bluuebunny/crossref_metadata_2025 huggingface dataset:
TMET-28. A METHOD FOR EX VIVO STABLE ISOTOPE TRACING IN SURGICALLY EXPLANTED GLIOMA ORGANOIDS
Authors: El Shami Mohamad , Savani Milan , Gattie Lauren , Hicks William , Richardson Timothy , McBrayer Samuel , Abdullah Kalil
Published: November 2022

Stable isotope tracing is a powerful method for elucidating tumor metabolism. While in vivo stable isotope tracing is one of the most faithful approaches for studying metabolic activity under pathophysiologically relevant conditions, this method is technically challenging and costly to perform. We therefore sought to optimize an ex vivo stable isotope tracing approach under conditions that recapitulate the in vivo tumor microenvironment (TME), focusing on three key features: cellular heterogeneity, nutrient availability, and oxygenation. Surgically eXplanted Organoids (SXOs) are 3-dimensional glioma models that retain the diverse cell populations in human brain tumors. We tested whether glioma SXOs can be cultured in Human Plasma-Like Medium (HPLM) under brain-relevant oxygen levels (5%) for the purpose of stable isotope tracing. We cultured SXOs in conventional media, HPLM for 24 hours, or HPLM for 120 hours under 5% oxygen. We observed no significant differences in markers of tumor architecture, cellular proliferation rates, or stemness profiles. We then performed stable isotope tracing in SXOs cultured in HLPM at 5% oxygen. We acclimated SXOs to HPLM for 24 hours or 120 hours before replacing media with either HPLM (unlabeled) or HPLM with 15N2 glutamine (labeled). 15N isotopic labeling patterns in various metabolites were compared between the 24- and 120-hour conditions by linear regression. These patterns were strongly concordant over time (r2 = 0.9544), indicating that steady-state metabolic fluxes are stable in these cultures. We then analyzed intermediates that occupy key nodes in amino acid metabolism, redox homeostasis, and nucleotide synthesis pathways and found similarly strong concordance between labeling patterns at the individual metabolite level. Our findings outline an effective approach for conducting ex vivo stable isotope tracing in heterocellular glioma models under conditions that mimic the TME. This approach may complement and enhance analogous in vivo methods to provide new insights into glioma metabolism.
TMET-31. CHOLESTEROL ESTERIFICATION IN A PATIENT WITH DIFFUSE ASTROCYTOMA PROGRESSING TO GLIOBLASTOMA
Authors: Ijare Omkar , Baskin David , Pichumani Kumar
Published: November 2022

Metabolic profiles of diffuse astrocytoma and recurrent tumor tissues were compared in a patient with diffuse astrocytoma who showed tumor recurrence and progressed to glioblastoma (GBM). The patient presented with a diffuse infiltrating tumor and was operated in April 2018. The pathology report showed that the tumor was a diffusely infiltrating astrocytoma with Ki-67 of 5% (WHO Grade II). In addition, the molecular testing results indicated that the tumor was IDH1wt with TERTp mutation and showed an indeterminate MGMT promoter status. Given diffuse brain involvement and IDH1wt status, an aggressive behavior was suggested for this tumor. After ~3.5 years (January 2022), the MRI examination showed tumor recurrence and the patient underwent craniotomy for the resection of the recurrent tumor. The pathology report showed a high-grade glial neoplasm composed of ovoid tumor cells with nuclear pleomorphism (GBM, WHO Grade IV). We collected the tumor specimens from both surgeries and compared the metabolic profiles using 1H NMR spectroscopy. The tumor specimens were extracted using methanol-chloroform (2:1 ratio) extraction-method. The methanol-layer of the recurrent tumor showed decreased levels of N-acetylaspartate (NAA) and myo-inositol, and the elevated levels of phosphocholine (PC) and glycine, compared to the diffuse astrocytoma. Decreased NAA and elevated PC and glycine are metabolic features of high-grade gliomas, suggesting progression of the low-grade astrocytoma to GBM. The chloroform-layers of both tumors showed the presence of cholesterol; however, cholesteryl ester (CE) was detected only in the recurrent tumor (GBM). The ratio of CE to cholesterol was 0.44, suggesting that 44% of cholesterol pool was esterified in GBM. Cholesterol esterification is dysregulated in GBM and targeting this metabolic pathway using AcylCoA:cholesterol acyltransferase (ACAT) inhibitors would be promising in the treatment of GBM and astrocytomas with TERTp mutation which transform to GBM.
TMET-33. ELONGATION CONTROL OF MRNA TRANSLATION SUPPORTS GROUP 3 MEDULLOBLASTOMA ADAPTATION TO NUTRIENT DEPRIVATION
Authors: Delaidelli Alberto , Yao Betty , Negri Gian Luca , Huang Yue Zhou , Huang Albert , Leprivier Gabriel , Sorensen Poul
Published: November 2022

Group 3 affiliation and MYC genetic amplification are associated with poor life expectancy and substantial morbidity in children suffering from medulloblastoma (MB). However, the high metabolic demand induced by MYC-driven transformation sensitizes MYC-overexpressing MB to cell death under conditions of nutrient deprivation (ND). Additionally, MYC-driven transformation is known to promote mitochondrial oxidative phosphorylation (OXPHOS). We previously reported that eukaryotic Elongation Factor Kinase 2 (eEF2K), the master regulator of mRNA translation elongation, promotes survival of MYC-overexpressing tumors under ND. Interestingly, eEF2K is overexpressed in MYC-driven MB and our preliminary proteomics data highlight large-scale alterations in OXPHOS components affecting eEF2K deficient MB cells. We therefore hypothesized that eEF2K activity is required for the selective translation of mRNAs needed for efficient OXPHOS, and for the progression of MYC-driven MB. We performed Multiplexed enhanced Protein Dynamic Mass Spectrometry in eEF2K knockdown MYC-overexpressing D425 MB cells to identify mRNAs selectively translated upon eEF2K activation. Messenger RNAs encoding multiple (9 out of 10 detected) components of the mitochondrial OXPHOS pathway are selectively translated upon eEF2K activation. Inactivation of eEF2K by genetic KO leads to the disassembly of electron transport chain (ETC) complexes I-IV without affecting mRNA levels of their respective components. Consistently, eEF2K KO MB cells display decreased mitochondrial membrane potential and 20% increased proton leak thorough the mitochondrial membrane. In addition, eEF2K inactivation results in increased Group 3 MB cell death under ND and doubles survival of MB bearing mice fed with calorie restricted diets (p< 0.05). Control of mRNA translation elongation by eEF2K is critical for mitochondrial ETC complex assembly and efficient OXPHOS in MYC-overexpressing MB, likely representing an adaptive response by which MYC-driven MB cells cope with acute metabolic stress. Future therapeutic studies will aim to combine eEF2K inhibition with caloric restriction mimetic drugs as eEF2K activity appears critical under metabolic stress conditions.
TMET-34. RADIATION METABOLOMICS IN PRIMARY HUMAN MENINGIOMA AND SCHWANNOMA: EARLY EXPERIENCE AND INITIAL RESULTS
Authors: Dougherty Mark , Taylor Eric , Hansen Marlan
Published: November 2022

Introduction

Meningiomas and schwannomas account for 45% of primary CNS tumors. Yet when surgery and radiation fail, no further treatments exist. Metabolomics has been used to discover new cancer therapies; however, to date few have used metabolomics to study meningiomas and schwannomas. Here we present initial results and lessons learned from this novel endeavor.

METHODS

Primary tumors were obtained from patients during surgery and immediately taken for culturing or xenograft implantation. Upon reaching >90% confluence, cultures were treated with 0gy, 3gy, 10gy, or 20gy gamma radiation, then flash frozen 6 or 72 hours post-treatment. Xenograft tumors were implanted in nude mice. MRI 4 weeks post-implantation confirmed tumor viability. Mice were then given 10gy, 20gy, or sham radiation treatment. Xenografts were harvested 72 hours post-treatment. Metabolites were measured with a ThermoISQ gas chromatography-mass spectrometer.

RESULTS

Eleven meningiomas and nine schwannomas were successfully cultured. Unsupervised hierarchical clustering of cultures demonstrated greater influence from tumor of origin than from radiation. Univariate analysis of schwannoma xenografts demonstrated elevated ornithine following radiation (fold change 1.62; P = 0.008). However, principal component analysis did not show significant between-group differentiation. Orthotopic meningioma xenografts did not produce sufficient tissue for metabolomics; however, subsequent subcutaneous implants have been successful (data forthcoming).

CONCLUSION

Standard cell cultures did not reveal significant metabolic changes following radiation; it is unclear whether this was due to culture technique or inter-tumor heterogeneity. In radiated schwannoma xenografts, elevated ornithine may implicate related pathways such as ornithine decarboxylase-mediated polyamide synthesis for DNA double-strand break repair. Compared to other ‘-omics’ studies, metabolomics requires more tissue per sample ( >10mg) and is more sensitive to environmental conditions. Thus, large sample sizes are needed to detect significant changes, and xenografts are likely superior to cell culture. Future plans include increased xenograft sample size and stable isotope tracing for pathway analysis.
TMET-36. ACID CERAMIDASE INHIBITION EXPLOITS SPHINGOLIPID VULNERABILITIES IN IDH MUTANT GLIOMAS
Authors: Zaibaq Faris , Dowdy Tyrone , Larion Mioara
Published: November 2022

The presence of the IDH mutation in gliomas is a major classifier of brain tumor subtypes and has several important implications for cancer growth. Our recent work uncovered that IDH-mutant tumors are susceptible to increased apoptosis via alterations of the sphingolipid pathway due to their excess production of pro-apoptotic ceramides over pro-proliferative sphingosine 1-phosphate (S1P). To that end, we proposed that this rheostat can be modulated to induce cell death in IDHmut tumors by targeting acid ceramidase, a critical sphingolipid enzyme in gliomas. We hypothesize that pharmacological inhibition of acid ceramidase will increase ceramide levels and therefore induce apoptosis in IDHmutgliomas. Using a preliminary drug screen, we have identified a group of haloacetate C2-ceramide derivatives known as SOBRACs that potently inhibit acid ceramidase. We selected five candidate compounds from this family and assessed the effectiveness of each drug in 3 I IDHmut (BT142, TS603, & U251mut) and 3 IDHmut (GSC923, GSC827, U251wt) patient-derived glioma cell lines, as well as non-immortalized normal human astrocytes, using the CCK8 cell viability assay. Our results indicate that the SOBRAC drugs are nearly 10 times more potent in IDH-mutant tumors compared to IDHmut cell lines. Additionally, the SOBRAC drugs are more effective than other known acid ceramidase inhibitors, making them attractive as potential novel therapeutics. To date, azide-SOBRAC is the most potent drug in the family, with EC50 value of 300 nM in BT142 cells (IDHmutmut
TMET-39. INHIBITION OF THE TCA-CYCLE IS SYNTHETICALLY LETHAL WITH LOSS OF FUNCTION OF BCL-XL IN GLIOBLASTOMA
Authors: Nguyen Trang , Shang Enyuan , Torrini Consuelo , Shu Chang , Westhoff Mike-Andrew , Karpel-Massler Georg , Siegelin Markus
Published: November 2022

Glioblastoma (GBM) remains an incurable disease, requiring more effective therapies. Through CRISPR and RNAi library screening interrogation we identified several TCA-cycle enzymes as essential for GBM growth. By combining a transcriptome and metabolite screening analyses we discovered that inhibition of the TCA-cycle by the clinically validated drug compound, CPI-613, is synthetically lethal with Bcl-xL loss of function (genetically and through the clinically validated BH3-mimetic, ABT263) in patient-derived xenograft as well neurosphere GBM cultures. Carbon tracing experiments (U-13 C-glucose and U-13 C-glutamine) and extracellular flux analysis showed that CPI-613 interferes with mitochondrial respiration. In turn, CPI-613 mediated energy deprivation drives an integrated stress response with an up-regulation of the BH3-only domain protein, Noxa in an ATF4 dependent manner as demonstrated by genetic loss of function experiments. Consistently, silencing of Noxa rescued from cell death induced by CPI-613 as well as by the combination treatment of ABT263 and CPI-613 in model systems of GBM. In patient-derived xenograft models of GBM in mice, the combination treatment of ABT263 and CPI613 suppressed tumor growth more potently than each compound on its own. Therefore, combined inhibition of Bcl-xL along with interference of the TCA-cycle might be a novel treatment strategy for GBM.
TMIC-02. PRE-SURGICAL IMMUNE CHECKPOINT BLOCKADE RESHAPES THE IMMUNE LANDSCAPE AND SPATIAL ARCHITECTURE OF METASTATIC BRAIN TUMORS
Authors: Sun Lu , Kienzler Jenny , Reynoso Jeremy , Lee Alexander , Owens Geoffrey , Everson Richard G , Nathanson David , Cloughesy Timothy , Liau Linda M , Hugo Willy , Kim Won , Prins Robert
Published: November 2022

Brain tumors reside in a modified microenvironment where inflammatory processes are heavily regulated. Nevertheless, our recent study showed a significant immune infiltration in recurrent glioblastoma (rGBM) treated with neoadjuvant PD-1 checkpoint blockade therapy. In comparison to GBM, the intracranial response of brain metastases (BrM) to immunotherapy has been less well studied. To test this question, we investigated the effect of pre-surgical immune checkpoint blockade (ICB) on the immune compartments of BrM, using multiplex immunofluorescence, single-cell RNA sequencing and spatial transcriptomics on a total of 17 patients. We found that ICB naïve BrM featured an immune exclusion phenotype with accumulation of immune cells in the fibrovascular stroma, necrotic and hemorrhagic peritumoral areas. Following ICB treatment, T cells and monocyte-derived macrophages were then observed within the tumor parenchyma. Computational receptor-ligand analysis suggested that the tumor-infiltrating T cells and macrophages were highly engaged by checkpoint receptor-ligand pairing, which may drive the T cells into an advanced exhausted state. To further explore the organization and interactions between the immune, stroma and tumor cells in BrM, we utilized the sequencing-based spatial transcriptomics technology. Our spatial transcriptomics data further highlighted the distinct spatial architectures between ICB naïve and –treated BrM tumors. It also pinpointed a unique macrophage subset that are preferentially co-localized with the brain vasculature cells and confined the immune infiltrates to the perivascular space. In summary, our study revealed ICB-induced marked changes in the immune composition and spatial organization in BrM and offers insights into new combination therapies that can help improve the clinical efficacy of immunotherapy for BrM patients.
CNSC-24. THE EPILEPTIC LANDSCAPE OF IDH MUTANT GLIOMAS
Authors: Drumm Michael , Wang Wenxia , Burdett Kirsten B , Sears Thomas , Javier Rodrigo , Cotton Kristen , Webb Brynna , Stolz Jacob , Byrne Kayla , Unruh Dusten , Walshon Jordain , Steffens Alicia , McCortney Kathleen , Bushara Omar , Weston Alyssa , He Suning , Lukas Rimas , Finan John , Franz Colin , Kurz Jonathan , Templer Jessica , Swanson Geoff , Horbinski Craig
Published: November 2022

Tumor-associated epilepsy (TAE) is a frequent complication of diffusely infiltrative gliomas, and impairs quality-of-life. We previously showed that isocitrate dehydrogenase 1 mutant (IDHmut) gliomas were associated with preoperative seizures, and that D2HG increased synchronized bursts in cultured neurons (PMID: 28404805). But the mechanism whereby this occurs, whether IDHmut inhibitors can block this, and whether seizure risk varies by IDHmut status post-operatively, are unknown. We discovered that exogenous 3 mM D2HG had a 150% greater effect on the firing rate of cultured mouse cortical neurons when nonneoplastic glia cells were present versus when they were absent (P=0.002). Coculture with patient-derived IDHmut glioma cells increased the firing rate of human cortical neuron/astrocyte spheroids by up to 272%, and the IDHmut inhibitor AG881 reduced the excitatory effect of IDHmut glioma cells on spheroids by 79% (P=0.0008). Using a novel in vivo model of TAE, wherein EEG recordings were taken of immunocompetent mice engrafted with isogenic mouse glioma lines (NRAS/ATRX/TP53mut ± IDHmut), we found that IDHmut gliomas produced 7.6-fold more epileptiform spikes than IDHwt gliomas (P=0.004). RNA-Seq analysis of the peritumoral mouse brain tissue showed that this increase in spikes was associated with significantly increased expression of key genes known to be upregulated in epilepsy, including SLC12A5, THSB1, VEGFA, FOSL2, and SYNPO. Treatment with 5 mg/kg AG881 by daily oral gavage reduced those spikes in IDHmut-engrafted mice by 51% within three days (P=0.027), whereas vehicle control had no effect (P=0.33). Among 247 patients with grade 2–4 adult-type diffuse gliomas, multivariable time-to-event analysis showed that post-operative seizure risk was positively associated with pre-operative seizures, subtotal resection, and IDHmut astrocytoma. Together, these data show that (i) the D2HG product of IDHmut gliomas increases neuronal excitation in a glial-dependent manner; (ii) IDHmut also affects post-operative seizure risk; (iii) IDHmut inhibitors may improve TAE control.
TMIC-08. FRIEND AND FOE: RADIATION THERAPY MODULATES GLIOBLASTOMA IMMUNE EVASION VIA EXTRACELLULAR VESICLES
Authors: Schweiger Markus , Amoozgar Zohreh , Tannous Bakhos
Published: November 2022

Glioblastoma (GBM) is the most common form of malignant primary brain tumor and despite optimal treatment, long-term survival remains incredibly rare. Radiation therapy (RT) leads to successful initial tumor regression but recurrence is inevitable. Previous studies have shown that ionizing radiation changes the composition of the tumor microenvironment and alters the expression of immune-related markers on tumor cells. Extracellular vesicles (EVs) are vesicular bodies of cellular origin and secreted by nearly every cell. They have been shown to carry a variety of cargo (DNA, proteins, RNAs, lipids) which can be taken up by other cells. Notably, previous studies highlight that EVs drive GBM progression and immune evasion by acting as multifunctional signaling complexes. Here we show that ionizing radiation of GBM cells results in an altered EV secretome and further facilitates the uptake of EVs in recipient cells. In addition, EVs secreted by GBM cells following radiation modulate the tumor microenvironment by manipulating the innate as well as the adaptive immune response. Macrophages exposed to EVs from irradiated GBM cells switched to the tumor supportive M2 phenotype and exhibited decreased phagocytotic ability.We dissected a novel mechanism by which GBM evades the immune system with the help of EVs following RT, pointing towards novel therapeutic strategies to prevent GB recurrence.
TMIC-14. DIFFUSE P16INK4A EXPRESSION IS ASSOCIATED WITH TUMOR CELL SENESCENCE AND SENESCENCE-ASSOCIATED SECRETARY PHENOTYPE RELATED TO IMMUNE MICROENVIRONMENT IN GLIOBLASTOMAS
Authors: Roh Tae Hoon , Kim Young Hwa , Park So Hyun , Eom Soyeong , Park Tae Jun , Kim Jang-Hee , Kim Se-Hyuk
Published: November 2022

Cellular senescence (CS) is a state of irreversible cell cycle arrest, and the expression of p16INK4a in cells is one of the reliable markers for CS. However, senescent cells are metabolically active with the senescence-associated secretory phenotype (SASP), which can influence the tissue microenvironment by paracrine signaling to the adjacent tumor, non-tumor, and immune cells. In the present study, we evaluated p16INK4a expression in glioblastoma and investigated its association with CS and SASPs. We analyzed the expression of p16INK4a in 73 glioblastomas by immunostaining. To examine the association of p16INK4a expression and CS, we performed senescence-associated β-galactosidase (SA-β-Gal) staining, a standard marker of CS, using glioblastoma cell lines and fresh frozen tumor tissues. For SASPs analysis, RNA sequencing with Gene Set Enrichment Analysis (GSEA) was performed. Among 73 glioblastomas, twenty-eight cases (38.4%) revealed diffuse strong p16INK4a expression in tumor cells. The glioblastoma with diffuse p16INK4a expression (GMDP) patients were younger (52.4 vs. 59.2 years) and showed prolonged overall survival (median: 25.5 vs. 12.3 months) compared to those harboring negative expression. In vitro analysis, p16INK4a over-expressed glioblastoma cell line showed increased expression of SA-β-Gal which indicates CS. In addition, fresh frozen tissues from GMDP also revealed SA-β-Gal positivity. RNA sequencing analysis revealed that splicing or protein biosynthetic genes were enriched in GMDP, and GSEA showed significant enrichment of SASP genes (false discovery rate < 0.05), especially chemokines associated with monocytes and macrophages. Our data suggest that increased expression of p16INK4a in glioblastoma is associated with tumor cell senescence, and SASPs from senescent tumor cells could be one of the crucial modulators in the tumor immune microenvironment.
CNSC-25. METABOLISM ACROSS BRIDGE OF SIGHS TO ONCOHISTONES
Authors: S.Senga Sasi
Published: November 2022

Brain tumours continue to be one of the leading causes of childhood mortality related to cancers. Oncohistones such as H3K27M, and H3G34R mutations have been linked to childhood brain tumours. The reason for the incidence of Oncohistone mutation in a developmental setting provides some novel insight for better therapeutics. The maintenance of the progenitor state might be driven by metabolism dictated Oncohistone mutations at the expense of differentiation in the developing brain paving way for tumourigenesis. I propose methods to target metabolic disturbances in the developing brain even prior to the rise of such somatic oncohistone mutations. Additionally, I provide insight into the reason for selective co-mutations that occur with specific Oncohistones in childhood brain tumours providing a rationale for effective therapeutics. In 2021, I proposed neuronal signalling as an Hallmark of cancer, in the context of childhood brain tumours, the nervous system functions as a fulcrum between metabolic reprogramming and the rise of oncohistone mutations leading to epigenetic reprogramming. The nervous system is a lucrative target in inhibiting the metabolic cues that support oncohistones. Metabolic reprogramming may underpin epigenetic reprogramming driving the developing brain across the Bridge of Sighs towards tumourigenesis.
TMIC-16. INTRATUMORAL THROMBOSIS INITIATES HYPOXIA AND NECROSIS TO DRIVE TUMOR PROGRESSION THROUGH DRAMATIC TUMOR MICROENVIRONMENTAL ALTERATION
Authors: Markwell Steven , Olson Cheryl , Li Jiabo , Shih Ling-Kai , Ross James , Brat Daniel
Published: November 2022

All GBM molecular subsets share the common trait of accelerated progression following necrosis which cannot be adequately explained by cellular proliferation arising from accumulated genetic alterations. We suggest that development of necrosis is much more than a passive phenomenon related to rapid growth but rather is a driving force behind TME restructuring responsible for sustaining accelerated expansion. Current tumor models fail to adequately mimic the magnitude of post-necrotic restructuring within the microenvironment and remain overly reliant on post-mortem analyses, obligating researchers to extrapolate causal relationships between necrosis and progression phenomena during tumor evolution. In GBM (WHO grade 4), the most malignant primary brain tumor, vascular pathology and central necrosis precede rapid, radial expansion. Mechanisms enabling selective fitness within a hypoxic/anoxic GBM setting remain poorly understood. Nanostring GeoMX spatial profiling demonstrates M1-like TAM enrichment in non-necrotic human samples and M2-like TAMs in perinecrotic regions. Our immunocompetent RCAS/tv-a model aptly captures events seen in human gliomas, exposing dynamic temporal and spatial changes that facilitate GBM progression, incorporating unique microenvironmental stressors typically absent from GBM animal models, specifically emerging central necrosis. Simultaneously, our in vitro models scrutinize how hypoxia-dependent signaling between GBM cells, microglia and monocytes alter TAM accumulation and function in the TME. TAMs increase dramatically with the onset of necrosis, with a preferential localization to the hypoxic zone in the perinecrotic niche, which supports their survival. Flow cytometry on digested pre- and post-necrotic GBMs, showed increased TAM accumulation at 6 weeks vs. 2 weeks (necrosis emerges in week 4-5). Our in vivo data along with in silico analysis of Ivy GAP and TCGA datasets suggests GBM cells within the peri-necrotic niche attract and polarize TAMs through MIF secretion, necrotic DAMP (adenosine, S100B) release, and arginase secretion to suppress T cells.
CNSC-26. NETWORK SIGNATURES OF SURVIVAL IN BRAIN TUMOUR GENETICS
Authors: Ruffle James , Mohinta Samia , Pombo Guilherme , Gray Robert , Kopanitsa Valeriya , Lee Faith , Brandner Sebastian , Hyare Harpreet , Nachev Parashkev
Published: November 2022

Tumor heterogeneity is increasingly recognized as a major obstacle to therapeutic innovation across neuro-oncology. Gliomas typically exhibit a spectrum of possible genetic mutations, reflecting multiple, potentially complex interactions across distinct molecular pathways. Predicting disease evolution and prescribing individually optimal treatment plausibly requires statistical models complex enough to capture the intricate genetic structure underpinning oncogenesis. Here we formalize this task as the discovery of distinct patterns of connectivity within network representations of the interactions between genetic loci. Evaluating multi-institutional clinical, genetic, and outcome data from 7847 consecutive patients over a 14-year period, we employ Bayesian stochastic block modelling to reveal a hierarchical network structure of tumor genetic interactions spanning (molecularly confirmed) IDH-wildtype glioblastoma, IDH-mutant oligodendroglioma and astrocytoma. We show that this structure not only identifies the patients’ diagnosis, but predicts individual survival with greater fidelity (Cox’s proportional hazard cross-validated (CPH-CV) concordance = 0.78; Bayesian 12, 24 and 36-month survival models = R2 0.31, 0.40 and 0.46, respectively) than the latest WHO tumor classification (CPH-CV concordance = 0.77; Bayesian 12, 24 and 36-month survival models = R2 0.26, 0.36 and 0.39, respectively) or linear models of genetic features (CPH-CV concordance = 0.76; Bayesian 12, 24 and 36-month survival models = R2 0.26, 0.35 and 0.37, respectively). For example, a diagnosis of IDH-wildtype glioblastoma yielded median survivals varying from 289 days in patients with the signature of marked EGFR amplification, MGMT methylation, and histone mutations, to 425 days in those with the signature of TERT mutations, histone and EGFR wild-types, and variable MGMT methylation. These findings suggest network-based analysis can reveal distinct signatures of survival with better prognostic fidelity than current gold standard diagnostic categories.
TMIC-30. EFFICIENT, QUANTITATIVE MAPPING OF TUMOR-IMMUNE NEIGHBORHOOD COMPOSITION IN GLIOBLASTOMA USING CASSATT
Authors: Brockman Asa , Khurana Rohit , Bartkowiak Todd , Hayes Madeline , Mobley Bret , Irish Jonathan , Ihrie Rebecca
Published: November 2022

The microenvironment surrounding brain cancer cells, and their position relative to anatomical features such as the vasculature, is a critical contributor to tumor malignancy. Immunohistochemical staining is widely used to examine such multicellular interactions, and approaches like cyclic immunohistochemistry (cycIHC), multiplexed immunofluorescence, or mass-based imaging can include up to 100 channels of interest. However, large (often > 50 GB) datasets from cycIHC or comparable approaches currently require an expert data scientist to concatenate open-source tools for each step of image pre-processing, registration, and segmentation, or the use of expensive proprietary software. We generated an open-source, unified, and user-friendly workflow for processing and analyzing cycIHC data - Cyclic Analysis of Single-Cell Subsets and Tissue Territories (CASSATT). CASSATT registers scanned slide images across all rounds of staining, segments individual nuclei, and scores marker expression on each detected cell. Beyond straightforward data analysis outputs such as dimensionality reduction, clustering, cell population identification and quantification, CASSATT explores the spatial relationships between cell populations via a statistical analysis module that can be applied to other high-dimensional imaging data types. In brief, it helps users identify populations of cells that interact - or do not interact - at frequencies that are greater than those occurring by chance. It also identifies specific ‘neighborhoods’ based on the cell types that surround each cell in the sample. The presence and location of these neighborhoods can be compared across slides or within distinct regions within a tissue. Within a newly generated cycIHC dataset consisting of 6 glioblastoma tissue sections processed through 8 cycles of chromogenic IHC staining, CASSATT successfully identified populations detected in parallel flow cytometry analyses and revealed specific associations between tumor and immune populations. Additional successful benchmarking was completed on a published tissue microarray dataset consisting of 107 cores processed through 18 cycles of staining and imaging.
TMIC-31. ADI-PEG20 RESTORES IMMUNITY IN THE TUMOR MICROENVIRONMENT AND ERADICATES GBM TUMORS IN MICE WHEN COMBINED WITH RADIATION
Authors: Symington Jake , Perryman Richard , Morse Sophie , O'Neill Kevin , Want Elizabeth , Syed Nelofer
Published: November 2022

Glioblastoma (GBM) is a primary brain tumour with poor prognosis and limited treatment options. We previously demonstrated that arginine deprivation by ADI-PEG20 is effective in GBM tumors which are auxotrophic for arginine. We also reported that ADI-PEG20 has efficacy in arginine non-auxotrophic GBM when combined with radiation in the presence of an immune competent environment. Here, we present detailed mechanistic insight into our latter findings using multiplex Imaging Mass Cytometry (Fluidigm) and Spatial Transcriptomics (10x Genomics) of tumor samples treated with ADI-PEG20. ADI-PEG20 enhanced the expression on MHC class II on infiltrating CD11c+ dendritic cells and these cells colocalised specifically with CD4+ T cells. We also observed changes in the expression of PD-1/PD-L1 with ADI-PEG20 and this was further enhanced when ADI-PEG20 was combined with radiation. Moreover, combination therapy increased the expression of chemokines involved in immune cell recruitment and activation. Our findings demonstrate that arginine deprivation restores immune function in the tumor microenvironment of arginine non-auxotrophic GBM tumors and suggests that combinations with immunotherapies will further enhance efficacy for GBM tumors.
TMIC-33. ARGINASE-1 AND ITS ROLE IN THE GLIOBLASTOMA MICROENVIRONMENT
Authors: Zamler Daniel , Shingu Takashi , Jiang Shan , Yu Fei , Jiang Yingda , Draetta Giulio , Hu Jian
Published: November 2022

An average GBM has roughly 30% infiltration of myeloid cells, the highest reported case at 70% infiltration, with some variation dependent on subtype. When we consider this fact in combination with the advent and success of immunotherapies in similar cancer types, the logical next step follows that myeloid cells, which are of the immune lineage, have the potential to clear this aberrant growth. Known for their incredible phagocytic capacity, myeloid cells are among the first responders to an injury to control pathogens and clear apoptotic cells from the site of an insult. Myeloid cells, through poorly understood mechanisms, undergo a switch after 3-5 days from attacking pathogens and clearing dead cells to secretion of growth cytokines to promote wound healing. We posit these myeloid cells may be tricked into erroneously supporting the tumor cells in an attempt to “heal the wound” instead of mounting an immune response. The enzyme responsible for this switch from pathogen response to wound healing is called Arginase-1 (Arg1). When Arg1 is upregulated it has a two pronged effect, the first is creation of peroxynitrite which is actively immunosuppressive to both myeloid and T-cells. The second is an increase in ornithine when Arg1 cleaves arginine into ornithine and urea, which can be used to create the building blocks for cellular growth as well as to create collagen, which is necessary for wound repair and found in high levels in brain tumors. Our hypothesis is that through manipulation of the Arg1 axis in myeloid cells we will increase anti-tumor response. We show the groundwork for evaluating whether or not this is a viable therapeutic target in GBM as well as assess currently available compounds for inhibition or Arg1 in the CNS.
CNSC-27. AN IDO-PROTAC HIGHLIGHTS A NOVEL TRYPTOPHAN METABOLISM- INDEPENDENT ROLE FOR IMMUNOSUPPRESSIVE IDO IN HUMAN GLIOBLASTOMA
Authors: Bommi Prashant , Monsen Paige , Bommi Lakshmi , Zhai Lijie , Bell April , Lauing Kristen , Mi Xinlei , Kocherginski Masha , Horbinski Craig , Lukas Rimas , Schiltz Gary , Wainwright Derek
Published: November 2022

INTRODUCTION

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

METHODS

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

RESULTS

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

CONCLUSIONS

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

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

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

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

BACKGROUND

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

OBJECTIVE

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

METHODS

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

RESULTS

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

DISCUSSION

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

Background

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

METHODS

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

RESULTS

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

CONCLUSION

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