bluuebunny/crossref_metadata_2025_split huggingface dataset:
TAMI-73. GLIOBLASTOMA CELL POPULATIONS WITH DISTINCT ONCOGENIC PROGRAMS RELEASE PODOPLANIN AS PROCOAGULANT EXTRACELLULAR VESICLES
Authors: Tawil Nadim , Bassawon Rayhaan , Meehan Brian , Montermini Laura , Nehme Ali , Choi Dongsic , Spinelli Cristiana , Adnani Lata , Mohammadnia Abdulshakour , Couturier Charles , Petrecca Kevin , Rak Janusz 
Published: November 2021

Abstract

BACKGROUND
Vascular anomalies, including thrombosis, are a hallmark of glioblastoma (GBM) and an aftermath of dysregulated cancer cell genome and epigenome. Up-regulation of podoplanin (PDPN) by cancer cells has recently been linked to an increased risk of venous thromboembolism in glioblastoma patients. Thus, regulation of this platelet activating protein by transforming events and release from cancer cells is of considerable interest.

AIMS
I. Investigate the pattern of PDPN expression and characterize PDPN-expressing cellular populations in GBM. II. Evaluate the contribution of oncogenic drivers to PDPN expression in GBM models. III. Investigate the potential involvement of extracellular vesicles (EVs) as a mechanism for systemic dissemination of PDPN and tissue factor (TF). IV. Examine the role of PDPN in intratumoral and systemic thrombosis.

METHODS
Bioinformatics (single-cell and bulk transcriptome data mining), GBM cell lines and stem cell lines, xenograft models in mice, ELISA assays for PDPN and TF, platelet (PF4) and clotting activation markers (D-dimer), EV electron microscopy, density gradient fractionation, and nano-flow cytometry.

RESULTS
PDPN is expressed by distinct glioblastoma cell subpopulations (mesenchymal) and downregulated by oncogenic mutations of EGFR and IDH1 genes, via changes in chromatin modifications (EZH2) and DNA methylation, respectively. GBM cells exteriorize PDPN and/or TF as cargo of exosome-like EVs shed both in vitro and in vivo. Injection of glioma PDPN-EVs activates platelets. Increase of platelet activation (PF4) or coagulation markers (D-dimer) occurs in mice harboring the corresponding glioma xenografts expressing PDPN or TF, respectively. Co-expression of PDPN and TF by GBM cells cooperatively increases tumor microthrombosis.

CONCLUSION
Distinct cellular subsets drive multiple facets of GBM-associated thrombosis and may represent targets for diagnosis and intervention. We suggest that the preponderance of PDPN expression as a risk factor in glioblastoma and the involvement of platelets may merit investigating anti-platelets for potential inclusion in thrombosis management in GBM.
TMOD-08. DEVELOPING DERIVATIVE GBM PDX, IN VIVO, FROM TREATMENT NAÏVE SOURCES
Authors: James David , Horbinski Craig , Stupp Roger , Ahmed Atique 
Published: November 2021

Abstract

PURPOSE
Post-therapy recurrent glioblastoma (GBM) patient-derived xenografts (PDX), developed from corresponding treatment-naïve PDX, could serve as useful resources for identifying therapeutics with activity against recurrent GBM. The goal of this study was to determine whether treatment-naïve intracranial GBM PDX, in mice receiving radiotherapy (RT) and/or temozolomide (TMZ), acquire the same mutations that occur in post-RT+TMZ GBMs from patients.

METHODS
Luciferase-modified, treatment-naïve GBM PDX were engrafted in the brains of athymic nude mice, followed by treatment with RT only (2 Gy/day x 5), TMZ only (10 mg/kg/day x 5), or RT+TMZ. Bioluminescence imaging was used to monitor intracranial tumor growth, response to treatment, and recurrence from treatment. Some mice with recurrent tumors received additional TMZ treatment. When mice became symptomatic, intracranial tumors were resected and engrafted subcutaneously in a new mouse host, then sequentially propagated subcutaneously into additional host mice. After the third passage, whole-exome sequencing (WES) was done, comparing post-therapy with treatment-naïve PDX sequence variants.

RESULTS
Analysis of PDX WES showed the following: 1) TMZ consistently caused more genes to incur coding sequence mutations than RT, as much as 13x more; 2) TMZ-treated tumor mutations were mostly G-C to A-T transitions (71-92%), consistent with the known mutagenic effect of TMZ; and 3) post-therapy PDX acquire similar mutations as do recurrent GBMs in patients, for example involving DNA mismatch repair gene MSH6. One of the derivative PDX with MSH6 mutation has been retested for response to RT and TMZ, with results showing its having become TMZ, but not RT resistant.

CONCLUSIONS
The mutation profiles of RT+TMZ-treated PDX are similar to those reported for GBMs that recur after RT+TMZ in patients. The new PDX resources described here may prove useful for identifying effective treatments against recurrent GBM.
TMOD-09. CREATION OF A P53-INDEPENDENT IN VITRO AND IN VIVO MODEL SYSTEM FOR THE STUDY OF H3.1K27M DIPG
Authors: Lagas Joseph , Yang Lihua , Becher Oren , Rubin Joshua 
Published: November 2021

Abstract
Diffuse Intrinsic Pontine Glioma (DIPG) is a devastating pediatric high-grade glioma that occurs in the brainstem with a median survival of less than 1 year. A greater understanding of the early tumorigenic events is essential for the development of effective therapeutics. DIPG is characterized by founder mutations in histone H3, either H3.1K27M or H3.3K27M. These mutations cause global hypomethylation, resulting in aberrant gene expression. It is unknown how this mechanism contributes to tumorigenesis. Interestingly, H3.1K27M DIPG show an increased incidence in females, whereas H3.3K27M DIPG shows no sex difference. This illustrates that the tumorigenic potential of H3.1K27M may be different between the sexes. Few models of DIPG incorporate the study of H3.1K27M despite the fact that it represents a unique opportunity to obtain valuable information on the tumorigenesis of DIPG through the study of the sex difference. Thus, we have created an in vitro and in vivo model system for H3.1K27M DIPG utilizing the RCAS mouse model system. This system utilizes RCAS vectors and a RCAS-ntva transgenic mouse line to deliver specific mutations to nestin expressing cells in the brainstem, including oligodendrocyte progenitor cells (OPCs), the predicted cell of origin. Delivering H3.1K27M, ACVR1 R206H, and PDGFaa at postnatal day 7 produces DIPG-like tumors in vivo, confirmed by H and E staining, between 60 – 110 days post injection. Additionally, confirmed through immunofluorescence staining, we can isolate a pure population of OPCs via immunopanning and infect them with RCAS vectors in vitro to produce stable expression of H3.1K27M. Introduction of H3.1K27M alone into male and female OPC cultures provides an opportunity to compare the early tumorigenic effects of H3.1K27M between the sexes in vitro. These results demonstrate that we have created an in vitro and in vivo H3.1K27M DIPG model system for the study of sex differences and tumorigenesis in DIPG.
TMOD-10. THE ROLE OF IDH1 R132H MUTATION IN GLIOMA – AN INVESTIGATION BY GENOME-EDITING IN HUMAN INDUCED PLURIPOTENT STEM CELLS
Authors: Köpp Alexander , Gawehn Luzie , William Doreen , Preussler Matthias , Richter Susan , Heide Michael , Marrone Lara , Thieme Anne , Borgmann Felix Kleine , Mittelbronn Michel , Capper David , Schröck Evelin , Klink Barbara 
Published: November 2021

Abstract

BACKGROUND
Hot-spot mutations in the Isocitrate dehydrogenase 1 (IDH1) cause a new catalytic function resulting in the production of 2-HG, a hallmark in the development of low-grade glioma. The tumorigenic mechanism of this mutation as well as the cell of origin are not known and there is a lack of suitable disease models. Thus, we aim to create a model mimicking glioma development by introducing the IDH1 R132H into human induced pluripotent stem cells (hiPSC) and investigate the influence on stem cell properties and cell differentiation in neuronal progenitor cells.

MATERIAL AND METHODS
We use CRISPR/Cas9 based genome editing to induce the IDH1 R132H mutation into healthy-control-derived hiPSCs. Successful introduction of the mutation was confirmed on DNA, RNA and protein level. The hiPSCs are then differentiated into cerebral organoids and characterized using transcriptome sequencing and methylation arrays.

RESULTS
We successfully introduced the IDH1 R132H mutation into hiPSCs and confirmed expression of the mutated protein by Western Blot. Metabolite measurement using liquid chromatography tandem mass spectrometry (LC-MS/MS) showed a forty times increased concentration of 2-HG in IDH-mutated compared to the wildtype hiPSCs, proving that the mutated enzyme is functional. To investigate effects of IDH1 R132H on cell differentiation, we generated cerebral organoids from our iPSC-models. The IDH1 R132H mutation did not inhibit cell differentiation or maturation of cerebral organoids but led to a downregulation of splicosome, proteasome and DNA repair enzymes as well as an upregulation of ECM components.

CONCLUSION AND OUTLOOK
hiPSCs with R132H mutation pose a promising model for investigations on early glioma development. We are currently step-wise including TP53 and ATRX loss of function mutations in our hiPSC models to recapitulating tumor development in vivo.
TMOD-13. IDENTIFYING DRIVERS IN THE CONVERGING SYNTENIC REGIONS OF SPONTANEOUS CANINE AND PEDIATRIC HIGH-GRADE GLIOMA USING IMAGING BASED CRISPR-CAS9 ARRAY SCREEN
Authors: Amin Samirkumar , Kang Wonyeong , Gujar Amit , Maher Leigh , Courtois Elise , Robson Paul , Dickinson Peter , Packer Rebecca , Lee Charles , Verhaak Roel 
Published: November 2021

Abstract
Gliomas occur in companion dogs at rates comparable to humans, with short-snouted breeds such as boxers being more susceptible than others. The natural progression of cancer in the immuno-competent host allows companion dogs diagnosed with sporadic glioma as an optimal model for preclinical testing of therapeutic approaches with human relevance, including immunotherapies. We have recently performed comprehensive genomic and epigenetic characterization of glioma in dogs to their human counterparts and found strong convergent evolution – shared somatic mutations and aneuploidies - among syntenic regions, including those of known pediatric glioma drivers, e.g., PDGFRA, MYC, PIK3CA. Here, using arrayed CRISPR-Cas9 imaging based phenotypic screen, we will probe potential oncogenic drivers and tumor suppressor genes within syntenic aneuploidies and thus outline functional versus non-functional heterogeneity of cancer aneuploidy. Specifically, we are conducting arrayed knockout screen (one gene per well) of 400+ genes within syntenic aneuploidies across canine (n=2) and pediatric (n=2) high-grade glioma cell lines. We will first capture images by high-speed confocal imaging system at three time points post-transduction of single guide RNAs (2 per gene) targeting each of 400+ genes in their separate wells. Then, using high-throughput image analysis and semi-supervised machine learning methods, we will measure well-based phenotypic features (viability, growth, and morphology) from these images. Genes will be ranked per cross-validated predicted probability in yielding either proliferating or slow-growing cell type based on learned phenotypic features using image data of knockout cells from and across wells. The top ranked genes will then be linked to oncogenes and tumor suppressors based on pathway and ontology analysis. We expect that we will see convergence of the most impactful molecular abnormalities (based on their knockout phenotypes) on mechanisms or candidate signaling pathways for the development of new drugs and repurposing of existing drugs for kids and dogs with high-grade glioma.
TMOD-14. CONJOINED CLASS I AND II EPITOPES ENHANCE NEOANTIGEN-TARGETED ACTIVE IMMUNOTHERAPY
Authors: Swartz Adam , Congdon Kendra , Nair Smita , Li Qi-Jing , Herndon James , Suryadevara Carter , Riccione Katherine , Reedy Jessica , Cook Sarah , Archer Gary , Norberg Pam , Sanchez-Perez Luis , Sampson John 
Published: November 2021

Abstract

INTRODUCTION
Cancer vaccines involve the administration of tumor-associated antigens into the host to generate anti-tumor T cell responses. Glioblastoma (GBM) is a disease with poor prognosis. GBM has a limited number of immunotherapeutic targets due to low mutational load, and is also highly heterogeneous; targeting a single antigen leads to antigen escape and tumor growth.

METHODS
VMDK mice were subcutaneously implanted with 750,000 SMA560 cells and on days 1 and 8 post implantation, mice were treated with 15 nmol of the universal helper epitope, P30, conjoined to the MHCI-restricted neoepitopes Odc1MHCI-P30 or Topbp1MHCI-P30. Human CD27 (hCD27) transgenic mice were intracranially implanted with CT2A-Odc1, followed by anti-CD27 and 15 nmol of Odc1MHCI-P30. B16.OVA or B16.F10 tumor cells were intracranially implanted in hCD27 mice and received SIINFEKL-P30 or Trp2-P30 conjoined peptides. Tumor growth, survival, or IFNγ secretion of splenic or tumor-infiltrating cells was assessed.

RESULTS
Unlike Odc1MHCI mixed with P30, conjoined Odc1MHCI-P30 had equivalent immunogenicity and anti-tumor efficacy to that observed with native long Odc1 peptide. Native long peptide of Topbp1 did not elicit an antitumor response, yet Topbp1MHCI-P30 caused an increase in numbers of IFNγ-secreting splenocytes and a decrease in tumor growth and similar to that seen with Odc1MHCI-P30 . Anti-CD27 treatment significantly increased numbers of IFNγ secreting splenocytes in Odc1MHCI-P30 vaccinated hCD27 mice, and the use of anti-CD27 with Odc1MHCI-P30 achieved long-term survival in 90% of tumor bearing hCD27 mice. Anti-CD27 synergized with SIINFEKL-P30 and Trp2-P30 to significantly improve survival after administration of these peptides.

CONCLUSIONS
Our work shows that poorly immunogenic neoantigens can be conjoined to P30 and used to generate an anti-tumor response in mouse models of GBM, and anti-tumor responses of conjoined peptides can be enhanced with anti-CD27 treatment. Together, these data demonstrate the efficacy of neoantigen vaccines for the treatment of heterogeneous GBM.
TMOD-16. HYPOXIA RESHAPES THE GLIOBLASTOMA MICROENVIRONMENT INVOKING NECROSIS AND TRIGGERING DISEASE PROGRESSION
Authors: Markwell Steven , Olson Cheryl , Ross James , Brat Daniel 
Published: November 2021

Abstract
Emerging necrosis within solid tumors corresponds with malignant progression. Current tumor model systems fail to adequately mimic the magnitude of post-necrotic restructuring within the microenvironment and remain overly reliant on post-mortem and descriptive analyses, obligating researchers to extrapolate causal relationships between necrosis and progression phenomena that emerge during tumor evolution. In glioblastoma (GBM; WHO grade 4), the most malignant primary brain tumor, vascular pathology and central necrosis precede rapid, radial expansion. Despite extensive genetic characterization in GBM, mechanisms enabling selective fitness within a hypoxic/anoxic setting remain poorly understood. Persistent nutrient deprivation culminates in necrosis, dramatically altering the tumor microenvironment (TME). We established mouse models that more aptly capture events found in human gliomas, exposing the dynamic temporal and spatial changes that facilitate expansive progression while including unique microenvironmental stressors typically absent from GBM animal models, specifically central necrosis. This model combines hypoxia-induced focal necrosis within GBM with real time intravital microscopy to capture TME restructuring and elucidate its impact on glioma progression. Our studies use genetically characterized patient-derived orthotopic GBM xenografts, alongside an immunocompetent RCAS/tv-a model, to determine how these processes impact disease progression and outcomes across multiple GBM molecular subtypes. Simultaneously, we employ in vitro models to scrutinize how hypoxia-related crosstalk between GBM, microglia and circulating monocytes alter tumor-associated macrophage (TAMs) recruitment and reprogramming. Our preliminary data suggests microenvironmental cues significantly alter microglia behavior and have demonstrated increased 3D invasion under hypoxic conditions as compared to normoxia. Monocyte invasion varies based on signals emanating from specific GBM subtypes, yet the signaling events that elicit differential responses remains unknown. However, exposure to any GBM conditioned media uniformly upregulates immunosuppressive TAM programming. Our ongoing investigations seek to uncover the mechanisms driving post-necrotic GBM evolution and reactive neuroinflammation
TMOD-19. FROM PATIENT TO PETRI DISH: INCREASING PATIENT-DERIVED GLIOBLASTOMA CULTURE EFFICIENCIES TO 95%
Authors: Verheul Cassandra , Fabro Federica , Ntafoulis Ioannis , Beerens Cecile , Hoogstrate Youri , Kers Trisha , Van den Burg Judith , Van Stanley , French Pim , Chien Miao , Dirven Clemens , Leenstra Sieger , Lamfers* Martine ,   GLIOTRAIN Consortium Biobank
Published: November 2021

Abstract

INTRODUCTION
The search for effective therapies for gliomas is progressively moving towards patient-specific medicine. In order to test patient-tailored therapies, it is vital to develop protocols for reliable establishment of patient-derived glioma cultures. We present a method for reliable culture establishment, with a 95% success rate in 114 consecutive high-grade samples.

METHODS
Cell cultures were established from either traditionally-resected tumor tissue or ultrasonic surgical aspirator (CUSA) derived tissue fragments, and expanded in serum-free culture, with selection of astrocytic populations if required. Cultures were started from single cells or small tumor fragments of 0.5-3mm (3D). Whole exome and RNA sequencing were carried out with the Illumina Novaseq and HiSeq platforms. Methylation profiling was performed with the Infinium MethylationEPIC array. Cultures and tumors were compared through analysis of single nucleotide polymorphisms and copy number profiles with the Infinium Global Screening Array. Intra-tumoral heterogeneity in cultures was investigated with single-cell transcriptomic sequencing (SORT-seq). We studied tumor-initiating potential by orthotopic injection of cultures in NOD-SCID mice.

RESULTS
Cultures started from single cells were established from CUSA material more efficiently (92%) than from traditional resection material (70%). 3D-derived cultures had a higher overall efficiency (95% for CUSA, 85% for traditional resection material). We confirmed high concordance in driver mutations, copy number and methylation profiles between tumors and derived cultures. Transcriptomics analysis, comparing tumors and derived cultures, revealed high consistency in gene expression distribution as demonstrated by correlation analysis (r=0.88). Singe-cell RNA-seq shows increased heterogeneity in CUSA derived-cultures, and decreased heterogeneity with passaging over time. Cultures faithfully produce tumors after orthotopic injection in NOD-SCID mice.

CONCLUSION
We present a highly successful method for the establishment of glioma cultures from patient material, with CUSA-derived cultures revealing greater heterogeneity. Cultures faithfully represent important molecular characteristics of parental tumors and can be used to test potential therapies in vitro.
TMOD-24. PATIENT-DERIVED ORGANOIDS TO MODEL AND CHARACTERIZE TUMORAL HETEROGENEITY OF PEDIATRIC BRAIN CANCERS
Authors: Lago Chiara , Ballabio Claudio , Miele Evelina , Tiberi Luca 
Published: November 2021

Abstract
Central nervous system (CNS) tumors are the most common solid tumors in the pediatric population and the main cause of death among childhood cancers. They present common features related to the development and produce symptoms based on their location, rate of tumor growth and age of the child. Tumors of embryonal origin, such as medulloblastoma or primitive neuroectodermal tumor, have a higher incidence in younger patients, whereas older ones tend to present tumors of glial origin. Most of CNS-embryonal tumors are highly malignant and consequently leading to poor prognoses. Despite the understanding of the underlying molecular landscape of these tumors that has helped in clinical advances to improve life expectancies of the patients, there is still an emerging need to minimize morbidity and manage the long-term therapy effects. Developing new models to better understand the heterogeneity of these tumors and to test personalized treatment strategies remains a clinical challenge. We are trying to answer to this medical need with Patient-Derived Organoids (PDOs) established through the direct in vitro culture of primary tumors and patient derived xenograft (PDX)-derived tumors. We have developed a method for generating PDOs from a variety of pediatric brain cancers that recapitulates the cellular heterogeneity, histological features and mutational profiles of the corresponding parental tumors. We have obtained promising results from the DNA methylation profile analysis, nowadays used for an accurate routine CNS tumors diagnosis. Through this new model, we confirmed the action of some clinically used drugs, obtaining a proof of concept of their possible application as a reliable tool for drug screening. Another interesting application derives from the enrichment of PDOs culture media for possible tumor-specific antigens: their identification could be of pivotal importance for the definition of tumor markers identifiable with diagnostic tests or as candidates for new therapies.
TMOD-28. MYC GENERATES AGGRESSIVE MEDULLOBLASTOMA BY HSP90 PATHWAY ACTIVATION AND ARF SILENCING
Authors: Mainwaring Oliver , Weishaupt Holger , Hutter Sonja , Zhao Miao , Borgenvik Anna , Rosén Gabriela , Breinschmid Laura , Verbaan Annemieke D , Sundström Anders , Annusver Karl , Kasper Maria , Swartling Fredrik 
Published: November 2021

Abstract
Medulloblastoma, the most common malignant pediatric brain tumor, often shows amplification or overexpression of the MYC transcription factor and arises in the presence of a functional p53 tumor suppressor protein. To elucidate the mechanism behind this inexplicable tumor development we generated an inducible, immunocompetent transgenic mouse model of MYC-expressing medulloblastoma. Aggressive tumors developed clonally in the presence of an unaltered p53 gene that molecularly resembled Group 3 medulloblastoma. Compared to MYCN-expressing medulloblastoma driven from the same promoter, we instead discovered pronounced and MIZ1-independent silencing of the ARF suppressor, which was also suppressed in MYC-amplified as compared to MYCN-amplified human medulloblastoma. While MYCN-driven tumor malignancy was more sensitive to ARF depletion, it dramatically increased metastatic spread of MYC-driven tumors. DNMT inhibition could restore ARF levels in MYC-expressing tumors but did not show any therapeutic advantage in tumors in vivo. Bioinformatics analysis further showed a strong correlation of the HSP90 pathway with MYC in human Group 3 MB and in the MYC-driven mouse model. The HSP90 inhibitor Onalespib showed significant selectivity for targeting MYC-driven as compared to MYCN-driven tumors. The drug promoted ARF restoration and increased the survival in our animal model which suggests that it could be potentially used in the treatment of MYC-driven ARF-silenced brain cancer patients.