bluuebunny/crossref_metadata_2025 huggingface dataset:
TMET-03. REPROGRAMMING OF GLIOBLASTOMA GLUCOSE METABOLISM FUELS TUMOR GROWTH
Authors: Scott Andrew J , Mittal Anjali , Al-Holou Wajd , Palavalasa Sravya , Meghdadi Baharan , O'Brien Alexandra , Zhou Weihua , Yang Annabel , Zhao Zitong , Lin Angelica , Tarnal Vijay , Lyssiotis Costas , Nagrath Deepak , Wahl Daniel R
Published: November 2023

Metabolic adaptation can promote oncogenic phenotypes in glioblastoma (GBM), but the metabolic pathways utilized by GBM and how they differ from the pathways utilized in normal cortex are poorly understood. Here, we utilize in vivo stable isotope tracing in mice and patients with GBM to define carbon fate and metabolic rewiring in cancer. We infused uniformly-labeled 13C glucose into mice bearing orthotopic GBM patient-derived xenografts or into patients undergoing surgical resection for likely GBM. Tumor and cortex were physically separated in mice by fluorescence-guided microdissection. In humans, samples of enhancing tumor, non-enhancing tumor and normal cortex were separated intraoperatively using MRI guidance. Samples were subsequently analyzed by liquid chromatography mass spectrometry to determine the downstream metabolic fates of infused 13C glucose. Of eight patients infused with 13C6-glucose, six were confirmed to have GBM while the others had non-GBM high grade gliomas. In mice and in patients, glucose-derived carbon effectively entered glycolysis and labeled glycolytic intermediates equivalently in tumor and cortex. Compared to GBM tissue, cortical tissue preferentially oxidized glucose-derived carbon in the TCA cycle and used glucose-derived carbon to synthesize neurotransmitters. Cortical tissue utilized glucose-derived carbon to synthesize the amino acid and neurotransmitter precursor serine, while GBM tissue derived serine from extracellular sources. By contrast, GBM tissue preferentially utilized glucose-derived carbon for the synthesis of purines, pyrimidines and NAD+/NADH. Using time-course isotope tracing studies in mice with GBM and a novel metabolic flux analysis framework, we quantified absolute metabolic fluxes in vivo and confirmed elevated nucleotide synthesis and serine salvage in GBM. Consistent with these results, eliminating dietary serine in mice slowed GBM PDX growth. These studies are the first measurements of numerous metabolic pathways in humans and indicate that GBMs suppress the physiologic utilization of glucose carbons to fuel biosynthesis, which represents a targetable metabolic liability.
TMET-06. METABOLOGENOMIC CHARACTERIZATION UNCOVERS HETEROGENEITY AMONGST IDH MUTANT
Authors: Ajisebutu Andrew , Nassiri Farshad , Mamatjan Yasin , Patil Vikas , Wang Justin , Voisin Mathew , Mansouri Sheila , Zadeh Gelareh
Published: November 2023

INTRODUCTION

Mutations in isocitrate dehydrogenase (IDH) enzymes are recognised to drive the molecular footprint of diffuse gliomas through the accumulation of oncometabolite R-2-hydroxyglutarate which drives the widespread restructuring of the DNA methylome; however, beyond R-2-hydroxyglutarate , a comprehensive metabologenomic characterization of IDH-mutant gliomas has yet to be performed. We aimed to characterize the global metabolic profile of IDH-mut and IDH-wt gliomas, identify clinically relevant subgroups based on distinct metabolic differences, and utilize matched epigenetic and transcriptional data to characterize these distinct groups. Method: Glioma samples from a cohort of 154 patients underwent multiplatform molecular analysis, including metabolomic studies, genome-wide DNA methylation profiling and bulk RNA sequencing. A integrative analysis was performed, including hierarchical clustering and principal component analysis of identified metabolites, differentially methylated probes, copy number alteration and bulk mRNA data. Survival analysis as well as multivariable hazard ratios for clinical variables were calculated by fitting Cox Proportional Hazards Models.

RESULTS

We discovered a group of IDH-mutant gliomas whose metabolic profile highly resembled IDH wildtype tumors. Notably, these IDH-mutant gliomas with dysregulated metabolism were distinguished from their IDH-mutant counterparts by significantly shorter overall survival (median OS 118.9 months vs 173.6 months, p=0.048). The IDH-mutant tumors with dysregulated metabolism harbored distinct epigenetic alterations that converged to drive proliferative and stem-like transcriptional profiles. The prognostic relevance of dysregulated metabolism complements, but was not wholly explained by canonically recognized prognostic classifications in IDH-mutant gliomas including 1p/19q codeletion, glioma CpG Island Hypermethylator (GCIMP) status and CDKN2A homozygous deletion.

CONCLUSION

Utilizing a cross-platform analysis we have uncovered a novel subtyping of IDH-mutant gliomas with dysregulated cellular metabolism with similar survival to IDH-wildtype tumors. The metabolic profile provides unique information on glioma phenotypes, which may facilitate a more comprehensive understanding of glioma biology, and provide a window to target novel dependencies.
TMET-11. BERBERINE AS A POTENTIAL ENHANCER FOR 5-ALA-MEDIATED FLUORESCENCE IN GLIOBLASTOMA: INCREASING DETECTABILITY OF INFILTRATING GLIOMA STEM CELLS TO OPTIMIZE 5-ALA-GUIDED SURGERY
Authors: Suehiro Satoshi , Ohtsuka Yoshihiro , Inoue Akihiro , Ohnishi Takanori , Yamashita Daisuke , Nishikawa Masahiro , Watanabe Hideaki , Kunieda Takeharu
Published: November 2023

OBJECTIVE

The prognosis of glioblastoma multiforme correlates with residual tumor volume. In fluorescence-guided surgery, 5-aminolevulinic acid (5-ALA) has been used to maximize safe resection. We searched for repositioning drugs that affect mitochondrial functions and energy metabolism, identifying berberine (BBR) as a potential enhancer of 5-ALA-mediated protoporphyrin IX (PpIX) fluorescence (5-ALA-fluorescence). Here, we investigated whether BBR can be applied to clinical practice as a 5-ALA-fluorescence enhancer.

METHODS

Effects of BBR on 5-ALA-fluorescence in glioma cells and GSCs were evaluated by flow cytometry (fluorescence-activated cell sorting; FACS) analysis. As 5-ALA is metabolized for heme synthesis, effects of BBR on mRNA expressions of seven enzymes in the heme-synthesis pathway were analyzed. Enzymes showing significantly higher expression than control in all cells were identified and protein analysis was performed. To examine clinical availability, the detectability and cytotoxicity of BBR in tumor-transplanted mice were analyzed.

RESULTS

Fluorescence microscopy revealed much more intense 5-ALA-fluorescence in both GSCs and non-stem cells with 5-ALA and BBR than with 5-ALA alone. FACS disclosed that BBR greatly enhanced 5-ALA-fluorescence compared to 5-ALA alone and enhancement was much higher for GSCs than for glioma cells. Among the seven enzymes examined, BBR upregulated mRNA expressions of ALA synthetase 1 (ALAS1) more highly in all cells, and activated ALAS1 through deregulating ALAS1 activity inhibited by the negative feedback of heme. In vivo study showed that 5-ALA-fluorescence with 5-ALA and BBR was significantly stronger than with 5-ALA alone, and the sensitivity and specificity of BBR-enhanced fluorescence were both 100%. In addition, BBR did not show any cytotoxicity for normal brain tissue.

CONCLUSIONS

BBR enhanced 5-ALA-mediated PpIX fluorescence by upregulating and activating ALAS1 through deregulation of negative feedback inhibition by heme. BBR is a clinically used drug with no side effects. BBR is expected to augment fluorescence-guided surgery and photodynamic therapy.
TMET-12. CDKN2A DELETION REMODELS LIPID METABOLISM TO PRIME GLIOBLASTOMA FOR FERROPTOSIS
Authors: Minami Jenna , Morrow Danielle , Bayley Nicholas , Fernandez Elizabeth , Salinas Jennifer , Tse Christopher , Zhu Henan , Su Baolong , Sammarco Alessandro , Liau Linda , Graeber Thomas , Williams Kevin , Cloughesy Timothy , Dixon Scott , Bensinger Steven , Nathanson David
Published: November 2023

Glioblastomas (GBM) are universally lethal brain tumors that exhibit extensive genetic and transcriptional heterogeneity. GBMs acquire metabolic adaptations in response to a multitude of intrinsic (oncogenotype) and extrinsic (tumor microenvironment) factors to fuel neoplastic progression. Large-scale multi-omic studies in cancer cell lines have further advanced our understanding of the intersection between molecular diversity and metabolic heterogeneity, indicating distinct metabolic vulnerabilities may exist within defined molecular subsets of cancer. However, the translatability of these observations into effective cancer therapies remains limited due to gaps in our understanding of the physiologic fidelity of metabolic studies conducted only in vitro. Thus, accounting for both the molecular diversity and environment of human tumors is crucial to identify translatable metabolic targets.
TMET-15. THE COMBINATIONAL EFFECT OF INHIBITOR FOR A FATTY ACID DESATURASE AND A FATTY ACID TRANSPORTER ON GLIOMA GROWTH
Authors: Yamasaki Tomohiro , Zhang Lumin , Dowdy Tyrone , Lita Adrian , Kurozumi Kazuhiko , Larion Mioara
Published: November 2023

BACKGROUND

It is not well understood how much exogenous fatty acids uptake and de novo fatty acid synthesis affect the cell proliferation in glioma. In this study, we examined the combinational effect of inhibitor for a fatty acid desaturase (SCD) and a fatty acid transporter (CD36) on glioma growth.

METHODS

Normal human astrocytes (NHA) were used as glial cells, and U251 transfected with IDH wild-type or IDH mutant gene and patient-derived glioma cells (BT142, TS603, GSC923S) were used as glioma cells. The function of SCD was suppressed by SCD inhibitor and SCD siRNA, and Sulfo-N-succinimidyl oleate (SSO) was used to inhibit the CD36 function.

RESULTS

We treated U251 cells with palmitic acid (C16:0), stearic acid (C18:0), palmitoleic acid (C16:1) or oleic acid (C18:1) after SCD inhibitors treatment or SCD knockdown, and cell proliferation was rescued only after the treatment with C16:1 and C18:1. NHA was not affected by SCD inhibitor, although CD36 inhibitor suppressed cell proliferation of both U251 and NHA. The combination of SCD inhibitor and CD36 inhibitor under the addition of exogenous fatty acid, oleic acid, suppressed the proliferation of U251 cells more effectively than SCD inhibitor or CD36 inhibitor alone. Discussion: The SCD inhibitory effect on gliomas was attenuated by the presence of exogenous monounsaturated fatty acids, whereas the CD36 inhibitor enhanced the cell-killing effect. On the other hand, it should be noted that CD36 inhibitors affect the proliferation of glial cells.
TMET-21. PROTON MAGNETIC RESONANCE SPECTROSCOPY (1H MRS) FOR THE DIAGNOSIS OF AGGRESSIVE MENINGIOMAS WITH RECURRENCE POTENTIAL
Authors: Ijare Omkar , Baskin David S , Powell Suzanne , Pichumani Kumar
Published: November 2023

In our previous study, we have observed elevated steady state levels of both alanine and glutamine in surgically resected tissue specimens from atypical meningioma (grade-II) patients compared to the benign (grade-I) meningioma patients. We have shown that meningioma cells utilize glutamine for the biosynthesis of alanine and other amino acids which may help in the cellular proliferation. While the role of elevated alanine in meningiomas is not well understood, this metabolite may serve as a metabolic marker for the diagnosis of aggressive meningiomas which may recur in the future. Here, we present a case of a large meningioma (7 cm x 4 cm x 8 cm) in a male patient (age: 68 years) who was diagnosed with an anterior cranial fossa, dural based extra-axial enhancing mass with the sinonasal extension compatible with a meningioma. The patient underwent multiple surgeries for the resection of the original tumor mass. Tumor tissue was collected from 2 regions: Nasal cavity and deeper tumor. Surgical pathology classified the tumor as a grade-I meningioma with MIB-1 index of ~10%. However, 1H MRS analysis of the tumor specimen showed elevated levels of alanine (Nasal cavity = 9.57 mM/g, tissue; Deep tumor = 5.50 mM/g, tissue), which are greater than the alanine levels reported for grade-II meningiomas (4.23 ± 1.92 mM). The elevated levels of alanine detected by 1H MRS suggested aggressive behavior of the tumor. After about 11 months, the repeat MRI examination showed tumor recurrence, and the patient underwent surgery again for the resection of the recurrent tumor. Surgical pathology of the recurrent tumor classified the tumor as a grade-I meningioma with increased proliferation rate (MIB-1 index of 10-12%). In conclusion, 1H MRS has potential to predict aggressive behavior of meningiomas and alanine can be used as a molecular marker for the differential diagnosis of meningiomas.
TMET-22. MULTI-OMIC SCREENING OF INVASIVE GBM CELLS IN ENGINEERED BIOMATERIALS AND PATIENT BIOPSIES REVEALS TARGETABLE TRANSSULFURATION PATHWAY ALTERATIONS
Authors: Choudhary Nikita , Garcia Joseph , Akins Erin , Jain Saket , Wolf Kayla , Zhang Jason , Lad Meeki , Shukla Poojan , Gill Sabraj , Carson Will , Carrete Luis , Zheng Allison , Kumar Sanjay , Aghi Manish
Published: November 2023

While the poor prognosis of glioblastoma arises from the invasion of a subset of tumor cells, little is known of the metabolic alterations within these cells that fuel invasion. We integrated spatially addressable hydrogel biomaterial platforms, patient site-directed biopsies, and multi-omics analyses to define metabolic drivers of invasive glioblastoma cells. Metabolomics and lipidomics revealed elevations in the redox buffers cystathionine, hexosylceramides, and glucosyl ceramides in the invasive front of both hydrogel-cultured tumors and patient site-directed biopsies, with immunofluorescence indicating elevated reactive oxygen species (ROS) markers in invasive cells. Transcriptomics confirmed upregulation of ROS-producing and response genes at the invasive front in both hydrogel models and patient tumors. Amongst oncologic ROS, hydrogen peroxide specifically promoted glioblastoma invasion in 3D hydrogel spheroid cultures. A CRISPR metabolic gene screen revealed cystathionine gamma lyase (CTH), which converts cystathionine to the non-essential amino acid cysteine in the transsulfuration pathway, to be essential for glioblastoma invasion. Correspondingly, supplementing CTH knockdown cells with exogenous cysteine rescued invasion. Pharmacologic CTH inhibition suppressed glioblastoma invasion, while CTH knockdown slowed glioblastoma invasion in vivo. Our studies highlight the importance of ROS metabolism in invasive glioblastoma cells and support further exploration of the transsulfuration pathway as a mechanistic and therapeutic target.
TMIC-02. SEX DIFFERENCES IN IMMUNE RESPONSE IN A MURINE GLIOBLASTOMA RESECTION MODEL CONFER A FEMALE SURVIVAL ADVANTAGE
Authors: Volovetz Josephine , Lee Juyeun , Lathia Justin
Published: November 2023

Glioblastoma (GBM) is the most common primary malignant brain tumor with a median survival of 15-20 months, despite standard of care treatment, which includes surgical resection, radiation, and chemotherapy. GBM is 1.6 times more common in males and overall survival can be up to 10 months shorter in males. Females are more responsive to standard of care therapy, but the mechanism behind these differences is not clear. The immune system is known to be suppressed in GBM patients, and recent studies have found sex differences in immune response. While pre-clinical GBM models are used to interrogate pathophysiology and response to therapeutic interventions, few of these models include surgical resection, despite its role in the standard of care for patients and as a defining therapy in prolonging survival. We developed a pre-clinical GBM resection model to evaluate whether there is a sex bias in survival and to interrogate sex-specific immune changes after surgery. We found that C57BL/6 mice implanted with syngeneic GBM cells demonstrate a sex bias after surgical resection, with males surviving for a median of 24 days compared to 33 days for females. To understand how the immune response is affected by surgical resection in males and females with GBM, we performed flow cytometry to characterize the immune phenotype and found that female mice who underwent resection of their tumors had significantly higher levels of CD4+ T cells in their resection cavity than male mice who underwent resection. The immune phenotype will be further characterized over time to evaluate how it evolves in each sex. These data support prior observations that the mechanisms of immunosuppression are different in males and females. They further underscore the complexity of sex bias in GBM and the need to account for surgical resection and sex in pre-clinical models to investigate next-generation therapeutic strategies.
TMIC-14. CLEC5A REGULATES TAM POLARIZATION, TME IMMUNOSUPPRESSION AND DISEASE PROGRESSION IN GLIOBLASTOMA
Authors: Li Jiabo , Wang Xuya , Tong Luqing , Markwell Steven , Shih Ling-Kai , Olson Cheryl , Carneiro-Lobo Tatiana , Yang Xunjun , Brat Daniel
Published: November 2023

Glioblastoma, IDH-wildtype (WHO grade 4) is the most common primary malignant brain tumor of adults and is characterized by an immunosuppressive tumor microenvironment (TME) and poor clinical outcomes. CLEC5A is a gene whose expression enhances immune response in inflammatory and infectious diseases, yet its role in GBM immune regulation is unclear. Seurat analysis of scRNA-seq data and CIBERSORTx analysis of GBM RNA-seq data showed that tumor-associated macrophages (TAMs) account for the large majority of immune cells in the GBM, and that CLEC5A expression in this population (CD163+) is highest in hypoxic, peri-necrotic zones. Notably, among all immunosuppressive TAM related genes, CLEC5A expression shows the strongest association with poor clinical outcomes. Monocytes (THP-1 cells) exposed to GBM conditioned media exhibit enhanced CLEC5A and CD163 expression. Moreover, overexpression of CLEC5A by THP-1 cells enhances TAM polarization, migration toward glioma cells, and increased secretion of cytokines that mediate immunosuppression, including IL-1β, IL-4, IL-6, IL-8, IL-10 and IL-13. CLEC5A triggers the activation and phosphorylation of the Syk-Jak2-STAT3 signaling through its interaction with Syk, offering a potential therapeutic intervention. In addition, CLEC5A acts as a receptor, binding to Podoplanin (PDPN) expressed on GBM cells in the peri-necrotic TME, triggering CLEC5A-Syk-JAK-STAT3 signaling and highlighting their role in TAM polarization and the immunosuppressive TME. In vivo, silencing CLEC5A delayed glioma growth, prolonged survival and was associated with reduced CD163 and neoplastic Ki-67 expression. Collectively, we found that CLEC5A expression by TAMs is regulated by PDPN-CLEC5A-Syk-JAK-STAT3 signaling and enhances TAM polarization, TME immunosuppression and GBM growth.
TMIC-15. GENETIC AND SPATIAL HETEROGENEITY OF PEDIATRIC HIGH-GRADE GLIOMAS CONFER UNIQUE INFLAMMATORY MICROENVIRONMENTS
Authors: Ross James , Pinero Gonzalo , Puigdelloses-Vallcorba Montserrat , Soni Nishant , Angione Angelo , Becher Oren , Tsankov Alexander , Hambardzumyan Dolores
Published: November 2023

Significant progress has been made molecularly defining pediatric high-grade gliomas (pHGG), including diffuse midline gliomas (DMG), yet little progress has been made with respect to delineating the inflammatory microenvironment. We utilize molecularly defined human samples and immunocompetent genetic mouse models to study how tumor location and genetic driver mutations influence the tumor microenvironment (TME). We report human DMGs have a significant enrichment of Elane+ neutrophils compared to their hemispheric counterparts. We validate this utilizing the RCAS-Tva mouse model, which histologically and genetically recapitulates human pHGGs. Using this model we demonstrate each distinct pHGG/DMG entity confers unique transcriptional identities as made evident by NanoString RNA expression profiles and whole-tumor single-cell RNA sequencing. H3.3WT DMGs and H3.3K27M DMGs cluster together and have high expression of inflammatory genes such as Ptprc, Trem2, Lag3, and Cd274 while H3.3WT and H3.3G34R hemispheric tumors, and H3.1K27M DMGs cluster together with low expression of these genes. Several genes were found to significantly correlate with median survival in human bulk RNA sequencing data including IL13RA2. Flow cytometry and immunohistochemistry demonstrate H3WT DMGs are enriched for monocytes and lymphocytes while H3.3K27M DMGs are enriched for microglia. Genetic perturbations were made to prevent TAM or neutrophil infiltration, including targeting Ccl3, Cxcl1, and Ccl8/12. Only Ccl8/12 knockout resulted in significant extension of survival in H3K27M DMGs, which was met with an increase in CD4+ and CD8+ T-cells and decreased neutrophil infiltration. CD4+ T-cell depletion and anti-PD1 therapy was performed to further study the role of lymphocyte infiltration in DMGs. Lastly, CCR1/CCR5 inhibitors were utilized to abrogate TAM infiltration in DMGs, resulting in decreased microglia infiltration and significant survival extension comparable to radiation therapy. Together, this work provides the foundation for developing or improving immunotherapies designed at specific subgroups of pHGG and DMGs, such as CAR-T-cell, oncolytic viral therapy, and checkpoint blockade.
TMIC-17. THE ROLE OF ICAM1 IN GLIOBLASTOMA TUMORIGENESIS UNDER HYPOXIC CONDITIONS
Authors: Mansouri Sheila , Devaraja Kaviya , Zadeh Gelareh , Singh Olivia , Ali Hafsah , Wei Qinxqia , Sosa Julio , Patil Vikas
Published: November 2023

Glioblastoma (GBM) is a deadly brain cancer in adults with limited treatment options. Cell adhesion molecules (CAMs) mediate cell-cell interactions and migration. Intercellular adhesion molecule 1 (ICAM-1) is a CAM expressed by various cell types in solid tumors, including macrophages, tumor and endothelial cells. We previously demonstrated that ICAM-1 expression is linked to shorter overall survival in GBM patients. We observed elevated ICAM-1 levels in hypoxic regions of GBMs. Considering the role of tumor-associated macrophages (TAMs) in GBM tumorigenicity, our objective was to investigate how ICAM-1 expression in TAMs contributes to GBM growth within the hypoxic tumor microenvironment (TME). We employed a comprehensive approach utilizing in vitro and in vivo models, as well as genomic analyses at both bulk and single-cell levels, to elucidate the association between ICAM-1 and TAMs in tumorigenicity. Our findings demonstrated that injecting GBM cells into ICAM-1 knockout mice led to prolonged survival, smaller tumors, reduced macrophage infiltration, and enhanced M1-like polarization of TAMs. Additionally, bone marrow reconstitution experiments confirmed the tumor-promoting role of ICAM-1-expressing TAMs. Cell deconvolution, immunohistochemical, and flow cytometry analyses of mouse tumors supported these findings. Gene set enrichment analysis of differentially expressed genes in xenografted tumors revealed that ICAM-1 presence upregulated key tumorigenic pathways, including epithelial-mesenchymal transition, IL2-STAT5, TNF-α via NFκB, IL6-JAK-STAT3, and RAS/MAPK. In vitro analysis indicated that ICAM-1 promoted macrophage migration and polarization towards M2-like state, which was further augmented by hypoxia-induced ICAM1 expression. Flow cytometry, cytometry by time of flight (cyToF), and single-cell RNA sequencing analysis of human and mouse GBMs revealed elevated ICAM-1 expression in hypoxic TAMs, suggesting the hypoxic microenvironment promotes the tumorigenic functions of TAMs. In conclusion, ICAM1 likely enhances GBM tumorigenesis by promoting TAM migration into the TME. Furthermore, environmental factors such as hypoxia contribute to increased ICAM-1 expression in TAMs, further exacerbating GBM tumorigenicity.
TMIC-20. A SPATIALLY RESOLVED HUMAN GLIOBLASTOMA ATLAS REVEALS DISTINCT CELLULAR AND MOLECULAR PATTERNS OF ANATOMICAL NICHES
Authors: Shah Nameeta , Park Hyun Jung , Sonpatki Pranali , Han Kyung Yeon , Yu Hyeon Jong , Kim Shin Wook , Chowdhury Tamrin , Byun Yoon Hwan , Kang Ho , Lee Joo Ho , Lee Soon-Tae , Won Jae-Kyung , Kim Tae Min , Choi Seung Hong , Shin Young-Kyoung , Ku Ja-Lok , Lee Sungyoung , Yun Hongseok , Park Sung Hye , Park Chul-Kee , Park Woong-Yang
Published: November 2023

Glioblastoma (GBM) is a highly plastic ecosystem where the complex interplay between different cellular components contributes to disease progression. Although single-cell RNA (scRNA)-seq has revealed remarkable cellular heterogeneity of GBM, our knowledge regarding the spatial organization of its cellular components is currently lacking. Here we created a comprehensive dataset of 115,914 spatial transcriptomes across 32 tissue sections with matched multi-omics profiling on a set of genotyped glioma samples. We present spatial maps of 56 fine-grained cellular components, including previously unrecognized subtypes of oligodendrocytes and stromal cells, and their spatial interaction networks in each GBM- associated anatomical niche. Furthermore, the deconvolution of bulk RNA-seq data using the integrated spatial and single-cell atlas revealed clinically relevant GBM ecotypes. Our data provides comprehensive insights into the cellular architecture of GBM at high spatial resolution. It will be a valuable resource to develop effective combinatorial therapies to target all tumor-fostering niches simultaneously.
TMIC-21. SPHINGOSINE-1-PHOSPHATE RECEPTOR 3 (S1PR3) IN THE GLIOBLASTOMA TUMOR MICROENVIRONMENT MAY PLAY A ROLE IN IMMUNOSUPPRESSION
Authors: Michelhaugh Sharon , Munson Jennifer
Published: November 2023

Glioblastoma remains an intractable clinical problem with few efficacious treatments and poor patient outcomes because of the challenge in treating the tumor cells that have invaded into the normal brain. These invasive tumor cells typically remain after standard-of-care surgical resection and likely persist to generate recurrent tumors. In our previous studies of the glioma tumor microenvironment (TME), we identified sphingosine-1-phosphate receptor 3 (S1PR3) as upregulated in glioma TME regions with increased fluid flow. In order to efficiently study S1PR3 in the normal brain/ glioblastoma tumor interface, we engaged the Wake Forest University Brain Tumor Center of Excellence to construct a tissue microarray (TMA) including glioblastoma tissue cores specifically of the normal brain/ tumor interface as determined by pathologist review. The TMA included FFPE tissue cores from 42 GBM patients (average age 67.7 ± 10.9 years, 25 male, 17 female). Immunohistochemistry to detect S1PR3 was performed using an antibody that targets the amino-terminal domain followed by spatial profiling using neuroscience, neuroinflammation, and immune antibody panels (Akoya Biosciences). Preliminary analyses of the spatial profiling revealed 34 evaluable tissue cores, with no difference in the percentage of neurons, glial cells, vascular cells, or T cells between the S1PR3 positive and negative cores, however a significant difference in the percentage of M1 macrophages/ antigen presenting cells was found between the two groups (S1PR3-positive 0.646 ± 0.569%, n=13; S1PR3-negative 2.95 ± 3.28%, n=21, p=0.0178 two-tailed t-test. These findings suggest that the presence of S1PR3 may be associated with reduced M1 macrophages/ antigen presenting cells and therefore S1PR3 may contribute to glioblastoma tumor associated immunosuppression.
TMIC-22. IDENTIFICATION OF EXOSOME MIRNA-MRNA INTERACTIONS IN THE GLIOMA TUMOR IMMUNE MICROENVIRONMENT
Authors: Xu Emily , Krueger Katharine , Sussman Jonathan , Akca Ebrar , Matache Irina-Mihaela , Halthore Amitha , Patterson Jonathan , Amankulor Nduka
Published: November 2023

INTRODUCTION

Exosomes have emerged as key local and systemic immune modulators in glioma, particularly in tumors with an IDH mutation. However, the exact mechanism by which these vehicles communicate with the tumor immune microenvironment warrants further exploration. We sought to identify exosomal miRNA differences in discrete genomic subsets in glioma and predict potential mRNA targets in tumor immune cell populations.

METHODS

Exosomes were isolated from IDH wild-type (IDHwt) and IDH mutant (IDHm) glioma organoids using ultracentrifugation. Nanoparticle Tracking Analysis and western blot were used to validate exosome capture. Exosomal miRNA was sequenced using the Illumina Novaseq platform and targets were predicted using TargetScan. Peripheral blood mononuclear cells (PBMCs) were isolated from glioma patients and processed for scRNA-seq.

RESULTS

The characteristics of isolated exosomes were consistent with previously published data. Six miRNAs were over-expressed in IDHm exosomes compared to IDHwt. Interestingly, none of these miRNAs were significantly up-regulated in IDHm tumor tissue. Comparison between predicted targets of IDHm exosome miRNA and scRNA-seq of circulating tumor PBMCs identified a set of expressed genes involved in the FOXO pathway, which is implicated in microglia activation and polarization. Furthermore, scRNA-seq analysis showed that the FOXO3 pathway is activated in CD14+AREG+ monocytes and CD14+RNA-lo monocytes, which are two cell populations that are fractionally down-regulated in IDHm glioma.

CONCLUSION

Our group and others have defined mutation-dependent mechanisms of immunosuppression in glioma. Here, we characterized the effects of tumor mutational status on exosome miRNA cargo and conducted a miRNA-mRNA network analysis to identify potential targets in tumor-associated myeloid cells. Our results suggest that exosomes may play a role in suppressing specific populations of monocytes via the FOXO pathway in an IDH-mutation dependent manner. This data sets the stage for mechanistic exploration of glioma exosomes on peripheral myeloid tumor immunity.
TMIC-28. TARGETING TUMOR-ASSOCIATED SMOOTH MUSCLE ACTIN (ACTA2) EXPRESSION IN MOUSE PRE-CLINICAL GLIOMA MODELS REDUCES TUMOR PROGRESSION AND EXTENDS SURVIVAL
Authors: Comba Andrea , Varela Maria Luisa , Abel II Cliford , Faisal Syed , Castro Maria , Lowenstein Pedro
Published: November 2023

The ACTA2 gene, which encodes α-smooth-muscle actin, is known to be involved in contractility and oncogenic transformation. However, the specific role of tumor-associated ACTA2 mesenchymal cells in gliomas is unknown. Using genetically engineered mouse models (GEMM) of glioma, we discovered that ACTA2 expression was increased within oncostreams, which are malignant multicellular structures found in high-grade gliomas. Furthermore, we observed reduced expression of ACAT2 mRNA in IDH-Mut mouse gliomas vs WT tumors. Analyzing human glioma TCGA and CCGA databases revealed a negative correlation between tumor ACTA2 expression and overall survival in glioma patients. ACTA2 mRNA expression was also higher in grade IV and recurrent tumors. To investigate the role of ACTA2 in glioma cells, we created ACTA2 knockdown GEMM of glioma. These mice showed very large increases (~300%) in median survival (NPDS: 253 days, NPAS: 329 days) in comparison to their respective control groups (NPD: 89 days, NPA: 97 days). Glioma neurospheres derived from knockdown tumors confirmed downregulation of ACTA2, and showed reduced cell proliferation compared to controls. Moreover, orthotopic models revealed a decreased potential for tumor development in vivo. To explore the mechanism mediating the effects of ACTA2 downregulation, we conducted RNA-Seq analysis of ACTA2 knockdown neurospheres. This analysis identified approximately 5000 differentially expressed genes, with gene ontology analysis highlighting significant enrichment in biological processes such as cell adhesion, cell communication, and regulation of locomotion. Pathway analysis identified cytokine-receptor interactions, focal adhesion, and NFK-B signaling as the most significant signaling pathways affected by ACTA2 downregulation. Finally, we evaluated the release of cytokines into the cell-conditioned media and found significantly decreased levels of GSCF and GMSCF cytokines in ACTA2 knockdown compared to controls. These findings indicate that ACTA2 expression is a pro-tumoral gene involved in glioma progression and plays an essential role in altering the TME.
TMIC-38. MITOCHONDRIAL ATP BIOGENESIS REGULATED BY VDAC1 IN TMEM119+ TUMOR-ASSOCIATED MICROGLIA AND MACROPHAGES MEDIATES HIGH-GRADE GLIOMA GROWTH
Authors: Wu Caren , Chen Yiyun , Lin Ya-Jui , Wei Kuo-Chen , Chang Kwang-Yu , Feng Li-Ying , Wu An-Chih , Chen Ko-Ting , Ren Alexander Liang , Nitta Ryan , Wu Janet , Pant Ayush , Cho Kwang Bog , Mackall Crystal , Chuang Jian-Ying , Huang Chiung-Yin , Li Gordon , Jackson Christopher , Chen Pin-Yuan , Lim Michael
Published: November 2023

Patients with high-grade glioma have a poor prognosis and an average survival of less than 15 months, which is associated with an increase in tumor-associated microglia and macrophages (TAMs). TAMs in the glioma microenvironment are traditionally thought to suppress antitumor immune responses and are metabolically reprogrammed by glioma. However, it is unknown whether metabolic processes, like ATP synthesis, in TAMs can directly affect glioma growth. Through RNAseq, we identified a novel subpopulation that had elevated metabolic expression patterns. These TAMs (TMEM119+) had increased protein expression of ATP synthases and VDAC1, and surprisingly only TAMs located within the tumor center had increased mitochondrial energy potential. In vitro and ex vivo co-culture assays determined that activated TAMs increased the expression of metabolic, growth, and survival genes in high-grade glioma cells. These activated TAMs produced higher levels of extracellular ATP, which in turn increased glioma cell viability. Inhibition studies identified P2X purinoceptor 7 (P2X7R) as a key player in the TAMs ATP-induced glioma survival. Our findings demonstrate for the first time that a subpopulation of TAMs induced a more tumorigenic microenvironment through the secretion of ATP.
TMIC-52. HISTONE H3K27ME3 LEVEL GOVERN TUMOR-ASSOCIATED MICROGLIA REPROGRAMMING IN RESPONSE TO GBM
Authors: Kang Sangjo , Sattiraju Anirudh , Ughetta Mary , Tsankov Alexander , Friedel Roland , Zou Hongyan
Published: November 2023

Glioblastoma (GBM) is the most common malignant primary brain tumor and its invasive nature makes complete dissection difficult. However, how GBM invade in the brain is poorly understood. Although, microglia are the resident immune cells in the brain that can support GBM invasion, their roles are yet elusive. Here, we retraced early steps of GBM invasion and their interaction with tumor-associated microglia (TAM) in a highly infiltrative murine GBM model in immunocompetent background. We show that microglia are activated in a wide tumor field ahead of GBM invasion, followed by glial net formation, encircling invading tumors. Next, physical contacts with GBM cells initiate an astounding morphological, and functional transformation of microglia and marrow-derived macrophages to form collectively organized migration streams with intertwined GBM cells. Here we show that Kdm6b, a H3K27me3 demethylase, is induced in TAMs during this process. Intriguingly, GBM disseminated more widely in juvenile host brains, which was associated with highly induced Kdm6b expression. Moreover, we show that Kdm6b inhibition with small chemical inhibitor GSKJ-4, perturbs TAM reprogramming, and GBM invasion. Together, our data reveal that microglia reprogramming provide directional cues for GBM invasion through epigenetic mechanisms and this may give insights to halt GBM invasion.
TMIC-57. CHARTING THE SPATIAL ARCHITECTURE OF GLIOBLASTOMA USING GBMAP: INSIGHTS FROM IN SITU SEQUENCING
Authors: Ruiz Cristian , Marco-Salas Sergio , Samuelsson Erik , Grillo Marco , Brandner Sebastian , Roy Ananya , Forsberg-Nilsson Karin , Kranendonk Mariëtte , Nilsson Mats , Stunnenberg Hendrik
Published: November 2023

Glioblastoma (GB), an aggressive brain malignancy, is associated with poor prognosis and quality of life. Single-cell RNA sequencing has helped unravel the complexity of GB cell states. However, to comprehensively unravel the pathobiology of GB, it is essential to consider the spatial context in which these cells reside within the tumor microenvironment (TME). In this study, we aimed to understand the spatial architecture of GB tumors and its implications on cellular and molecular heterogeneity. To achieve this, we developed GBmap, a comprehensive resource that harmonizes 26 datasets comprising 239 patients and over 1.1 million cells. Leveraging computational advancements, we utilized GBmap to create a GB-specific panel for charting the spatial architecture of tumors using an in situ sequencing (ISS)-based platform (HybISS) in an independent cohort of 13 tumors. We curated a panel of 209 genes based on the GBmap, capturing cell phenotypic features, intra- and inter-tissue heterogeneity, and in silico L-R analysis predictions. The panel includes markers for specific cellular processes such as inflammation, extracellular matrix remodeling, hypoxia, and angiogenesis, providing an additional layer of functional information. Our optimized pipeline incorporates a new method for ISS-based transcriptomics data (direct RNA targeting), which enhances sensitivity and tolerance for tissue specimens of poorer RNA quality. This new approach enables improved cell type/state resolution and classification confidence. Using this advanced approach, we observed heterogeneous cell-cell colocalization patterns that vary among patients, revealing spatiotemporal distributions of specific cancer phenotypes and distinct co-localization patterns with neighboring cells in the TME. Our findings showcase the unprecedented spatial and cellular resolution achieved, surpassing previous GB studies. Our study sheds light on the spatial architecture of GB tumors, unveiling previously unexplored cellular and molecular interactions. GBmap and our optimized pipeline offer a powerful resource for researchers to investigate GB heterogeneity, generate hypotheses, and contribute to advancing GB research.
TMIC-68. SIRPΑ BLOCKADE AS AN IMMUNOTHERAPEUTIC STRATEGY TO TREAT BREAST CANCER BRAIN METASTASIS
Authors: Tsai Yu-Ting , Wilson Adam , Cook Katherine , Zhao Dawen , Lesser Glenn , Soto-Pantoja David R
Published: November 2023

Triple-negative breast cancer (TNBC) has a high incidence of brain metastasis, with no standard therapeutic strategy for treatment. Therefore, new targets are urgently needed to overcome disease mortality. The CD47/SIRPα signaling pathway is implicated in tumor progression due to bypassing innate and adaptive immune surveillance. Most strategies targeting this pathway focus on targeting the receptor CD47; however, direct targeting SIRPα as a potential approach to mitigate metastatic burden remains understudied. Single-cell-RNA-sequencing indicates that SIRPα expression on basal epithelial cells, and TCGA data shows SIRPα expression is associated with a significant reduction in survival in basal-like breast cancer. Our immune staining against SIRPα in breast cancer patient biopsies shows a 3.5-fold increase in expression in metastatic lesions compared to the primary tumor (n=19; p ≤ 0.01). Staining of 4T1 parental and brain-trophic 4T1-Br3 cells showed an 84% increase in SIRPα in the metastatic cells (n=3; p ≤ 0.05). Furthermore, Real-time cell impedance analysis revealed that SIRPα blockade inhibits TNBC 4T1br3 cell migration (n=4; p≤0.01), suggesting a link between SIRPα expression and metastatic potential. Furthermore, in vivo SIRPα blockade reduced brain metastatic burden by approximately 90%, (n=4-7; p ≤ 0.05). Digital spatial profiling of tissues revealed that SIRPα blockade was associated with reductions in immune checkpoints, including CTLA-4, PD-1, PD-L1, and LAG3, and immunosuppressive T-cell/monocyte populations. Furthermore, our spatial analysis indicated that brain lesions of WT mice have increases in the extracellular matrix protein fibronectin in the immune enriched regions, potentially serving as a barrier for immune cell infiltration contributing to invasion, metastasis, and immune evasion, was reduced by 70% in SIRPα treated brain lesions (n-3-6; p ≤ 0.05). Thus, these data suggest that SIRPα blockade may influence the tumor microenvironment to limit brain metastatic breast cancer growth and potentially enhance patient survival to metastatic brain disease.
TMIC-70. A TRANSCRIPTIONALLY ACTIVE EPIGENETIC IMPRINTING OF THE JAK/STAT PATHWAY MEDIATED BY H3.3-G34 MUTATIONS IN PEDIATRIC HIGH-GRADE GLIOMA (PHGG) INDUCES AN IMMUNOREACTIVE TME
Authors: Haase Santiago , Fabiani Maria Belen Garcia , Banerjee Kausjik , Mujeeb Anzar Abdul , Li Yingxiang , Zhu Ziwen , Nunez Felipe , Yu Jennifer , Lowenstein Pedro , Castro Maria
Published: November 2023

Pediatric high-grade gliomas (pHGGs) stand as the primary cause of cancer-associated fatalities among children. Sixteen percent of pHGGs found in children and young adults present Gly34Arg/Val substitutions within the histone H3.3 (H3.3-G34R/V). Using a syngeneic, genetically engineered mouse model (GEMM) and human pHGG cells encoding H3.3-G34R, we showed that this mutation leads to downregulation of the DNA repair pathways and enhanced susceptibility to DNA damage. DNA damage response (DDR) inhibitors in vivo, further increased genetic instability, and activation of the cGAS-STING pathway, which induces the release of immune-stimulatory cytokines. We carried out an epigenetic analysis using ChIP-seq in our GEMM, and our findings indicate that this mutation triggers transcriptionally active chromatin states within genes linked to immune reactivity, particularly JAK-STAT signaling pathway genes. Correspondingly, G34-mutant pHGG cells release immune-activating cytokines that reprogram the immune cells within the tumor microenvironment (TME). Myeloid derived suppressor cells from G34-mutant pHGG are phenotypically less immunosuppressive than the MDCS form histone wild type pHGG. Moreover, G34-mutant pHGG TME exhibit higher frequency of CD8 T cells. Based on these findings, we assessed the efficacy of an adenoviral-mediated immunostimulatory gene therapy (GT). This therapy mediates expression of Flt3L within pHGG TME, which aids dendritic cell recruitment. Thymidine kinase (TK), converts ganciclovir (GSV) into a cytotoxic compound, leading to pHGG cells’ death. GT resulted in 60% of long-term survival in mice harboring G34R pHGG. The animals that survived due to the GT did not develop tumors after a rechallenge with G34R pHGG cells, demonstrating that the treatment induces antitumoral immunological memory. In conclusion, our findings suggest that G34-mutant pHGG patients could benefit from combined therapies that target genetic instability and stimulate the immune system. Funded by NIH/NINDS.
TMIC-73. UNRAVELING THE ALTERED EXTRACELLULAR MATRIX LANDSCAPE IN GLIOBLASTOMA AFTER RADIATION TREATMENT
Authors: Manou Dimitra , Rosberg Rebecca , Pietras Alexander
Published: November 2023

BACKGROUND

Extracellular matrix (ECM) is a complex network of macromolecules, influencing cellular behavior and tissue integrity. Abnormal alterations in the composition and organization of the extracellular matrix have been implicated not only in the pathogenesis of numerous diseases, including cancer but are also related to the state of the disease. Despite advancements in treatment, glioblastoma (GBM) exhibits a high rate of recurrence. Tumor microenvironment (TME) plays a critical role in the regulation of cell behavior, and one important understudied tumor determinant is the ECM. Previous studies of our laboratory revealed that the matrix derived from irradiated astrocytes positively influences the stemness capacity and radio-resistance of GBM cells.

MATERIAL AND METHODS

The first aim of this project is to elucidate the landscape of important extracellular matrix hits in the TME of primary and recurrent GBM, using an in-house single seq database and an ECM-focused proteomic analysis in decellularized tissues. The second aim is to spatially map important neighborhoods with common crucial cell populations including astrocytes and macrophages and specialized regions such as perivascular and hypoxic niches using multiplex analysis.

RESULTS AND CONCLUSIONS

Preliminary data indicate a significant imbalance between fibrous components and glycans in the ECM after irradiation. In light of these considerations, our goal is to understand the TME interactions of cell populations with the matrix and the post-irradiation effect on matrix composition, aiming for an opportunity window for combinational ECM-targeted therapies.
TMIC-74. SPATIAL LANDSCAPE OF MELANOMA BRAIN METASTASES WITH AND WITHOUT PRE-SURGICAL IMMUNE CHECKPOINT BLOCKADE
Authors: Sun Lu , Ding Lizhong , Monteleone Amber , Liau Linda , Hugo Willy , Kim Won , Prins Robert
Published: November 2023

We recently characterized the immune populations of brain metastases samples with or without immune checkpoint blockade (ICB) treatment. In that study, we utilized single cell RNAseq (scRNAseq) and multiplex immunofluorescence (mIF) to demonstrate that ICB induced T cell infiltration and the migration of a unique MRC1+ macrophage subset from the perivascular space into the tumor parenchyma. However, scRNAseq’s tissue dissociation step loses spatial information of cells while mIF has low dimensionality, covering only a few markers for each tissue section. Thus, we applied sequencing-based Visium spatial transcriptomics (ST) to study the spatial architecture of melanoma brain metastases with/without ICB treatment and developed a pipeline that leverages public scRNAseq data as a reference to resolve fine-grained cell types on each Visium spot. ICB was associated with an increased CXCL13+ terminally exhausted T cell subset in close spatial proximity to tumor cells, suggestive of tumor specificity. ICB also reduced multiple MRC1+ macrophage subtypes in the blood vessel regions, supporting our mIF observation. Furthermore, we discovered that MRC1+ macrophages in close proximity to blood vessels expressed elevated TIMP1. TIMP1 is a known inhibitor of MMP9 that prevents ECM degradation and blood vessel damage, thus limiting T cell extravasation. In contrast, MRC1+ macrophages that migrated into the tumor had higher interferon activation and expressed CXCL9 and CXCL12 chemokines, which could recruit T cells via CXCR3 and CXCR4. Such findings could explain the increased T cell infiltration with ICB even in metastatic brain tumors. However, these MRC1+ macrophages also had an M2 signature and expressed multiple immune checkpoint ligands. Such data suggest these cells might also increase immunosuppressive interactions with T cells. In summary, our study reveals how ICB affects the spatial architecture and crosstalk of different cell types within melanoma brain metastases and will provide insights into new therapeutic strategies for brain metastases patients.