bluuebunny/crossref_metadata_2025 huggingface dataset:
TMET-20. TERT ACTS VIA FOXO1 TO ALTER REDOX METABOLISM IN BRAIN TUMORS
Authors: Batsios Georgios , Taglang Celine , Tran Meryssa , Gillepspie Anne Marie , Viswanath Pavithra
Published: November 2022

Telomere maintenance is a fundamental hallmark of cancer. Most tumors maintain telomere length via reactivation of telomerase reverse transcriptase (TERT) expression. Identifying imaging biomarkers of TERT can enable non-invasive assessment of tumor proliferation and response to therapy. Deuterium magnetic resonance spectroscopy (DMRS) following administration of 2H-labeled substrates recently emerged as a novel, clinically translatable method of monitoring metabolic activity in vivo. The goal of this study was to delineate metabolic reprogramming associated with TERT expression and to leverage this information for non-invasive imaging of tumor burden and treatment response in gliomas. Our results indicate that TERT expression is associated with elevated levels of the redox metabolite NADH in glioblastomas and oligodendrogliomas. Mechanistically, TERT expression is associated with inhibitory phosphorylation and cytosolic sequestration of FOXO1. FOXO1, in turn, negatively regulates nicotinamide phosphoribosyl transferase (NAMPT), which is the rate-limiting enzyme in NAD+ biosynthesis. As a result, TERT upregulates NAMPT, resulting in elevated steady-state pools of NAD+ and NADH. Concomitantly, FOXO1 negatively regulates the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, which converts NAD+ to NADH. As a result, TERT upregulates the NADH/NAD+ ratio. Since elevated NADH and NADH/NAD+ ratio drive pyruvate conversion to lactate, we then examined whether DMRS-based imaging of [U-2H]-pyruvate metabolism reports on TERT expression in gliomas. Our results indicate that doxycycline-inducible TERT silencing significantly reduces lactate production from [U-2H]-pyruvate in tumor-bearing mice. Importantly, [U-2H]-pyruvate metabolism to lactate differentiates tumor from normal brain in vivo, including at clinical field strength (3T). Furthermore, [U-2H]-pyruvate reports on early response to treatment with TERT inhibitors or with radiochemotherapy in mice bearing orthotopic patient-derived gliomas at early timepoints before radiographic alterations can be visualized by magnetic resonance imaging. Collectively, our studies integrate a mechanistic understanding of TERT biology with innovative imaging that has the potential to improve assessment of tumor burden and treatment response for glioma patients.
TMET-26. PRIMARY OLIGODENDROGLIOMA CELL CULTURE VIABILITY: AN IN VITRO STUDY WITH METABOLIC MODULATORS
Authors: Villaverde Marcela , Arias Elsa Hincapié , Merenzon Martin , Mazzon Alejandro , Seoane Eduardo , Belgorosky Denise , Eiján Ana Maria
Published: November 2022

Oligodendrogliomas are tumors that develop from oligodendrocytes, the myelinating cells of the central nervous system. Oligodendrocytes are highly metabolically active cells that synthesize and transfer metabolites to neighboring cells. Given its intimate metabolic relation with neurons, we aim to investigate oligodendrocyte metabolism as an antitumoral target. A recurred oligodendroglioma WHO grade III was surgically removed from a 50-year patient after 10 years of progression-free disease. The tumor sample was mechanically digested and cultured at 37oC, 5% CO2, in DMEM: F12, 10 % FBS and antibiotics. Primary oligodendroglioma cells were trypsinized and seeded in a 96-well plate. After 24 hs, cells were treated with metabolic modulators: metformin (MET, mitochondrial complex II inhibitor, 5 mM), 2 deoxyglucose (2DG, hexokinase inhibitor, 1 mM), 6-aminonicotinamide (6AN, pentose phosphate pathway inhibitor, 50 µM) and/or 1400W and S-methylisothiourea (both iNOS inhibitors, 5 µM, and 50 µM respectively). Standard treatment with temozolomide (TMZ, 200 µM) was also performed. After 5 days of treatment, cells were stained with violet crystal. Two weeks after tumor sample digestion, a primary oligodendroglioma culture was successfully established. In vitro, proliferating cells appeared mostly undifferentiated with reduced branching complexity. Hexokinase inhibition by 2DG notoriously affected the viability of oligodendroglioma cells. Similar results were obtained with standard TMZ treatment. On the other hand, the inhibition of the pentose phosphate pathway by 6AN did not affect the cell monolayer. However, 6AN was able to increase the effect of MET as monotherapy. Both, MET and 2DG altered oligodendrocyte morphology inducing a more fusiform shape. Finally, iNOS inhibition modestly disrupted cell's monolayers and this effect did not seem to be improved by combinatory therapies. Glycolytic inhibitor 2DG resulted effective against oligodendroglioma cells. Whereas further studies are needed to validate these results, a better understanding of metabolic susceptibility could allow the development of better-targeted and more-effective therapeutic approaches.
TMET-27. DEXAMETHASONE-ASSOCIATED GENE EXPRESSION AND METABOLITE MODIFICATIONS IN GLIOMA TISSUE AND CELL CULTURES
Authors: Trautwein Christoph , Zizmare Laimdota , Maise Louise , Becker Hannes , Skardelly Marco , Tatagiba Marcos , Merk Daniel , Pichler Bernd J , Tabatabai Ghazaleh
Published: November 2022

Altered metabolism is amongst the hallmarks of cancer including diffuse glioma. We have recently presented a large database with tissue metabolites that are modulated by IDH1 mutation in diffuse glioma. Furthermore, we suggested potential candidate biomarkers for prospective validation that could serve for further refinement of prognostic groups in IDH1-mutated glioma. Glucocorticoids, particularly dexamethasone, are widely used in neuro-oncology and trigger a variety of metabolic effects, e.g. systemic adverse events including steroid-induced diabetes and truncal weight gain. In this subsequent study, we investigated dexamethasone-associated modifications of metabolite profiles in human glioma tissue (n=24 treated with dexamethasone, n=77 without) and in glioma cell lines. We performed ex-vivo Nuclear Magnetic Resonance (NMR) spectroscopy and RNA sequencing to discover comparison-specific metabolites and pathways. Dexamethasone-associated pathways involved increased energy and growth metabolism. Furthermore, we characterized dexamethasone-induced metabolite modulations in glioma cell lines to investigate dexamethasone effects in a rather homogeneous glioma cell population. We investigated the cellular effects of selected significant metabolites in acute cytotoxicity and clonogenic survival assays. Our data provide further insight into dexamethasone-associated metabolic effects in cell lines and tissue that might be relevant for further studies and its use in clinical practice.
TMET-29. THE GLUTAMINE TRANSPORTER SLC1A5 IS ASSOCIATED WITH REWIRING OF AMINO ACID, LIPID, AND IMMUNE PATHWAYS AND TUMOR PROGNOSIS IN PEDIATRIC BRAIN CANCERS
Authors: Kraya Adam A , Jin Run , Zhao Chao , Familiar Ariana , Storm Philip B , Wellen Kathryn , Resnick Adam C , Nabavizadeh Ali
Published: November 2022

Glutamine transporters play an important role in supporting increased tumor nutritional demands, often through overexpression of the solute carrier (SLC) family of membrane transporters. We aimed to understand the relationship of SLC transporter expression with pediatric brain tumor subtypes, lipid metabolism and their potential prognostic significance using data from the Pediatric Brain Tumor Atlas (PBTA). Using the expression of amino acid transporter genes, we found that elevated expression of glutamine transporters (SLC1A5, SLC7A5, SLC7A11, SLC38A5, SLC38A3) predicted shorter progression-free survival (PFS) in low-grade gliomas (LGGs) and poorer overall survival in pediatric ependymomas, high-grade gliomas (HGGs), and medulloblastomas. We focused specifically on SLC1A5 given the availability of imaging probes (18 F-Fluoroglutamine and 18F-Fluciclovine) for the corresponding amino acid transporter (ASCT2). Kaplan-Meier analysis found that higher expression of SLC1A5 was associated with shorter OS in ependymoma and medulloblastoma (p = 0.032 and p = 9.8e-4) and shorter PFS in LGG (p = 0.022). We found a universally higher expression of ATP citrate lyase (ACLY) and Acetyl-CoA carboxylase 1 (ACC) relative to normal brain tissue across all histologies. BRAF driven low grade gliomas and SHH-driven medulloblastomas showed differential expression of ACLY compared to other histologies. In addition, Acyl-CoA synthetase short chain family member 1 and 2 (ACSS1 and 2) were universally downregulated in pediatric gliomas, indicating that pediatric CNS tumors seem to rely on ACLY over ACSS for lipid synthesis. Gene set analysis showed higher expression and network rewiring of amino acid, lipid, and immune pathways in SLC1A5-high expressing clusters. SLC1A5 correlated negatively with pathways associated with lipid metabolic breakdown/degradation and correlated positively with pathways associated with lipid biosynthesis. In conclusion, our work demonstrates that glutamine transporters, particularly SLC1A5, represent compelling targets in pediatric brain tumors and can be exploited with theranostic approaches and amino-acid PET imaging.
TMET-30. ENHANCED RADIOSENSITIZATION OF GLIOMAS BY METABOLIC TARGETING OF SALVAGE PATHWAY OF NAD SYNTHESIS THROUGH NAMPT INHIBITION
Authors: Sharma Pratibha , Xu Jihong , Huo Longfei , Ju Zhenlin , Li Ziyi , Ban Kechen , Landesman Yosef , Sampath Deepa , Puduvalli Vinay
Published: November 2022

BACKGROUND

Pathway-specific targeted agents have failed to improve outcome in patients with high grade gliomas. Tumor recurrence is driven by glioma heterogeneity and emergence of therapy-resistant subpopulations. We have previously reported that metabolic targeting of NAMPT, the rate limiting enzyme in the NAD salvage synthesis pathway, circumvents glioma heterogeneity and induces cytotoxicity by mitochondrial membrane depolarization and generation of reactive oxygen species. Based on these data, we examined the potential for NAMPT inhibition to synergize with ionizing radiation to enhance glioma cytotoxicity in vitro and in vivo.

METHODS

Using a panel of genetically heterogeneous patient-derived glioma stem-like cells (GSC), we examined the effects of NAD targeting using KPT9274, a potent NAMPT inhibitor, in combination with ionizing radiation on cell proliferation and clonogenicity using Cell Titre Glo assay, colony formation assays and secondary sphere formation. Changes in DNA damage-related proteins were visualized by immunofluorescence assay and confocal microscopy. Effects on gene expression and protein levels were assessed using western blot, reverse phase protein array (RPPA) and RNA Seq.

RESULTS

NAMPT inhibition reduced cell proliferation rate, self-renewal and clonogenic capacity in vitro. Combining KPT-9274 with ionizing radiation caused synergistic inhibition of secondary sphere and colony formation of GSC. RPPA and RNASeq results showed downregulation of several DNA repair proteins which was further confirmed using immunofluorescence staining indicating a crucial role for these proteins in glioma resistance to therapies. Results of ongoing in vivo studies to assess the PK/PD, drug toxicity and survival benefits of this combination therapy in orthotopic GSC intracranial glioma mouse model will be reported.

CONCLUSIONS

Our results suggest that metabolic targeting through NAMPT inhibition sensitizes glioma cells to radiotherapy. Ongoing studies delineating the mechanistic basis of these effects and their impact in vivo as a potential rationale for clinical assessment will be reported.
TMET-38. LOSS OF FUNCTION OF CDK7 IS SYNTHETICALLY LETHAL WITH FATTY ACID OXIDATION INHIBITION IN GLIOBLASTOMA
Authors: Nguyen Trang , Shang Enyuan , Shu Chang , Akman Hasan , Izar Benjamin , Westhoff Mike-Andrew , Karpel-Massler Georg , Bruce Jeffrey , Canoll Peter , Siegelin Markus
Published: November 2022

CDK7 has been identified as a potential drug target for glioblastoma (GBM), a highly lethal primary brain tumor. However, resistance to therapy develops quickly, which may be facilitated by drug-induced reprogramming of metabolism. By combination of a transcriptome and metabolite screening analyses followed by carbon tracing (U-13C-Glucose, U-13C-Glutamine and U-13C-Palmitic acid) and extracellular flux analysis we demonstrated that both genetic and pharmacological (YKL-5-124 and THZ1) CDK7 inhibition elicited substantial metabolic reprogramming. Specifically, CDK7i elicited an increase of oxygen consumption rate fueled by enhanced fatty acid oxidation (FAO) manifested by enhanced labeling of citric acid cycle intermediates from palmitic acid. Consistently, the combination treatment of CDK7i inhibitors with blockers of FAO (etomoxir) or cellular respiration (gamitrinib) exerted substantial synergistic growth inhibition in patient derived xenograft as well as neurosphere GBM cultures, which was mainly driven by a collapse of oxidative energy metabolism. In turn, exogenous administration of adenosine triphosphate partially rescued from the cell death induced by the combination treatment. Moreover, the combination treatment activated intrinsic apoptosis through a reduction of both Mcl-1 and Bcl-xL as demonstrated by rescue experiments. Finally, the combined administration of YKL-5-124 and etomoxir extended overall in an orthotopic patient-derived xenograft model of GBM. In summary, these data support that simultaneous targeting of CDK7 and FAO might be a potential novel therapy against GBM.
TMIC-04. TARGETING AN ECM PROTEIN INVOLVED IN BREAST CANCER BRAIN METASTASES WITH AN ONCOLYTIC HSV-1
Authors: Rivera-Caraballo Kimberly , Yoo Ji young , Hong Bangxing , Kaur Balveen
Published: November 2022

The beta-1 subunit of integrins (CD29) is important for cell-extracellular matrix interactions. CD29 expression is increased in breast cancer cells, and it promotes their invasion, migration, and intravasation, all of which can result in breast cancer brain metastasis (BCBM). While CD29 expression can be correlated to poor prognosis of breast cancer patients, the median survival of BCBM patients predicted to have better prognosis is 36 months. Despite current BCBM treatments such as craniotomy, reaching the metastatic site is often challenging. Modified viral vectors, such as oncolytic herpes simplex virus (oHSV), have been clinically approved for the treatment of malignant melanoma and brain tumors, as they infect and selectively kill tumor cells. Previously, we identified that combination of oHSV and humanized CD29 blocking antibody (OS2966, aCD29) enhanced viral replication by reducing antiviral immune response, increased tumor cell killing, and rendered survival benefit to tumor bearing mice with intra-tumoral, but not systemic, oHSV administration. These findings suggested that the antitumor efficacy of the therapeutic combination required intratumoral delivery of anti-CD29, since the antibody could not penetrate the blood brain barrier and reach intracranial tumors. To overcome this, we report the generation of an oHSV encoding the scFv-Fc fragment of this antibody (HSV-aCD29), which retains its oncolytic function, and evaluate the impact of HSV-aCD29 on the CD29 downstream signaling. We will further characterize the safety, antitumor efficacy, and assess the immune response to HSV-aCD29 in vitro and in vivo. We anticipate that compared to our control virus, HSV-aCD29 will block CD29 activity which will reduce antiviral immune responses and enhance tumor cell lysis, thus improving oHSV efficacy. Together, this study proposes the blockade of CD29 with the targeted delivery of a CD29 blocking antibody mediated by a novel oHSV as a potential synergistic therapy against BCBM.
TMIC-05. SINGLE-CELL MULTI-OMIC ANALYSIS OF SPORADIC VESTIBULAR SCHWANNOMAS REVEALS SCHWANN CELLS WITH AN INJURY PHENOTYPE AND AN ASSOCIATION BETWEEN TUMOR SIZE AND MACROPHAGE INFILTRATE
Authors: Patel Bhuvic , Barrett Thomas , Pugazenthi Sangami , Khan Saad , Yim Aldrin , Buchman Craig , Patel Akash , Petti Allegra , Puram Sid , Kim Albert
Published: November 2022

Vestibular schwannomas (VS) comprise 8% of primary brain tumors, with rising prevalence due to increased detection by imaging. How intertumoral heterogeneity and differences within the tumor microenvironment (TME) contribute to the pathogenesis of VS remains poorly understood. We performed single-cell RNA sequencing (scRNA-seq) on 15 sporadic VS with paired single-nucleus assay of transposase accessible chromatin sequencing (snATAC-seq) on 7 tumors. Dimension reduction analysis revealed diverse Schwann, immune—predominantly macrophage, and stromal cell populations. Schwann cells adopted multiple unique functional states, such as repair-like, myelinating, and hypoxia-response, reflecting undescribed intratumoral heterogeneity within VS-associated Schwann cells. Remarkably, VS-associated Schwann cells were found to be phenotypically similar to Schwann cells in the setting of peripheral nerve injury, which is robustly associated with macrophage recruitment. Indeed, using our scRNA-seq atlas to perform deconvolution analysis of a broader cohort of VS, including newly sequenced (n = 22) and published bulk RNA-sequencing datasets (n = 153), we found variation in proportion of immune cells was strongly correlated with the proportion of macrophage infiltrate (R,2 = 0.86, p < 0.001). Furthermore, we found that macrophage/monocyte lineage cells represent a disproportionately large number of cycling cells in the VS TME, a finding corroborated by immunohistochemistry. Clinically, tumors with high macrophage infiltrate were associated with larger tumor size (Fisher’s exact test p = 0.007). Together, these findings suggest macrophages play an important role in VS pathogenesis. We therefore sought to characterize potential cell-to-cell interactions between Schwann cells and macrophages in our scRNA-seq atlas. Ligand-receptor analysis revealed several cytokines expressed by Schwann cells with cognate receptors expressed by macrophages, including MIF, SPP1, MDK, SEMA3C and IL34, which may be responsible for recruiting macrophages. In summary, we describe previously uncharacterized cellular diversity within VS, highlight an association between macrophage infiltration and clinical phenotypes, and identify potential therapeutic targets for VS treatment.
TMIC-07. PYK2 SIGNALING IS UPREGULATED IN RECURRENT GLIOBLASTOMA TUMORS IN C57BL/6/GL261 GLIOMA IMPLANTATION MODEL
Authors: Ortiz-Rivera Jescelica
Published: November 2022

Glioblastoma (GBM) is the most aggressive type of brain cancer with no successful treatment available. Microglia makes up to 30% of total GBM tumor mass. Previously we demonstrated that microglia within the tumor stimulate glioma cell growth and invasion through the proline rich tyrosine kinase 2 (Pyk2). Tumor resection is the first line treatment for GBM. Our previous studies with use of mouse glioma implantation model indicate that microglia in a site of tumor resection undergo significant modulations in cytokines expression profile. We hypothesize that microglia activated by tumor resection induces Pyk2 signaling in glioma cell and promotes malignant properties of the tumor cells that fail to be eliminated by resection. Using C57BL/6/GL261 mouse glioma implantation model, we analyzed primary implanted tumors and re-grown tumor after surgical resection. Two weeks after glioma cells implantation, tumor resection was performed, and two weeks later regrown tumors were used for western blot analysis. Total Pyk2 and phosphorylated Pyk2[Y579/50] were analyzed. The study revealed that Pyk2 phosphorylation is upregulated in re-grown tumor compared to primary implanted tumor. Treatment with PF-562271 (Pyk2/FAK inhibitor, once/daily, 50mg/kg, orally) beginning the 2ndday after tumor resection, reduced Pyk2 phosphorylation in regrown tumors compared to animals that did not receive treatment with PF-562271. In conclusion we can state that tumor resection procedure induces the upregulation of Pyk2 phosphorylation in regrown tumors. This effect is eliminated with oral administration of PF-562271.
TMIC-09. HV1 PROTON CHANNELS PROMOTE MYELOID INFILTRATION AND GLIOMA PROGRESSION
Authors: Zheng Jiaying , Wang Lingxiao , Ayasoufi Katayoun , Zhao Shunyi , Goddery Emma , Fain Cori , Johnson Aaron , Wu Long-Jun
Published: November 2022

Myeloid cells comprising up to 50% of total tumor mass in glioblastoma (GBM) contribute to tumor progression and immunosuppression. Restraining glioma-induced myeloid cell infiltration increases functional T cells within the tumors, improves the efficacy of checkpoint blockade, and significantly extends mouse survival. Here, we focus on an ion channel, voltage-gated proton channel Hv1, which is mainly expressed in myeloid cells and shapes the physiological functions of myeloid cells. Our bioinformatics analysis demonstrated that elevated Hv1 expression in the tumor mass correlates with poor disease prognosis in patients. Using the leading immunocompetent model of GBM in mice, we showed that Hv1 knockout mice (Hv1-/-) exhibit slower glioma progression and prolonged survival. Notably, in vivo two-photon imaging, immunofluorescence, and full-spectrum flow cytometry revealed that Hv1-/- mice have reduced monocyte/macrophage infiltration, higher frequency of MHCII+ among all infiltrating myeloid cells, and increased PD-1+CD4+ T cells in the tumor-burdened hemisphere. As a result, we combined anti-PD-1 treatment with Hv1 knockout in the middle stage of glioma, and found that approximately 30% of Hv1-/- mice were cured. Together, our results demonstrated that Hv1 regulates myeloid infiltration and could be a novel therapeutic target for the treatment of GBM.
TMIC-10. IDENTIFICATION, VALIDATION AND BIOLOGICAL CHARACTERIZATION OF NOVEL GLIOBLASTOMA TUMOUR MICROENVIRONMENT SUBTYPES: IMPLICATIONS FOR PRECISION IMMUNOTHERAPY
Authors: White Kieron , Connor Kate , Meylan Maxime , Bougoüin Antoine , Salvucci Manuela , Bielle Franck , O’Farrell Alice , Sweeney Kieron , Weng Linqian , Bergers Gabriele , Dicker Patrick , Ashley David , Lipp Eric S , Low Justin , Zhao Junfei , Wen Patrick Y , Prins Robert , Verreault Maite , Idbaih Ahmed , Prehn Jochen , Varn Frederick , Verhaak Roel , Sautès-Fridman Catherine , Fridman Wolf , Byrne Annette
Published: November 2022

New precision medicine therapies are urgently required for glioblastoma (GBM). However, to date, efforts to subtype patients based on molecular profiles, have failed to direct treatment strategies. We hypothesized that interrogation of the GBM tumor microenvironment (TME) and identification of novel TME-specific subtypes could inform new precision treatment strategies. To this end, a refined and validated microenvironment cell population (MCP)-counter method was applied to > 800 GBM patient tumours and validated by multiplex-immunohistochemistry. The MCP-counter deconvolution method interrogates the TME composition from transcriptomic data. Using this refined method, we classified the GLIOTRAIN(www.gliotrain.eu) IDHwt GBM cohort (n=123) into 3 novel clusters characterised by differences in TME composition and subsequently validated findings in the TCGA (n=69), CGGA (n=72) and DUKE (unpublished)(n=162) cohorts. TMEHigh tumours (30%) displayed elevated immune populations, functional orientation markers, immune checkpoint genes, and upregulated immunoregulatory pathways. Moreover, tertiary lymphoid structures were a feature of TMEHigh/mesenchymal+ patients. TMEMed (46%) tumours displayed heterogeneous immune populations and upregulated neuronal signalling pathways. TMELow (24%) tumours represented an ‘immune-desert’ group, high EGFR mutation frequency and upregulated EGFR signalling pathways. Longitudinal analysis of the GLASS cohort revealed TME-subtype transitions upon recurrence, influenced by TME composition changes. Finally, assessment of three GBM immunotherapy clinical trial cohorts revealed that TMEHigh patients treated with neo-adjuvant anti-PD1 have a significantly improved survival (P=0.04). Moreover, TMEHigh patients treated with anti-PD1 and an oncolytic virus (PVSRIPO) in the adjuvant setting, showed a trend towards improved survival (P=0.15 and P=0.056 respectively). Overall, we have established a novel TME-based classification system for application in intracranial malignancies. This system may be used to better inform a precision targeting approach in the brain tumour setting. For example, we hypothesise that patients bearing TMELow tumours may be amenable to neoadjuvant anti-TIM3 + EGFR inhibitor, TMEMed to anti-angiogenic immunotherapy, and TMEHigh patients to neoadjuvant anti-PD1 + anti-CTLA4.
TMIC-17. DISCOVERING DOMINANT TUMOR CHARACTERISTICS IN CYSTIC GLIOBLASTOMA
Authors: Chen Zhihua , Miao Yifeng , Yao Shun , O’Meally Denis , Frankhouser David , Tang Biao , Li MingHui , Frankel Paul , Shahmanyan Mari , Qiu Yongming , Rockne Russell C
Published: November 2022

BACKGROUND

As a rare subtype of glioblastoma (GBM), the genetic features and outcomes in cystic GBM (cGBM) are largely unknown. This study aimed to evaluate the survival of cystic features, investigate genomic patterns and the immune microenvironment in patients with cGBM.

METHODS

Gd T1-weighted, T2-weighted, T2-Flair, DWI or ADC images were used to classify cGBM or noncystic GBM (non-cGBM). Clinical information from IvyGAP, EGA databases and Chinese cohort were used for survival analysis. Fresh cystic fluid and residual tumor tissue were collected to evaluate immunological components, pathological and genomic differences between cGBM and non-cGBM. Data from IvyGAP were included to determine difference of molecular feature among substructure.

RESULTS

143 cases with cGBM and non-cGBM were screened to compare survival outcomes. Cystic features were to confer a survival benefit. It was an independent prognostic factor irrespective of IDH, MGMT and therapeutic regimen. cGBM patients have a hypomethylation state compared to non-cGBM. Active immune cells such as CD4+, CD8+ T cells were present in the cystic fluid and infiltrated into tumor. 10 cytokines/chemokines were only detected in cystic fluid, 8 cytokines/chemokines increased two-fold in cystic fluid than in peripheral blood serum (PBPs). Much differential methylated position (DMPs), differentially expressed genes (DEGs), Gene Set Enrichment Analysis (GSAE), Functional Epigenetic Modules (FEM) networks and molecular feature of substructural regions were different between cGBM and non-cGBM.

CONCLUSION

These results imply that cGBM could be a new subtype and has distinctive immune microenvironment which may have potential value correlating with prognosis.
TMIC-20. SERINE AND ONE-CARBON METABOLISM ADAPTING GLIOMA CELLS TO LOW GLUTAMINE MICROENVIRONMENT
Authors: Tanaka Kazuhiro , Nagashima Hiroaki , Iwahashi Hirofumi , Hashiguchi Mitsuru , Sasayama Takashi
Published: November 2022

Cancer cells optimize nutrient utilization to supply energetic and biosynthetic pathways. This metabolic process also includes redox maintenance and epigenetic regulation through nucleic acid and protein methylation, which enhance tumorigenicity and clinical resistance. However, less is known about how cancer cells exhibit metabolic flexibility to sustain cell growth and survival from low nutrient microenvironment. Here, we find that serine and glycine levels were higher in low-nutrient regions of tumors in glioblastoma multiforme (GBM) patients than they were in other regions. Metabolic and functional studies in malignant glioma cells demonstrated that serine availability and one-carbon metabolism support glioma cell survival following glutamine deprivation. Serine synthesis was mediated through autophagy rather than glycolysis. Gene expression analysis identified upregulation of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) to regulate one-carbon metabolism in malignant glioma cells and GBM patient-derived sphere cells. In clinical samples, MTHFD2 expression was highest in the nutrient-poor areas around “pseudopalisading necrosis.” Genetic and pharmacological suppression of MTHFD2 caused tumor cell death and growth inhibition of glioma cells upon glutamine deprivation as well as autophagy inhibition did. These results may have important implications for serine-dependent one-carbon metabolism for glioma cells to survive glutamine starvation and suggest a new therapeutic strategy for patients with malignant glioma.
TMIC-22. INTERPLAY BETWEEN GBM AND IMMUNE CELLS IN THE MOUSE GLIOMA MICROENVIRONMENT AT SINGLE-CELL RESOLUTION
Authors: Lenkiewicz Anna , Jacek Karol , Ghosh Mitrajit , Cyranowski Salwador , Pilanc Paulina , Szadkowska Paulina , Ochocka Natalia , Kaminska Bozena
Published: November 2022

Glioblastoma (GBM) is the most common primary tumor of the central nervous system in adults. GBM patients have a particularly poor prognosis and extremely short survival time due to lack of effective therapies and rapid tumor relapse. Neoplastic cells should be effectively recognized and destroyed by the immune system, but its antitumor activity is often inhibited by tumor-secreted factors that contribute to the tumor immunosuppressive microenvironment (TME). As TME plays a key role in cancer progression and immune evasion, understanding the interplay between GBM cells and myeloid and lymphoid populations is pivotal in creating new therapeutic strategies for GBM patients. To identify subtypes and functional diversity of immune cells in glioma TME we employed single-cell RNA and protein sequencing (CITEseq, Cellular Indexing of Transcriptomes and Epitopes by Sequencing) and Visium (10XGenomics) spatial transcriptomics. We characterized the populations of myelo- and lymphoid cells, and examined their unique transcription profiles, functional diversity and localization in TME. By combining analysis of CITE-seq with spatial transcriptomics we characterized and described 35 phenotypes of immune cells, which then we localized spatially within TME in GL261 mouse gliomas. Our results indicated that the peripheral monocytes/macrophages (Cd49d) localized in the tumor core, while microglia (Tmem119) accumulated at the invasive edge. Moreover, we observed a ring of activated astrocytes and rare T lymphocytes dispersed around the tumor. Finally, Ligand-Receptor and CellChat analysis of our CITE-seq results revealed the interplay between GBM, myeloid cells and lymphocytes, indicated on potential factors responsible for accumulation and tumor-evoked reprograming of immune cells. Single-cell technologies provide high-resolution insights into cellular and functional heterogeneity of gliomas, the analysis of which, in the future, will provide us with new therapeutic strategies for GBM patients. Studies were supported by NSC grant 2020/39/B/NZ4/02683 (BK) and PACIFIC Call 1 PAS (MG).
TMIC-25. SPATIAL TRANSCRIPTOMIC LANDSCAPE OF DIFFUSE GLIOMA
Authors: Wu Zhichao , Dazelle Karen , Chung Hye-Jung , Aldape Kenneth
Published: November 2022

Single-cell studies have suggested heterogeneity of neoplastic cell states in diffuse gliomas. IDH-mutant astrocytoma and oligodendrogliomas (IDH-glioma) is described by cell states resembling including oligodendrocytes(OC), astrocytes (AC), and cycling neural progenitor cells (NPC); and IDH-wildtype glioblastoma (GBM-IDHwt) is marked by cell states resembling AC, NPC, oligodendrocyte progenitors (OPC) and mesenchymal (MES). Collective experience indicates that cell states are dynamic and can transition, at times recapturing brain development and inflammatory wound response. However, the spatial architecture of cell states, cell types and their interactions are yet to be investigated. We quantified the transcriptomics of 1,941 regions of interest (ROIs) from 19 IDH-glioma (non-1p/19q-codeleted n = 11, 1p/19q-codeleted n = 8) and 11 GBM-IDHwt tumors using the NanoString GeoMx Cancer Transcriptome Atlas (CTA, n = 1,811 genes) to study their spatial heterogeneity. Where appropriate, ROIs were selected to represent tumor core, infiltration edge, pseudopalisading necrosis, microvascular proliferation, and histological normal areas, if available. CTA profiling showed strong inter- and intra-tumor heterogeneity. Genes involved in oncogenesis and extra-cellular matrix such as VEGFA and VCAN, and macrophages (including CD74 and CCL3/1) showed the highest spatially variation. We identified substantial tumor immune environmental differences across ROIs including immune code, monocyte-like rich and lymphocyte-rich groups. The GBM-IDHwt cell states OPC and NPC were highly positively correlated (p-value = 5.65e-122) across ROIs suggesting spatial co-localization, but MES cell states were negatively correlated with the other three GBM cell states. M0 macrophage abundance was positively correlated with the MES state but negatively correlated with other cell states in GBM-IDHwt. The MES state was significantly lower in infiltrating edge of the tumor (p-value = 1.39e-34) but higher in the pseudopalisading necrosis region (p-value = 7.66e-30). Overall, this spatial transcriptomics dataset provides spatially informed relationships of neoplastic cell states and the immune microenvironment in diffuse glioma.
TMIC-39. TARGETING EXTRACELLULAR MATRIX HYALURONIC ACID-CD44 SIGNALING REDUCES TUMOR STEMNESS AND SENSITIZES TUMOR TO VIROTHERAPY AND ENHANCES THERAPEUTIC POTENTIAL FOR CANCER TREATMENT
Authors: Sahu Upasana , Mullarkey Matthew P , Hong Evan , Guangsheng Pei , Zhongming Zhao , Hong Bangxing , Kaur Balveen
Published: November 2022

The tumor microenvironment (TME), including the non-tumor cells and the extracellular matrix (ECM) plays a crucial role in tumor progression and metastasis. Hyaluronic acid (HA), the major glycosaminoglycan present in brain ECM, has long been associated with the progression and invasiveness of brain tumors. HA signals primarily thorough CD44, an adhesion/homing receptor leading to the induction of cellular AKT, MEK and HIF signaling, thereby promoting tumor cell proliferation, aggressiveness and therapy resistance. While HA in the ECM has been shown to interfere with infection and spread of oncolytic viruses, the impact of tumor-ECM interaction induced signaling on oncolytic virotherapy is heavily understudied. RNA sequencing and gene set enrichment analysis of glioma cells infected with an oncolytic Herpes Simplex Virus-1 (oHSV) demonstrate an enrichment of pathways related to tumor-ECM interaction. Immunostaining of brain sections from intracranial tumor bearing mice also reveals increased HA after oHSV treatment. Our results further demonstrate that HA/CD44-mediated signaling inhibits virus replication in vitro. Herein, to evaluate the impact of blocking tumor-ECM interactions without altering the secreted ECM, we created oHSV-sCD44, an oHSV that encodes for extracellular soluble CD44 (sCD44) that can function as a dominant negative receptor for membrane bound CD44. oHSV-sCD44 significantly reduces the stemness of glioblastoma stem cells (GSCs), induces DNA damage and sensitizes the GSCs to radiation therapy. Intra-tumoral injection of oHSV-sCD44 into patient-derived primary GBM xenograft model significantly inhibits tumor growth accompanied by reduced stemness and decreased HA expression, and increased oHSV replication and tumor cell lysis in TME. Moreover, blocking HA-CD44 signaling with a single dose of oHSV-sCD44 in murine glioma syngeneic model induces the development of a significant anti-tumor immune response with enhanced T cell infiltration. Collectively, our findings implicate oHSV-sCD44 as a potential oncolytic and immune-stimulating anticancer therapeutic.
TMIC-42. LEVERAGING B CELL IMMUNITY TO PROMOTE IMMUNOTHERAPY IN GLIOBLASTOMA
Authors: Hou David , Castro Brandyn , Zolp Andrew , Dapash Mark , Arrieta Victor , Zhao Junfei , Zhang Peng , Rashidi Aida , Burnham Emma , Patel Ronit , Lesniak Maciej , Miska Jason , Lee-Chang Catalina
Published: November 2022

Immunotherapy has revolutionized cancer treatment but has yet to be translated into brain tumors. Studies in melanoma and sarcoma, amongst other models, have revealed the accumulation of germinal-center-like B cells as a key survival predictor post-PD1 blockade. We seek to leverage B cell immunity to enhance immunotherapy effectiveness in glioblastoma (GBM). In human GBM and murine glioma models, we found that B cells in the tumor microenvironment (TME) are activated, but the expression of co-inhibitory molecules such as CD32 and CD72 blocks downstream effector function. Transcriptomic analysis showed high expression of inhibitory TGFβ receptors on B cells and high levels of TGFβ1 cytokine in the TME. We showed direct inhibition of B cell function through TGFβ signaling that could be prevented with TGFβ receptor blockade. Spatial multiplex immunofluorescence analysis of the TME revealed that tumor and myeloid cells express high levels of TGFβ and are also near B cells, allowing for TGFβ-mediated B cell inhibition. Blocking the TGFβ pathway via transgenic mice with TGFβ receptor knockout on B cells or TGFβ cytokine knockouts in myeloid cells, or generation of a CT2A tumor line with TGFβ cytokine knockdown, all demonstrated a survival benefit and more germinal-center-like B cells. There was also increased T cell proliferation and anti-tumor cytotoxicity. Finally, inhibiting αVβ8 integrin, a required factor that releases active TGFβ, is a translatable approach that also increased B cell proliferation and animal survival. Dual treatment with αVβ8+PD1 blockade showed the most potent survival as well as immunological memory against tumor re-challenge. Analysis of the B and T cell compartments after dual treatment showed synergy, with robust cellular proliferation and functional differentiation of plasmablasts and effector T cells. Collectively, our study highlights the importance of B cells in the TME and a remodeled approach to boost the effects of immunotherapy against GBM.
TMIC-49. CHITINASE-3-LIKE 1 PROTEIN COMPLEXES REGULATE PD-1 SIGNALING-MEDIATED IMMUNOSUPPRESSION IN GLIOBLASTOMA
Authors: Srivastava Rashmi , Chen Apeng , Dodda Meghana , Zou Han , Hu Baoli
Published: November 2022

Glioblastoma (GBM), the most common and lethal brain tumor with a median survival rate of only 15 months, remains largely incurable despite intensive multimodal treatment, including immunotherapeutic strategies being tested in clinical trials. GBM is highly immunosuppressive and resistant to immunotherapy because glioma cells escape from effective antitumor immunity through programing the tumor microenvironment (TME). Owing to the tremendous heterogeneity and plasticity of tumor cells and the surrounding TME, understanding the mechanisms of immune evasion by GBM remains elusive. We have recently discovered that the Chitinase-3-like-1 (CHI3L1)-Galectin-3 (Gal3) protein binding complex can selectively promote tumor-associated macrophage migration and infiltration with a protumor M2-like phenotype and T cell-mediated immunosuppression, which are governed by a transcriptional program of NF-κB/CEBPβ in the CHI3L1/Gal3-PI3K/AKT/mTOR axis. The immunoprecipitation coupled to liquid chromatography-mass spectrometry analysis revealed that galectin 3–binding protein (Gal3BP) competes with Gal3 to bind with CHI3L1 for negative regulation of the CHI3L1-Gal3 mediated processes. Interestingly, a newly-developed Gal3BP mimetic peptide can disrupt CHI3L1-Gal3 interaction, resulting in decreasing migration of M2-like bone marrow-derived macrophages (BMDMs), increasing CD8+ T cell infiltration, reversing immunosuppression, and inhibiting tumor progression in vitro and in vivo. Analyzing PD-1 signaling activation, we found that the Gal3BP mimetic peptide significantly decreased PD-L1 expression in tumor cells. Correlation analysis showed that CHI31L and Gal3 (encoded by LGALS3 gene) are significantly associated with both PD-L1 and PD-L2 in GBM patient samples. Furthermore, overexpression of CHI3L1 increased expression levels of PD-L1 and PD-L2, and CHI3L1 deletion decreased their expression in GBM patient-derived neurosphere lines. The treatment with recombinant CHI3L1 protein significantly increased PD-L1 and PD-L2 expression in M2-like BMDMs (with high levels of endogenous Gal3). Collectively, these data suggest that CHI3L1 protein complexes control the GBM immunosuppressive microenvironment by PD-1/PD-L1/PD-L2 signaling, providing new immunotherapeutic strategies for this brain cancer.
TMIC-52. RELATIONSHIP BETWEEN MACROPHAGE AND RADIOSENSITIVITY IN HUMAN PRIMARY AND RECURRENT GLIOBLASTOMA: IN SILICO ANALYSIS WITH PUBLICLY AVAILABLE DATASETS
Authors: Kim In Ah , Jang Bum Sup
Published: November 2022

The glioblastoma microenvironment predominantly contains tumor-associated macrophages that support tumor growth and invasion. We investigated the relationship between tumor radiosensitivity and infiltrating M1/M2 macrophage profiles in public datasets of primary and recurrent glioblastoma. We estimated the radiosensitivity index (RSI) score based on gene expression rankings. Macrophages were profiled using the deconvolution algorithm CIBERSORTx. Samples from The Cancer Genome Atlas (TCGA), Chinese Glioma Genome Atlas (CGGA), the Ivy Glioblastoma Atlas Project dataset, a single-cell RNA sequencing dataset (GSE84465), Glioma Longitudinal Analysis Consortium (GLASS), and an immunotherapy trial dataset (GSE121810) were included. RSI-high radioresistant tumors were associated with worse overall survival in TCGA and CGGA than RSI-low tumors. M1/M2 macrophage ratios and RSI scores were inversely associated, indicating that radioresistant glioblastoma tumor microenvironments contain more M2 than M1 macrophages. In the single-cell RNA sequencing dataset, the mean RSI of neoplastic cells was positively correlated with high M2 macrophages proportions. A favorable response to programmed cell death protein 1 (PD-1) therapy was observed in recurrent glioblastomas with high M1/M2 macrophage ratios and low RSI scores. In patients with recurrent glioblastoma, fewer M2 macrophages and low RSI scores were associated with improved overall survival. High M2 macrophage proportions may be involved in radioresistant glioblastoma.
CNSC-29. INVESTIGATING THE INFLUENCE OF DOPAMINERGIC ACTIVITY ON THE GLIOBLASTOMA NICHE
Authors: Ware Matthaeus , Pontual Luiza Lopes , Yu Chunying , Kushida Michelle , Rastegar Naghmeh , Dolma Sonam , Dirks Peter
Published: November 2022

Glioblastoma (GBM) is an incurable disease of adults and children and the deadliest form of central nervous system (CNS) malignancy. Despite major advances in our understanding of GBM biology in recent years, the prognosis for patients who develop this disease has not improved. If we hope to find new treatments for GBM that are safe and effective, we desperately need to reform our thinking. The brain’s chemical milieu is rich with neurotransmitters, and our lab’s screen of 680 neuroactive compounds on patient-derived glioblastoma stem cells (GSCs) in vitro strongly implicates neurotransmitter pathways as critical regulators of the GSC niche. More specifically, disrupting dopamine signaling by inhibiting its receptor D4 on GSCs causes substantial GSC apoptosis in vitro and attenuates GBM growth in vivo (Dolma et al., Cancer Cell, 2016). Dopamine (DA) is a catecholamine neurotransmitter that is essential for reward learning and movement and has numerous roles in cognition. Consequentially, dysregulation of DA signaling is associated with a diversity of brain diseases ranging from drug addiction to schizophrenia to Parkinson’s. Our research aims to determine how DA signaling affects normal neural stem cell (NSC) and tumorigenic GSC populations, as we hypothesize that GSCs arising/residing in DA projection zones exploit dopaminergic (DAergic) activity for GBM growth. Toward these aims, we have developed in vivo model systems and harnessed them to study NSC and GSC niches in the context of either controlled activation or depletion of DAergic neurons. These manipulations of the brain's DAergic neurons are achieved using optogenetic stimulation, genetic depletion, and neurotoxin-mediated ablation in mice. Ultimately, unraveling the dopaminergic influence on GBM may contribute to a redeployment of existing treatments—that modulate DA signaling and are already approved to treat CNS disorders—to patients with this deadly brain cancer.
TMIC-56. TUMOR CELL ARCHITECTURAL HETEROGENEITY AND SPATIAL INTERACTIONS WITH THE TUMOR IMMUNE MICROENVIRONMENT IN GBM
Authors: Silver Aryeh , Feier Diana , Tian Guimei , Andrews Michael , Dajac Mariana , Yang Changlin , Ghosh Tanya , Fu Dongtao , Von Roemeling Christina , Rahman Maryam , Sarkisian Matthew , Huang Jianping , Harrison Jeffrey , Mitchell Duane , Deleyrolle Loic
Published: November 2022

INTRODUCTION

Glioblastoma (GBM) contains cell populations with distinct metabolic requirements, with fast-cycling cells harnessing aerobic glycolysis, and treatment-resistant slow-cycling cells (SCCs) preferentially engaging lipid metabolism. The interaction between immune and tumor cells, and how their metabolic heterogeneity shapes the immune landscape in GBM has yet to be understood. Objectives: The primary objective of this project is to spatially and molecularly decode the GBM microenvironment heterogeneity with a specific focus on unraveling the metabolic links that underlie the interaction of SCCs with the immune compartment.

METHODS

Multiple murine glioma cell lines coupled with geospatial profiling were used to establish metabolic heterogeneity and communications, while various genetic and pharmacological approaches were applied to assess the effect of disrupting the metabolic interplay between SCCs and the immune system.

RESULTS

We determined that SCCs exhibit distinct metabolic dependencies, involving preferential lipid metabolism supported by enhanced fatty acid uptake. We also found that SCCs exhibit specific geospatial distribution and that tumor progression is regulated by their interactions with immune suppressive cells, which in turn work against tumor immune rejection by inhibiting T cell anti-tumor activity. The immune microenvironment shaped by SCCs is marked by specific metabolic features enhancing lipid exchange capacities that are exploited by SCCs to support their survival and functions. Importantly, disrupting lipid metabolic exchange sensitized tumors to chemotherapy.

CONCLUSION

Our results reveal that metabolic interactions between SCCs and tumor-associated immune suppressive cells within the GBM microenvironment play a critical role in the development of drug and immune resistant tumors. This study delineates these metabolic communications and assesses the potential therapeutic effect of disrupting these interactions to treat GBM. The insights generated from this project uncover fundamental principles of the emerging connections between the tumor microenvironment, cell metabolism, anti-tumor immunity, and associated therapeutic vulnerabilities.