bluuebunny/crossref_metadata_2025_split huggingface dataset:
TMOD-23. PRECLINICAL DRUG EVALUATION IN A GENETICALLY ENGINEERED MINIPIG MODEL OF NEUROFIBROMATOSIS TYPE 1
Authors: Osum Sara , Stemmer-Rachamimov Anat , Widemann Brigitte , Dombi Eva , Vitte Jeremie , Dahiya Sonika , Rizvi Tilat , Ratner Nancy , Messiaen Ludwine , Gutmann David , Giovannini Marco , Moertel Christopher , Largaespada David , Watson Adrienne 
Published: November 2019

Abstract
We have employed gene-editing technology to create a Neurofibromatosis Type 1 (NF1) minipig that replicates the broad spectrum of disease that develops in NF1 patients and meets the National Institute of Health’s diagnostic criteria for NF1. The NF1 boars are fertile and the NF1 mutant allele is transmitted at a Mendelian rate with no reduction in fitness of offspring that inherit this allele. To date, we have observed 100% penetrance of café au lait macules, a phenotype that occurs in nearly every NF1 patient, but has never been demonstrated in any other animal model. The NF1 minipig develops cutaneous neurofibromas and optic pathway glioma, that histologically resemble human tumors. Additionally, we have observed other NF1-associated phenotypes including Lisch nodules, tibial dysplasia, white matter decompaction, hypopigmentation, and freckling of the skin. The FDA has emphasized the need for development and testing of new therapies in large animal disease models prior to human studies. Therefore, we have conducted pharmacological studies in our NF1 swine to look at the pharmacokinetic and pharmacodynamic properties MEK inhibitors, currently in clinical trials for NF1. We have demonstrated that oral administration of the MEK inhibitors results in clinically relevant plasma levels of the drug and inhibition of Ras signaling, and that certain MEK inhibitors can cross the blood brain barrier and have a pharmacodynamic effect, suggesting that they may be effective in treating NF1-associated brain tumors. We envision this large animal model of NF1 will become a standard in the evaluation of the safety and efficacy of new drugs prior to Phase I clinical trials. Further, an NF1 minipig may enable researchers to better understand the biological and genetic mechanisms underlying this complex disease, detect NF1-related tumors earlier, identify biomarkers, discover novel drug targets, and test new drugs and combination therapies for safety and efficacy.
TMOD-24. DEVELOPMENT OF A NOVEL IN VIVO MODEL OF LUNG-TO-BRAIN METASTASIS PROGRESSION
Authors: Singh Mohini , Nayyar Naema , Subramanian Megha , Brastianos Priscilla 
Published: November 2019

Abstract
Brain metastases (BM) are the most common neoplasm to affect the adult central nervous system, occurring ten times more frequently than primary brain tumor. BM develop in 40–50% of advanced lung adenocarcinoma (LUAD), and the lack of durable response to chemotherapy, immunotherapy, or targeted therapy will result in death within a year of BM diagnosis. Despite the high burden of disease, dismal prognosis, and the increase in incidence over time, the biological underpinnings of BM remain poorly understood due to both the difficulty of obtaining a sizable collection of metastatic tissue samples as well as a lack of clinically relevant models. As such, it is possible that the inability to properly study this disease may result in metastasis driver-genes remaining undiscovered. Typical models of BM utilize direct implantation of tumor cells into the mouse brain, or inoculation into the blood via intracardiac/intracarotid injections; these routes are invasive and cannot fully recapitulate the entirety of the metastatic cascade. Here we present a novel, non-invasive method to develop primary lung tumors and brain metastases. A 10µL tumor cell suspension in phosphate-buffered saline was applied to the nostrils of lightly anesthetized mice, allowing direct deposit of cells into the lungs. Mice were monitored with bioluminescence imaging bi-weekly and culled at 2.5–3 months post-inoculation. Lungs and brains were also removed and imaged, where tumors in both organs were observed. Further research is necessary to elucidate either the linear or parallel progression of tumor formation within this model. Nonetheless, our technique presents a novel preclinical tool to interrogate the metastatic process, allowing validation of genetic drivers as well as therapeutic screening.
TMOD-28. AUTHENTIC HUMAN GLIOMA MODELING USING GENETICALLY ENGINEERED INDUCED PLURIPOTENT STEM CELLS
Authors: Koga Tomoyuki , Chaim Isaac , Markmiller Sebastian , Benitez Jorge , Parisian Alison , Miki Shunichiro , Hessenauer Florian , Turner Kristen , Venneti Sriram , Malicki Denise , Wechsler-Reya Robert , Mischel Paul , Chen Clark , Yeo Gene , Furnari Frank 
Published: November 2019

Abstract
Many mouse and human glioma models have been utilized to study the genetic alterations involved in the genesis of these tumors, but they have not been fully evaluated for how closely they recapitulate pathobiology, including tumor heterogeneity, which is an inherent feature making patient treatment difficult. Here we present new glioma models using genetically engineered human pluripotent stem cells, in which authentic pathobiology is recapitulated through precision gene editing. Specifically, we show that neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs), with different combinations of genetic drivers introduced by CRISPR/Cas9-mediated editing give rise to distinct intracranial tumors recapitulating authentic pathobiology of the disease when engrafted in immunocompromised mice. NPCs deficient in PTEN and NF1, a genotype associated with the mesenchymal molecular subtype, and NPCs deficient in TP53 and expressing a PDGFRA activating mutation (PDGFRAΔ8–9), a genotype associated with the proneural glioblastoma molecular subtype, give rise to distinct tumors resembling glioblastoma. Both models presented inter and intra-tumor heterogeneity based on single-cell RNA sequencing, with the former model showing proneural signature and the latter a mesenchymal signature, and both having different degrees of cycling, and stem cell-enriched populations. The TP53/PDGFRA model had more clonal variability related with striking karyotype abnormalities including extrachromosomal DNA. Additionally, we expanded this approach to pediatric gliomas. Brainstem tumors derived from NPCs introduced with TP53 R248Q and H3F3A K27M mutations presented features of glial tumors with global expression of histone H3 K27M accompanied by suppression of histone H3K27 trimethylation, compatible with H3 K27M-mutant pediatric diffuse midline glioma. Using these isogenic human brain tumor models, we aim to advance our understanding of the pathobiology associated with different driver mutations and further, to provide a platform for development of targeted therapy.
ANGI-02. GENOME-WIDE shRNA SCREEN IDENTIFIES CANDIDATE GENES DRIVING GLIOBLASTOMA INVASION
Authors: Schuster Anne , Neirinckx Virginie , Klein Eliane , Nazarov Petr V , Oudin Anais , Muller Arnaud , Azuaje Fransisco , Herold-Mende Christel , Klink Barbara , Niclou Simone 
Published: November 2019

Abstract

BACKGROUND
A major hallmark of glioblastoma (GBM) is its invasive capacity, contributing to its aggressive behaviour. Invasive cells cannot be easily removed by surgery or irradiation and eventually result in lethal recurrence. A better understanding of the invasion process and the key molecular players underlying the invasive potential of GBM may lead to the identification of new therapeutic targets for GBM patients.

MATERIAL AND METHODS
To identify candidate genes responsible for invasion, a genome-wide shRNA screen was performed in patient-derived GBM cultures. The most promising candidate was validated in in vitro invasion assays, ex vivo brain slice cultures and in vivo orthotopic xenografts in mice. Gene knockdown in invasive GBM cells was compared with overexpression in non-invasive cells. RNAseq of knockdown cells, along with the generation of deletion constructs were applied to uncover the mechanisms regulating invasion.

RESULTS
A zinc-finger domain containing protein was identified as an invasion essential candidate gene. Knockdown of this gene confirmed a strong impact on invasion in highly invasive GBM cells. In contrast, gene overexpression switched non-invasive GBM cells to an invasive phenotype. Deletion of one or both zinc-finger motifs decreased invasion indicating that both are essential for regulating invasion. Mutation of the nuclear localisation signal resulted in retention of the protein in the cytoplasm and loss of the invasion phenotype demonstrating that the protein activity is required in the nucleus. Gene expression analyses revealed that invasion-related genes are significantly regulated by the candidate gene once it is localized in the nucleus.

CONCLUSION
We identified a zinc-finger containing protein as a novel driver of GBM invasion, presumably through transcription factor activity resulting in the induction of an invasive transcriptional program. This protein and its downstream pathway may represent novel promising targets to overcome invasive capacities in GBM.
TMOD-37. INTRAVITAL MONITORING DYNAMIC TUMOR MICRO-ENVIRONMENTAL EVOLUTION FOLLOWING HYPOXIA-INDUCED NECROSIS IN GLIOBLASTOMA
Authors: Markwell Steven , Ross James , Olson Cheryl , Nunez-Santana Felix , Arvanitis Constadina , Mukherjee Subhas , Brat Daniel 
Published: November 2019

Abstract
The onset of necrosis correlates with malignant progression in virtually all human cancers. Whether necrosis arises from or promotes rapid tumor expansion remains unknown, primarily due to insufficient model systems necessary for capturing these dynamic changes as they develop. In glioblastoma (GBM; WHO grade IV), the most malignant primary brain tumor, the development of central necrosis precedes rapid, radial expansion precipitously leading to patient mortality. While genetic alterations in GBM have been highly characterized, the resultant biological adaptations leading to accelerated tumor growth require further mechanistic investigation. The onset of necrosis dramatically changes the tumor microenvironment (TME), evolving from a sheet-like growth of infiltrating malignant cells undergoing gradual expansion to a complex 3-D microsystem comprising diverse cell types and spatially segregated signaling networks. To reveal the dynamic temporal and spatial changes promoting progression, we are generating mouse models that more appropriately capture events found in human gliomas, accounting for unique microenvironmental stressors often lacking in GBM animal models, specifically central necrosis. We developed a novel method combining hypoxia-induced focal necrosis within high-grade gliomas with intravital microscopy to study TME restructuring and its impact on glioma progression in real time. As translational applications, we are investigating how hypoxia and necrosis promote glioma stem cell (GSC) enrichment in their peri-necrotic niche and how the massive influx of tumor-associated macrophages (TAMs) promotes glioma progression. Our studies use both genetically characterized patient-derived orthotopic GBM xenografts in humanized mice, alongside an immunocompetent RCAS/tv-a model, to determine how antagonizing these processes impacts disease progression and outcomes across multiple GBM subtypes. Our preliminary data indicate substantial differences pre- and post-necrosis regarding GSC and TAM enrichment and their biological impact, however, these mechanisms have not yet been studied. Our models capture glioma growth dynamics, GSC enrichment, and TAM influx, facilitating innovative therapeutic interventions to improve patient outcome.
TMOD-38. PRODUCTION OF D-2-HYDROXYGLUTARATE IN SACCHAROMYCES CEREVISIAE LEADS TO GROWTH IMPAIRMENT AND DECREASED SURVIVAL
Authors: Fiola Sophie , Ganni Eli , Lo Rita , Yee Lok Ka , Kuzmin Elena , Diaz Roberto 
Published: November 2019

Abstract
High levels of D-2-hydroxyglutarate (D2HG) are found in several types of cancers, most notably low grade gliomas (LGGs). The accumulation of D-2HG contributes to tumorigenesis through a variety of mechanisms including decreased utilization of oxidative phosphorylation and histone hypermethylation. The use of the budding yeast Saccharomyces cerevisiae as a model system to study cancer allows for faster, more efficient elucidation of various molecular mechanisms, including functional genomics via genomic array screening. S. cerevisiae encodes two homologs of the human D-2HG dehydrogenase: the mitochondrial Dld2 and cytosolic Dld3. We detected an increase in the production of D-2HG in the dld3∆ knockout strain by LC-MS. In addition, the dld3∆ knockout strain shows decreased survival and a growth impairment in glucose-containing liquid media. However, this strain did not show a significant growth impairment on glucose or glycerol-containing solid media. Using publicly available Synthetic Genomic Array (SGA) analysis data from TheCellMap.org, we investigated the top negative gene interactions for our dld3 knockout strain. GO analysis of these negative gene interactions showed enrichment of targets locating to the mitochondria, suggesting that the increase of 2-HG leads to mitochondrial impairment, consistent with previous observations in other models of LGGs. The top two targets of the SGA screen were mdm35, a mitochondrial interspace membrane protein involved in assembly of the mitochondrial respiratory chain complex and cdc8, a component of the de novo pyrimidine biosynthesis pathway. Taken together, these results suggest that the dld3∆ knockout strain is an appropriate model in which to study the D-2HG-driven changes that occur during tumorigenesis.
SCIDOT-05. DEVELOPING VARIABLE LYMPHOCYTE RECEPTORS THAT TARGET PATHOLOGICALLY EXPOSED NEURAL ECM TO TREAT GLIOBLASTOMA
Authors: J Umlauf Benjamin , A Clark Paul , M Lajoie Jason , V Georgieva Julia , Bremner Samantha , R Herrin Brantley , V Shusta Eric , S Kuo John 
Published: November 2019

Abstract

INTRODUCTION
The median survival of gliobastoma (GBM) patients remains less than two years despite aggressive treatments. Current targeted GBM therapies demonstrate initial therapeutic benefit; however, patients relapse due to therapeutic resistance and failure to eliminate GBM cells at the invasive margin. Therefore, we propose a two-prong approach: first, target pathologic disruption of the blood brain barrier (BBB) via exposure of neural ECM rather than disease markers to overcome therapy-resistant GBM; and second, designing therapeutic payloads that extracellularly spread throughout the tumor volume.

METHODS
Variable Lymphocyte Receptors (VLRs, a lamprey-derived antigen recognition system) were identified with high specificity for neural ECM. Candidate VLRs underwent further refinement using ex vivo tissue staining. Utilizing pathologic disruption of BBB as an approach for targeting GBM was confirmed in vivo with intracranial murine glioblastoma models. Finally, an immunogenic peptide was attached via a cleavable linker to the neural ECM binding VLRs for conditional release extracellularly to spread throughout the tumor.

RESULTS
The lead neural ECM-binding VLR candidate, named P1C10, demonstrates diffuse binding to parenchymal neural ECM, without detectable binding to other tissues. P1C10 demonstrates nanomolar affinity for neural ECM, and preferentially accumulates in intracranial GL261 and U87 murine GBM models. Finally, P1C10-targeted doxorubicin-loaded liposomes significant increased survival of mice with intracranial GBM. In additional studies, treating murine GBM models with a P1C10 VLR linked to an immunogenic peptide reduced GBM proliferation and increased infiltration of cytotoxic T cells.

CONCLUSIONS
We present proof-of-concept demonstration for targeting intracranial GBM via neural ECM exposed at pathological BBB disrupted sites. Additionally, P1C10 neural ECM-targeting VLR delivers chemotherapy-loaded nanoparticles and immunogenic peptides designed to spread extracellularly throughout the tumor. Thus, this novel strategy links a physiological ECM targeting scheme with extracellular-released therapeutics to treat primary GBM, and has potential for delivering therapies to other CNS diseases with pathological BBB.
SCIDOT-21. IMPROVING DRUG DELIVERY TO GLIOBLASTOMA BY TARGETING CANONICAL WNT/β-CATENIN SIGNALING IN THE BLOOD-BRAIN BARRIER
Authors: Vezina Amelie , Jackson Sadhana 
Published: November 2019

Abstract

BACKGROUND
Glioblastoma (GBM) patient survival and therapy response is greatly hindered by the presence of invasive glioma stem cells (GSC) and the blood-brain barrier (BBB) which limits effective drug delivery. WNT/beta-catenin signaling is important in the development and maintenance of the BBB by mediating transcription of growth factors, multidrug resistance proteins, and junctional proteins. In WNT-subtype medulloblastoma, activating mutations of beta-catenin lead to reciprocal secretion of WNT antagonists such as WIF1 and DKK1 into the tumor microenvironment. These WNT antagonists can act upon the surrounding endothelium and induce a leaky BBB. Therefore, we hypothesize that pharmacological inhibition of WNT/beta-catenin signaling in brain endothelial cells will decrease BBB integrity, enabling enhanced paracellular drug delivery to infiltrative GSCs.

METHODS
We recapitulated the WNT-medulloblastoma phenotype in GBM by activating WNT/beta-catenin signaling in primary human GSCs, inducing secretion of downstream WNT antagonists. Conditioned-media (CM) from GSCs was then applied to human brain microvascular endothelial cells (HBMEC) to indirectly inhibit WNT signaling. Additionally, we directly inhibited WNT/beta-catenin signaling in HBMECs with the small molecule inhibitor ICG-001. Endothelial cell-cell interaction was measured by electrical impedance using the ACEA xCELLigence system. Fenestration and junctional expression were evaluated by immunoblotting and immunofluorescence.

RESULTS
ICG-001 or WNT-GSC-CM, but not control GSC-CM, upregulated fenestration related protein, PLVAP, and downregulated junctional proteins claudin-5, ZO-1, and VE-Cadherin in HBMECs. Endothelial cell-cell interaction was transiently decreased by ICG-001 or WNT-GSC-CM. Pre-clinical studies are underway to evaluate the functional impact of WNT/beta-catenin inhibition on BBB integrity and permeability in rodent glioma models. Altogether, these results support targeting WNT/beta-catenin signaling in brain endothelial cells to enhance drug delivery to CNS tumors.

CONCLUSION
Modulation of intratumoral Wnt/beta-catenin signaling, particularly in highly resistant GSCs, may enhance chemotherapy drug delivery, potentially expanding the drug portfolio and improving the prognosis of GBM.
SCIDOT-31. DELAYED CONTRAST MRI FOR HIGH SENSITIVITY DEPICTION OF SUBTLE BBB DISRUPTION IN BRAIN PATHOLOGIES AND AS MEANS FOR MONITORING/DEVELOPING BBB DISRUPTION TECHNOLOGIES
Authors: Sharabi Shirely , Last David , Daniels Dianne , Cooper Itzik , Bresler Yael , Shohami Ester , Liraz-zaltsman Sigal , Mardor Yael 
Published: November 2019

Abstract
Understanding the effects of BBB disrupting technologies on the neurovascular unit is important for safety and for optimal timing of therapeutic agents administration. We studied the effects of two novel BBB disrupting methods employing pulsed electrical fields (PEFs) on wash-in/wash-out rates and on vascular dilatation/constriction in mice and rats.
The first method uses high, minimal-invasive PEFs inducing electroporation in endothelial cells. The second employs low, non-invasive PEFs inducing tight-junction function alterations. Both were found to induce transient BBB disruption. Contrast-enhanced T1-weighted MRI (T1-Gd) and treatment response assessment maps (TRAMs) based on delayed-contrast MRI, were applied to depict BBB permeability, wash-in/wash-out rates and vascular dilatation/constriction. T1-Gd, acquired following high PEFs application, showed immediate enhancement in the treatment area, lasting for ~1 hour. However, TRAMs, obtained at different times post treatment (immediately-24 hours) indicated vasodilatation with fast wash-in/wash-out immediately post treatment (preventing efficient tissue accumulation of peripherally administered drugs) and delayed (~1 hour post PEFs) vasoconstriction, lasting several hours. This was followed by large volumes of subtle BBB disruption, visible mainly on TRAMs. These volumes exceeded the original volumes observed immediately post treatment, suggesting this may be the optimal time for drug administration. Low PEFs however, induced subtle BBB disruption, depicted only in the TRAMs, with slow contrast accumulation, reaching a plateau at ~40 min post treatment. The TRAMs showed significantly increased sensitivity to BBB disruption compared to T1-Gd also in mice post traumatic brain injury. Lesion volumes depicted in the TRAMs were x2.5 larger than the enhancing volumes on T1-Gd (p<0.02). The results significantly correlated with histological assessment of blood vessels coverage by astrocytes end-feet (r2=0.77, p<0.05), an histological indicator for BBB integrity. These results demonstrate the TRAMs advantages in depicting BBB disruption with high sensitivity/resolution, thus providing additional information regarding the neurovascular unit function.
SCIDOT-33. ULTRASOUND-FACILITATED DRUG DELIVERY DEVICE FOR LOCAL DRUG INFUSION AGAINST BRAIN TUMORS
Authors: Saito Ryuta , Jia Wenting , Sato Aya , Inoue Kenji , Okuno Saori , Ohashi Yuji , Kamada Kei , Yoshikawa Akira , Tominaga Teiji 
Published: November 2019

Abstract

OBJECT
Local drug delivery into brain tumor faces a major problem: limitations of distribution. Recently, we have been working to develop a novel treatment strategy delivering chemotherapeutic agents directly into the tumor. Previously, we developed an ultrasound facilitated drug delivery (UFD) system in order to achieve extensive drug distribution (J Neurosurg 124, 2016). Subsequently, we fabricated a second-generation device by analyzing optimal ultrasound generating condition. Here, we tested the intraparenchymal and intratumoral drug distribution using the device.

METHODS
Resonance frequency of the device was determined by measuring the strength of sound field using hydrophone in water. Using the UFD system, applying resonance frequency, evans blue dye was infused into the striatum of Fisher344 rats. Immediately after the infusion, brains were harvested, quickly frozen, and sectioned to evaluate the distributions of the dye. Similar study was then performed in 9L brain tumor model.

RESULTS
The second-generation device successfully distributed the dye to the volume 1.5~2 times larger than simple CED with using half the driving voltage than first-generation device. Moreover, the distribution instability, which was a major problem with the first-generation device, was resolved achieving smaller standard deviation. Using multiple resonance frequencies of the device, we found the difference in drug distribution; suggesting the existence of optimal frequencies for brain interstitial drug delivery. This drug delivery strategy also worked in brain tumor model increasing the coverage inside the brain tumor.

CONCLUSIONS
The second generation UFD device successfully and stably achieved enlarged distribution in the brain parenchyma and in the brain tumor.