bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

Abstract
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

Abstract

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

Abstract
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

Abstract
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 &gt; 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

Abstract
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

Abstract
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

Abstract

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 &lt; 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 &lt; 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 &lt; 1.0) and exhibits upregulation of the neurofilament polypeptides Nefl, Nefm (p &lt; 0.05, unpaired t-test) as well as the neuronal differentiation marker Stmn2 (p &lt; 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

Abstract

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

Abstract

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

Abstract
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

Abstract
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.