bluuebunny/crossref_metadata_2025 huggingface dataset:
SURG-54. RECURRENT BRAIN METASTASES (RBM) TREATED WITH SURGICALLY TARGETED RADIATION THERAPY (START): PRELIMINARY SAFETY EVALUATION FROM A MULTI-INSTITUTIONAL PROSPECTIVE REGISTRY
Authors: Kotecha Rupesh , Lee Stuart , Peach Matthew S , Patel Toral , Wardak Zabi , Floyd John , McGinity Michael , Hanft Simon , Robin Adam , Lee Ian , Sloan Lindsey , Shen Colette , Rodriguez Analiz , Bojanowski-Hoang Kimberly , Wanebo John , Smith Kris , Shah Mitesh , Richardson Angela , Chamoun Roukoz , Camarata Paul , Milligan Brian , Leng Lewis , Seid Patricia , Wasilewski Andrea , Monyak David , Nowlan Adam , McCracken David , Dunbar Erin , Ranjan Tulika , DiNapoli Vincent , Pham Huong , Ryan Robert , Mercado Catherine , Avgeropoulos Nicholas , Choutka Ondrej , Patel Akash , Jalali Ali , Haydon Devon , Deb Nimisha , Zhu James , Piccioni David , Hoeprich Mark , Ansari Michelle Aizenberg , Baskin David , Sevak Parag , Brachman David , Garcia Michael , Patel Sita
Published: November 2024

BACKGROUND

Resection and intraoperative brachytherapy for operable recurrent brain metastasis allows for pathologic confirmation of recurrent disease, mass effect relief, and immediate initiation of radiotherapy (RT). In this analysis, we report patterns-of-use and treatment-related adverse events (AEs) for rBM patients treated with Cs-131 collagen tiles, an FDA-cleared intracranial brachytherapy device.

METHODS

Patients with rBM who underwent resection and surgically-targeted radiation therapy (GammaTile, GT Medical Technologies Inc., Tempe, AZ USA) on a prospectively enrolling phase 4 registry study (NCT04427384) were analyzed. AEs were graded per CTCAE v5.0.

RESULTS

Between 11/2020 and 2/2024, 56 rBM in 51 consecutive patients underwent STaRT at 19 centers, with 5 patients having 2 metastases implanted concurrently. 44 patients (86%) had prior same-site RT (median interval 14.5 mo, range 3-56). Primary tumor histologies were lung (27), melanoma (8), breast (7), renal (4), colon (2), and “other” (3). Median pre-operative maximum diameter was 3.0 cm (range 1.4-5.7); age 63 (range 28-81); 53% females; KPS median 90 (range 40-100); and median implantation time 3 minutes. 26 patients were implanted at a 1st, 15 at a 2nd, and 10 at ≥ 3rd same-site recurrence (range 1-9). At a median follow-up of 6.2 months (range <1-35.1), 6/51 patients (11.8%) experienced ≥Gr 3 AEs at a median of 12 (range 1-69) days postoperatively (POD). No radiation necrosis (RN) events were observed, and no AEs occurred in multi-implant cases or where STaRT was the initial form of RT.

CONCLUSIONS

In this prospective multi-institutional study, STaRT demonstrated an excellent safety profile in a cohort of larger rBM, even in the setting of multi-recurrent disease. Accrual and follow-up are on-going and will provide data on tumor control and long-term RN rates.
TMET-08. RADIATION-INDUCED SLC25A22 CONTRIBUTES TO GLIOBLASTOMA RADIORESISTANCE ACQUISITION THROUGH REMODELING GLUTAMATE METABOLISM
Authors: Kim Byeongsoo , Lee Haksoo , Kim Dahye , Park Junhyeong , Park Sujin , Kang JiHoon , Youn HyeSook , Youn BuHyun
Published: November 2024

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.
TMET-11. IMPACT OF METABOLIC RISK FACTORS ON SURVIVAL IN GLIOBLASTOMA IDH-WILD TYPE
Authors: Paul Aboubechara John , Aboud Orwa
Published: November 2024

Altered cellular metabolism is a hallmark of cancer. Systemic metabolic dysregulation has been associated with the pathogenesis of cancer, including glioblastoma. In particular, the metabolic syndrome has been found to increase the risk of developing various systemic cancers, but it is not known how often the metabolic syndrome occurs in patients with glioblastoma or how it affects their clinical outcomes. This study retrospectively investigates the prevalence and impact of metabolic syndrome, and its component risk factors, on survival in patients with glioblastoma IDH-wild type. Interim analysis includes results from 73 patients with anticipation of over 100 patients who received treatment at UC Davis between 2018-2024. Metabolic syndrome prevalence prior to diagnosis was 41% (30/73), higher than the prevalence of 33% in the general population. Patients with metabolic syndrome showed a trend towards reduced overall survival. A significant dose-response association was found between the number of metabolic risk factors and overall survival (p=0.03). Hyperglycemia demonstrated worsened survival (p=0.04), aligning with prior studies. Obesity, hypertension, and dyslipidemia were not significantly associated with survival. These findings highlight the therapeutic potential for targeting systemic metabolic dysregulation, and hyperglycemia in particular, in patients with glioblastoma.
TMET-15. INVESTIGATING THE IL4I1-AHR AXIS IN GLIOBLASTOMA METABOLISM
Authors: Sayeeram Deepak , Holfelder Pauline , Prentzell Mirja T , Immler Lorena , Karabogdan Ivana , Hau Peter , Trump Saskia , Opitz Christiane A
Published: November 2024

Glioblastoma (GB) is the most frequent and malignant form of primary brain tumors in adults. Despite multi-modal therapy consisting of surgery, radio- and chemotherapy, tumors recur and overall survival remains poor. Previously, we identified that besides the classical tryptophan-catabolizing enzyme tryptophan-2,3-dioxygenase (TDO2), the L-amino acid oxidase interleukin-4-induced 1 (IL4I1) is able to activate the transcription factor aryl hydrocarbon receptor (AHR) via production of downstream metabolites. Activation of the AHR in GB may promote tumor progression e.g. by inhibiting anti-tumor immune responses. In this study, we aim to elucidate further the specific effects of the metabolic enzyme IL4I1 and its metabolites on AHR activation in GB. Further, we set out to identify and validate novel AHR target genes downstream of IL4I1. Harnessing the IL4I1-AHR axis might lead to the development of novel therapy strategies helping to overcome resistance in GB in the future.
TMET-28. RECIPROCAL LINKS BETWEEN METHIONINE METABOLISM, DNA REPAIR AND THERAPY RESISTANCE IN GLIOBLASTOMA
Authors: Korimerla Navyateja , Wilder-Romans Kari , Xu Jie , Haq Isra , Zhou Weihua , Kalev Peter , Qi Nathan , Evans Charles , Kachman Maureen , Zhao Zitong , Lin Angelica , Scott Andrew J , O’Brien Alexandra , Kothari Ayesha , Palavalasa Sravya , Peterson Erik , Hyer Marc L , Marjon Katya , Lyssiotis Costas A , Sleger Taryn , Wahl Daniel R
Published: November 2024

Glioblastoma (GBM) is uniformly lethal due to profound treatment resistance. Altered cellular metabolism is a key mediator of GBM treatment resistance. Uptake of the essential sulfur-containing amino acid methionine is drastically elevated in GBMs compared to normal cells, however, it is not known how this methionine is utilized or whether it relates to GBM treatment resistance.Here, we find that radiation acutely increases the levels of methionine-related metabolites in a variety of treatment-resistant GBM models. Stable isotope tracing studies further revealed that radiation acutely activates methionine to S-adenosyl methionine (SAM) conversion through an active signaling event mediated by the kinases of the DNA damage response. In vivo tumor SAM synthesis increases after radiation, while normal brain SAM production remains unchanged, indicating a tumor-specific metabolic alteration to radiation. Pharmacological and dietary strategies to block methionine to SAM conversion slowed DNA damage response and increased cell death following radiation in vitro. Mechanistically, these effects are due to depletion of DNA repair proteins and are reversed by SAM supplementation. These effects are selective to GBMs lacking the methionine salvage enzyme methylthioadenosine phosphorylase. Pharmacological inhibition of SAM synthesis hindered tumor growth in flank and orthotopic in vivo GBM models when combined with radiation. By contrast, methionine depletion does not reduce tumor SAM levels and fails to radiosensitize intracranial models, indicating depleting SAM, as opposed to simply lowering methionine, is critical for hindering tumor growth in intracranial models of GBM. These results highlight a new signaling link between DNA damage and SAM synthesis and define the metabolic fates of methionine in GBM in vivo. Inhibiting radiation-induced SAM synthesis slows DNA repair and augments radiation efficacy in GBM. Using MAT2A inhibitors to deplete SAM may selectively overcome treatment resistance in GBMs with defective methionine salvage while sparing normal brain.
TMIC-16. SPECIFIC TARGETING OF THE MRNA EXPRESSION OF TGFB2 LIGAND TO POTENTIALLY ENHANCE IMMUNE THERAPIES IN LOW-GRADE GLIOMAS
Authors: Qazi Sanjive , Chang Wen-Han , Maida Anthony , Trieu Vuong , Lee Cynthia
Published: November 2024

Low-grade glioma (LGG) tumors are characterized by a low infiltration of immune cells, requiring therapies augmenting the immune response in the tumor microenvironment (TME). The Transforming Growth Factor beta (TGFB) pathway contributes to an immunosuppressive environment. We propose targeting the specific inhibition of Transforming Growth Factor Beta 2, TGFB2 mRNA, as an attractive therapeutic strategy using antisense oligonucleotides. We conducted a bioinformatic-led investigation highlighting the ratio of TGFB2:TGFB1 mRNA expression levels in the cohort of 513 TCGA patients, revealing the TGFB2 ratio as a negative prognostic indicator through utilizing a multivariate Cox proportional hazards model that included the TGFB2 ratio and age at diagnosis as linear covariates to predict overall survival (OS). The model showed a 1.8-fold higher TGFB2 ratio relative to TGFB1 mRNA expression correlated to worse OS. Furthermore, the impact of the TGFB2 ratio was significantly affected by the age at diagnosis, in which older patients exhibited worse OS at increased TGFB2 ratios. Increasing the ratio of TGFB2 relative to TGFB1, TGFB3, TGFBR1, or TGFBR2 in LGG patients led to significant increases in multivariate Cox model determined hazards ratios, HR (HR (95% CI range)) of 1.372 (1.224-1.539), 1.434 (1.288-1.560), 1.327 (1.195-1.473), and 1.381 (1.233-1.547) respectively (P<0.0001 for all comparisons). Correlation of TGFB2:TGFB1/TGFB3/TGFBR1/TGFBR2 ratio across 1330 Reactome pathways using Gene Set Enrichment Analysis showed the most positively correlated genes were members of TME pathways including PD-1 signaling, Syndecan interactions, Non-integrin membrane-ECM interactions, EPH-Ephrin signaling, and Collagen biosynthesis and modifying enzymes. These results suggest that the specific targeting TGFB2 in LGG can remodel the TME for immune therapies to improve OS outcomes.
TMIC-17. TARGETING THE FLUIDIC FORCE-SENSING MECHANISM TO TREAT BRAIN TUMOR METASTASIS
Authors: Huang Xi
Published: November 2024

Biofluid flow generates fluid shear stress (FSS), a mechanical force widely present in tissue microenvironment. How brain tumor growth alters the conduit of biofluid, thereby impacting FSS-regulated cancer progression is unknown. Medulloblastoma (MB) is the most common malignant brain tumor in children. Dissemination of MB cells into the cerebrospinal fluid (CSF) initiates metastasis within the central nervous system. By simulating CSF dynamics based on magnetic resonance imaging of MB patients, we discovered that FSS is elevated at the cervicomedullary junction. Strikingly, we found that MB-relevant FSS promotes metastasis along mouse spinal cords. Mechanistically, FSS induces metastatic cell behaviours, including weakened cell-substrate adhesion, increased motility, cell clustering, and plasma membrane localization of glucose transporter 1 (GLUT1) to enhance glucose uptake. FSS is perceived by mechanosensitive ion channel PIEZO2, which drives actomyosin contractility-dependent GLUT1 recruitment at the plasma membrane. Genetic targeting of PIEZO2 or pharmacologic inhibition of GLUT1 mitigates metastasis. Collectively, these findings define a targetable FSS-activated mechano-metastatic cascade for the treatment of MB metastasis.
TMIC-18. ROLE OF NEUROTRANSMITTERS IN GLIOBLASTOMA PATHOPHYSIOLOGY: EMERGING INSIGHTS
Authors: Irshad Khushboo , Singh Sanjay , Ahmad Khalil Ali , Ban Kechen , Aghoghovwia Benjamin , Mahalingam Rajasekaran , Harris Momo , Alnassiry Sama , Bertrand Renae , Gargapati Aishani , Bui Nathan , Kuo Truc , Lang Frederick , Pan Yuan
Published: November 2024

INTRODUCTION

Glioblastoma (GBM) are the most common primary brain tumor in adults associated with a dismal patient outcome. While it is well-known that glutamatergic neurons can form functional synapses with glioblastoma cells to enhance tumor growth, lesser is known about the effects of activity of other neuronal types on glioblastoma biology, especially among different glioblastoma molecular subtypes. Here, we examined the effects of various neurotransmitters on the growth of glioblastoma cells characterized as classical, proneural and mesenchymal subtypes.

METHODS

Patient-derived glioblastoma cell lines were exposed to varying concentrations of different neurotransmitters and assessed for cell viability using CCK-8 assay. Transcriptomic data from TCGA (The Cancer Genome Atlas) database was analyzed for correlation of expression of neurotransmitter receptors (NTRs) with patient survival. Using IVY-GAP database, we compared the expression of NTRs at leading-edge versus tumor core of tumors to assess their possible involvement in cell migration and microenvironmental interactions.

RESULTS

Consistent with previous findings, we observed an increase in cell viability in all GBM cell lines after being incubated with L-glutamate. The classical subtype has a stronger growth response to L-glutamate compared to the other two subtypes. TCGA data revealed the expression of GRIK4 (glutamate ionotropic receptor kainate type subunit 4) as highest in the classical subtype as well as inversely correlated with worse patient survival in this GBM subtype, as opposed to proneural or mesenchymal patient groups. CCK8 assay also suggested that acetylcholine and dopamine increase glioblastoma cell growth. A subset of cholinergic and glutamatergic receptors (CHRM1, GRM2) was found upregulated in infiltrating/leading-edge regions of glioblastoma tumors compared to the tumor core, suggesting possible involvement in glioblastoma-neuron interactions.

CONCLUSION

Our findings suggest that GBM subtypes may differentially leverage neuro-modulatory mechanisms to support their growth. Future studies will investigate the differential neuronal responses in GBM subtypes using pharmacological/genetic approaches.
TMIC-21. PYK2/MAPK/ERK SIGNALING PATHWAY REGULATES EXTRACELLULAR VESICLE RELEASE IN GLIOBLASTOMA
Authors: Ramires Serrano Neisha , Perez Emir Perez , Kucheryavykh Yuriy , Kucheryavykh Lilia
Published: November 2024

Glioblastoma (GBM), the deadliest brain cancer, typically proves fatal within a year despite treatment. Tumor-associated myeloid cells (TAMs) support GBM growth, treatment resistance, and immune surveillance. Extracellular vesicles (EVs) facilitate communication between tumor and TAMs, exchanging proteins, lipids, RNA, and genetic material. As EVs can travel through the bloodstream to distant tissues, they trigger signaling pathways and metabolic changes in remote organs while also recruiting bone marrow-derived cells and modulating immune responses. Therefore, EVs serve as a crucial mechanism for the distant regulation of the tumor’s immune microenvironment. We hypothesize that Pyk2/MAPK/Erk signaling regulates EV release by modulating the actin cytoskeleton, impacting TAM infiltration and activation in tumors. This study aims to investigate the role of Pyk2/MAPK/Erk in specific EV release in GBM cells, potentially offering insights into therapeutic targets. Using confocal imaging and flow cytometric analysis of EVs, purified from medium conditioned from human primary GBM cell lines with and without Pyk2 CRISPR/Cas9 knock-out (Pyk2KO), the study identified that Pyk2KO cells do not release the population of EVs with diameter bigger then 600nm and up-regulate release of smaller EVs. Additionally, flow cytometric analysis of EVs, released from cells, fluorescently labeled with membrane dye carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), detected 14% of CFSE+ events in EV’s population, purified from Pyk2WT, vs. to 2.5% from Pyk2KO cells. Membrane labeling of cells enables the tracking of microvesicles (MV) shedding and was used to differentiate between MV and ES based on the process of their biogenesis. Considering that larger size and the transition of fluorescence from the plasma membrane are indicative of MVs, our data indicate that Pyk2 is involved in the regulation of MV release. FUNDING: PRSTRT2022 and NIH Grant 1R15CA287203
TMIC-24. BICARBONATE RECEPTOR SLC4A4 SENSITIZES INFILTRATING GLIOBLASTOMA CELLS TO CHANGES IN EXTRACELLULAR PH
Authors: Patel Kunal , Perryman Travis , Prabhu Ashna , Harper Stuart , Dudley Lindsey , Delgadillo Alondra , Gor Aidan , Bhaduri Aparna , Kornblum Harley
Published: November 2024

BACKGROUND

Glioblastoma progression involves changes in extracellular pH (pHe) related to rapid cell turnover and growth. There exist differences in pH between contrast-enhancing solid tumor and non-enhancing infiltrating tumor. SLC4A4 is a two-way bicarbonate transporter used in cortical astrocytes to buffer intracellular pH.

METHODS

In patients undergoing surgery for newly diagnosed IDH wild-type glioblastoma, we obtained tissue from solid tumor and infiltrating tumor. Single-nucleus RNA sequencing (n=15), immunohistochemistry (n=20) were carried out to identify SLC4A4 in infiltrating tumor beyond the contrast enhancing edge. SLC4A4 knock-down models were used to evaluate functional biology of this bicarbonate receptor using our tumor – cortical organoid model of infiltrating glioma and gliomaspheres. pH measurements were recorded using pH sensitive microelectrodes to evaluate changes on pH buffering ability of tumor cells during acid titration assays.

RESULTS

Single-nucleus RNA sequencing (n=15) and spatial RNA sequencing (n=3) showed SLC4A4 to be the highest expressed transmembrane receptor on infiltrating glioblastoma cells but not solid tumor. IHC (n=20) confirmed higher protein expression in infiltrating tumor (p=0.0390). Infiltrating cells were specifically sensitive to decreasing pHe (p=0.0105). In the infiltrating glioma cortical-organoid model, the slc4a4- had increased proliferation (GFP+ cell quantitation; n=10, p=0.0410). There was also increased proliferation in wild-type cells treated with SLC4A4 inhibitor DIDS (n=10, p=0.0016). Immunostaining of these models showed that downmodulation of SLC4A4 was associated with increased Ki67 and decreased caspase staining.

CONCLUSIONS

SLC4A4 plays a role in pH buffering in glioblastoma. We hypothesize that loss of SLC4A4 in solid tumor allows for desensitization of these cells to an acidic extracellular environment and allows for increased tumor growth and density while infiltrating tumor remains sensitivity to changes in pHe. An understand of pH regulation may lead to therapeutic efficacy through pH modulation of tumor.
TMIC-26. IMMUNE CELL DYNAMICS WITHIN GLIOMAS: THE INFLUENCE OF GRADE AND IDH STATUS
Authors: Grewal Eric P , GK Richardson Leland , Sun Jing , Ramapriyan Rishab , Martinez-Lage Maria , Miller Julie J , Carter Bob S , Cahill Daniel P , Curry William T , Choi Bryan D
Published: November 2024

Mutations in isocitrate dehydrogenase (IDH) are known to significantly impact the prognosis and biology of diffuse gliomas. A number of studies have compared the immune microenvironment of IDH-wildtype glioma versus IDH-mutant glioma, and have also characterized the impact of mutant-IDH on lymphocytes. However, few studies have explored how mutant-IDH may affect tumors as they progress and increase in grade, or have delved into the myeloid immune cell compartment. Thus, we sought to understand whether tumor progression affected intratumoral immune cell composition within IDH-mutant glioma, and aimed to compare IDH-mutant versus IDH-wildtype glioma in both humans and mice. Using bulk RNA sequencing, we compared paired samples from patients with IDH-mutant glioma before and after progression, focusing on differentially-expressed immunological genes and relative immune cell proportions. We also compared grade 4 IDH-mutant astrocytoma cases to IDH-wildtype glioblastoma, performing both transcriptomic and cellular analyses. Finally, we engineered murine glioma cells to test the effect of mutant-IDH alone in the tumor microenvironment. We found that multiple immune-related genes distinguish low- and high-grade IDH-mutant glioma, and that high-grade tumors contain greater proportions of suppressive myeloid cells. When comparing IDH-wildtype to IDH-mutant glioma, we found enrichment of suppressive macrophages and myeloid-derived suppressor cells (MDSC) in IDH-wildtype tumors. Finally, our murine model of IDH-wildtype versus IDH-mutant glioma mirrored many of the myeloid cell shifts observed in human tumors. Our data show that progression of IDH-mutant tumors is a significant immunological phenomenon, associated with marked alterations in the tumor immune microenvironment. We also show that IDH-mutant glioma harbors greater inflammatory signatures and fewer suppressive myeloid cells than IDH-wildtype glioma, and that engineering mutant IDH into murine tumors is sufficient to model these differences. These findings improve our understanding of key intratumoral cell populations that may be harnessed by future immunotherapeutic strategies.
TMIC-29. IMMUNE SPECIFIC IMAGING TECHNIQUES IN GLIOBLASTOMA: SELFI MRI IDENTIFIES AGGRESSIVE AND IMMUNOSUPPRESSIVE BIOLOGICAL FEATURES THAT CONTRIBUTE TO THERAPEUTIC RESISTANCE
Authors: Stoller Jared , Schwartz Daniel , Wongsawaeng Doonyaporn , Holland Samantha , Stateler Cooper , Kersch Cymon , Starkey Alicia , Muldoon Leslie , Ambady Prakash , Raslan Ahmed , Dogan Aclan , Sanusi Olabisi , Harrington Christina , Neuwelt Edward , Barajas Ramon
Published: November 2024

Gadolinium (Gd) enhanced MRI is nonspecific for identifying aggressive glioblastoma biology. Glioblastoma-associated macrophages (GAMs) are pivotal in regrowth. Segregation and Extravascular Localization of Ferumoxytol Imaging (SELFI) is a quantitative immune specific MRI technique. SELFI+ values quantify M2 GAMs by phagocytized Ferumoxytol (Fe), while SELFI- values delineate intravascular Fe, cerebral blood volume. The relationship between SELFI and biology driving therapeutic resistance remains unknown. We aimed to determine if SELFI identified immunosuppressive GAMs and biology contributing to poor outcomes, a capability not yet achievable in clinical practice. This prospective study included four patients with glioblastoma. All underwent preoperative Gd and Fe (7 mg/kg up to 510 mg) enhanced MRI allowing for image guided biopsy based on Gd enhancement and FLAIR hyperintensity. RNA was analyzed on GeneChip Clariom Arrays. Intraoperative biopsy location was referenced to MRI for placement of volumes of interest (VOIs). VOIs quantified MRI phenotype and SELFI values. Gene Set Enrichment Analysis (GSEA) assessed differential expression between MRI features. T-test assessed differences in SELFI (p <0.05) and GSEA significance was assessed by normalized enrichment score (NES) and false discovery rate (FDR) q-value. Four patients provided 36 image guided tissue samples with sufficient quality to assess the causal relationships between MRI features and transcriptomics. Stratification by patient MGMT methylation status (N= 3 unmethylated) demonstrated Gd+ regions had significantly elevated SELFI+ values (Mean±SD, Gd+ = 0.24±0.17, Gd- = 0.08±0.09, P<0.01). GSEA demonstrated that tissues with elevated SELFI+ and lowered SELFI- values were comprised of significantly upregulated hypoxia and transforming growth factor-beta (TGFβ) signaling; among others (NES>1.5, FDR q-value<0.05). SELFI MRI uniquely quantifies glioblastoma by localizing sites of biological aggressiveness known to contribute to therapeutic resistance and disease recurrence. Patient enrollment continues to further define the causal relationships between SELFI, GAMs, and glioblastoma biology.
TMIC-31. MOLECULAR PROGNOSTIC FACTORS OF THE PROGRESSION OF VESTIBULAR SCHWANNOMA AFTER PRIMARY TUMOR RESECTION
Authors: Mizuta Ryo , Makino Keigo , Otai Yoshihiro , Fujii Kentaro , Ishida Joji , Uneda Atsuhito , Tsuboi Nobushige , Hirano Shuichiro , Kemmotsu Naoya , Suruga Yasuki , Imoto Ryoji , Kegoya Yasuhito , Hokama Madoka , Ikemachi Ryosuke , Matsumoto Yuji , Tomita Yusuke , Shimazu Yosuke , Inoue Hirofumi , Yan Yuanqing , Wang Ziyi , Ono Mitsuaki , Kurozumi Kazuhiko , Ichikawa Tomotsugu , Yasuhara Takao , Tanaka Shota
Published: November 2024

BACKGROUND

Although vestibular schwannoma is a benign tumor, 10% of the tumor regrow after primary tumor resection. The factors associated with tumor progression from several reports remain controversial, and the role of the tumor microenvironment in vestibular schwannoma is still unclear. In this study, we analyzed the prognostic factors associated with the tumor progression after primary resection.

METHODS

We retrospectively reviewed the medical records of patients who underwent surgical resection and were diagnosed with vestibular schwannoma between January 2005 and June 2020. We performed RNA sequencing to identify the prognostic factors and immunohistochemistry to assess the tumor microenvironment, including analysis of the CD80 and CD206 expression of macrophages. We compared the progressed group whose tumor size got larger than 2mm after primary resection or who underwent additional treatment because of regrowth with the stable group.

RESULTS

Fifty-five patients underwent surgical resection for newly diagnosed vestibular schwannoma, and 12 of them had tumor regrowth. RNA sequencing revealed that the significantly upregulated pathways in the stable group was related to immune systems, while that of the progressed group was related to cell cycle. In addition, there were more myeloid cells, especially M2 macrophage infiltration in the stable group. Immunohistochemistry also showed higher M2 macrophages in the stable group.

CONCLUSION

In this study, multiomic analysis revealed the upregulation of immune-related pathways in the stable group and the cell cycle pathway in the progressed group. Altering the tumor microenvironment may offer a therapeutic strategy in vestibular schwannoma after primary resection.
TMIC-36. EVALUATING PHARMACOLOGICAL INHIBITION OF LSD1 IN MODELS OF DIFFUSE MIDLINE GLIOMA AND EFFECTS ON IMMUNE MICROENVIRONMENT SIGNALING
Authors: Nam Deokhwa , Hernandez Faith , Stitzlein Lea , Becher Oren , Chandra Joya
Published: November 2024

BACKGROUND

Diffuse midline glioma (DMG) is the leading cause of death for pediatric brain tumor patients. DMG is remarkably immunologically senescent, even in comparison to other “immune-cold” tumors like adult glioblastoma. Lysine specific demethylase-1 (LSD1) is a histone demethylase exerting context-specific mechanisms of gene repression and activation and can be inhibited pharmacologically. We found LSD1 regulates the expression of inflammatory cytokines such as Interleukin 18 (IL18) and immune signaling receptors in DMG. Here, we investigated whether IL18 is transcriptionally regulated by LSD1 in vitro and in vivo using clinically relevant small molecule inhibitors.

METHODS

LSD1 binding to the IL-18 promoter was evaluated using chromatin immunoprecipitation. Transcript and protein expression using qRT-PCR and western blotting assessed the impact of LSD1 inhibition on IL18-related pathways and immune signaling pathways. LSD1 inhibitors (IMG-7289 or GSKLSD1) were evaluated patient-derived xenograft models. Results. We found elevated LSD1 binding on the IL18 promoter in DIPG VI cells. Subsequent qRT-PCR and western blot analyses demonstrated increased IL18 protein levels following treatment with GSKLSD1 or IMG-7289 in DIPG VI cells, alongside heightened IL18 mRNA expression compared to DMSO-treated cells. Either GSKLSD1 or IMG-7289 treatment in DIPG IV cells also led to significantly elevated IL18 binding protein mRNA levels. Orthotopic xenografts using DIPG IV bearing mice dosed with LSD1 inhibitors over the course of 35 days several weeks showed a decrease in IL-18R, and an increase in IL-18 BP transcript levels alongside a significant increase in Slamf7 transcript levels. Some evidence of tumor inhibition was seen in male but not female mice treated with GSKLSD1.

CONCLUSIONS

Our investigation revealed IL18 as a direct target of LSD1 specifically in DIPG VI, and gender specific effects of GSKLSD1 treatment in vivo. Future studies will prioritize validating and elucidating the underlying mechanisms the therapeutic potential of LSD1 inhibition in DMG.
TMIC-37. GEMISTOCYTIC TUMOR CELLS AND IMMUNE REACTIVE MICROGLIA PROGRAMMED FOR GLIAL SCARRING CHARACTERIZE T CELL CONTAINMENT IN IDH-MUTANT ASTROCYTOMA
Authors: van Hijfte Levi , Geurts Marjolein , de Heer Iris , Ghisai Santoesha , Balcioglu Hayri , Hoogstrate Youri , Vallentgoed Wies , Head Rania , Westerman Bart , Wesseling Pieter , Joyce Johanna , Debets Reno , French Pim
Published: November 2024

Isocitrate dehydrogenase 1/2 mutant (IDHmt) astrocytoma is considered a T cell-deprived tumor, yet little is known regarding the phenotype that relates to T cell exclusion. To aid better understanding of this mechanism, we systematically characterized the IDHmt astrocytoma immune microenvironment in a large cohort by multiplex immunofluorescence (IF) (n=75), bulk RNA sequencing (n=158), single nucleus RNA sequencing (n=7) and spatial RNA and protein profiling (n=12). We integrated and analyzed all datasets to acquire a robust characterization of the microenvironment organization and validated our results using IF stainings. We demonstrated that in IDHmt astrocytomas, T cells were contained in the perivascular space (i.e., T cell cuffs). In the tumor stroma surrounding T cell cuffs, we found a dense accumulation of gemistocytic tumor cells (GTCs), a histologically distinct tumor cell subtype. GTC-high tumors demonstrated enhanced expression of immune cell migration and activation pathways that were reliably expressed by TAMs and T cells. Notably, we found that GTCs formed a unique transcriptional cell sub-cluster that expressed markers including CD44, TNC and CRYAB, which are specific for glial scarring, a central nervous system specific mechanism for immune exclusion. Surrounding T cell cuffs, GTCs co-localized with immune-reactive microglia that expressed the chemoattractants CCL3 and CCL4. Together, co-localizing GTCs and reactive microglia expressed receptor-ligand pairs that take part in reactive astrogliosis and glial scarring, including Osteopontin – CD44 and IL-1β – IL1R1. Validation whole-slide multiplex IF stainings for this spatial relationship showed specific local accumulation of CD44 positive GTCs and TAMs around T cell cuffs in GTC-high tumors, which was absent in GTC-low tumors. Together, we show that IDHmt astrocytomas organize a network of tumor cells and leukocytes that mimics glial scarring and can aid T cell exclusion. Elucidating such specific immune exclusion mechanisms is vital for more appropriate target selection in novel immune therapies.
TMIC-41. RBBP4/P300 COMPLEX CONTROLS GENES MODULATING ANGIOGENESIS AND IMMUNOSUPPRESSION IN GLIOBLASTOMA MICROENVIRONMENT
Authors: Mapunda Josephine , Htee Hser , Wang Li , Mortazavifarsani Seyedehsahar , Suzuki Yuta , Verma Vivek , Hoeppner Luke , Sarkaria Jann , Kitange Gaspar
Published: November 2024

The RBBP4/p300 complex was recently shown to control genes critical for DNA damage repair and may play a role in glioblastoma (GBM) therapy responsiveness, especially to temozolomide (TMZ). However, the role of this complex in the modulation of the GBM tumor microenvironment (GBM-TME) remains elusive. Because pro-tumorigenic GBM-TME is primarily due to angiogenesis and immune tolerance, we evaluated whether the RBBP4/p300 complex may control these critical processes. To that end, we used shRNA to silence each RBBP4/p300 complex member in GBM43 cells and RNAseq to identify angiogenesis and immune genes regulated by this complex. Our data demonstrated that silencing RBBP4 or p300 suppressed the expression of HIF1α and several other hypoxia response genes, including VEGFA and DDIT4, suggesting that RBBP4/p300 complex may regulate angiogenesis. Consistent with a role in regulating angiogenesis, culture medium conditioned by GBM43 cells expressing the control non-targeting shRNA (shNT) significantly induced angiogenic tube formation by human umbilical vein endothelial cells (HUVEC). In contrast, this effect was blocked by the medium conditioned by the shRBBP4 or shp300 expressing cells. Both shRBBP4 and shp300 expressing cells failed to activate HIF1α accumulation and other signaling in response to hypoxia. Additionally, the control shNT-conditioned medium inhibited IFN-γ production while the medium conditioned by the shRBBP4 or shp300 expressing cells supported IFN-ɣ production from CD8+ T cells. Intriguingly, silencing RBBP4 or p300 also suppressed the expression of CCL2 and PD-L1 (CD274). CCL2 protein is a chemokine that promotes TME infiltration by myeloid-derived suppressor cells (MDSCs), while PD-L1 interacts with PD1 to suppress cytotoxic CD8+ T cell activation. Although this is an ongoing investigation, we conclude that RBBP4/p300 complex mediates the expression of hypoxia response genes, including HIF1α, and immunosuppressive genes, including PD-L1, and this activity may enhance angiogenic and immunosuppressed GBM-TME, making this complex a potential target for GBM therapy.
TMIC-46. TRANSCRIPTIONAL HETEROGENEITY AND MECHANISTIC PATHWAYS OF RECURRENT GLIOBLASTOMA: INSIGHTS FROM A PRE-CLINICAL RECURRENT TUMOR MODEL
Authors: Fayyad Zaman Mohammad , Daou Marc , Phillips Lynette M , Singh Sanjay , Long Lihong , Gumin Joy , Ledbetter Daniel , Lang Frederick F
Published: November 2024

Despite aggressive therapy combining surgical resection, radiation and chemotherapy, glioblastoma (GBM) almost always recurs. Currently, there is no standard treatment for recurrent GBM, and patient inclusion in clinical trials is based on the genetic and molecular profile of the primary tumor even though the effects of chemoradiation on the transcriptional subtype in matched primary and recurrent GBM remain largely understudied. Paralleling the GLASS consortium, we sought to perform a longitudinal assessment of GBM recurrence after therapy based on molecular subtype with the hypothesis that recurrent tumors exhibit a distinct transcriptional landscape different from treatment-naive tumors. To address this, we utilized primary tumor-derived glioma stem cells (GSCs) representing classical and proneural subtypes. We then undertook in vivo intracranial implantation studies and performed standard concomitant treatment that mimics the Stupp protocol. Upon bulk RNAseq data analyses of untreated (n=21) and recurrent tumors (n=17) we identified that while the molecular subtype of recurrent tumors does not change post therapy, their transcriptional profile is more heterogenous compared to untreated tumors for both subtypes. Hierarchical cluster analyses revealed distinct sub-clusters of recurrent tumors. Common mechanisms of recurrence include upregulation of angiogenesis pathways and PI3k/Akt/mTOR signaling pathways. Interestingly, recurrent tumors exhibit upregulation of genes involved in cellular metabolism and membrane transport/ potential. The source of heterogeneity in recurrent tumors in both subtypes appears to be due to the diversity in expression of gene sets involved in hallmark of cancer pathways such as cell cycle, Notch and Wnt signaling pathways and heterogeneity in oncogenic signatures and cell states resembling fetal or neural cells. Further research into the recurrent tumor microenvironment by spatial multi-omics approaches using this pre-clinical recurrent GBM model will elucidate the linkage between cellular as well as molecular heterogeneity and enable identification of targets that can help drive therapeutic decisions for recurrent GBM patients.
TMIC-62. INVESTIGATING THE ROLE OF OSTEOPONTIN IN RADIORESISTANCE OF H3.3-G34R PEDIATRIC HIGH-GRADE GLIOMA
Authors: Mukherji Devarshi , Haase Santiago , Varela Maria Luisa , Welch Joshua , Lowenstein Pedro , Castro Maria
Published: November 2024

Pediatric high-grade gliomas (pHGGs) are currently the leading cause of cancer-related deaths in children. Around 15% of pHGGs harbor a glycine to arginine (G34R) mutation in histone H3.3. Gliomas in general, including H3.3-G34R gliomas, contain cell populations that are resistant to chemotherapy and radiation therapy. These resistant cell populations are responsible for tumor recurrence seen in over 90% of patients within two years after treatment. Uncovering molecular mechanisms responsible for radiation resistance is essential for the development of new targeted therapies against glioma. In our lab’s H3.3-G34R mouse model, we have observed mice that are both radioresistant and radiosensitive. In order to investigate potential mechanisms of resistance leading to this phenotype, we performed single-cell RNA sequencing (scRNA-seq) on 3 non-treated (NT) glioma-bearing mice, 3 mice resistant to treatment (R), and 3 mice susceptible to treatment (S). The scRNA-seq data was processed and filtered using FastQC, Cellranger, CellBender, and DoubletFinder before downstream analysis. Ligand-receptor interaction analysis using CellChat in conjunction with differential expression analysis demonstrated differential expression and secretion of a cytokine known as Secreted Phosphoprotein 1 (SPP1)/Osteopontin (OPN) from tumor-associated microglia and macrophages. Current literature on SPP1 has suggested that its expression may play a role in tumor aggressiveness. However, there is conflicting evidence regarding its mechanism of action when secreted from tumor-associated microglia and macrophages. Additionally, its role in radioresistance has yet to be fully explored. We will present data utilizing in vitro and in vivo experimentation to elucidate the role that differential expression of SPP1 may play in the radioresistance of our H3.3-G34R mouse model. Supported by grants from NIH-NINDS, the Pediatric Brain Tumor Foundation, and Ian’s Friends Foundation
TMIC-63. DUAL IMMUNE CHECKPOINT BLOCKADE IN A NOVEL EGFR-DRIVEN MURINE GLIOMA MODEL INCREASES PERIPHERAL T CELL PROLIFERATION BUT FAILS TO PROLONG SURVIVAL
Authors: Li Marissa , Pioso Marissa , Breunig Joshua , Grausam Katie , Nathanson David , Prins Robert
Published: November 2024

Patients with glioblastoma (GBM) survive only 15-18 months following standard-of-care surgical resection and radio-chemotherapy. Immune checkpoint blockade (ICB), effective in extracranial cancers, failed to improve survival in patients with recurrent GBM in a multi-center phase III clinical trial. As such, we are interested in the molecular mechanisms that drive ICB resistance. Oncogenic EGFR activation is observed in ~50% of GBMs and is associated with immunosuppression; however, whether EGFR signaling impairs T cell function in GBM resulting in limited ICB efficacy remains unclear. Therefore, we leveraged the novel MADR-mEGFRvIII glioma model, which expresses murine EGFRvIII under a tetracycline-off system, to investigate the relationship between EGFR activation and responsiveness to dual anti-PD-1 and anti-CTLA-4 (10mg/kg) in vivo. Mice were bled prior to enrollment and weekly thereafter, which confirmed anti-PD-1-PD-1 binding via competitive flow cytometric assay. Following two weeks on treatment, animals were euthanized for immunophenotypic analyses of tumors and tumor-draining lymph nodes. These analyses revealed a significantly increased prevalence of peripheral CD11b+ myeloid cells and enhanced peripheral T cell proliferation, indicated by Ki67 positivity, after treatment with dual ICB compared to isotype control. However, these shifts did not translate to effective ICB-mediated recruitment of peripheral T cells to the tumor microenvironment and no significant survival benefit was observed (p=0.68, n=15/treatment arm). Given our previous findings that EGFR ablation via doxycycline enhanced intratumoral T cell infiltration, we hypothesize the rational combination of EGFR-targeted therapy and ICB will have a synergistic effect; thus, future directions include a study combining the two treatment regimens consisting of a lead-in with doxycycline to increase intratumoral T cell trafficking, followed by treatment with dual ICB. This work will provide insight into the interplay between oncogenic signaling and immunosuppression, contributing to the development of novel combination therapies in treating this lethal disease.
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
Authors: Suarez Meade Paola , Whitehead Rachel , Clemenceau Jean , Norton Emily , Kulkarni Tanmay , Hong Changjin , Teeter Caroline , Aramburu Manuela , Chaichana Kaisorn , Rosenfeld Steven S , Kenchappa Rajappa , Guerrero-Cazares Hugo , Schiapparelli Paula V , Hwang Tae H , Konstantopoulos Konstantinos , Quinones-Hinojosa Alfredo
Published: November 2024

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.
TMIC-70. CORRELATION OF LLT-1 AND NLRC4 INFLAMMASOME AND ITS EFFECT ON GLIOBLASTOMA PROGNOSIS
Authors: Park JeongMan , Kim Yu Jin , Kim Dongkil , Sim JeongMin , bog Cho Kwang , cho Kyunggi , Moon Ju Hyung , Sung Kyoung Su , Lee Dong Hyun , Lim Jaejoon
Published: November 2024

PURPOSE

LLT-1 is a well-known ligand for the natural killer (NK) cell inhibitory receptor NKRP1A. Here, we examined NLRC4 inflammasome components and LLT-1 expression in glioblastoma (GBM) tissues to elucidate potential associations and interactions between these factors.

METHODS

Nine GBM tissues were collected for experiments and RNA sequencing, while fifty-two tumor core tissues were collected for RNA sequencing. Expression of LLT-1 and other proteins was assessed by immunofluorescence. Computational analyses utilized RNA-seq data from 296 and 52 patients from the Chinese Glioma Genome Atlas and CHA medical records, respectively, using survival, non-negative matrix factorization clustering, Gene Ontology enrichment, and protein-protein interaction analyses. Receptor-ligand interactions between tumor and immune cells were confirmed by single cell RNA sequencing analysis.

RESULTS

In GBM tissues, LLT-1 was predominantly colocalized with Glial fibrillary acidic protein (GFAP) -expressing astrocytes, but not with microglial markers like Iba-1. Additionally, LLT-1 and activated NLRC4 inflammasomes were co-expressed mainly in intratumoral astrocytes, suggesting an association between LLT-1, NLRC4, and glioma malignancy. High LLT-1 expression correlates with poor prognosis, particularly in the mesenchymal subtype, and is associated with TNF and NOD-like receptor signaling pathway enrichment, indicating a potential role in tumor inflammation and progression. At the single-cell level, mesenchymal-like malignant cells were highly enriched in the TNF, NLR, and IL-1 signaling pathways compared to other malignant cell types.

CONCLUSION

We revealed an association between NLRC4 inflammasome activity and LLT-1 expression, suggesting a novel regulatory pathway involving TNF, inflammasomes, and IL-1, potentially offering new anti-glioma approaches by enhancing NK cell-based treatments.