bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

Abstract

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

Abstract
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

Abstract
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

Abstract
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

Abstract

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

Abstract
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

Abstract

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&lt; 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

Abstract
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

Abstract
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

Abstract
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.