bluuebunny/crossref_metadata_2025_split huggingface dataset:
TMIC-46. SINGLE-CELL MAPPING OF HUMAN BREAST CANCER BRAIN METASTASES REVEALS PROGNOSTICALLY RELEVANT IMMUNE LANDSCAPES
Authors: Jassowicz Lena , Wong John , Soller Julia , Sor Furkan , Feng Fangyoumin , Barthel Mandy , Unterberg Andreas , Lichter Peter , Warta Rolf , Zapatka Marc , Seiffert Martina , Herold-Mende Christel 
Published: November 2023

Abstract
Brain metastases are associated with a particularly poor prognosis in breast cancer and improved treatment strategies are much needed. The patient-individual role of immunotherapy and anti-tumor immune control in breast cancer brain metastases (BCBM) is still poorly understood. Here, we characterized the niche-specific tumor microenvironment of BCBM at single-cell resolution in a clinically annotated patient cohort that underwent surgery at Heidelberg University Hospital (n = 156). Quantification of T cell infiltrates across whole tissue sections of n = 137 BCBM revealed strong inter-patient heterogeneity of T cell abundance. Of note, high CD8 T cell densities predicted prolonged overall survival independent of established prognostic factors. Deeper phenotyping of BCBM T cells and matched blood samples by flow cytometry showed a specific enrichment of a CD103+ tissue resident-like CD8 T cell subset expressing high levels of activation markers and checkpoint molecules in BCBM. In contrast to a general T cell enrichment in the stroma, this subset was detected more frequently within tumor islands, which might suggest its anti-tumor reactivity. We investigated potential regulators of T cell infiltration and function in the brain metastatic niche focusing on the triple negative breast cancer sub-cohort (n = 49) concluding the following: i) The quantification of a broad range of immune cells, incl. myeloid cells, NK, and B cells, in whole tissue sections revealed distinct immune ecotypes associated with T cell infiltrates. ii) An in-depth characterization of all major immune and stromal cells by single nuclei RNA-sequencing (n = 28/49) allowed for identification of potential cellular and molecular interaction partners, which was confirmed by iii) analysis of their neighborhoods by spatial transcriptomics in a targeted, single-cell resolving approach (n = 8/28). We anticipate that our findings will reveal immunoregulatory cell subsets and molecules and potentially contribute to improving immunotherapeutic approaches for BCBM patients in the future.
TMIC-53. TRANSGLUTAMINASE 2 EXPRESSION IN GLIOBLASTOMA: EVIDENCE FOR A ROLE IN EFFEROCYTOSIS
Authors: Lorimer Ian , Elgafarawi Mara , Lui Margarita , Sevinc Filiz , Leger Alexa , Munoz David , Keillor Jeffrey , Woulfe John 
Published: November 2023

Abstract
Glioblastoma is the most common type of brain cancer in adults. It is currently incurable and there is a clear need for more effective treatments. The heterogeneity of this cancer and its ability to promote both local and systemic immunosuppression are major obstacles to the development of effective therapies. Previous studies have explored the use of transglutaminase 2 (TGM2) inhibitors for glioblastoma therapy. These studies focussed on TGM2 that is expressed in a subset of glioblastoma cells. We show here that glioblastoma-associated macrophages are a major source of TGM2 in glioblastoma tumours. Analysis of bulk and single cell RNAseq data showed that TGM2 mRNA was expressed at high levels in glioblastoma-associated macrophages. Immunohistochemical and immunofluorescence analysis of TGM2 expression was performed in a mouse xenograft model in which human mesenchymal subtype glioblastoma cells were grown intracerebrally in nude mice. In these xenograft tumours, glioblastoma cells were negative for TGM2 expression, while infiltrating macrophages were strongly positive. The most intense staining for TGM2 was observed in macrophages in the vicinity of necrotic regions. Apoptotic cells were frequently present within the necrotic regions and highly TGM2-positive macrophages were observed to be phagocytosing these. The same patterns of TGM2 expression were also observed in samples from glioblastoma patients. Consistent with a role in phagocytosis, TGM2 co-localized with the endosomal protein CD68 in glioblastoma-associated macrophages. The phagocytosis of apoptotic cells by macrophages, known as efferocytosis, is tolerogenic, both because it clears potentially antigenic material and because it induces an immunosuppressive phenotype in macrophages. As TGM2 has a previously established role in efferocytosis and autoimmunity suppression, our findings suggest that TGM2 has a role in maintaining the glioblastoma immunosuppressive environment and that its inhibition might enhance immunotherapy for this cancer.
TMIC-63. SINGLE CELL SEQUENCING OF RECURRENT GLIOBLASTOMA DEFINES CELLULAR STATES ASSOCIATED WITH THERAPY RESISTANCE
Authors: Tripathi Arushi , Shankar Sunita , Ravikumar Visweswaran , Loper Jackson , Ji Sunjong , Hassan Zainab , Haydin Yacoub , Huang Dah-Luen , Bhadury Sagnik , Regier Jeffrey , Rao Arvind , Al-holou Wajd 
Published: November 2023

Abstract
Glioblastoma (GBM), an aggressive, primary tumor is composed of multiple clonal populations. Resistance to therapy is a hallmark of GBM, making it a deadly disease. Upon therapeutic stress, tumor heterogeneity and glial stem cell plasticity within the primary tumor contributes to reprogramming of the transcriptional activity. This alters the tumor landscape and generates therapy resistant clonal populations. Single cell RNA sequencing studies of primary GBM have defined various tumor cell states namely, the NPC-like (Neuronal precursor-like), OPC-like (oligodendrocyte precursor-like) and MES-like (mesenchymal-like) cellular states. These studies have also defined the immune components and the normal brain cells associated with primary GBM. Yet, there are no studies focused on the single cell analysis of recurrent GBM. To define the tumor landscape at recurrence, we performed single cell RNA sequencing of 180K cells from excised tumor tissue obtained from 11 patients after failure of therapeutic interventions. A corresponding cohort of 240K cells were sequenced from primary tumors obtained from 13 patients. We performed cellular state analyses of tumor cells in both the primary (~70K) and recurrent (~10K) samples. Primary GBM showed higher fractions of AC-like (0.55), compared to NPC-like (0.10), OPC-like (0.20) and MES-like (0.15). On the other hand, recurrent tissues have very low AC-like (0.09) but elevated NPC-like (0.23), OPC-like (0.40) and MES-like (0.27). This data shows a significant decrease in AC-like cells and corresponding increase in MES-like, OPC-like, NPC-like cells after recurrence. Within this dataset we also have matched longitudinal samples for analysis. We are currently performing trajectory analysis to identify genes and transcription programs activated during tumor recurrence. Experimentally, using known markers for the different cellular states we have also performed immunofluorescence analyses to validate the different cell states altered during recurrence. Our efforts to generate comprehensive recurrent GBM atlas will prove to be valuable to the GBM community.
TMIC-66. SPATIALLY RESOLVED METABOLIC INTERACTIONS IN THE GBM MICROENVIRONMENT
Authors: Chakraborty Avirup , Feier Diana , Silver Aryeh , Yang Changlin , Fu Dongtao , Rahman Maryam , Sarkisian Matthew , Huang Jianping , Harrison Jeffrey , Mitchell Duane , Deleyrolle Loic 
Published: November 2023

Abstract
Glioblastoma (GBM) contains cell populations with distinct metabolic requirements, with fast-cycling cells (FCCs) harnessing aerobic glycolysis, and treatment-resistant slow-cycling cancer stem cells (SCCs) preferentially engaging in lipid metabolism. The interaction between immune and tumor cells, and how tumor metabolic heterogeneity shapes the immune landscape in GBM has yet to be understood. Our study allows for the molecular and spatial decoding of the heterogeneity within the GBM microenvironment, with a specific focus on unraveling the metabolic links that underlie the interplay between SCCs and the immune compartment. Our work indicates a new paradigm of communication in the TME of GBM, where SCCs play a pivotal role in shifting the tumor milieu toward an immunosuppressive phenotype, in part by recruiting specific tumor-promoting immune cells that stimulate SCC-driven tumor progression and provide metabolic support to these treatment-resistant cells. Our study delineates these metabolic communications and assesses the potential therapeutic effect of disrupting these interactions to treat GBM. The insights generated from this project uncover fundamental principles of the emerging connections between the tumor microenvironment, cell metabolism, anti-tumor immunity, and associated therapeutic vulnerabilities. In conclusion, our study provides a novel understanding of the role of metabolic interactions in the development of drug and immune resistant GBM. These findings suggest that targeting metabolic interactions, specifically between SCCs and tumor-associated immune suppressive cells, may be a promising therapeutic strategy for GBM.
TMIC-82. SPATIAL AND SINGLE-CELL PROTEOMIC LANDSCAPING OF THE HYPOXIC MICROENVIRONMENT IN GLIOBLASTOMA
Authors: Mansouri Sheila , Gandhi Shreya , Zaidi Mark , Singh Olivia , Karimi Shirin , Lombard Phoebe , Dvorkin-gheva Anna , Valesquez Carlos , Ali Hafsah , Devaraja Kaviya , Sosa Julio , Patil Vikas , Wei Qinxqia , Wu Ronald , Li Mira , Cheung May , Voisin Mathew , Gao Andrew , Hedley David , Aldape Kenneth , Wouters Bradley , Zadeh Gelareh 
Published: November 2023

Abstract
Glioblastoma (GBM) is a fatal adult solid tumour with median overall survival of 18-20 months post-diagnosis; contributing factors to therapeutic inefficacy include acquisition of genomic alterations post-therapy, immune evasion, deregulated hypervascularization, and tumor microenvironmental factors such as hypoxia. Combined with extensive inter- and intra-tumoral heterogeneity at bulk and single-cell level, hypoxia contributes to a gradient of molecular alterations that are specific to different cell populations that make up tumour bulk and reside in specific niches. Hitherto, high-dimensional histopathologic analyses of hypoxic regions within GBM tissue have not been performed. We took a combined spatial and single-cell proteomic profiling approach to investigate the histopathologic features of hypoxia by leveraging a unique clinical study wherein the exogenous hypoxia marker, pimonidazole (PIMO), was administered to GBM-patients preoperatively. Tissue specimens were subjected to imaging mass cytometry, high-resolution imaging, and serial immunohistochemistry using a panel of markers associated with cellular hallmarks of hypoxia, metabolism, proliferation, stemness, angiogenesis, and immune cell types. Our findings showed that PIMO staining is associated with histopathologic features of hypoxia and correlates with specific metabolic, immune, and stemness markers in GBM; specific lymphocyte populations were depleted from hypoxic regions alongside alterations in macrophagic and microglial landscape in a niche-specific manner; hypoxia reduced the proportion of proliferating glioma initiating cells and altered the proliferative, transcriptional, and translational capacity of different cell populations; microvessel density was reduced in hypoxic microenvironment. Cytometry by time-of-flight further validated the altered proportions of specific immune cell types enriched in hypoxic populations of GBM microenvironment. Our study is the first to report use of PIMO to interrogate spatial, single-cell, and phenotypic architecture associated with tissue hypoxia and altered expression of biomarkers associated with hypoxia/glycolysis, immune infiltration, proliferation, and stemness. Identification of targetable biomarkers and mediators of hypoxia-driven habitats in GBM may provide direction for future immunotherapeutic research.
TMIC-86. SPATIAL ORGANIZATION OF THE PEDIATRIC HIGH-GRADE GLIOMA TUMOR-IMMUNE LANDSCAPE
Authors: van den Broek Thijs , Hoogendijk Raoull , Kranendonk Mariëtte , Ruiz Cristian , Megchelenbrink Wout L , Lammers Julie , Ries Mario G , Hoving Eelco , van der Lugt Jasper , Rios Anne C , Stunnenberg Hendrik , van Vuurden Dannis , Zomer Anoek 
Published: November 2023

Abstract

BACKGROUND
Advances in cellular immunotherapies have shown promising results for brain tumors, including pediatric-type diffuse high-grade gliomas (pHGG). A comprehensive understanding of the tumor-immune microenvironment (TME) is essential for efficient target identification and novel therapeutic strategies. The interplay between cells and their spatial localization within the TME may affect immune cell phenotype and function. We characterized these interactions and phenotypes by single-cell (SC) spatial proteomic analysis.

METHODS
Thirty-two tissue biopsies of pHGGs were collected from the Princess Máxima Center biobank consisting of 14 diffuse midline gliomas, H3K27-altered (DMG) and 18 other pHGGs. Three cores per biopsy were assembled in a tissue micro-array (TMA). A cyclical multiplex immunofluorescence antibody staining technique by means of chemical antibody-removal was applied to the TMA sections. This resulted in hyperplexed images enabling detailed mapping of the spatial organization. Survival data of patients was collected retrospectively. Clusters of interacting cells were detected using cellular neighborhood (CN) analysis and SC distance analysis.

RESULTS
SC analysis revealed a large fraction of myeloid cells in the TME, up to 21% of total cells within a tumor, characterized by expression of IBA1 and CD68. We also identified some significant differences in the composition and spatial organization of DMGs compared to other pHGGs. More specifically, DMG biopsies showed lower abundance of CD163+/CD206+ immunosuppressive myeloid cells compared to other pHGGs (p&lt; 0.001). Additionally, the presence of T cells was significantly lower in DMGs compared to other pHGGs (p&lt; 0.001). CN analysis revealed a higher occurrence of myeloid/astrocyte-like neighborhoods in pHGGs other than DMG. The presence of this cluster in these tumors is associated with higher risk of dying (p&lt; 0.05). Thus, the combination of our spatial proteomic SC technology framework and unique patient sample collection will offer essential insights into the cellular interplay in the TME, and its influence on patient outcomes.
MODL-04. ESTABLISHMENT OF TUMOR MICROENVIRONMENT-PRESERVING ORGANOID MODEL FROM PATIENTS WITH INTRACRANIAL MENINGIOMA
Authors: Ahn Stephen 
Published: November 2023

Abstract
Although meningioma is the most common primary brain tumor, treatments depend only on the surgery and radiotherapy, and recurrent meningiomas have no standard therapeutic options due to a lack of clinically relevant research models. Current meningioma cell lines or organoids cannot reflect biological features of patients’ tumors since they undergo transformation along culture and consist of only tumor cells without microenvironment. Here, we firstly established patient-derived meningioma organoids (MNOs) preserving diverse cell types of tumor microenvironment, including endothelial cells and macrophage/microglia cells. Exclusion of the enzymatic dissociation-reaggregation steps endowed MNOs with original histology and tumor microenvironment. In addition, we used liquid media culture system instead of embedding into Matrigel, thus easy-to-handle, cost-efficient, and time-saving. MNOs maintained their functionality and morphology after long-term culture ( &gt; 9 wk) and repeated cryopreserving-recovery cycles. The similarity between MNOs and their corresponding parental tumors were confirmed by both immunohistochemistry and whole-exome sequencing. As a representative application, we utilized MNOs in drug screening, and mifepristone, an antagonist of progesterone receptor, showed prominent antitumor efficacy in respect to viability, invasiveness, and protein expression. Taken together, our MNO model overcame limitations of previous meningioma models and showed superiority in terms of resemblance with parental tumors. Thus, we expect that our model can facilitate translational research of identifying and selecting drugs for meningioma in the era of precision medicine.
MODL-14. IMAGING, HISTOLOGICAL AND MOLECULAR CHARACTERIZATION AND COMPARISON OF POST-ABLATION RECURRENT TUMOR WITH THE PRIMARY TUMOR IN A PRECLINICAL GLIOBLASTOMA MODEL
Authors: Nagaraja Tavarekere , Datta Indrani , Morosini Natalia , Bartlett Seamus , Ayloo Bhargav , Cabral Glauber , Avritt Faith , Hasselbach Laura , Parasar Parveen , deCarvalho Ana , Singh Jaspreet , Knight Robert , Brown Stephen , Ewing James , Noushmehr Houtan , Lee Ian 
Published: November 2023

Abstract
Recurrent glioblastoma (rGBM) is highly aggressive and invasive. A reliable preclinical model that recapitulates these features is not presently available. The objective was to generate a preclinical rGBM model, characterize and compare its imaging, histological and molecular signatures in comparison to the primary tumor. Immune-suppressed, RNU/RNU female rats were implanted with U251N tumor cells in one brain hemisphere (n=33). Tumor progression in all rats was followed by longitudinal dynamic contrast enhanced-magnetic resonance imaging (DCE-MRI). In 24 rats the tumor was ablated under diffusion-weighted imaging (DWI)-guided laser interstitial thermal therapy (LITT) at post-implantation 2-weeks. Cohorts from twenty ablated rats were euthanized at post-LITT 24 h, 2- and 4-weeks and, along with 5 unablated controls, used for hematoxylin and eosin (H&E) and Ki67 staining. Tissues from 4 other unablated and 4 recurrent tumors at post-LITT 2-weeks were used for RNAseq. All the rats survived the LITT procedure. Unablated controls showed increased tumor burden by post-implantation 2 weeks and were euthanized. In the LITT group, MRI showed little tumor tissue at 24 h, evidence of recurrence at 2 weeks and significant tumor tissue at 4 weeks and matched with histological evidence for tumor recurrence. Compared to the primary tumor, H&E staining showed increased vascular hyperplasia, mitotic bodies and hypoxic regions with pseudopalisading necrosis in the recurrent tumor. Increased KI67 staining in recurrent tumors suggested higher rates of proliferation and evidence of infiltration into host tissue. Pathway analyses demonstrated differentially expressed genes in the canonical pathways of hypoxia-inducible factor-alpha (HIF-1α), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and OX40 signaling. The genes for the following functions were significantly affected: cell cycle, cellular movement and cell morphology. Reliable preclinical rGBM models are few. These data suggest that this model replicates the features of human rGBM and can be useful in testing putative anti-glioma therapies.
MODL-30. UTILIZING SERUM-FREE CO-CULTURE MODELS FOR IN-VITRO MODELING OF TUMOR-ASSOCIATED MACROPHAGES IN GLIOBLASTOMA
Authors: Alrefai Hasan , Kumar Manoj , Nassour Lauren , Schanel Taylor , Beierle Andee , Anderson Joshua , Hicks Patricia , Willey Christopher 
Published: November 2023

Abstract

BACKGROUND
The GBM tumor microenvironment consists of various cancerous and non-cancerous cells that influence GBM growth, invasion, angiogenesis, and resistance to chemoradiation. In-vitro models of GBM often lack multiple cell types. Some addressed this by using multicellular models with macrophages and astrocytes; however, these models employ serum-containing media, which causes GBM brain-tumor initiating cells to lose their stem-like properties. Moreover, these models use IL-4/13 polarized macrophages (M2), despite tumor-associated macrophages (TAMs) not adhering to the M2 phenotype. Thus, there is a need to develop physiologically accurate in-vitro models of GBM.

METHODS
We transitioned THP-1 monocytes from serum-containing RPMI 1640 to serum-free TheraPEAKTM X-VIVOTM-15 media. Monocytes were stimulated towards a macrophage-like state with PMA and polarized by co-culturing with GBM patient-derived xenograft (PDX) lines using transwell inserts. Macrophage polarization was characterized through cytokine arrays, invasion, angiogenesis, and phagocytosis assays, comparing them to resting (M0), pro-inflammatory (M1), and M2 macrophages.

RESULTS
THP-1 monocytes grown in X VIVO-15 media exhibited similar proliferation rates to those in RPMI 1640 for up to 48 hours. However, at 72 hours, there was a slight decrease in proliferation rates in X VIVO-15 media (p&lt; 0.0001). Macrophages polarized by the GBM PDX line, JX39P-RT, did not exhibit the cytokine expression profile of any other group. M2 macrophages displayed higher phagocytic capacity (25%) of JX39P-RT compared to M0, M1, and TAMs (&lt; 15%, p&lt; 0.01). M1 macrophages showed significantly higher invasion rates than M0, M2, and JX39P-polarized TAMs.

CONCLUSIONS
We have demonstrated a serum-free method by which we can polarize macrophages towards a GBM-supportive phenotype that differs molecularly and functionally from M0, M1, and M2 macrophages. This high-fidelity method of modeling TAMs in GBM will aid in the development and testing of innate immunomodulatory therapeutics that may one day enter the clinic in hopes of improving GBM outcomes
MODL-35. BIOENGINEERED HYDROGEL AND BLOOD BRAIN BARRIER-ON-A-CHIP SYSTEM TO STUDY GLIAL CELL ACTIVATION AND BLOOD BRAIN BARRIER DYSFUNCTION IN GLIOBLASTOMA
Authors: dePalma Thomas , Hughes Kennedy , Fraas David , Tawfik Marie , Hisey Colin , Skardal Aleksander 
Published: November 2023

Abstract
Treatments for glioblastoma (GBM) are limited leading to short patient life expectancies after diagnosis. Current preclinical models of GBM fail to accurately replicate the function of the blood brain barrier (BBB) and the extreme heterogeneity of the GBM tumor hindering the drug development process. Therefore, there is a pressing need for improved model systems to study how different GBM subpopulations interact with glial cells and the BBB. To build such a model, we first focused on designing a 3D extracellular matrix-based hydrogel biomaterial that would support human astrocytes and a functional BBB. The hydrogel chosen for these studies is composed of functionalized hyaluronic acid (HA) and gelatin and has similar mechanical properties to the human brain. Using confocal imaging and qPCR, we have shown that astrocytes encapsulated in this hydrogel have greater branching and express fewer markers of activation compared to astrocytes grown in commonly used 3D biomaterials such as collagen I and Matrigel. Treatment with inflammatory cytokines and GBM cell derived extracellular vesicles (EVs) revealed that astrocytes in this environment display the activation and inflammatory phenotypes observed in vivo in GBM. Next, this hydrogel biomaterial was deployed in a bioengineered BBB-on-a-chip. We have shown that this system supports the formation of 3D microvessel structures with human brain endothelial cells, pericytes, and astrocytes that accurately mimic the function of the human BBB. Studies using this BBB-on-a-chip have shown that invasive glioma stem cells alter BBB permeability influencing the transport of certain therapeutics to the tumor site. We are now beginning to test how various subpopulations of GBM tumor cells behave in this model and how changes to glial cells and the BBB impact drug delivery and efficacy. This study shows that engineered in vitro models can provide critical insight into disease mechanisms and inform treatment design in GBM.