bluuebunny/crossref_metadata_2025 huggingface dataset:
TMIC-60. THE ROLE OF ICAM1 IN GLIOBLASTOMA TUMORIGENESIS UNDER HYPOXIC CONDITIONS
Authors: Devaraja Kaviya , Singh Olivia , Mansouri Sheila , Ali Hafsah , Zadeh Gelareh
Published: November 2022

Glioblastoma (GBM) is a fatal brain cancer in adults with ineffective existing treatment methods. Cell adhesion molecules (CAMs) are proteins that are expressed on the surface of cells and enable them to interact with one another and the surrounding microenvironment. Intracellular adhesion molecule 1 (ICAM-1) is a cell adhesion molecule expressed by macrophages, tumor cells and endothelial cells in GBM. Preliminary data showed that ICAM-1 is associated with poorer overall and progression free survival in GBM patients and ICAM-1 expression levels increase in recurrent GBM tumors. TAMs enhance tumor growth and proliferation, within GBM. The goal of this study was to determine whether and how ICAM-1 expression on TAMs contributes to GBM tumorigenesis, specifically within the hypoxic tumor microenvironment. We hypothesized that ICAM1 facilitates TAM migration, proliferation and phagocytosis, thus enhancing tumor growth. We found that upon incubation of human and mouse primary macrophages in hypoxia, ICAM-1 expression levels increased and were further exasperated upon culturing with tumor cell-conditioned medium. Incubation of ICAM-1 deficient macrophages in hypoxia resulted in lower migration and proliferation in ICAM-1 deficient cells compared to wild type cells. When cultured with tumor cell condition media, migration and proliferation of ICAM-1 deficient macrophages was lower than wild type macrophages. Further, intracranial injection of tumor cells into ICAM-1 deficient and wild type mice revealed that ICAM1 deficient mice survived longer and had lower overall tumor volume than wild type mice. In conclusion, the expression of ICAM1 in TAMs likely promotes GBM tumorigenesis by enhancing their proliferation and migration into the tumor microenvironment. ICAM-1 plays a role in macrophage migration, proliferation, and its expression on TAMs, resulting in more aggressive and invasive tumors. In addition, certain environmental factors like hypoxia further enhance the expression of ICAM-1 in macrophages which is associated with more aggressive features of GBM.
TMIC-61. LOSARTAN PREVENTS IMMUNOTHERAPY-ASSOCIATED EDEMA AND ENHANCES SURVIVAL IN GLIOBLASTOMA
Authors: Datta Meenal , Chatterjee Sampurna , Perez Elizabeth , Gritsch Simon , Roberge Sylvie , Duquette Mark , Chen Ivy , Naxerova Kamila , Kumar Ashwin , Ghosh Mitrajit , Emblem Kyrre , Ng Rosa , Ho William , Kumar Pragya , Krishnan Shanmugarajan , Dong Xinyue , Speranza Maria , Neagu Martha , Reardon David A , Sharpe Arlene , Freeman Gordon , Suva Mario , Xu Lei , Jain Rakesh
Published: November 2022

Immune checkpoint blockers (ICBs) have revolutionized the treatment of some solid cancers but have failed to benefit the majority of glioblastoma (GBM) patients. Two reasons underlying limited ICB benefit are: 1) immune-related adverse events, and 2) resistance conferred by the tumor microenvironment. Here, we show that ICBs induce cerebral edema in patients and GBM mouse models. This edema results from an inflammatory response to ICB treatment that disrupts the blood-tumor-barrier, as confirmed by intravital imaging, mechanistic blocking studies, and single-cell RNA sequencing. Losartan – a commonly prescribed antihypertensive agent – controls ICB-induced edema, reprograms the immunosuppressive tumor microenvironment, and improves survival under ICB therapy. In combination with a standard of care regimen in mice mimicking clinical treatment (surgical resection, chemoradiation), losartan increases the percent of long-term surviving (cured) mice under ICB therapy from 16% to 43%. Finally, a bihemispheric “resect-and-response” model to establish predictive biomarkers from the tumor microenvironment reveals that cured mice have an immunostimulatory (“hot”) immune tumor compartment prior to therapy. These results provide the basis for clinical testing of adding to losartan to ICB treatment for GBM patients.
TMIC-63. SELECTIVE TARGETING OF IMMUNE-SUPPRESSIVE LEUKOCYTES TO REPROGRAM THE GBM IMMUNE LANDSCAPE
Authors: Abdelfattah Nourhan , Maldonado Jose , Liu Jia-Shiun , Tran Nhat , Yun Kyuson
Published: November 2022

A significant barrier to immunotherapy efficacy is GBM’s immunosuppressive microenvironment composed of few tumor-infiltrating lymphocytes (TILs; < 5%) but abundant myeloid cells, making it an “immune cold” tumor. To enhance immunotherapy efficacy, a better molecular and functional understanding of the heterogeneous cell types in the GBM microenvironment and their function is urgently needed. Single cell RNA-sequencing offers high-resolution cellular and molecular data to elucidate cancer and stromal cell phenotypes at the single cell level to identify the most relevant cell types to target to enhance immune activation. We recently reported an integrated, multi-regional and -dimensional single-cell transcriptomic analysis of 201,986 human glioma and immune cells derived from 44 tissue fragments from 18 human glioma patients. In doing so, we mapped GBM cellular heterotypia and spatial, molecular, and functional heterogeneity of glioma associated immune cells. We observed an extensive spatial and molecular heterogeneity of glioma cells, microglia, macrophages, and T cells within the same tumor samples in low-grade gliomas (LGGs), primary GBMs, and recurrent GBMs. Importantly, our analysis of 83,479 glioma infiltrating myeloid cells identifies 9 molecularly distinct myeloid subtypes: 4 microglia, 4 bone marrow-derived macrophage (BMDM), and dendritic cell subtypes. Importantly, five of these myeloid cell subtype gene signatures were independent predictors of patient survival in multiple datasets, demonstrating the importance of immune cells in disease progression and aggressiveness. Here, we report evidence for heterogeneous cell:cell communication between glioma and immune cells in different regions within a patient, using CellChat, Cellphone DB, and NicheNet tools. We also nominate immune modulators other than PD1/PDL1 and CTLA3 as more promising targets in GBM. Finally, we provide evidence that S100A4 is a promising novel immunotherapy target since its high-level expression is required for immune-suppressive macrophages and Tregs to block T cell infiltration and DC maturation.
TMIC-64. EXTRACELLULAR VESICLE TRAFFICKING IN GBM
Authors: Maire Cecile , Salviano-Silva Amanda , Kolbe Katharina , Westphal Manfred , Lamszus Katrin , Ricklefs Franz
Published: November 2022

Extracellular vesicles (EVs) are secreted by all cell types, including tumor cells, and are found in increased numbers in the plasma of GBM patients. EVs may contain high-value genetic material that can be useful for tracking tumor development, as well as membrane proteins that affect other cells. This prompted us to investigate how tumor EVs might influence immune cells in glioma, and in primary and secondary lymphoid organs as well as in the circulation. To this end we used a syngeneic GBM mouse model and tracked tumor EVs from the brain to the meninges, cervical lymph nodes, plasma, bone marrow and spleen. Interestingly, we were able to identify tumor EVs mostly in the cervical lymph nodes by ImageStream imaging flow cytometry just 30min after tumor EV injection into the brain. However, when tumor EVs were produced by a large gliomas transfected with dTomato, we found them mainly in plasma, less frequently in bone marrow and never in the spleen. We confirmed these data by extracting DNA from EVs and detecting specific dTomato sequences using digital droplet PCR. In addition, we detected CD11b+ macrophages in the meninges that likely travel through the lymphatics that have taken up tumor EV or tumor material. We confirm that tumor EVs are capable of eliciting an immune response by activating T cells. However, prolonged contact and large number of EVs could also block antigen recognition by T cells and thus contribute to the propagation of an immunosuppressive environment in GBM.
TMIC-65. DISSECTING THE EXPRESSION AND FUNCTION OF ANGIOPOIETIN-1 IN BRAIN TUMORS
Authors: Langhnoja Jaldeep , Wei Xin , Arounleut Phonepasong , Phoenix Timothy
Published: November 2022

Angiopoietin-1 (Angpt1) is a secreted protein that can promote angiogenesis and vascular stability in development and certain disease states through activation of the Tie2 receptor. However, little is known about its expression or function across brain tumor types. Surveying its expression across pediatric and adult brain tumors we show Angpt1 is upregulated in glial brain tumors, including high-grade gliomas and ependymomas, and that expression patterns correlate with cell-type expression patters we recently described in the normal developing and adult brain. To examine the cell type and location of Angpt1 expression within tumors we have generated electroporation based high-grade glioma and ependymoma mouse models in Angpt1-GFP reporter knock-in mice. Angpt1-GFP expression is found mainly in tumor cells within both tumor models, with varying expression in high-grade glioma models based on driver mutations and molecular subtype. Single-cell transcriptomic studies in these mouse models are underway to further delineate expression across tumor and non-tumor cell types. Moreover, we are leveraging this flexible electroporation-based mouse brain tumor modeling platform to examine Angpt1 function within brain tumors. Preliminary data shows that including a DNA plasmid encoding Cre-recombinase induces recombination and loss of Angpt1 in our electroporation-based brain tumor models, and ongoing studies aim to delineate its function in tumor pathogenesis, including tumor angiogenesis and blood-brain barrier function.
TMIC-76. HUMANIN RELEASE FROM TUMOR ASSOCIATED MYELOID CELLS PROMOTES GLIOMA CHEMORESISTANCE
Authors: Cheng Jiying , Li Min , Flüh Charlotte , Synowitz Michael , Bhat Krishna , Balasubramaniyan Veerakumar , Kaelin Roland , Glass Rainer
Published: November 2022

Transcriptomic screens of brain tumor (glioblastoma; GBM) parenchymal cells indicated tumor supporting traits of the GBM microenvironment, but largely excluded mitochondrially enriched gene-sets. Here, we show that a mitochondrial transcript of GBM parenchymal cells contributes to therapy resistance. We inspected the non-coding transcriptome of human GBM associated myeloid cells (GAM) and observed an upregulation of the mitochondrial ribosomal subunit MT-RNR2, which contains an open reading frame for the signaling peptide humanin. Immunohistology disclosed that humanin was preponderant in GAM. In vitro assays with a range of human stem-like GBM cells (GSCs) revealed that nanomolar concentrations of humanin can drive tumor cell expansion and chemoresistance to temozolomide. A series of genetic, pharmacological and Western blotting experiments showed that extracellular humanin increased GSC viability via stimulation of the GP130/IL6ST receptor and MAPK (Erk) activation. Humanin responsiveness was subject to inter-individual heterogeneity and predominated in GSC with high expression levels of the IL6ST subunit. Mechanistically, humanin promoted the ATR-dependent DNA-repair machinery in GSCs via induction of the DNA-clamp component HUS1. A slice culture model containing human microglia and GSCs confirmed these observations. Humanin is absent from the mouse brain. Exogenous delivery of humanin into orthotopic GBM mouse models or over-expression specifically of a secreted humanin variant recapitulated the in vitro findings. Blockade of the trimeric interleukin receptor with the brain permeant, FDA-approved drug bazedoxifene blocked humanin-mediated chemoresistance in vitro and in vivo. Overall, we identified a clinically applicable compound together with a predictive marker to prevent chemoresistance in a human-specific GBM model.
TMIC-78. CIRCADIAN REGULATOR CLOCK DRIVES IMMUNOSUPPRESSION IN GLIOBLASTOMA
Authors: Xuan Wenjing , Hsu Wen-Hao , Khan Fatima , Dunterman Madeline , Pang Lizhi , Wainwright Derek , Ahmed Atique , Heimberger Amy , Lesniak Maciej , Chen Peiwen
Published: November 2022

The symbiotic interactions between cancer stem cells and the tumor microenvironment (TME) are critical for tumor progression. However, the molecular mechanism underlying this symbiosis in glioblastoma (GBM) remains enigmatic. Here, we show that circadian locomotor output cycles kaput (CLOCK) and its heterodimeric partner brain and muscle ARNT-like 1 (BMAL1) in glioma stem cells (GSCs) drive immunosuppression in GBM. Integrated analyses of the data from transcriptome profiling, single-cell RNA sequencing, and TCGA datasets, coupled with functional studies, identified legumain (LGMN) as a direct transcriptional target of the CLOCK–BMAL1 complex in GSCs. Moreover, CLOCK-directed olfactomedin-like 3 (OLFML3) upregulates LGMN in GSCs via the hypoxia-inducible factor 1-alpha (HIF1A) signaling. Consequently, LGMN promotes microglial infiltration into the GBM TME via upregulating CD162, and polarizes infiltrating microglia towards an immune-suppressive phenotype. In GBM mouse models, inhibition of the CLOCK–OLFML3–HIF1A–LGMN–CD162 axis reduces intratumoral immune-suppressive microglia, increases CD8+ T cell infiltration, activation and cytotoxicity, and synergizes with anti-PD1 therapy. In human GBM, the CLOCK-regulated LGMN signaling correlates positively with microglial level and poor prognosis. Together, these findings uncover the CLOCK–OLFML3–HIF1A–LGMN axis as a molecular switch that controls microglial biology and immunosuppression, thus revealing potential new therapeutic targets for GBM patients.
TMIC-80. SPATIOTEMPORAL GENOMICS OF HUMAN MEDULLOBLASTOMA WITHIN SUBGROUPS
Authors: Liu Hailong , Qiu Xiaoguang , Jiang Tao
Published: November 2022

Whereas evidences of single-cell studies have suggested the cellular diversity of medulloblastoma, the most common malignant brain tumor in childhood, the understanding of spatial status for specific cell populations and gene modules remains rudimentary. Herein, we comprehensively analyze single-nucleus RNA expression, chromatin accessibility and spatial transcriptomic profiling from 52 human medulloblastomas spanning four molecular subgroups, complemented with the bulk whole genome and RNA sequencing data across 300 samples. We comprehensively interrogated the composition of medulloblastoma microenvironment and spatially described the developmental trajectory from progenitor-like to differentiated malignant cells. Furthermore, distinct heterogeneity within molecular subgroups leads to the identification of twelve subtypes of medulloblastoma, which unfortunately lacks the cellular biology. By deciphering the copy number variation and transcriptional programs at single-cell resolution, we in-depth characterized medulloblastoma and mapped the activity of gene expression and transcription factor binding according to individual subtype. The WNT-β, SHH-γ, Group 3-β and Group 4-γ were observed in early differentiation stage. Harmony alignment revealed subtype-dependent modules recapitulated the neurodevelopmental gene enrichment, strongly correlating to clinical outcomes. Collectively, our findings provide spatiotemporally resolved insights into transcriptome and epigenome of twelve-subtype medulloblastoma allowing further refinement regarding clinical risk stratification strategies.
TMIC-83. SEX AND ANTI-GLIOMA IMMUNITY
Authors: Kaminska Bozena , Ochocka Natalia , Segit Pawel , Wojnicki Kamil , Jacek Karol , Grajkowska Wieslawa
Published: November 2022

Glioblastomas (GBMs) are aggressive, inevitably lethal brain tumors that are massively infiltrated by myeloid cells supporting tumor growth. The main myeloid cell populations encompass brain-resident microglia, bone marrow (BM)-derived monocytes/macrophages and dendritic cells. Interestingly, patients with glioblastoma show sex-dependent differences in the incidence rate (male-to-female ratio of 1.6:1 in GBMs and 2:1 in most malignant mesenchymal GBMs), transcriptomic profiles and patient responses to a standard therapy. Sex-related differences such as higher expression of pro-inflammatory genes in female microglia and stronger upregulation of MHCII coding genes in microglia from male GL261-bearing mice were detected (Ochocka et al. 2021). We employed Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) to reliably dissect myeloid cell identities, states and evaluate dynamics of myeloid infiltration during progression of murine GL261 gliomas. We demonstrate the diversity of myeloid cells within the glioma microenvironment: glioma-activated microglia are the major source of cytokines attracting other immune cells, whereas BM-derived cells show the monocyte-to-macrophage transition and immunosuppressive phenotypes. This transition is coupled with a phenotypic switch from the IFN-related to antigen-presentation and tumor-supportive signatures. Moreover, we found striking sex-dependent differences in transcriptional programs and composition of myeloid cells in gliomas. Higher abundance of protumor macrophages in males correlated with greater tumor size. Re-analysis of single-cell omics data from human GBMs revealed the predominance of inflammatory monocytes in female GBMs and abundance of protumor macrophages in male GBMs. Our findings expand understanding of the complexity of anti-tumor immune responses in gliomas and may guide future therapies in consideration of patient sex. Studies supported by National Science Centre Poland research grants PRELUDIUM16 2018/31/N/NZ3/01696, OPUS 14 2017/27/B/NZ3/01605 and 2020/39/B/NZ4/02683.
TMIC-84. MULTIREGIONAL ANALYSIS OF GLIOBLASTOMA REVEALS TOPOLOGICAL DIVERGENCE OF CANCER CELLS AND THEIR SURROUNDING MICROENVIRONMENT
Authors: Salatino Roberto , Onubogu Ugoma , Klemm Florian , Joyce Johanna , Szentirmai Oszkar , Janiszewska Michalina
Published: November 2022

Tissue organization plays an important role in tumor development and evolution. Systematic investigation of the differences in the local composition of the tumor microenvironment (TME) in glioblastoma (GBM) in distinct regions of the tumor is needed for identification of traits driving tumor recurrence. Thus, we performed an analysis of three tumors, six spatially distant biopsies each, representing tumor surface, core, and deep margin. GBM cell fluorescence generated by the 5-aminolevulinic acid (5-ALA) metabolic labeling prior to surgery allowed us to enrich for the tumor cell fractions and the cells of the microenvironment, respectively, from each biopsy. Using RNA-seq and scRNA-seq we compared the transcriptomic profiles of GBM and tumor microenvironment (TME) at distinct locations within the tumor. We observed a gradient of GBM subtypes across the tumor regions, with a molecular identity progressively less defined from the tumor core towards the margins. Interestingly, the single-cell based copy number analysis revealed a subset of GBM cells that appeared to evade the 5-ALA labelling. The high prevalence of these 5-ALA-negative GBM cells within the deep tumor margins points to a metabolically distinct subpopulation that could be responsible for local tumor recurrence. The phenotypic gradient of cancer cells from tumor core towards the margins was also mirrored by tumor microenvironment-enriched fractions. Deep margin samples from the profiled tumors were transcriptionally more divergent than biopsies from other regions. Moreover, they were depleted from major populations of TME cells. In situ analysis revealed that TERT promoter mutant cells are not equally distributed across the tumor indicating the coexistence of GBM sub-clones with distinct evolutionary potential. Altogether, our results provide a novel insight into co-evolution of tumor microenvironment and GBM clonal heterogeneity within the context of the tumor tissue.
MODL-03. IDH-MUTANT GLIOMA GWAS ALLELE RS55705857 ON 8Q24 IS CHANGING THE DYNAMICS OF UNPATTERNED CEREBRAL ORGANOID DEVELOPMENT
Authors: Drucker Kristen , Kollmeyer Thomas , Yanchus Connor , De Lorenzo Silvana , Eckel-Passow Jeanette , Lachance Daniel Honore , Schramek Daniel , Jenkins Robert
Published: November 2022

BACKGROUND

The topologically associated domain (TAD) on 8q24 surrounding MYC is important for many types of cancer. The region is also important in the formation of IDH-mutant glioma; the risk allele rs55705857 being associated with the development of these tumors. The rs55705857 risk allele significantly increased the formation of tumors in an IDH-mutant mouse model. Cerebral organoids have been found to recapitulate the early development of the brain. We hypothesized that the rs55705857 risk allele may alter the phenotype of cerebral organoids.

METHODS

Isogenic induced pluripotent stem cells (iPSCs) were developed with (n=3) and without (n=2) the rs55705857 risk allele from the parental cell line PGP-1 (GM23338). These cells were cultured and differentiated into unpatterned cerebral organoids and observed throughout differentiation. Organoids were fixed, sectioned, and stained by H&E and immunofluorescence.

RESULTS

Organoids containing the risk allele were smaller throughout development, starting at the embryoid body stage and continuing up to over 75 days in culture, compared to two isogenic non-risk lines. At 4 weeks the risk allele containing organoids also contained a larger number of small rosette-like neuroepithelia, instead of the more continuous neuroepithelia observed in the non-risk allele organoids. At 75 days, the organoids with the risk allele maintained a larger proportion of Sox2 positive cells, which are enriched in the tight clusters of small cells with round nuclei.

CONCLUSIONS

The risk allele rs55705857 in the MYC TAD at 8q24 is altering the development of unpatterned cerebral organoids. This difference is visible as early as embryoid body development and persists through neuroepithelial development. Further studies of these differences may help our understanding of how rs55705857 accelerates IDH-mutant glioma development.
MODL-05. DISCOVERY OF DYNAMIC MINIMAL RESIDUAL DISEASE STATES IN ADULT GLIOBLASTOMA USING SINGLE CELL TECHNOLOGY
Authors: Zador Zsolt , Mikolajewicz Nicholas , Han Hong , Voisin Mathew , Venugopal Chitra , Chokshi Chirayu , Maich Will , Jason Moffat , Zadeh Gelareh , Singh Sheila
Published: November 2022

Persister states are proposed oncogenic substrates of disease recurrence in cancer. Recent concepts suggest persisters may occur in dynamic states of the cell cycle (such as cycling and non-cycling states). We therefore investigated biological programs at the post-treatment minimal residual disease (MRD) state following standard chemoradiotherapy in patient-derived xenograft models of GBM. Our analysis of single cell RNA sequencing (scRNA-seq) data from 2704 tumor cells (929 cells post treatment, 1775 matched controls) yielded a cellular profile for non-cycling and cycling persister states. We validated these programs in 3 independent scRNA-seq datasets of human glioblastoma specimens consisting of over 16,000 cells from various genetic backgrounds, including an internal two patient-matched primary and recurrent GBM pairs with over 13,000 cells. Finally, we determined that clones identified based on large scale chromosomal rearrangements converge on previously identified persister states including the dynamic states discovered in our study. Our results provide new evidence towards dynamic persister states in glioblastoma, further analysis of these dynamic states is critical to targeting this incurable disease.
MODL-08. ESTABLISHING IN VITRO MODELS TO RECAPITULATE PROTEIN-LEVEL PROGNOSTIC CELL PHENOTYPES FROM PRIMARY HUMAN GLIOBLASTOMA
Authors: McDonald Taylor , Chotai Silky , Bartkowiak Todd , Medina Stephanie , Hayes Madeline , Brockman Asa , Mobley Bret , Sills Allen , Morone Peter , Thompson Reid , Chambless Lola , Irish Jonathan , Ihrie Rebecca
Published: November 2022

Glioblastoma (GBM) is a highly aggressive primary central nervous system tumor for which median survival continues to be low, despite multimodal treatment and surgical resection. Heterogeneity at many levels (genomic, cytoarchitectural, and metabolic) likely contributes to these poor outcomes, and single-cell approaches can help to identify prognostically meaningful cells and features within heterogeneous tumors at each of these levels. Recent studies using mass cytometry (CyTOF) and the accompanying analysis pipeline Risk Assessment Population Identification (RAPID) measured per-cell protein levels in > 2 million cells from 28 IDH1/2-wildtype primary glioblastoma tumors and identified 2 phenotypes within these tumors that are present in different abundances across patients and have contrasting prognostic value. Increased abundance of Glioblastoma Negative Prognostic (GNP) cells within these tumors is predictive of shorter overall survival (OS HR = 1.07 [95% CI 1.02–1.12], p = 0.003), while the converse is true for Glioblastoma Positive Prognostic (GPP) cells (OS HR = 0.93 [0.87–1.0], p = 0.05). To effectively study these phenotypes, reproducible in vitro models are needed that recapitulate the total protein expression and phosphorylation events that distinguish GNP and GPP cells. Using available RNA transcript data, a candidate subset of glioma stem cell models were identified and are now being profiled using standardized mass-tagged antibody panels. These culture models will be quantitatively compared against established GNP and GPP populations in patient tissue samples using the Marker Enrichment Modeling statistic as well as other machine learning tools. Development of these models will be critical to future experiments aimed at identifying possible mechanistic therapies that effectively eliminate each of these identified populations, as well as aligning these protein-level phenotypes to other genomic and functional data in glioblastoma samples and models.
MODL-09. MODELLING MIGRATION OF GLIOBLASTOMA PATIENT-DERIVED CELLS USING HUMAN IPSC-DERIVED NEURAL SPHEROID AND HIGH CONTENT IMAGING
Authors: Tsang Victoria , Danovi Davide , Lieberam Ivo
Published: November 2022

Glioblastoma multiforme (GBM) is the most common and aggressive brain tumour in adults. Despite current advances, the existing standard of treatment is ineffective, and the survival prognosis remains 18 months on average from diagnosis. Migrating tumour cells have been implicated in the therapeutic resistance of GBM. They spread by interacting with structures such as white matter tracts and inevitably cause recurrence of the tumour. Valuable cell models able to capture the invasiveness of GBM are critically needed to develop innovative therapies targeting migrating GBM cells. We established an in vitro model mimicking the GBM microenvironment by co-culturing patient-derived GBM cells and human-induced pluripotent stem cell-derived cortical neural spheroids with radiating axons. Using high content imaging, we developed a robust workflow to quantify the GBM cells' infiltration of the neural spheroid in endpoint assays. Images were acquired on the Operetta CLS and analysed using Harmony Imaging and Analysis Software. We also performed live imaging assays using the Phasefocus Livecyte, in which we studied the directionality, displacement and speed of the GBM cells engaged on axons. Our data indicate that GBM cells change morphology when cultured on axons and that they migrated towards the neural spheroid once engaged on axons. We showed that patient derived-cell lines representing different subtypes vary in migratory properties as well as in levels of infiltration capability. Finally, we used this model to test antagonists inhibiting pathways involved in the migration of GBM cells. To summarise, we developed a novel model able to mimic the GBM migration on axons and able to screen for compounds affecting cell migration which could potentially offer innovative precision-medicine therapies.
CNSC-33. TRANSCRIPTOMIC NEURON-TUMOR INTERACTION MAPPING OF EXTRACRANIAL TUMORS AND BRAIN METASTASES
Authors: Schubert Marc , Wick Wolfgang , Kuner Thomas , Winkler Frank , Venkataramani Varun
Published: November 2022

Ongoing efforts in the field of Cancer Neuroscience seek to improve the understanding of neuron-cancer interactions and their effects on tumor progression. In glioma, synaptic neuronal input to tumor cells drives tumor progression and invasion. Even though neuron-cancer interactions have been described in brain metastases and extracranial tumors as well, the mechanisms of action between the nervous system and tumor cells are incompletely understood. Systematic transcriptomic analyses to detect overarching principles of neuron-tumor interaction have been lacking so far. Here, we performed focused integrative transcriptomic and proteomic analyses in more than 10 different extracranial tumor entities and brain metastases to analyse potential neuron-tumor interactions.Neurotransmitters receptor genes were heterogeneously expressed across extracranial tumor entities and brain metastases. We used Markov Affinity-based Graph Imputation of Cells to recover gene-gene relationships to robustly identify more than 50 initial gene co-expression networks (GCNs) associated with neurotransmitter genes. Molecular pathways that are associated with neurotransmitter receptor gene expression show heterogenous and overlapping neuronal gene expression programs in tumor cell subpopulations. While some GCNs showed overarching expression patterns of neuronal interactions across many entities including brain metastases as well as extracranial tumors, some GCNs were specialised signatures for certain tumor types. To characterise underlying gene regulatory networks, we performed transcription factor analyses. Certain GCNs were clearly associated with specific transcriptional regulators and known tumor biological functions. To validate our results, we further analysed the GCNs in bulk RNA datasets as well as on a protein level. We were able to identify neuronal gene expression patterns that were associated with worse survival across certain tumor entities.The results suggest that brain metastases and extracranial tumors have multiple biologically relevant mechanisms of interaction with the nervous system. These analyses are the foundation for further validation and clinical translation.
MODL-12. A NOVEL GENETICALLY ENGINEERED H3.3G34R MODEL REVEALS COOPERATION WITH ATRX LOSS IN UPREGULATION OF HOXA CLUSTER GENES AND PROMOTION OF NEURONAL LINEAGE
Authors: Abdallah Aalaa , Cardona Herminio , Gadd Samantha , Brat Daniel , Wadhwani Nitin , Shen Chen , Picketts David , Li Xiao-Nan , Becher Oren
Published: November 2022

BACKGROUND

Pediatric high-grade gliomas (pHGGs) are an aggressive CNS tumor which are often characterized by mutations in H3F3A, the gene that encodes Histone H3.3 (H3.3). A substitution of the Glycine at position 34 of H3.3 with either Arginine or Valine (H3.3G34R/V), was recently described in a large cohort of pHGG samples and has been characterized as occurring in anywhere between 5-20% of pHGGs. Attempts to study the mechanisms of H3.3G34R have proven difficult due to the developmental nature of the disease and the requirement of co-occurring mutations for model development.

METHODS

We utilized the RCAS/tv-a system to develop a genetically engineered mouse model (GEMM) that incorporates PDGF-A activation, TP53 loss and the H3.3G34R mutation both in the context of ATRX loss and ATRX presence in nestin expressing progenitor cells.

RESULTS

Transcriptomic analysis revealed that ATRX loss in the context of H3.3G34R upregulates the Hoxa cluster genes Hoxa2, Hoxa3, Hoxa5, and Hoxa7 (p < 0.05, unpaired t-test). H&E staining and EMA immune-staining indicate that ATRX loss is correlated with a higher incidence of ependymal differentiation in H3.3G34R expressing samples (p < 0.05, unpaired t-test). GSEA and RT-qPCR analysis demonstrate that H3.3G34R expression in the context of ATRX loss promotes increased expression in genes associated with neuronal lineage (FWER pval < 1.0) and exhibits upregulation of the neurofilament polypeptides Nefl, Nefm (p < 0.05, unpaired t-test) as well as the neuronal differentiation marker Stmn2 (p < 0.001, unpaired t-test).

CONCLUSION

Our study proposes a model in which cooperation between ATRX loss and H3.3G34R expression mediate major transcriptomic and histopathological changes in H3.3G34R pHGGs. Broadly, our work highlights the importance to study mechanisms of co-occurring genetic events separately and in combination.
MODL-14. MODELING THE INTRATUMORAL HETEROGENEITY OF AGGRESSIVE GLIOBLASTOMA ON ORGANOTYPIC BRAIN SLICES TO OPTIMIZE TUMOR-HOMING TUMORICIDAL INSC TREATMENT
Authors: Zhang Xiaopei , Dunn Denise , Valdivia Alain , Buckley Andrew , Floyd Scott , Hingtgen Shawn , Satterlee Andrew
Published: November 2022

BACKGROUND

Tumor-homing tumoricidal neural stem cell (tNSC) therapy is a promising new strategy that recently entered human patient testing for glioblastoma (GBM). Developing strategies for tNSC therapy to overcome intratumoral heterogeneity, variable cancer cell invasiveness, and differential drug response of GBM will be essential for efficacious treatment response in the clinical setting. We sought to create novel hybrid tumor models and investigate the impact of GBM heterogeneity on tNSC tumor response and treatment durability.

METHODS

We utilized organotypic brain slice explants and human cells with varied properties to generate heterogeneous GBM models ex vivo and in vivo. We first investigated the treatment response and durability of mono- and combination therapy with primary NSCs and fibroblast-derived human induced neural stem cells (iNSCs) engineered with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or enzyme-prodrug therapy. Next, we utilized the ex vivo models and molecular assays to explore mechanisms driving tumor adaption and escape.

RESULTS

Non-invasive imaging, molecular assays, and immunohistochemistry showed the new hybrid GBM models recapitulated key aspects of the clinical disease. Testing in multiple in vivo models showed that tNSC-TRAIL therapy induced robust initial inhibitions in tumor growth and significantly increased survival. However, tumors rebounded in multiple models and patterns of tumor regrowth varied with therapeutic, dose, and route of administration. We found that adjusting iNSC delivery strategies increased spatiotemporal TRAIL coverage and significantly decreased GBM volume throughout the brain, reducing tumor burden 100-fold as quantified in live ex vivo brain slices and resulting in varied impact on treatment durability and median survival across models of both solid and invasive tumors.

CONCLUSIONS

These studies report new hybrid models that accurately capture key aspects of GBM heterogeneity which markedly impact treatment response while demonstrating the ability of tNSC mono- and combination therapy to overcome certain aspects of heterogeneity for robust tumor kill.
MODL-16. SOMATICALLY ENGINEERED MOUSE MODELS RECAPITULATING CELLULAR AND MOLECULAR FEATURES OF HUMAN GBM
Authors: Pyczek Joanna , Ahn Bo Young , Seo Heewon , Lawn Samuel , Snelling Shannon , Osz Katalin , Flynn Robyn , Morrissy Sorana , Chan Jennifer
Published: November 2022

INTRODUCTION

Mouse models are instrumental in advancing our understanding of glioblastoma (GBM), however many commonly used models have features that limit their practicality or applicability. These limitations can include an incomplete immune context, a requirement for complex breeding strategies, high cost of specialized lines, and unpredictability in where tumors arise. We sought to develop and validate a mouse modeling system for some of the major human GBM subtypes that overcame these practical challenges.

METHODS

In vivo electroporation was used to introduce genetic alterations into periventricular cells of early postnatal C57Bl6/j mice. PiggyBac transposon/transposase and CRISPR-Cas9 systems were used to overexpress (OE) or knock out (KO) genes associated with mesenchymal (Nf1-KO/Pten-KO/p53-KO), classical (EGFRvIII-OE/Cdkn2a-KO/Pten-KO), and proneural (Pdgfra-OE/Cdk4-OE/p53-KO) IDH-WT GBM subtypes. KO or OE was confirmed by Indel Detection by Amplicon Analysis and immunofluorescent staining (IF). Tumours were analyzed for histologic, immunohistochemical, and transcriptional (RNAseq) features.

RESULTS

Tumors arose with near complete penetrance and median survivals of 36 to 116 days amongst models. All tumors were GFAP-expressing high-grade gliomas, with distinct phenotypes associated with different genetic combinations. Nf1-KO/Pten-KO/p53-KO tumors were composed of spindle cells. EGFRvIII-OE/Cdkn2a-KO/Pten-KO and Pdgfra-OE/Cdk4-OE/p53-KO tumors showed prevalence of small, round cells. Molecularly, Nf1-KO/Pten-KO/p53-KO tumors were enriched for human mesenchymal signature and displayed more differentiated phenotype. The Nf1-KO/Pten-KO/p53-KO model also showed increased stromal cells and macrophages. The other models had mixed proneural and classical profiles and were enriched for OPC- and NPC-like gene signatures found in human GBM. Once tumors formed, cells from each model were transplantable into C57Bl6/j mice to generate subsequent tumors.

CONCLUSION

We developed and validated a rapid, versatile, and reproducible system to model GBMs. These models allow for controlled study of GBM pathogenesis, progression, and treatment response, and allow for robust generation of syngeneic implantable mouse models that can serve as valuable tools for preclinical testing.
MODL-18. ORGANOTYPIC BRAIN SLICE CULTURES: A COMPREHENSIVE MODEL FOR PRIMARY CNS TUMOR STUDIES
Authors: Reid Alexandra
Published: November 2022

Inducing an immunosuppressive environment has been one of the most notable subversive tactics employed by aggressive primary brain tumors like Glioblastoma (GBM). It is vital that we garner a better understanding of the cell-cell interactions occurring within the tumor microenvironment (TME) in situ. Current in vitro and in vivo models sorely limit our ability to study the cell-cell interactions within the TME while accounting for its complexity and the spatial orientation of its components. Unfilled, this gap in physiologically relevant models greatly hinders the translation of impactful research findings to successful treatment options for patients diagnosed with GBM and other primary CNS tumors. We have devised a novel organotypic brain slice culture (BSC) model study using tumor-bearing C57BL/6J mice which brings the potential to not only determine the cell-cell interactions within the TME but also investigate the underlined mechanisms which govern them. Obtaining BSCs from tumor-bearing mice at various time points post-implantation also allows for analysis of the trafficking of various immune cells across the blood-brain barrier (BBB). We demonstrated outcomes similar to those seen in vivo following the introduction of parallel conditions, further supporting the physiological relevancy of this model. Thus, organotypic brain slice cultures from tumor-bearing mouse models may serve as a credible and incredibly versatile model for the study of primary CNS tumors which may bridge the gap between in vitro and in vivo studies.
MODL-20. REDUCTION OR LOSS OF MSH6 CONFERS RESISTANCE TO TEMOZOLOMIDE IN GLIOBLASTOMA
Authors: Vallcorba Montserrat Puigdelloses , Pinero Gonzalo , Hambardzumyan Dolores
Published: November 2022

Glioblastoma (GBM) is the most common and aggressive primary brain tumor. The only intervention that has improved the survival rate of GBM patients over the past several decades has been combining temozolomide (TMZ) with radiotherapy (RT), which increased median survival by only ~2.5 months to where it currently stands at ~15 months. Unfortunately, all GBM patients eventually die due to tumor recurrence. Intrinsic or acquired resistance to TMZ is a significant contributing factor to tumor progression, and various mechanisms have been suggested, including deficiencies in DNA mismatch repair (MMR) genes such as MSH6. To test the biological significance of Msh6 in GBM growth and resistance to TMZ and immunotherapy, we have generated immunocompetent genetically engineered mouse models (GEMMs) of GBM with germline loss of Msh6. To generate tumors with reduced or abrogated Msh6, we utilized GEMMs based on the RCAS/tv-a gene transfer system in combination with mice that exhibited heterozygous and homozygous loss of Msh6. When PDGFB was overexpressed in combination with the silencing of Tp53 to induce tumors, tumor-bearing mice with reduced or deficient Msh6 demonstrated increased tumor growth and shorter survival time compared to wild-type tumor-bearing mice. Our data demonstrate that two weeks of TMZ treatment at a clinically relevant dose of 25 mg/kg provides a significant survival advantage in WT-tumor-bearing mice, while no efficacy was observed in tumor-bearing mice with either heterozygous or homozygous loss of Msh6. We are currently evaluating the mechanism by which of Msh6 confers TMZ-resistance.
CNSC-35. CANCERS GOT THE NERVE
Authors: S.Senga Sasi
Published: November 2022

Brain cancers carry a bleak prognosis despite the increase in our understanding of the disease. Brain tumours are in some ways a distinct entity in comparison to other types of tumours due to the distinct type of cell population in the brain tumour microenvironment. Sympathetic and parasympathetic nervous systems have been shown to exert a context-dependent effect on tumourigenesis. In 2021, I proposed neuronal signalling as a hallmark of cancer, albeit resection of specific nerves to hinder tumourigenesis is both challenging and may not result in the desired outcome. In order to add the nervous system to our armamentarium of therapeutic strategies against cancer, we may need to look beyond conventional methods of reducing synaptic plasticity such as with the use of anti-epileptic drugs. One among the strategy I propose is to utilize epigenetics to our advantage. Using lysine demethylase 5 specifically KDM5C to alter the synapticity in the brain tumour microenvironment is one way of exploiting the synaptic plasticity to our advantage against cancers. Additionally, metabolism intertwines the effects mediated by the nervous system which can be exploited to pivot the tumour-promoting effects of the nervous system towards tumour elimination.
MODL-27. PRECLINICAL INTERROGATION OF IMMUNOTHERAPY TREATMENT STRATEGIES IN GLIOBLASTOMA (GBM) USING A CLINICALLY RELEVANT DISEASE MODEL
Authors: Clerkin James , Connor Kate , White Kieron , Sweeney Kieron , Shiels Liam , Van Brussel Thomas , Arijs Ingrid , Lambrechts Diether , Maher Stephen , Marignol Laure , Prehn Jochen , O'Brien David , Byrne Annette
Published: November 2022

Clinically faithful pre-clinical models are essential for screening therapies and studying resistance mechanisms. Historically models have failed to predict response in the clinical setting. Current models seldom incorporate surgical resection, and commonly use young animals whose immune contexture differs from older patients. Here, we have established an orthotopic model, employing the syngeneic, mesenchymal-NFpp10a-cell line, in both young and aged mice. We have characterised the model's response to standard of care resection and temozolomide (TMZ) treatment. We have further studied response to anti-PD1 therapy (adjuvant and neoadjuvant). NFpp10a-Luc2 expressing cells were orthotopically implanted into C57BL/6 mice (aged { > 18months} and young {6-8weeks}) and weekly bioluminescence imaging (BLI) performed to monitor tumour growth. Several therapeutic interventions were assessed to study tumour growth and survival: (1)surgical resection, (2)TMZ, (3)anti-PD1 (4)neoadjuvant anti-PD1. RNAseq, murine microenvironment cell population (mMCP) counter and gene ontology analyses characterised treatment related changed in the TME. We demonstrated survival advantage in aged mice undergoing resection (Resection:33.5 days vs Non-Resection:18 days;p=0.0166). Subsequently, we observed that TMZ and anti-PD1 monotherapy had no impact on NFpp10a-Luc2 growth (TMZ-overall:p=0.9001, and anti-PD1-overall:p = 0.7933) or survival (TMZ-overall:p = 0.3035, anti-PD1-overall:p= 0.6328). Neoadjuvant anti-PD1 treated mice demonstrated no survival advantage compared to IgG control (33-days vs 35-days:p = 0.9429). or BLI signal (p = 0.1703). NFpp10a-Luc2 forms immune-cold TME relative to commonly employed models. mMCP counter analysis demonstrates neoadjuvant anti-PD1 induced influx of CD8+-T cells, B cells, and monocytes into the TME compared to IgG control, and was associated with significantly upregulated CXCR3 chemokine receptor binding pathway on gene ontology analysis (p = 0.0045). Overall, we have, for the first time established and characterised response of the NFpp10a-Luc2-C57BL/6 model to resection, TMZ and anti-PD1 therapies. NFpp10a-Luc2 tumours demonstrate similar therapeutic resistance and TME profile compared to human mesenchymal GBM. The model may therefore be employed in future pre-clinical studies to guide clinical trials in mesenchymal GBM.
MODL-30. AN ENGINEERED TRANSGENIC MOUSE MODEL OF RETINOBLASTOMA
Authors: Liu Xiao , Pei Yanxin
Published: November 2022

Retinoblastoma, the most common intraocular tumor of childhood, was recently shown to comprise two major molecular subtypes. Subtype 1 includes most heritable forms, with germline mutation of RB1 and a cone-differentiated state. Subtype 2 is often non-hereditary, exhibits stemness features, increased expression of MYCN and its target genes, and a high risk of metastasis. Relative to subtype 1, subtype 2 tumors have a worse prognosis. To date, no mouse model has been available for studying subtype 2 disease. It is known that SHH pathway proteins including SHH, GLI1 and GLI2 are abnormally elevated in human retinoblastoma. We recently discovered that activation of GLI2 in the developing mouse retina leads to intraocular tumors that exhibit cellular and molecular features resembling human subtype 2 retinoblastoma with aberrant upregulation of MYCN and its target genes. Because MYCN is a downstream target of the SHH pathway, we hypothesize that inhibiting the SHH-GLI2-MYCN signaling axis may effectively treat subtype 2 disease. Using our GLI2 based mouse model of retinoblastoma, we will elucidate mechanisms underlying tumor initiation and progression, and evaluate the therapeutic potential of SHH and GLI2 inhibitors in preventing retinoblastoma. We will validate our mouse model findings using human retinoblastoma cell lines and patient-derived tumor xenografts. Our studies will yield new mechanistic insights into the development of subtype 2 retinoblastoma and identify novel molecular targets for early detection, treatment, and prevention of ocular tumors and associated metastatic disease.
MODL-31. PHOTORECEPTOR-POSITIVE PROGENITORS PUTATIVE CELLS OF ORIGIN IN MYC-DRIVEN GROUP 3 MEDULLOBLASTOMA
Authors: Zhao Miao , Mainwaring Oliver , Rosén Gabriela , Olsen Thale Kristin , Rijpkema Sanne , Sundström Anders , Weishaupt Holger , Furukawa Takahisa , Swartling Fredrik
Published: November 2022

The cell of origin of Group 3 medulloblastoma, the most malignant medulloblastoma subgroup, is currently unknown and remains controversial. Transcriptional profiling has revealed that Group 3 medulloblastomas are characterized by elevated expression of a photoreceptor program, which has not been described in the normal cerebellar development but is well characterized in the developing retina and pineal gland. We used lineage tracing and single-cell sequencing to compare normal brain development with tumor development in our previously generated MYC-driven transgenic mouse model (GMYC), where mice spontaneously develop Group 3 medulloblastoma after 4-6 months of age. We found that tumor cells emerged from progenitor cells where MYC overexpression drove the transformation of immature progenitor cells with photoreceptor pathway activity. Our data suggest that MYC-driven Group 3 medulloblastoma originates from progenitor cells expressing a photoreceptor program, which has implications for future research and the development of novel treatments targeting this devastating childhood malignancy.