cre- littermates. In contrast, depletion of MG significantly reduces the survival in Gl261 and CT-2A engrafted mice. ScRNAseq confirms the effective depletion of MG and the subsequent emergence of a MG-like cell cluster that transcriptionally remains distinct. The iTME in MG-depleted mice is characterized by a decreased abundance of exhausted and naïve CD8-Tcells and an increased infiltration of MdCs. GSEA demonstrates a dampened inflammatory profile of infiltrating MdCs.

CONCLUSION

The presence of MG orchestrates the development towards a more antitumorigenic iTME by shaping the antitumoral response of incoming myeloid cells and furthermore directly or indirectly influencing T-cell immunity. Understanding this interplay of MG and MdCs and its impact on instruction of antitumoral T-cell immunity could help guiding the development of novel immunotherapeutic strategies.
TMIC-44. THY1 CONFERS PROLIFERATIVE ADVANTAGES AND THERAPY RESISTANCE WITHIN LOCALIZED NICHES OF GLIOMA
Authors: Ravikumar Visweswaran , Shankar Sunita , Ji Sunjong , Hassan Zainab , Haydin Yacoub , Huang Dah-Luen , Fehmi Ziad , Rehemtulla Alnawaz , Rao Arvind , Al-holou Wajd
Published: November 2023

Resistance to therapy is a hallmark of Glioblastoma (GBM), making it a deadly disease. Earlier, using a longitudinal mouse model, we reported that the immunoglobulin-like cell surface receptor THY1, was significantly upregulated in recurrent GBM tumors. Here, we used single cell and spatial transcriptomics data to track THY1 expression through treatment resistance. Single cell analysis of primary and recurrent GBM shows that THY1 expression is significantly associated with multiple tumor cell states, namely, the NPC-like (Neuronal precursor-like), OPC-like (oligodendrocyte precursor-like) and MES-like (mesenchymal-like) cellular states. Further, spatial analysis of primary GBM shows that tumor cells resident in highly vascularized regions and at invasive edges express significantly high levels of THY1. Specifically, high THY1 levels at the invasive edge is associated with NPC-like state, known to migrate through white matter tracts in vascular regions. In contrast, THY1 expression is high in MES-like cells within recurrent GBM tumor cells. Single cell analysis of a matched primary and recurrent tissue shows a remarkable increase in THY1 expression upon recurrence coincident with a switch to predominantly MES-like state. Using protein markers that define different cellular states we performed immunofluorescence analyses to track the expression of THY1 across both primary and recurrent tumors. We show evidence of high THY1 protein expression associated with NPC-like cells in different niches within primary tumors. In recurrent GBM tissues, THY1 protein expression is significantly higher and present along the palisading regions surrounding necrosis. Finally, we show that dox inducible knockout of THY1 completely abrogates the ability of a patient derived organoid to establish tumors in mice. We are currently exploring this model to study the role of THY1 in therapy resistance. Together, these data suggest that THY1 is critical for tumor growth and upon therapeutic stress can contribute towards shift in tumor cell identity to generate therapy-resistant clonal populations.
TMIC-47. INHIBITION OF MIR-146A-5P DECREASES PROGRESSION OF MELANOMA BRAIN METASTASIS VIA NOTCH PATHWAY DYSREGULATION IN ASTROCYTES
Authors: Rigg Emma , Wang Jiwei , Xue Zhiwei , Lunavat Taral , Liu Guowei , Hoang Tuyen , Parajuli Himalaya , Han Mingzhi , Bjerkvig Rolf , Nazarov Petr , Nicot Nathalie , Kreis Stephanie , Margue Christiane , Nomigni Miléne Tetsi , Utikal Jochen , Miletic Hrvoje , Ystaas Lars , Sundstrøm Terje , Li Xingang , Thorsen Frits
Published: November 2023

Melanoma has a high propensity for brain metastasis, occurring in 40% of late-stage patients. The progression of secondary tumors in the brain depends on the support of the surrounding microenvironment, emphasizing the importance of modulating neighboring cells for successful metastasis. Tumor-derived extracellular vesicles (EVs) play a crucial role in creating a pre-metastatic niche for tumor growth by transferring proteins, RNA, and DNA. This study aims to explore the involvement of melanoma brain metastasis (MBM)-derived EVs in MBM development and identify a target miRNA for potential therapeutic intervention. In an in vivo metastatic model, MBM-EV pre-treated mice exhibited significantly increased MBM tumor burden compared to non-primed mice. In vitro co-cultures of MBM-derived EVs with astrocytes led to astrocyte activation characterized by enhanced proliferation, invasion, cytokine production, and upregulation of GFAP. Microarray analysis revealed significant upregulation of miR-146a-5p in MBM-EVs compared to healthy astrocyte and melanocyte EVs. In situ hybridization of clinical MBM samples showed significantly elevated miR-146a-5p expression compared to healthy brain controls. Sequencing of NHA after co-culture with miR-146a-5p mimic revealed NUMB, a Notch signaling pathway inhibitor, as a miR-146a-5p target. Expression levels of Numb and other downstream Notch proteins were significantly altered in NHA in the presence of MBM-derived EVs and miR-146a. Deserpidine was identified as a potential inhibitor of miR-146a-5p through a drug docking study, and qPCR analysis confirmed reduced miR-146a-5p expression in deserpidine-treated MBM cells and EVs in vitro. In an MBM in vivo model, deserpidine-treated mice exhibited significantly increased survival, reduced tumor burden, and decreased astrocyte activation in both tumor and non-tumor brain tissues. In conclusion, EVs are important regulators of MBM and establish tumor-supporting reactive astrocytes by delivery of miR-146a. Elevated miR-146a levels in patients suggests a potential clinical intervention, and our results indicate deserpidine as a promising candidate for adjuvant therapy.
TMIC-48. SYNAPTIC PROTEINS BASSOON, DLG4, DLG1, MAGI2, SHANK1, AND HOMER1 IN GLIOMA
Authors: Timmer Marco , Lauer Nina , Kuhl Saskia , Goldbrunner Roland
Published: November 2023

To thrive, glioma cells exploit pathways that are active in normal CNS cells, as well as in normal neurotransmitter signaling. In healthy excitatory synapses the postsynaptic submembrane space consists of multiprotein complexes called the postsynaptic density (PSD). The PSD is responsible for the structural organization, the functioning, and the regulation of neuronal signaling. The five MAGUK Proteins DLG4, DLG1, Magi2, Shank1, and Homer1 are part of the healthy PSD complex and play a crucial role not only in NMDA and AMPA receptor signaling. Investigating the role of these MAGUK proteins in glioma biology could bring further insight into which principal neurobiological features, including organization in communicating networks, are recapitulated by tumor cells of different stages in gliomas. qPCR was used to determine expression levels of DLG4, DLG1, Magi2, Shank1, and Homer1 in 7 different glioma groups (GII, GIII, primary GBM before and after chemotherapy, secondary glioblastoma recurrence with and without chemotherapy). Western Blot was done for DLG4, DLG1, and Homer1. Immunohistochemical staining of frozen sections, paraffin sections and in vitro cells was performed. The expression levels of DLG4, DLG1, Magi2, Shank1, and Homer1 were reduced especially in high grade glioma compared to control tissue. A tendency of decreasing protein levels with increasing malignancy is visible for the majority of proteins. In cell culture all proteins were also present. siRNA experiments are ongoing. The presence of these synaptic proteins in glioma tissue and moreover also primary cell culture could indicate a role in tumor biology and the connection of glioma cells with neuronal tissue.
TMIC-50. SIGLEC-15 IS A NEW MYELOID CHECKPOINT IN GBM, WHOSE SUPPRESSION LEADS TO EXTREMELY DURABLE RESPONSES WITH ONCOLYTIC THERAPY
Authors: Kesarwani Ashwani , Costa Amanda De Andrade , Hu Tong , Rich Jeremy , Kim Albert , Shi Pei-Yong , Diamond Michael S , Chheda Milan
Published: November 2023

Zika virus (ZIKV) significantly increases survival in multiple syngeneic glioma mouse models; like other oncolytic therapies, it generates a potent, anti-tumor CD8+ T-cell response. Post-ZIKV, we observe a 32-fold increase in myeloid cells in the tumor microenvironment. Siglec-15 is a newly identified myeloid immune checkpoint in cancer, which suppresses T-cell activation; an antibody targeting Siglec-15 is currently in clinical trial for lung cancer. Using immunostaining, for the first time, we report Siglec-15 is expressed on myeloid cells in human GBM specimens. In syngeneic murine models, anti-Siglec-15 alone does not improve survival. However, in combination with ZIKV, anti-Siglec-15 significantly improves survival in the CT2A, SB28, and GL261 models. For example, in the CT2A model, the combination increased long-term survivors (at least 90 days) to 60%, compared to 20% with ZIKV alone and 0% with no treatment. To further confirm the role of Siglec-15, we used Siglec-15 knockout (KO) mice and observed 70% long-term survivor rate in Siglec-15 KO mice treated with ZIKV, compared to 40% in wild-type mice (B/6) with ZIKV. ZIKV treatment in Siglec-15 KO mice led to activation of CD8 T-cells and increased expression of cytotoxic molecules granzyme B and perforin, indicating enhanced anti-tumor activity. Additionally, we detected high levels IFN-gamma and TNF-alpha, further supporting the anti-tumor response. We further improved efficacy by targeting PD-1. This combination resulted in 90% long-term survivors. Moreover, in a model for recurrence, upon contralateral rechallenge with CT2A in long-term survivors, we observed long-term memory T-cell responses, with increased numbers of CD8+ T-cells displaying effector and resident memory cell phenotypes, compared to age-matched controls. We obtained similar results with immune-resistant SB28 model. This work suggests that oncolytic therapy combined with blocking myeloid checkpoint Siglec-15, using an agent already in humans, and blocking CD8+ T cell checkpoint with anti-PD1, is worthy of study in humans.
TMIC-51. THE RELEASE OF INFLAMMATORY CYTOKINES ARE REGULATED BY PYK2 SIGNALING IN GBM CELLS
Authors: Morales-Colón Emely , Kucheryavykh Lilia
Published: November 2023

Glioblastoma (GBM) is a highly aggressive brain cancer that is typically fatal within a year of diagnosis. Previous research has shown a positive relationship between the activation of a protein called proline-rich non-receptor tyrosine kinase (Pyk2) in GBM cells in addition to the expression of cytokines by tumor-infiltrating myeloid (TIM cells). Leading to a sustained increase in the expression levels of monocyte chemoattractant protein 1 (CCL2), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL10, IL12, and vascular endothelial growth factor (VEGF), as well as an increase in the CD86+/CD206+ TIM population. We hypothesize that Pyk2 signaling in GBM cells is involved in the regulation of the release of inflammatory cytokines, which in turn suppresses the immune response in the tumor microenvironment. We used CRISPR/Cas9 knockout Pyk2 (Pyk2KO) and wild type (Pyk2WT) GL261 mouse glioma cells in this study. Analysis of the cytokines present in the cell culture medium conditioned by Pyk2KO and Pyk2WT cells identified a decrease in the release of CCL2 and CCL5 in the Pyk2KO cells compared to the Pyk2WT cells. RT-PCR analysis of myeloid cell population, isolated from Pyk2WT and Pyk2KO tumors in the GL261-C57Bl/6 GBM model, identified a significant down-regulation of key pro-tumorigenic factors such as CCL2, CCL12, CCL5, tumor necrosis factor (TNF), VEGF and epidermal growth factor (EGF) in the Pyk2KO tumors compared to the Pyk2WT tumors. Additionally, flow cytometry analysis revealed a reduction in myeloid-derived suppressor cells (MDSCs), an increase in lymphoid dendritic cells (DC), and up-regulation in TNF/IFNg expressing and antigen-specific CD8+ T cells in Pyk2KO tumors. Furthermore, a significant decrease in pro-tumorigenic Ly6C+/CD206+ and an up-regulation of inflammatory Ly6C+/CD86+ myeloid cells were found in the Pyk2KO tumors. These findings suggest that Pyk2KO tumors display an immunocompetent microenvironment with enhanced phagocytic and cytotoxic function compared to Pyk2WT tumors. NIH Grant 1SC1GM122691 and PRSTRT2022 supported this study
TMIC-56. DECIPHERING THE INTRICATE GLIOBLASTOMA TUMOR MICROENVIRONMENT AND EGFRVIII TRANSCRIPT DISTRIBUTION THROUGH SINGLE-CELL SPATIAL PROFILING
Authors: Gregory Simon , Jain Vaibhav , Hocke Emily , Abramson Karen , Thomas Elizabeth , Satterfield Diane , Walsh Kyle , Brown Michael , McLendon Roger , Ashley David
Published: November 2023

INTRODUCTION

Profiling the tumor microenvironment (TME) in glioblastoma (GBM) poses challenges due to its complex and heterogeneous nature. Analyzing diverse cell populations, deciphering dynamic cell-cell interactions, and overcoming spatial-temporal variations present significant challenges to characterizing the GBM TME. These challenges can, in part, be overcome using in situ sequencing (ISS) approaches that offer the advantage of directly analyzing gene expression within intact tissue, providing spatial and molecular context for understanding complex biological processes and tumor heterogeneity. Method/

RESULTS

Here, we report a ‘first in world’ application of 10xGenomics Xenium ISS platform to profiling RNA expression profiles within GBM tissue, including primary and recurrent tumors, to establish single cell-level profiling of the TME. Our panel of canonical and custom GBM genes allowed us to establish profiles of high-resolution cell neighborhoods, intratumoral cell-types and cell-states, and the distribution of infiltrating tumor cells throughout the tissue. Exploiting the chemistry that underlies the Xenium platform, we also developed a novel ISS isoform expression assay that semi-quantitatively profiles a mutated variant of the epidermal growth factor receptor (EGFR). Data show that EGFRvIII, which has been shown to promote tumor growth, invasion, and resistance to therapies, is preferentially expressed within tumor-subsets within the GBM. Our EGFRvIII assay not only has the potential to act as a high sensitivity method of tumor diagnosis but its distribution will allow for correlation of this tumorigenic marker with activated pathways across the tissue.

CONCLUSION

ISS is a valuable tool for profiling the complex TME in GBM, enabling analysis of intratumoral cell types, cell states, and EGFRvIII distribution. Our study demonstrates its pioneering application, offering insights into the molecular landscape and potential for novel diagnostic signatures and improved treatment strategies for this aggressive cancer.