bluuebunny/crossref_metadata_2025 huggingface dataset:
TMIC-56. ROLE OF HUMAN BRAIN TUMOR STEM CELLS-DERIVED EXTRACELLULAR VESICLES ON THE PHENOTYPIC TRANSDIFFERENTIATION OF HUMAN NEURAL PROGENITOR CELLS
Authors: Zarco Natanael , Norton Emily , Lara-Velazquez Montserrat , Carrano Anna , Quinones-Hinojosa Alfredo , Guerrero-Cazares Hugo
Published: November 2019

Glioblastoma (GBM) is the most aggressive of all the brain tumors with a median patient survival less than 15 months. Despite of surgical resection, radiotherapy, and chemotherapy, recurrence rate is almost 100%. A great percentage of GBM tumors (~60%) infiltrate and contact the ventricular-subventricular zone (V-SVZ). Interestingly, these tumors are the most aggressive, and invariably lead to higher distal recurrence rates, shorter time to tumor progression, and lower overall survival of the patient. The reason for this role of V-SVZ-proximity on the outcome of GBM patients is unknown. We suggest that a potential explanation is the interaction of GBM with the V-SVZ. This region is the largest neurogenic niche in the adult brain where neural stem cells (NSCs) give rise to newborn neuroblasts that migrate toward the olfactory bulb. In GBM there is a cell subpopulation called brain tumor stem cells (BTSCs) with NSCs-like characteristics, but with added potential for tumor initiation, recurrence and invasiveness. Tumor microenvironment plays an important role in migration and invasion process. In the present work, we used the total exosome isolation kit to purify Extracellular Vesicles (EVs) from human primary cultures of BTSCs. We determined that BTSCs-derived EVs contain specific information that is transfer to primary cultures of human Neural Progenitors Cells (NPCs) modulating their proliferation rate, cell viability, and migration. In addition, we identify that NPCs taken up BTSCs-derived EVs and significantly increase the expression levels of stemness-related genes such as Nestin, Nanog, and Sox2, suggesting that a phenotypic transdifferentiation is being carry out. These results support our hypothesis that GBM modulate the tumor microenvironment close to the V-SVZ by releasing EVs that target cellular components in this region and promote their phenotypic transformation, highlighting that NPCs biology changes in the context of tumor environment.
TMIC-61. ROLE OF CX3CR1 SIGNALING IN MALIGNANT TRANSFORMATION OF GLIOMAS
Authors: Lee Sungho , Latha Khatri , Rao Ganesh
Published: November 2019

High-grade gliomas (HGGs), including the most common primary brain tumor, glioblastoma (GBM), may arise from malignant transformation of low-grade gliomas (LGGs). While LGGs are often clinically indolent, GBMs have dismal outcomes despite maximal therapy. Accumulating data suggest that chemokine signaling directly contributes to malignant transformation of LGGs by altering tumor behavior or impacting the immune microenvironment. Here, we examined the role of CX3CR1 signaling in the malignant transformation of LGGs. First, patients with malignantly transformed LGGs were genotyped for the presence of the common loss-of-function CX3CR1 V249I polymorphism, and median overall survival was compared between the genotypes. Second, RNA sequencing data was analyzed for differential gene expression based on genotype. Third, surgical samples were examined for altered expression of M2 macrophage markers and microvessel density between the genotypes. Finally, a genetically-engineered murine model was leveraged to model endogenous intracranial gliomas with targeted expression of CX3CL1 and CX3CR1, individually or in combination. Our data demonstrate that heterozygosity (V/I) or homozygosity (I/I) for the loss-of-function CX3CR1 polymorphism was associated with significantly better median overall survival in patients with LGGs that have transformed to HGGs, compared to the wild type genotype (V/V). In addition, HGGs from V/I and I/I genotypes exhibit significantly decreased levels of CCL2, important for the recruitment of M2 macrophages, as well as decreased levels of ANGPT1 and MMP9, which mediate angiogenesis. This correlates with reduced intratumoral accumulation of CD204 positive macrophages and microvessel density in tumors from V/I and I/I patients. Finally, in the RCAS-PDGFB driven model of LGG, co-expression of CX3CL1 and CX3CR1 promotes more malignant tumor phenotype and shorter tumor-free survival. Taken together, our results show that CX3CR1 signaling promotes malignant transformation of LGGs via accumulation of glioma associated M2 macrophages and microglia and increased angiogenesis.
ACTR-69. FEASIBILITY STUDY OF THE EMULATE THERAPEUTICS™ VOYAGER SYSTEM IN PATIENTS WITH NEWLY DIAGNOSED GLIOBLASTOMA
Authors: Barkhoudarian Garni , Blondin Nicholas , Chowdhary Sajeel , Fonkem Ekokobe , Vaillant Brian , Kesari Santosh
Published: November 2019

BACKGROUND

The EMulate Therapeutics Voyager system is an investigational non-sterile, non-invasive, non-thermal, non-ionizing, portable, home-use medical device that uses a specific, localized ultra-low radio frequency energy (ulRFE®) cognate for the treatment of brain cancer.

METHODS

This open-label, multi-center study (NAT-109) enrolled adults newly diagnosed with GBM. Following surgical debulking, patients were enrolled and treated concurrently with temozolomide, radiotherapy, and Voyager. The objective of the study is to assess if the Voyager is a safe and feasible treatment for newly diagnosed GBM when combined with standard of care. The primary outcome measure is safety, assessed by the incidence and evaluation of any adverse events (AEs) associated with the Voyager. The secondary outcome measure is clinical utility, assessed by progression-free survival and overall survival.

RESULTS

Enrollment is closed, and treatment and long-term follow-up is ongoing. A total of 37 patients were enrolled and treated. 27 patients reported 282 AEs, none of which required withdrawal from the study. One AE was reported as probably related to the device - i.e., mild dysesthesia, which resolved without interruption or cessation of treatment with the device. 15 patients reported 28 SAEs, and none were reported as related to the device. 56% of patients were progression-free at 6 months, and 43% were progression-free at 12 months. 89% of patients were still alive after 6 months, 71% were still alive after 12 months.

CONCLUSIONS

The Voyager system appears to be safe and feasible for the treatment of newly diagnosed GBM. Given that therapy is delivered non-invasively and no device-related serious adverse events were reported, further prospective study of the investigational device is planned.
TMOD-07. IN SITU MICROSCOPY OF DRUG AND IMMUNE CELL INTERACTIONS AGAINST BIOFABRICATED TUMOR MODELS
Authors: McGhee Eric , Uruena Juan , Levings Padraic , van Meter Kylie , Smolchek Ryan , Hood Derek , Flores Catherine , Mitchell Duane , Gregory Sawyer W
Published: November 2019

BACKGROUND & SIGNIFICANCE

Cancer is a disease in 3-dimensions and there is a desperate need for infrastructure and models to study immuno-oncology treatment strategies in 3D. A new culture system for long-duration maturation of patient derived microtumors has been developed. This system enables in situconfocal imaging and movies of T-cells and tumor model interactions, including measurements of T-cell migration, infiltration, and killing. HYPOTHESIS: Biofabrication of 3D microtumors using bioprinting in transparent soft granular gel media can facilitate the precise arrangement and stability of extra-cellular matrix components, tumor, and immune cells while maintaining continuous liquid perfusion.

METHODS

The design, development, and validation of a modular negative pressure perfusion system controls the transport of liquid growth medium, drugs, antibodies, growth factors, and metabolic waste management in 3D. In situ confocal fluorescent microscopy measures cell positions, velocities, and viability during experiments. Steady perfusion velocities are controlled through negative pressure, and velocities from 1–100 nm/s can be achieved.

RESULTS

Cell motility, adhesion, and dynamic rearrangement of fibroblasts and endothelial cells within a 3D co-culture of microtumors evolved dramatically over the first 72 hours. Tracking of activated CD8+ T-cells revealed super-diffusive motion in the presence of 3D tumors with a range of 250 µm. Activated T-cell migration speeds have been measured to be between 1.3–2.0 um/min in the 3D media, and preliminary estimates of T cell migration forces are on the order of 1 nN. Arrival of CD8+ T-cells to the tumors within the first 30 minutes revealed cell killing which continued for over 3-hours and resulted in a 2-fold reduction in tumor cell numbers.

CONCLUSIONS

This integrated system of 3D bioprinting, perfusion culture plates, and confocal microscopy enables in situ3D studies of cancer biology, immunotherapy, and drug treatment regimens and provides unique insights and measurements of immune cell invasion dynamics in 3D microtumors.
TMOD-08. SERUM ESTABLISHED CANCER STEM CELL CULTURES REPRESENT UNIQUE MODELS OF GLIOBLASTOMA INTER-TUMOR HETEROGENEITY
Authors: Maturi Nagaprathyusha , Tan E-Jean , Xie Yuan , Sundström Anders , Bergström Tobias , Casar-Borota Olivera , Jiang Yiwen , Uhrbom Lene
Published: November 2019

Glioblastoma (GBM) is the most frequent primary malignant brain tumor. The invasive and heterogeneous nature of GBM stem cells (GSCs) provide the basis for extreme therapy resistance of GBM and the majority of patients survive less than one year from diagnosis. Relevant models of GBM are important for basic research and drug screening and development, and the current consensus in the field is that GSCs are most faithfully maintained under serum-free neural stem cell (NSC) conditions. We have used serum-free NSC media to explant 230 patient-derived GBM samples, and have in parallel also explanted the same samples in regular serum-containing media. We found, accordingly, that serum-free media was most effective in generating sustainable cultures (137/230, 60%), called serum-free (SF) cultures. Interestingly, we also found that that there was a subgroup of GBM specimens that could only be established in serum-containing media (31/230, 13%), called exclusive serum-containing (eSC) cultures. We characterized a number of the eSC cultures and found that they expressed typical GSC markers such as MSI-1, BMI-1, nestin, CD44 and SOX2. They also displayed all functional characteristics of GSCs, i.e. extended proliferation, sustained self-renewal and orthotopic tumor initiation. Molecular analyses showed that the eSC cultures were most closely related to but separated from mesenchymal subtype GBM. In summary we have established GSC cultures that would have evaded modeling under serum-free conditions, representing unique cell models of GBM inter-tumor heterogeneity. Key words: Glioblastoma, serum cultures, stem-like cells, GBM heterogeneity
TMOD-09. MODELING TERT PROMOTER MUTATION IN ISOGENIC NEXT GENERATION GBM MODELS
Authors: Miki Shunichiro , Koga Tomoyuki , Parisian Alison , Furnari Frank
Published: November 2019

Telomere reverse transcriptase (TERT) promotor mutations increase TERT expression and are known to be the most common single genetic abnormality in Glioblastoma (GBM). Recent studies have revealed this mutation to be an early event in gliomagenesis but it is detected subclonally in a considerable number of cases. Due to the long telomere length and TERT expression in somatic cells of mice, genetically engineered mouse models of GBM do not address the function of this mutation and thus an ideal heterozygous TERT promotor mutation model is yet to be established. Recently, our lab established strategies for generating GBM models using neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (hiPSCs) that have been CRISPR/Cas9 engineered with different combinations of authentic GBM-related genetic drivers which can be used for introduction of mutant TERT promotor (MTP) sequence and examination of its function. We obtained multiple heterozygous MTP hiPS clones in the context of GBM genetic alterations, CDKN2A/B and PTEN deletion, using a two-step targeting approach. Two single-strand guide RNAs (sgRNAs) were used to delete the TERT promotor and a portion of exon 1 followed by repair of the locus with wild type TERT promoter (WTP) and MTP sequence. Following promoter reconstitution, TERT promotor function was tested in iPSCs, NPCs, and astrocytes. This analysis revealed successful TERT expression silencing in WTP astrocytes. Furthermore, orthotopic injection of WTP and MTP NPCs into immunodeficient mice formed pathologically similar GBM-like tumors in same period of time. TERT expression was only detected in cell lines derived from MTP tumors, indicating TERT promotor mutation has little impact in initiation of tumor formation from NPCs, explaining its subclonality in patient tumors. Our model enables further research on MTP function and dependency during gliomagenesis.
TMOD-15. IDENTIFYING THE SPATIAL AND TEMPORAL DYNAMICS OF GLIOBLASTOMA SUBPOPULATIONS WITHIN INDIVIDUAL PATIENTS
Authors: Morris Bethan , Curtin Lee , Hawkins-Daarud Andrea , Bendok Bernard , Mrugala Maciej , Li Jing , Tran Nhan , Hu Leland , Rahman Ruman , Smith Stuart , Auer Dorothee , Hubbard Matthew , Owen Markus , Swanson Kristin
Published: November 2019

Glioblastomas (GBMs) are known to be complex tumors comprising multiple subpopulations of genetically-distinct cancer cells; it is thought that this genetic variation is a major factor in the lack of observed survival benefit of treatment regimes that target one of these subpopulations. The field of radiogenomics seeks to study correlations between MRI patterns and genetic features of GBM tumors. Spatial radiogenomic maps produced using machine-learning (ML) methods that are trained against information from image-localized patient biopsies identify regions where particular cancer sub-populations are predicted to occur within a GBM, thus non-invasively characterizing the regional genetic variability of these tumors. These tumor subpopulations may also interact with one another, in ways which may be of a competitive or cooperative nature to varying degrees. It is important to ascertain the nature of these interactions, as they may have implications for treatment response to targeted therapies, and characterization of the spatio-temporal dynamics of these co-evolving sub-populations will shed light on why some therapies fail. Here we combine mathematical modeling techniques and spatially-resolved radiogenomic maps to study the nature of these interactions between molecularly-distinct GBM subpopulations. We model the interactions between cell populations using a partial differential equation based formalism. The model is parameterized using radiogenomic ML maps from which we infer the nature of interactions between subpopulations. Furthermore, using maps as inputs, the model turns static maps into dynamic information, thus providing insight into how these subpopulations composing the tumor change over time and the effect this has on observed treatment response for individual patients.
TMOD-18. EXPLORING THE FACTORS LEAD TO SUCCESS OF CAR T-CELL THERAPY IN GLIOBLASTOMA WITH COMPUTATIONAL MODELING AND IN VITRO DATA
Authors: Sahoo Prativa , Yang Xin , Abler Daniel , Maestrini Davide , Adhikarla Vikram , Brown Christine E , Rockne Russell
Published: November 2019

Chimeric antigen receptor (CAR) T-cell therapy is an emerging targeted immunotherapy which has shown success in liquid cancers such as leukemias. CAR T-cells are also being used for the treatment of solid tumors such as glioblastoma, which is a primary brain tumor. Ongoing phase I trials have been designed to evaluate CAR T-cell dosing, scheduling, and route of administration in order to understand and improve the efficacy of CAR T-cell therapy. A better understanding of factors leading to the success of CAR T-cell immunotherapy for solid tumors will be necessary to improve outcomes for patients with solid tumors and to advance the field of CAR T-cell immuno-oncology. Here we use mathematical model to explore factors in determining a successful response to CAR T-cell therapy: proliferation, persistence, and killing capacity of CAR T-cells. Using a novel in vitro experimental apparatus, we are able to measure the density of cancer cells over several days in 15 minute interval time resolution. This highly temporally resolved data provides a unique opportunity to confidently estimate parameters of the model and to provide insights into the dynamics of CAR T-cell proliferation, persistence, and killing capacity. Furthermore we explore the relationship between these factor with CAR T-cell dose level. We will show results from experiments using patient-derived cancer cell lines as well as cancer cells engineered to express specific levels of the target antigen (IL13Rα2) to quantitatively evaluate the roles of proliferation, persistence, and killing in cells with different levels of antigen expression. We will discuss the interpretation of the model parameters and demonstrate the clinical value of this analysis through an application of CAR T-cell treatment tailored to the dynamics of an individual patient’s cancer growth rate.
TMOD-20. COMBINING TARGETED INHIBITOR AND RADIOTHERAPY IN TREATING ANAPLASTIC MENINGIOMA
Authors: Wang Yufen , Horbinski Craig , James Charles , Sathiaseelan Vythialingam , Kalapurakal John
Published: November 2019

BACKGROUND

Anaplastic meningiomas pose a serious health concern due to their propensity for recurrence and brain invasion. Five-year overall survival of patients with anaplastic meningiomas is less than 10%, similar to that of glioblastoma patients. Standard treatment of recurrent anaplastic meningioma is radiotherapy (RT), but radioresistance is common. New therapeutic strategies, used singularly or in combination with RT, are urgently needed. Since deletion of the gene encoding the p16 cell cycle checkpoint protein is common in anaplastic meningioma, we hypothesized that such tumors would be sensitive to Palbociclib, a cdk4/6 inhibitor, and that Palbociclib could enhance RT efficacy.

METHODS

CH157 anaplastic meningioma cells were used in vitro for investigating tumor cell proliferation response to RT and Palbociclib, either alone or in combination, and as indicated by MTT assay results. CH157 cells were also used to establish intracranial xenografts in mice treated with RT or Palbociclib. Accuracy of RT dose was verified with calibrated MOSFET\OSLD dosimeters. Mice were monitored for intracranial tumor growth and response to treatment, as indicated by bioluminescence imaging. Length of survival data was collected and analyzed by log-rank test.

RESULTS

At two days post RT, the in vitro anti-proliferative effect of 2, 6, and 10 Gy radiation was significantly enhanced with concurrent 1mM Palbociclib (p < 0.001). Daily administration of Palbociclib (150 mg/kg) for 1 week significantly extended the survival of mice with intracranial CH157 xenografts (group mean value of 18.4 vs. 13.6 days with vehicle control, p < 0.001). A single dose of 10 Gy RT was superior to 6 Gy administered 1x and 2Gy/day administered on 5 consecutive days (group mean values of 37.4 vs. 24.0 vs. 24.6, respectively: p < 0.001).

CONCLUSIONS

These data support further investigation of RT + Palbociclib for treating p16-deficient anaplastic meningiomas.
TMOD-21. TARGETING ANAPLASTIC MENINGIOMA WITH PANOBINOSTAT IN PATIENT-DERIVED ORTHOTOPIC XENOGRAFT (PDOX) MODELS DERIVED FROM A MATCHING PAIR OF PRIMARY AND RECURRENT TUMORS
Authors: Zhang Huiyuan , Qi Lin , Du Yuchen , Braun Frank , Kogiso Mari , Huang Lei , Klisch Tiemo , Zhao Yanling , Guo Lei , Li Can , Zhao Sibo , Lindsay Holly B , Injac Sarah , Baxter Patricia , Su Jack , Stephan Clifford , Keller Charles , Yang Jianhua , Li Xiao-Nan , Patel Akash
Published: November 2019

BACKGROUND

Meningioma is the most common brain tumor in adults. Despite the overall benign nature of meningioma, skull base tumors can be difficult to completely resect while others exhibit progression and aggressive profiles. The lack of clinically relevant animal models is blocking the development of novel therapies.

MATERIAL AND METHODS

Twelve surgical specimens (1 × 105) from 11 adult meningioma patients were implanted into the frontal cranial-base of the brain of SCID mice. Mice were then followed and assessed for tumor formation. Tumor growth was confirmed by small animal MRI. Pathologic features of the PDOX models and the matched patient tumors were compared with standard H&E and immunohistochemical staining. RNAseq was performed to examine the molecular fidelity of PDOX tumors and to identify new therapeutic targets. A panel of 60 clinically-relevant drugs was developed for screening drug sensitivity. In vivo examination of therapeutic efficacy of Panobinostat was performed in two models by treating preformed PDOX tumors with i.p. injection (10 mg/kg), 5 days on, 5 days off for 2 cycles.

RESULTS

Intracranial xenograft formation was confirmed in two samples derived from the same patient, the first an atypical meningioma (K029MEN-P) and the second, which progressed to anaplastic meningioma at recurrence (K029MEN-R). MRI scanning revealed that the PDOX tumors grew from the skull base. These patient tumor cells can be cryopreserved for long-term maintenance of tumorigenicity. The xenograft tumors replicated histopathological features of parental tumors. Overall gene expression profiles of PDOX were similar to the original patient tumors. Using MEN primary culture cells, we screened 60 drugs and identified 12 (20%) active compounds. Panobinostat also significantly prolonged survival of mice bearing orthotopic meningiomas.

CONCLUSION

A set of meningioma PDOX models derived from primary and recurrent tumor was established. Our data further demonstrate that panobinostat exerts potent antitumor activity against high-grade meningioma.
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

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

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

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

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

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

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

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

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

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

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.
SCIDOT-35. A NOVEL MODEL FOR THE OPTIMIZATION OF DRUG-DEVICE COMBINATIONS FOR THE TREATMENT OF BRAIN TUMORS
Authors: Killick-Cole Clare , Woolley Max , Johnson David , Lewis Owen , Moore Patrick , Fletcher Julia , Skinner Paul , Bienemann Alison , Gill Steven
Published: November 2019

BACKGROUND

Failure of many brain tumor treatments has been attributed to the inability of a compound to cross the blood-brain barrier. Convection-enhanced delivery (CED) offers a method to administer drugs directly to the tumor site, thereby mitigating this limitation. CED has been used in several preclinical and clinical studies, though an approved treatment is yet to come to fruition. A medical device suitable for repeated intraparenchymal delivery is valuable for brain tumor treatment strategies, but a lack of appropriate preclinical models capable of infusing clinically relevant volumes has hindered progress in clinical translation. Currently utilized preclinical models include porcine, which have limited use due to their growth rates and canine and non-human primates, which are restricted by ethical considerations. This study investigates the functional characteristics and stability of the Renishaw clinical Neuroinfuse™ drug delivery system in an ovine model.

METHODS

A head fixation frame was refined for ovine stereotactic surgery. Eight Romney ewes were implanted with the drug delivery system, comprising of four catheters and a transcutaneous port allowing chronic re-access infusions without repeated surgery. A maximum of four infusions were performed per subject, at four to eight-week intervals.

RESULTS

The implantation of the Neuroinfuse™ drug delivery system was well-tolerated in all subjects, without serious device related adverse events. Artificial CSF/gadolinium infusions were performed on five subjects. Three subjects were maintained for a follow-up study. Putaminal and thalamic distribution volumes remained stable over the re-access period of up to six months post-implantation.

CONCLUSION

Romney ewes are suitable for the stereotactic implantation of the Neuroinfuse™ chronic drug delivery system. Transcutaneous ports remained integrated for the study duration, allowing repeated intraparenchymal delivery. This novel preclinical model provides a stable platform for infusion regime optimization of drug-device combinations for chronic CED, which will undoubtably de-risk clinical translation in the neuro-oncology CED field.