bluuebunny/crossref_metadata_2025 huggingface dataset:
TMET-32. INTRAOPERATIVE MICRODIALYSIS FOR GLIOMA METABOLIC RECONNAISSANCE AND BIOMARKER DISCOVERY
Authors: Riviere-Cazaux Cecile , Carlstrom Lucas , Rajani Karishma , Munoz-Casabella Amanda , Rahman Masum , Burns Terry
Published: November 2022

Gliomas are genomically heterogeneous tumors that may harness a convergent and therapeutically targetable set of metabolic pathways. At present, the metabolic landscape of in situ human gliomas remains incompletely characterized, hampering translational progress. To that end, we are leveraging the previously untapped potential of high molecular weight microdialysis to determine the global extracellular metabolic profiles of live human gliomas. Under an investigational device exemption, HMW microdialysis (< 100 kDa) was performed at 2.0 μL/min in an initial discovery cohort of five patients in glioma and adjacent brain during neurosurgical resection; a subsequent cohort of five patients was independently analyzed to critically evaluate results from the discovery group. Untargeted metabolomics via ultra-performance liquid chromatography-tandem mass spectrometry revealed over 300 named metabolites and five drugs from only 20 μL of microdialysate, representing a short and feasible 10 minutes of intraoperative collection time. Enrichment analysis of each patient’s tumor vs. brain ranked extracellular metabolome highlighted marked metabolic convergence within the most aggressive regions of molecular diverse tumors (FDR = 0). Pathway analysis revealed significant enrichment for large neutral amino acid pathways, including valine, leucine, and isoleucine biosynthesis (p=1.6E-9) and degradation (p=0.001) and glycine, serine, and threonine metabolism (p=4.7E-5). Notably, this amino acid signature was not as abundantly present in nonenhancing tumor when compared to enhancing tumor (Average tumor/brain: 1.9x vs. 4.3x, respectively), suggesting upregulation of neutral amino acid transporters in enhancing tumor or delivery from plasma into the CNS via a disrupted BBB. Interestingly, guanidinoacetate (GAA) was our most highly conserved and upregulated metabolite (128.9x in tumor vs. brain). Given its co-production with ornithine, the precursor to protumorigenic polyamines, we posit that GAA may serve as a biomarker of increased ODC activity in live human gliomas. In conclusion, intraoperative HMW microdialysis feasibly offers improved opportunities to perform glioma metabolic biomarker and therapeutic discovery.
TMET-37. SODIUM-DEPENDENT GLUCOSE TRANSPORTER 2 (SGLT2) IS OVEREXPRESSED IN THE MAJORITY OF ATRTS AND A SUBSET OF PEDIATRIC PATIENTS WITH HIGH-GRADE GLIOMA; A POTENTIAL IMAGING AND THERAPEUTIC TARGET
Authors: Nabavizadeh Ali , Zhao Chao , Martinez Daniel , Familiar Ariana , Koptyra Mateusz , Ippolito Joseph , Rubin Joshua , Storm Philip B , Resnick Adam C , Santi Mariarita
Published: November 2022

Sodium glucose cotransporters (SGLT) are a class of glucose transporters independent of the conventional glucose transporters (GLUT). Like the GLUT, SGLT2 plays an important role in glucose uptake in many solid cancers. Importantly, SGLT2 inhibitors are a new class of anti-diabetic drugs and have recently been investigated as a potential anti-cancer therapy in a variety of solid cancers. In fact, a recent study in adult high-grade gliomas showed that an SGLT-specific PET imaging probe ([18F] Me-4FDG) is sensitive for visualization of high-grade glioma (HGG) with superior tumor-normal brain uptake relative to 18F-FDG-PET. We aimed to investigate the expression of SGLT2 within pediatric brain tumor subtypes using RNA sequencing data from PedcBioPortal and immunohistochemistry of tissue microarrays (TMAs). Using the RNA sequencing data from 603 patients with pediatric brain tumors, we found that HGGs have the highest SGLT2 expression (Z score=0.13± 1.03) and were significantly higher compared to low-grade gliomas (Z score= -0.12, p=0.049), medulloblastomas (Z score=-0.28, p=0.001), and ependymomas (Z score=-0.30, p=0.002). Atypical teratoid rhabdoid tumors (ATRT) also had significantly higher Z score (0.10±0.67) compared to medulloblastomas (p=0.038), and ependymomas (p=0.049). There was no significant difference between HGGs and ATRTs in SGLT2 expression (p=0.899). Analysis of 4 TMAs including HGGs, medulloblastomas, ependymomas, and ATRTs after SGLT2 immunohistochemistry demonstrated strong membranous staining in a small subset and moderate-weak staining in the majority of HGGs. The majority of ATRTs were positive. The majority of medulloblastomas and ependymomas were negative, noting that the membranous nature of the staining may be a limiting factor in evaluation of tumors with less cytoplasm. In conclusion, our work demonstrates that SGLT2 has heterogeneous expression in pediatric brain tumors and is overexpressed in the majority of ATRTs, and a subset of pediatric HGGs, and may represent a compelling paradigm that integrates metabolic imaging and therapy of these tumors.
TMIC-12. COMPLEMENT SIGNALING IN HYPOXIC NICHES OF THE GLIOMA MICROENVIRONMENT
Authors: Rosberg Rebecca , Smolag Karolina , Sjölund Jonas , Pantazopoulou Vasiliki , Pietras Kristian , Blom Anna , Pietras Alexander
Published: November 2022

Glioblastoma is the most common and aggressive primary brain tumor in adults. Despite treatment through surgery, irradiation and chemotherapy, all patients suffer recurrence of treatment-resistant tumors. Previous studies from our lab showed that astrocytes become reactive and generate tumor-supportive conditions for glioma cells, when exposed to hypoxia or irradiation. Reactive astrocytes are known to upregulate proteins of the complement system, especially complement component 3 (C3), in several neurological disorders. However, it remains unexplored how these complement proteins are expressed in stromal astrocytes in glioblastoma. Tissue sections from a glioma mouse model showed presence of C3 in the invasive front, and in hypoxic, and perivascular spaces of the tumors, where there is an abundance of astrocytes. C3 expression co-localized with Nestin and CD44, two markers of mesenchymal and/or stem-like glioma cells. Primary human astrocytes grown in hypoxic conditions upregulated C3 as well as other proteins associated with a more extensive infiltrative phenotype of glioblastoma. In general, a strong correlation between hypoxia (195 genes) and complement (200 genes) gene signatures were found in TCGA GBM dataset (R= 0.82, p-value= 0). Single-cell sequencing data from primary GBM tumors showed a subpopulation of highly C3-expressing astrocytes, which were enriched for cellular pathways comprising epithelial mesenchymal transition (EMT), TNF-alpha, complement, hypoxia, and interferon signaling. Human glioma cell lines from proneural, classical and mesenchymal subtypes all showed increased CAIX and GLUT1 expression grown under lower oxygen tensions, while two out of three subtypes showed upregulation of stemness markers (OCT4 and NANOG1) as well as C3 and C3aR. Overall, our data indicate a strong link between hypoxia and complement expression in the brain tumor microenvironment, where a local expression of complement proteins could possibly lead to tumor promoting signaling, which will be further explored.
TMIC-13. GLIOMA-DRIVEN DENDRITIC CELL DYSFUNCTION AS AN IMMUNE EVASION STRATEGY AGAINST ADOPTIVE CELLULAR THERAPY
Authors: Jin Dan , Dean Bayli DiVita , Francis Connor , Font Laura Falceto , Reid Alexandra , Figg John , Sebastian Mathew , Flores Catherine
Published: November 2022

BACKGROUND

Our previous studies has developed an adoptive cellular therapy (ACT) against high grade gliomas in both human and murine systems. ACT significantly improves survival in murine models of CNS malignancies. Our studies have shown that efficacy is associated with the observed increase in tumor-associated dendritic cells (DC) which arise from transferred hematopoietic stem cells (HSC). HSC-derived DCs play a pivotal role in mounting the anti-tumor immune response. In this study, we have identified that treatment resistance may be due to glioma-driven dendritic cell dysfunction. We observed significant down regulation of co-stimulatory markers on tumor-associated dendritic cells in mice that escaped ACT and subsequent capacity to activate tumor-reactive T cells.Method: KR158B bearing mice received HSC and tumor reactive T cells one day before 9 Gy irradiation followed by 3 does of BMDC vaccine. Tumor associated DCs were sorted from tumors evaded from ACT and co-cultured with primary KR158B reactive T cells or escaped KR158B reactive T cells. T cell proliferation and interferon gamma were detected for comparison. PCR array was performed to identify dysregulated functional genes.Results and

CONCLUSIONS

Functional evidence demonstrated that dendritic cells from resistant tumor had significantly decreased capacity in activating tumor-reactive T cells against either primary KR158B glioma cells or T cells generated to target tumor cells that evaded ACT. Gene expression data showed this may be due to significant decreases in genes associated with co-stimulation, T cell engagement, and antigen presentation.
TMIC-19. NEURODEVELOPMENTAL SUBTYPES SHAPE LIPID METABOLIC REPROGRAMMING IN GLIOMAS
Authors: Minami Jenna , Bayley Nicholas , Tse Christopher , Zhu Henan , Salinas Jennifer , Morrow Danielle , Yong William , Cloughesy Timothy , Liau Linda M , Graeber Thomas G , Nathanson David
Published: November 2022

Gliomas have been classified into molecular (proneural, classical, mesenchymal) and neurodevelopmental (astrocyte, mesenchymal, neural progenitor cell (NPC), oligodendrocyte progenitor cell (OPC)) subtypes describing inter- and intra-tumoral heterogeneity; however, the functional outcomes and therapeutic implications of these subtypes has yet to be fully described. Metabolic reprogramming is a hallmark of cancer, and malignant cells, including gliomas, acquire metabolic adaptations in response to a multitude of intrinsic (oncogenotype, mutations) and extrinsic (tumor microenvironment) factors to fuel neoplastic progression. Altered metabolism in glioma is of particular interest given the extensive molecular heterogeneity of tumors, while also developing in the brain microenvironment, a tissue known for its unique metabolic milieu. It is unknown whether neurodevelopmental subtypes influence metabolism in gliomas. Preliminary comprehensive lipidomic and transcriptomic analysis of over 200 patient-derived glioma samples revealed that distinct lipid signatures were linked to neurodevelopmental subtypes. Specifically, proneural-like gliomas (OPC, NPC, Neuron) had a lipid metabolic profile enriched in ether lipids. Conversely, mesenchymal-like gliomas (radial glia, MES.progenitor, vascular) have a lipid metabolic profile enriched in triacylglycerides (TAGs). Intriguingly, these differences in lipid metabolic programs between subtypes were associated with environmental dependencies; in contrast to more mesenchymal like gliomas, which could grow irrespective of tumor microenvironment (brain or in vitro cell culture), the proneural-like gliomas required features of the brain microenvironment to accumulate complex fatty acids and grow. Collectively, these data emphasize the metabolic heterogeneity within gliomas, and reveal a subset of gliomas that lack metabolic plasticity in fatty acid biosynthetic programs, indicating a potential brain-microenvironment specific metabolic dependency linked to transcriptional identity that may be targeted for therapy.
TMIC-24. SINGLE-CELL ANALYSIS OF TUMOR-ASSOCIATED MICROGLIA AND MACROPHAGES FROM HUMAN GLIOBLASTOMA
Authors: Anand Atul , Olsen Rikke Frydendahl Sick , Burton Mark , Harwood Dylan Scott Lykke , Poulsen Frantz Rom , Halle Bo , Pedersen Christian Bonde , Kruse Torben , Thomassen Mads , Kristensen Bjarne Winther
Published: November 2022

INTRODUCTION

Patients with glioblastoma have a very poor prognosis. Tumor-associated microglia and macrophages (TAMs) constitute up to 30 % of the cells in glioblastoma, and they secrete cytokines, chemokines and growth factors that influence the microenvironment. The existence of different TAM subtypes appears to be more complex than the established M1 and M2 phenotypes, but their role in glioblastoma is not fully understood and rarely considered therapeutically. This could explain why many clinical trials fail despite of promising preclinical results. This project aims to interrogate the existence and characteristics of different TAM subtypes in human glioblastoma in order to identify novel subpopulations and therapeutic targets. MATERIALS AND

METHODS

Live CD11b+ TAMs were isolated from patient glioblastoma tissue, and single-cell RNA sequencing (scRNA-seq) was performed using the 10X Genomics Chromium platform. The data was processed and analyzed with the R-package Seurat.

RESULTS

We have sequenced 74,000 TAMs/microglia from three glioblastomas and two control brain biopsies. In the controls, we detected mostly microglia, while the primary glioblastomas showed a predominance of monocyte-derived TAMs. We identified 11 TAM subtypes, such as hypoxic, proliferating, interferon-induced, chemokine-producing and TNF-producing TAMs, as well as a novel subtype potentially involved in tumor progression.

CONCLUSION AND PERSPECTIVES

We have detected a spectrum of TAM subtypes, which is more complex than the established M1 and M2 phenotypes. Our findings confirm a recent TAM scRNA-seq study, and in addition, we identify a novel subpopulation, which express known tumor-promoting genes, normally expressed by cancer cells. We are currently validating our findings. This study contributes to the elucidation of the glioblastoma microenvironment. It will potentially lead to identification of novel clinically relevant targets affecting the TAM-glioblastoma interactions.
TMIC-27. SPATIALLY RESOLVED T CELL RECEPTOR SEQUENCING (SPTCR-SEQ) UNCOVERS REGIONAL DIVERSITY OF ANTI-TUMOR IMMUNE RESPONSES
Authors: Benotmane Jasim Kada , Kueckelhaus Jan , Beck Juergen , Schnell Oliver , Heiland Dieter Henrik
Published: November 2022

Regional diversity of anti-tumor immunity in glioblastoma (GBM) has been poorly investigated due to technical limitations. To overcome these hurdles, we developed SPTCR-seq (SPatial TCell Receptor sequencing) to spatially profile T cell immune response. Here, we performed 10X-Visium spatial transcriptomics and SPTCR-seq on 9 de-novo IDH wt glioblastoma. For SPTCR-seq, we captured enriched T-cell receptor (TCR) sequences by hybridization followed by Oxford nanopore (ONT) long-read sequencing. We established a computational pipeline for spatial demultiplexing, error correction and TCR reconstruction using a Seq2Vector model, followed by a long-term memory autoencoder to deconvolute and compare CDR3 sequence similarities across patients. The on-target rate for detected TCR genes reached over 85%, of which our long-TCR algorithm could completely reconstruct TCRs in over 70%. To decipher the regional diversity of T-cell clonality, we performed spatially weighted regression to estimate the overlap of T-cell clonal expansion to the recently described regional transcriptional niches. We found that myeloid cell infiltration was highly correlated with increased T cell diversity. We integrated single cell RNA-seq data and annotated T cell subtypes through the “natural barcode” of the CDR3 sequence, revealing that CD4+ T cells interact with myeloid cells via MHCII in segregated areas, also described as the Reactive Immune niche. In contrast, CD8+ T cells exhibit a dysfunctional/exhausted phenotype at the same location. In comparison to the myeloid-infiltrated regions, the hypoxia-associated niches showed increased CD8+ T cells with greater clonal expansion, suggesting an immune response anti-cancer immunity. Global spatial vector field analysis revealed a spatial co-occurrence of metabolic vulnerability and a mutational burden with CD8+ T effector cells negatively associated with myeloid cell infiltration. Integration of CDR3 sequences in different patients revealed higher similarity of motifs of CD4+ compared with CD8+ T cells in hypoxia areas. Our data provide new insights into the regional heterogeneity of cancer immunity.
TMIC-29. CHARACTERIZING THE ROLE OF TRIGGERING RECEPTOR EXPRESSED ON MYELOID CELLS 2 (TREM2)-EXPRESSING MICROGLIA/MACROPHAGES IN GLIOBLASTOMA
Authors: Peshoff Mekenzie , Gupta Pravesh , Oberai Shivangi , Milam Nancy , Ahmed Aml , Dang Minghao , Wang Linghua , Bhat Krishna
Published: November 2022

The tumor microenvironment in glioblastoma (GBM) contains numerous cell types, notably a rich immune compartment dominated by myeloid cells, such as brain-resident microglia and infiltrating macrophages. However, current immunotherapies target lymphoid components like T cells, which are sparse in the brain and the tumors originating there. Therefore, immune checkpoint drugs designed for systemic cancers have not yielded significant benefits for patients with glioblastoma, and the efficacy of immunotherapy for central nervous system malignancies lags behind treatments for other solid tumors. Further understanding of the myeloid compartment is necessary for developing glioblastoma immunotherapies. Our recent single cell RNA-seq (scRNA-seq) analyses have identified microglial clusters that are enriched for antigen presentation and phagocytic gene expression modules. These clusters were restricted to microglia and macrophages seen only in glioma patients, but not in myeloid cells derived from non-glioma brain controls. Triggering receptor expressed on myeloid cells 2 (TREM2), a receptor abundant on microglia and vital for the detection and phagocytosis of pathogens, was highly expressed in these clusters. Because microglia expressing TREM2 play a protective role in neurodegenerative diseases like Alzheimer's disease, we examined the function of TREM2+ myeloid cells in GBM using mice lacking TREM2. Implantation of GL261 cells in TREM2-/- mice showed significantly reduced survival relative to wild-type mice, suggesting a protective role of TREM2+ myeloid cells in glioma. Data on immunophenotyping of glioma xenografts from WT and TREM2-/- mice brains using state of the art technologies (e.g. Cytek) will be presented. We conclude that TREM2 is a phagocytic immunoregulator in glioblastoma, manifesting anti-glioma properties to TREM2+ myeloid cells.
TMIC-36. REPROGRAMMING THE GLIOBLASTOMA IMMUNE MICROENVIRONMENT WITH CONVECTION ENHANCED GENE THERAPY REVEALS INTRATUMOR IL6 DRIVES GLIOBLASTOMA IMMUNOSUPPRESSION AND GROWTH
Authors: Young Jacob S , Cho Nam Woo , Lucas Calixto-Hope , Seo Kyounghee , Santos Raquel , Liu S John , Phillips Joanna J , Ozawa Tomoko , Bhaduri Aparna , Aghi Manish , Berger Mitchel S , Raleigh David
Published: November 2022

The glioblastoma microenvironment is an immunosuppressive barrier to therapeutic innovation. We hypothesized intratumor convection enhanced delivery (CED) of gene therapy vectors could reprogram the glioblastoma immune microenvironment and elucidate therapeutic vulnerabilities. To test this, SB28 or GL261 glioblastoma allografts were implanted into immunocompetent mice and treated with CED of attenuated adeno-associated virus 9 vectors (AAV9) encoding experimental cytokines (Il1b, Ccl4, or Apoa1) underlying infiltration or activation of anti-tumor immune cells in other intracranial tumors. Serial intracranial bioluminescence was used to assess glioblastoma growth and animals were monitored for survival. The impact of gene therapy perturbations on the glioblastoma immune microenvironment was assessed using histology, immunohistochemistry, single-cell mass cytometry (CyTOF), and multiplexed cytokine assays. Serial body weight and systemic cytokine measurements showed no evidence of treatment toxicity. AAV9-APOA1 or AAV9-IL1B gene therapy CED treatments attenuated SB28 growth and prolonged survival, decreasing immunosuppressive macrophage infiltration and increasing CD8 T cell and microglia infiltration of the glioblastoma microenvironment compared to control AAV9 vectors. Gene therapy CED treatments did not attenuate GL261 growth or prolong survival, but CyTOF of human glioblastomas (n=6) in comparison to preclinical models revealed untreated GL261 glioblastomas were endogenously enriched in CD8 T cells and other lymphoid lineages compared to untreated SB28 or human glioblastomas. Multiplexed cytokine assays demonstrated suppression of intratumor IL6 is a conserved mechanism of action underlying glioblastoma gene therapy responses. Single-cell RNA sequencing analysis of 32,877 cells from human glioblastomas (n=11) showed IL6 is predominantly produced by radial glial like cancer stem cells or endothelial cells in the tumor microenvironment. In support of these findings, survival from intracranial SB28 glioblastomas was prolonged in Il6 knockout C57BL/6J mice compared to wildtype mice. In summary, we report a novel strategy using gene therapy and CED to reprogram the glioblastoma immune microenvironment, revealing IL6 drives glioblastoma immunosuppression and growth.
TMIC-38. CD39 EXPRESSION IN GLIOBLASTOMA TUMOR MICROENVIRONMENT DOES NOT AFFECT SURVIVAL OR T-CELL EXHAUSTION
Authors: Zheng Allison , Pappu Ananya , Gill Sabraj , Stelter Isabella , Aghi Manish
Published: November 2022

Glioblastoma (GBM) is the most common malignant brain tumor with a median survival of 15 months. GBM is very difficult to treat due to numerous factors, including the paucity of immune infiltration of the tumor itself. Efforts to overcome this have led to use of immune checkpoint inhibitors such as PD-1 inhibitor, which have revolutionized treatment of many other solid tumors, and yet have not been successful in the treatment of GBM. This has led to many studies investigating various other similar markers. One such marker is CD39, which is highly upregulated in GBM and functions as the first enzyme in the metabolic pathway of ATP breakdown to adenosine. Additionally, CD39 is thought to contribute to T-cell exhaustion. Like PD-1, CD39 inhibition has had preliminary success in the treatment of other solid tumors. However, when tested on GBM, blocking the ATP breakdown pathway did not improve survival in mice, even in conjunction with PD-1 inhibition. Our team sought to determine if the expression of CD39 on the cell surface functioned as an exhaustion marker by injecting two different cell lines of GBM into CD39 global null mice. There was no survival benefit seen in either cell line when compared to wild type mice. Next, expression of known exhaustion markers such as PD-1, TIM3, and LAG3 on tumor infiltrating lymphocytes were analyzed. Expression of TIM3 and 2B4 were found to be increased in CD39KO mice in comparison to wild type (p < 0.01). Additionally, numerous exhaustion markers such as LAG3, TIGIT, and CTLA4 were decreased on tumor in comparison to peripheral blood (p < 0.01). These results demonstrate the complex nature of immune signaling in GBM tumor microenvironment that our team continues to investigate.
TMIC-41. ARGININE DEPRIVATION ALTERS DNA DAMAGE RESPONSE IN ARGINOSUCCINATE SYNTHETASE NEGATIVE GLIOBLASTOMA AND POTENTIATES STANDARD OF CARE
Authors: Ghosh Avik , Perryman Richard , O'Neill Kevin , Syed Nelofer
Published: November 2022

Glioblastoma (GBM) is the most aggressive and prevalent primary brain malignancy with an average life expectancy of 18 months despite the standard of care which includes resection, temozolomide and radiotherapy. Thirty percent of GBMs show transcriptional loss of arginosuccinate synthetase 1 (ASS1), displaying arginine auxotrophy and hence, are amenable to arginine deprivation therapy by ADI-PEG20. Moreover, these tumours are more aggressive than their non-arginine auxotrophic counterparts. In this study we demonstrate that this may lie in part due to their enhanced ability to repair damaged DNA. ADI-PEG20 significantly altered the expression of DNA repair genes in vitro and in vivo. We observed downregulation of PCNA, ATR (p=0.0063) and CHEK1 (p=0.0150) and upregulation of CHEK2 (p=0.0033) and RAD51 (p=0.0230). PARP1 was also downregulated, albeit not statistically significant. Addition of ADI-PEG20 to temozolomide and radiation also caused significant downregulation of BRCA1 (p=0.0340) . With DNA repair gene upregulation contributing to treatment resistance and DDR inhibitors shown to potentiate current therapies in the literature, not only do our results help elucidate the mechanisms of radiosensitisation with ADI-PEG20, targeting this pathway with DDR inhibitors alongside the pleiotropic effects of ADI-PEG20 may provide a novel therapeutic combination for ASS1 negative GBM.
TMIC-43. HEDGEHOG LIGANDS FROM GLIOBLASTOMA CELLS INDUCE ASTROCYTES INHIBITION OF CANCER STEM CELLS
Authors: Seo Kyounghee , Liu S John , Daggubati Vikas , Phillips Joanna J , Clarke Jennifer , Prados Michael , Bhaduri Aparna , Lim Daniel , Ullian Erik , Raleigh David
Published: November 2022

BACKGROUND

The Hedgehog pathway directs gene expression programs that are essential for glioblastoma stem cells in vitro, but a clinical trial suggested Hedgehog pathway inhibition does not improve glioblastoma outcomes (ABTC-0904). In pancreatic and bladder cancer, Hedgehog signaling through the tumor microenvironment induces stromal morphogens to inhibit cancer stem cell proliferation. Thus, we hypothesized Hedgehog signaling through the glioblastoma microenvironment may inhibit glioblastoma stem cell proliferation in vivo.

METHODS

Hedgehog signaling was studied using patient-derived, fluorescently-labeled IDH wild-type glioblastoma cells (GBM6, SF11956, SF11907, GPMP004, GPMP005) in vitro, in vivo after intracranial implantation in mice, or in 3D co-culture with iPSC-derived organoids comprised of human astrocytes or neurons. Genetic loss-of-function experiments were performed using CRISPR interference. Cell proliferation, stem cell marker expression, Sonic Hedgehog (SHH) expression, or primary cilia architecture were assessed using immunofluorescence, confocal microscopy, or live-cell imaging. Hedgehog signaling or morphogen expression were assessed using QPCR or single-cell RNA sequencing. Pharmacologic experiments were performed using vismodegib to inhibit the Hedgehog pathway, or FK506 to induce stromal morphogen expression.

RESULTS

Vismodegib increased glioblastoma cell proliferation and stem cell marker expression in vivo or in co-culture with astrocyte organoids. Single-cell RNA sequencing demonstrated glioblastoma cell co-culture with astrocyte organoids induced Hedgehog target genes and stem cell markers in glioblastoma cells, or stromal morphogens in astrocytes, which also expressed primary cilia. CRISPRi suppression of SHH in glioblastoma cells increased glioblastoma cell proliferation and stem cell marker expression in co-culture with astrocyte organoids. Neither vismodegib nor SHH suppression induced glioblastoma cell proliferation or stem cell marker expression in vitro or in co-culture with neuron organoids. FK506 induced astrocyte morphogen expression, inhibiting glioblastoma cell proliferation, stem cell marker expression, and the effects of vismodegib in co-culture with astrocyte organoids.

CONCLUSIONS

SHH from glioblastoma cells signals through astrocytes to inhibit cancer stem cell proliferation.
CNSC-28. IDENTIFICATION OF EAG2-KVß2 POTASSIUM CHANNEL COMPLEX IN NEURON-GBM COMMUNICATIONS REVEALS GBM VULNERABILITY TO DESIGNER INTERFERENCE PEPTIDE
Authors: Dong Weifan , Chen Xin , Huang Xi , Fekete Adam , Wang Lu-yang , Liu Hongwei
Published: November 2022

Glioblastomas (GBMs) are the most aggressive brain tumors. GBM cells form extensive tumoral networks to communicate with each other and with surrounding neurons. Neuronal activity promotes GBM cell proliferation by secreting protumorigenic factors and triggering neuronal-activity-dependent Ca2+ transients, which are persisted and transmitted via tumor networks to promote overall tumor growth and therapy resistance. While how these processes are regulated is largely unknown. Here we show the GBM networks and Ca2+ transients are regulated by a voltage-gated potassium channel complex comprised of EAG2 and Kvβ2. GBM cells selectively overexpress Kvβ2 isoform 4 to facilitate tumor-specific Kvβ2-EAG2 interaction, which regulates neuron-GBM contact-dependent localization of EAG2. Rationally designed interfering peptide K90-114TAT blocks EAG2-Kvβ2 interaction, leading to reduced tumor cell proliferation, increased apoptosis, and prolonged survival of patient-derived xenograft mouse models with no evidence of toxicity to normal tissue. Single-cell RNA sequencing revealed a subgroup of GBM cells is highly sensitive to K90-114TAT treatment. These cells exhibit neuronal signatures and are highly associated with TMZ resistance and worse prognosis. In accordance with the notion, we found neurons promote TMZ resistance of GBM cells, which is regulated by the EAG2-Kvβ2 complex. Treating TMZ-resistant GBM xenograft mice with K90-114TAT yielded significant tumor burden reduction and prolonged survival. Together, our findings revealed the EAG2-Kvβ2 channel complex as a key regulator of neuron promoted GBM progression and designed an interfering peptide with anti-GBM efficacy and low general toxicity, which may have significant clinical impact.
TMIC-47. SINGLE-CELL TRANSCRIPTOMICS REVEALS ANTITUMOR IMMUNE RESPONSES OF PROTON TREATMENT IN GLIOBLASTOMA
Authors: Berry Kalen , Ogurek Sean , Rao Rohit , Lu Richard
Published: November 2022

Proton therapy is an innovative radiation therapy modality that has been shown to induce higher cytotoxicity to tumor cells compared to standard x-ray therapy while sparing normal tissue. However, the impact of proton radiotherapy on the growth and progression of glioblastoma (GBM) has not been defined. We hypothesize that understanding tumor evolution and micro environmental responses to radiotherapy is critical for developing and targeting interventions. In this study, we investigate the effect of proton versus standard photon radiotherapy on tumor growth and the microenvironment in an orthotopic syngeneic GBM model over the course of treatment. We characterized tumor tissues at early, progressing, and terminal phases post irradiation by single cell transcriptomics, bulk RNA sequencing, and histology. We find that proton irradiation led to a drastic reduction in tumor volume at early stages before eventual recurrence. Proton treatment exhibits equivalent if not superior, tumor control when compared to normal photon radiation. Single cell transcriptomics analyses show a dramatic increase in immune cell populations including T cell and macrophage populations which peak shortly before tumor recurrence. In addition, we identify an increase in exhausted T cell markers such as PD1, Tim-3, and Lag-3 in T cells post irradiation. Treatment with therapeutic agents that reprogram microglia/macrophages to an anti-tumor phenotype and activate T cell effector functions effectively eradicate GBM recurrence post irradiation. Thus, our study demonstrates a powerful combined treatment strategy with radiation against GBM and provides valuable information about how tumor growth and microenvironment respond to proton therapies, as well as potential mechanisms of tumor response and recurrence in GBM.
TMIC-50. AN INNOVATIVE THREE-DIMENSIONAL GELATIN FOAM CULTURE SYSTEM FOR IMPROVED STUDY OF GLIOBLASTOMA STEM CELL BEHAVIOR
Authors: Yang Meng-Yin , Chang Chun-Pi
Published: November 2022

Three-dimensional (3-D) tissue engineered constructs provide a platform for examining how the local extracellular matrix contributes to the malignancy of various cancers, including human glioblastoma multiforme. Here, we describe a simple and innovative 3-D culture environment and assess its potential for use with glioblastoma stem cells (GSCs) to examine the diversification inside the cell mass in the 3-D culture system. The dissociated human GSCs were cultured using gelatin foam. These cells were subsequently identified by immunohistochemical staining, reverse transcriptase-polymerase chain reaction, and Western blot assay. We demonstrate that the gelatin foam provides a suitable microenvironment, as a 3-D culture system, for GSCs to maintain their stemness. The gelatin foam culture system contributes a simplified assessment of cell blocks for immunohistochemistry assay. We show that the significant transcription activity of hypoxia and the protein expression of inflammatory responses are detected at the inside of the cell mass in vitro, while robust expression of PROM1/CD133 and hypoxia-induced factor-1 alpha are detected at the xenografted tumor in vivo. We also examine the common clinical trials under this culture platform and characterized a significant difference of drug resistance. The 3-D gelatin foam culture system can provide a more realistic microenvironmentthrough which to study the in vivo behavior of GSCs to evaluate the role that biophysical factors play in the hypoxia, inflammatory responses and subsequent drug resistance.
TMIC-55. IMMUNE DIGITAL-SPATIAL PROFILING AND CHARACTERIZATION OF GLIOMA MICROENVIRONMENT AFTER ADOPTIVE CELLULAR THERAPY
Authors: Font Laura Falceto , Jin Dan , Dean Bayli DiVita , Francis Connor , Reid Alexandra , Sebastian Mathew , Figg John , Long-James Kaytora , Deleyrolle Loic , Flores Catherine
Published: November 2022

INTRODUCTION

Our group has previously shown that Adoptive Cellular Therapy (ACT) is efficacious against CNS malignancies including medulloblastoma, brain stem glioma, and glioblastoma. In this study, we characterized the spatial distribution of immune cells within the tumor microenvironment after ACT and conducted regional genomic analysis in situ to obtain insights into the underlying cellular dynamics. This method enabled us to define specific differences in gene expression within the T cells in the treated versus untreated tumors including changes in gene expression of regulatory T cell (Treg) associated genes, such as Runx1 and TGF-β. We also found a significant increase in Batf3 expression associated with therapy.

METHODS

We performed in situ GeoMx Digital Spatial Profiling (DSP) and whole genome transcriptomics (RNA) of murine glioma KR-158B-luciferase tumors after adoptive cellular therapy. ACT was conducted in orthotopic KR158B tumor-bearing mice. Histological slides were sent to be stained for CD45, CD3, GFP (to mark hematopoietic stem cell-derived cells) and nuclei, and processed following GeoMx DSP workflow for RNA. Overall gene expression clustering profiles were conducted using a Principal Component Analysis (PCA), and relative gene expression differences were analyzed by unpaired t-tests.

RESULTS/CONCLUSIONS

We observed differential expression of a substantial number of genes in the ACT-treated group compared to control. Our findings also indicate that tumors treated with ACT cluster separately from control tumors in PCA analysis. Furthermore, we found downregulation of genes associated with the suppressive properties of regulatory T cells (Runx1 and TGF-β), and upregulation of dendritic cell characteristic genes (Batf3) and antigen presentation genes (H2-Ab1) in ACT-treated tumors vs control. These findings provide insights into the immune cell dynamics and specific genes that are differentially expressed within the tumor microenvironment. This could help improve current adoptive cellular therapies to treat brain tumors.
TMIC-58. PATTERNS OF CELLULAR SUBPOPULATION COHABITATION DEFINE GLIOBLASTOMA STATES
Authors: Bond Kamila , Curtin Lee , Hawkins-Daarud Andrea , Urcuyo Javier , De Leon Gustavo , Sereduk Christopher , Singleton Kyle , Langworthy Jazlynn , Jackson Pamela , Krishna Chandan , Zimmerman Richard , Patra Devi , Bendok Bernard , Smith Kris , Nakaji Peter , Donev Kliment , Baxter Leslie , Mrugala Maciej , Al-Dalahmah Osama , Hu Leland , Tran Nhan , Rubin Joshua , Canoll Peter , Swanson Kristin
Published: November 2022

Characterizing intra- and inter-tumoral heterogeneity of glioblastoma has historically relied on discrete classifications of malignant cell populations leaving immune and other cell populations, known to exist admixed with the malignant tumor cells, relatively neglected. Manifold learning algorithms can manage deconvolving multiple cell populations and are often used to track cell state transitions in single cell transcriptomics. We applied a manifold learning approach to TCGA microarray data (Nf525) and bulk transcriptomics of 134 image localized biopsies across 30 patients with primary and 9 with recurrent glioblastoma to further elucidate how to organize biopsies across a spectrum of possible tissue states. The algorithm revealed a low-dimensional manifold graph for which each biopsy lives across 3 polarizing tissue states - one that is associated with diffusely invaded brain, one that is enriched in mesenchymal genes, and one that is enriched in classical proliferative tumor signatures. We deconvolved the bulk transcriptomics of the image-localized biopsies to reveal the relative abundance of 18 malignant, immune, and other cell subpopulations in each biopsy. Overlaying the cellular decomposition onto the manifold graph visualizing the tissue state distributions revealed that transitions between states correlate with changes in cellular cohabitation composition. The tumor cellular cohabitation ecologies have the lowest diversity, as inferred by ecological measures such as Shannon entropy and evenness, at the distal poles of the graph when compared to the transitional arms. Further, we found that the relationship between imaging appearance of contrast enhancement on T1-weighted MRI and the biopsy cellular composition varies with sex and primary vs recurrent biopsy status. The limited spectrum of possible tissue states revealed by the manifold learning is suggestive of a limited continuum along which tumor and non-tumoral cell populations can cohabitate. Such a limited low-dimensional biological space may constrain the dynamics of tumor biology in a predictable manner.
TMIC-66. FIBROBLAST ACTIVATION PROTEIN: TISSUE EXPRESSION, POSITRON-EMISSION TOMOGRAPHY UPTAKE AND ITS PROGNOSTIC SIGNIFICANCE IN GLIOSARCOMAS AND GLIOBLASTOMAS
Authors: Oster Christoph , Keyvani Kathy , Blau Tobias , Schmidt Teresa , Feldheim Jonas , Lazaridis Lazaros , Kleinschnitz Christoph , Sure Ulrich , Schildhaus Hans-Ulrich , Scheffler Björn , Herrmann Ken , Kebir Sied , Glas Martin
Published: November 2022

BACKGROUND

Although histologically distinct, gliosarcomas and glioblastomas are treated according to the same standards. Fibroblast-activation-protein (FAP) is a component of a tumor-specific subpopulation of fibroblasts that play a critical role in tumor growth and invasion. Few case studies suggest an elevated expression of FAP in glioblastoma and a particularly strong expression in gliosarcoma, possibly for its predominant mesenchymal differentiation. However, the prognostic impact of FAP in glioblastoma and gliosarcoma is unclear. Question:What is the clinical relevance of FAP-expression in gliosarcoma and glioblastoma and how does it correlate with FAPI46-PET-uptake?

METHODS

In a retrospective analysis, patients diagnosed with glioblastoma and gliosarcoma were enrolled. Histological examination was performed using immunohistochemical FAP-staining and quantitative analysis with standardized FAP-Score (0 to 3,with higher values indicating stronger expression). In a subset of patients, FAPI46-PETs have been performed. The SUV values are planned be correlated with FAP-expression in the tumor tissue. Data obtained from immunohistochemical analysis and SUV values are planned to be brought into perspective with clinically relevant prognostic factors, including survival estimates.

RESULTS

A total of 45 patients have been enrolled. On immunohistochemical staining, gliosarcomas expressed significantly more FAP (average FAP-Score,2.3) than glioblastomas (average FAP-Score,0.7,p< 0.0001). While in glioblastoma FAP-expression was confined to the perivascular space, in gliosarcoma the neoplastic cells themselves -in addition to the perivascular extracellular matrix- expressed FAP. Initial analyses indicate that tissue FAP-expression is correlated with tracer uptake on FAPI46-PET. Further studies on clinical relevance using correlation to clinical data are currently underway.

CONCLUSIONS

This study indicates that FAP-expression is much more abundant in gliosarcomas as opposed to glioblastomas. This could open not only a diagnostic but also a therapeutic gap. The prognostic role and the association of FAP-tissue expression with FAPI46-PET tracer uptake will be reported at the time of abstract presentation.
TMIC-69. MITOCHONDRIAL TRANSFER FROM ASTROCYTES ENHANCES METABOLISM AND DRIVES PROLIFERATION OF GLIOBLASTOMA
Authors: Watson Dionysios , Bayik Defne , Lauko Adam , Sprowls Samuel , Deshpande Gauravi , Wilifford Sarah , Troike Katie , Silver Daniel , Wang Sabrina , Seder Juliana , Kay Kristen , Hjelmeland Anita , Lathia Justin
Published: November 2022

Mitochondrial transfer occurs both in stroke (central nervous system) and inflammatory pain (peripheral nerves). However, its role in glioblastoma (GBM) remains poorly understood. We hypothesized that mitochondrial transfer from non-malignant to GBM cells supports tumor metabolism and growth. Using transgenic mice expressing fluorophore-tagged mitochondria, we found that ~50% of orthotopically-implanted mouse GBM cells acquire mitochondria. Brain-resident cells, especially astrocytes, were the primary mitochondrial donors in vitro and in vivo. Mitochondrial transfer also occurred from immortalized human astrocytes to patient-derived xenograft (PDX) models in vitro at rates of 15-35%. GBM cells that acquired mitochondria expressed higher levels of the ATP-synthase subunit ATP5A and produced more ATP, while metabolomics revealed multiple upregulated pathways in recipient cells. These data point to increased metabolic activity in recipient cells. In vivo, mouse GBM cells that acquired mitochondria were more likely to be in S/G2/M cell cycle phases. We observed a similar effect in PDX that acquired astrocyte mitochondria in vitro, suggesting that transfer drives GBM proliferation. Using sorted mouse and human GBM cells with/without in vitro astrocyte mitochondrial acquisition, we found that mitochondrial transfer promoted in vitro self-renewal and in vivo tumorigenicity, leading to significant reduction in survival and increased penetrance in orthotopic GBM models. Transfer in mouse and human systems was contact-dependent and was abrogated by physical separation of donor and recipient cells by transwell inserts. Pharmacologic inhibition of cytoskeleton and gap junctions did not affect transfer rate, while blocking growth-associated protein 43 (GAP43) function by c-Jun N-terminus kinase inhibition decreased transfer rate by 15-30%, suggesting a potential role of GAP43. Taken together, mitochondrial transfer comprises a fundamental, protumorigenic mechanism of GBM, enhancing metabolic activity and driving tumor cell proliferation. Elucidating the molecular machinery regulating astrocyte mitochondrial transfer and its downstream protumorigenic effects will lead to therapeutic opportunities targeting this understudied tumor microenvironment interaction.
TMIC-71. SPATIAL ANALYSIS OF GENETIC HETEROGENEITY AND TUMOR MICROENVIRONMENT INTERACTIONS IN GLIOBLASTOMA
Authors: Onubogu Ugoma , Salatino Roberto , Szentirmai Oszkar , Vaubel Rachael , Janiszewska Michalina
Published: November 2022

Genetic variation can be considered a driver of somatic evolution in glioblastoma (GBM); however, the tumor microenvironment (TME) is a key contributor to the selection process and evolutionary potential of cancer cells. Although diverse ecosystems exist in GBM which impact tumor cell fitness and characterize an aggressive pathophysiology, little is known about how genetically distinct subpopulations of GBM cells interact with the surrounding TME. Therefore, we sought to investigate how the varying clonal genotypes of GBM cells are influenced by different types of selective pressures within the TME. Our imaging-based strategy is centered around Specific-to-Allele PCR Florescence In Situ Hybridization (STAR-FISH), an in situ PCR based probe hybridization method for detection of point mutations and copy number variations at the single-cell level. Using STAR-FISH to illuminate the spatial distribution of clones with mutations in the TERT promoter, we integrated multicolor immunofluorescent staining, DNA-FISH, reflectance imaging, and computational cellular analysis methods to achieve highly-multiplexed single cell spatial profiling of intact GBM specimens. With this strategy we are able to simultaneously evaluate tumor tissues for the presence of hypoxic, angiogenic, and inflammatory niches, extracellular matrix organization, nuclear morphology, mutational status, and several oncogenic amplifications on the same slide and location. We analyzed 20 matched primary and recurrent GBM samples, imaging several distinct regions of the same tumor. Our initial results have revealed interesting and potentially novel distributions of subclones residing within specific microenvironments. These results include TERT mutation positive clones enriched in areas possessing more structured extracellular matrix, as well as unique arrangements of infiltrating immune cells in the angiogenic tumor niche. This research underscores the importance of the TME in selecting for certain phenotypes that characterize somatic evolution, highlighting novel genetic and spatial dependencies which sustain heterogeneity in GBM.
TMIC-73. HIGH-DIMENSIONAL HISTOPATHOLOGIC EVALUATION OF THE HYPOXIC MICROENVIRONMENT IN GLIOBLASTOMA
Authors: Mansouri Sheila , Zaidi Mark , Singh Olivia , Ali Hafsah , Karimi Shirin , Lombard Phoebe , Dvorkin-Gheva Anna , Velasquez Carlos , Devaraja Kaviya , Sosa Julio , Patil Vikas , Wei Qingxia , Wu Ronald , Li Mira , Cheung May , Voisin Mathew , Gao Andrew , Hedley David , Aldape Kenneth , Wouters Bradly , Zadeh Gelareh
Published: November 2022

Rapidly growing solid tumors such as glioblastoma (GBM) are characteristically hypoxic, displaying large areas of necrosis surrounded by hyperproliferative pseudopalisading cells. Intra-tumoral hypoxia develops over time in the three-dimensional space and the degree of tissue oxygenation is a dynamic process that varies continuously. Combined with the extensive inter- and intra-tumoral heterogeneity associated with GBM at the bulk and single cell level, hypoxia contributes to a gradient of molecular alterations that are specific to the different cell populations that make up the bulk of the tumor and reside in specific niches. To date, high dimensional histopathologic analyses of the hypoxic regions within GBM tissue have not been performed. Here, we took a combined spatial and single-cell proteomic profiling approach to investigate the histopathologic features of hypoxia by leveraging a unique clinical study where the exogenous hypoxia marker pimonidazole (PIMO) is administered to patients with GBM prior to surgery. Tissue specimens were subjected to imaging mass cytometry and serial immunohistochemistry using a panel of 27 markers associated with cellular hallmarks of hypoxia, metabolism, proliferation, stemness, angiogenesis, and immune cell types. We took high-resolution imaging and statistical approaches to explore the interplay of the different markers within hypoxic regions of primary and recurrent GBMs, in addition to IDH-mutant gliomas. Our findings elucidate the expression pattern of key biological markers relative to one another, altered composition of different cell types, along with differential proliferative, transcriptional, and translational activation states associated with each cell type within the hypoxic regions of GBM.