bluuebunny/crossref_metadata_2025_split huggingface dataset:
TMIC-57. PRO-TUMORAL EFFECTS OF INTRA-TUMORAL NEUTROPHILS IN THE GLIOBLASTOMA MICROENVIRONMENT
Authors: Shah Sumedh , Yagnik Garima , Nguyen Alan , Wadhwa Harsh , Spatz Jordan , Safaee Michael , Cheng Justin , Aghi Manish 
Published: November 2019

Abstract
While macrophage enrichment and lymphocyte depletion have been described in glioblastoma, intratumoral neutrophils and their effect on glioblastoma have been under-characterized. While tumor-associated neutrophils (TANs) were initially regarded as passive bystanders due to their short-lived nature, investigation of TANs in other cancer types revealed pro-tumoral roles. Therefore, we sought to characterize TANs in the glioblastoma microenvironment using transcriptomic analysis and define their oncologic effects. Flow cytometric analysis of patient samples for neutrophils (CD11b+/CD15+/CD66b+) revealed higher percentages of TANs in glioblastoma compared to low-grade gliomas (1.76% [n=13] vs. 0.33% [n=6], p=0.03). Using the Transwell migration assay with glioblastoma tumor conditioned-media (CM), we found that recruitment of circulating neutrophils to tumor sites is mediated by leukotriene-B4 chemoattraction and that this interaction can be blocked with the addition of LtB4 receptor antagonist, LY293111. TANs were morphologically activated, unlike circulating neutrophils from GBM patients (P< 0.05) and, while not intravascular, were close to blood vessels. We performed single-cell RNA sequencing of isolated TANs and found a distinct transcriptomic profile relative to circulating neutrophils from these patients, particularly upregulated osteopontin. Osteopontin concentration was significantly higher in TAN CM than in patient-matched peripheral blood neutrophil CM (3.2ng/mL [n=3] vs. 0.02ng/mL [n=3], p< 0.05). Because osteopontin is linked to GBM stem cell-like phenotype maintenance and TANs localized to the perivascular niche where GBM stem cells reside, we investigated TAN-GBM stem cell interactions and osteopontin as a potential mediator. We found TAN CM increased proliferation and stem cell markers (Nanog, Oct4, Sox2) of stem cell-containing GBM neurospheres (p< 0.01). These effects were blocked by osteopontin-neutralizing antibodies (p< 0.01). Our work defines neutrophil-mediated pro-tumoral effects and their mechanisms and identifies a novel approach to target GBM stem cells—by disrupting the immune cell mediators that create their supportive microenvironment in the perivascular niche.
TMIC-58. THE CELLULAR AND MOLECULAR BASIS FOR MESENCHYMAL TRANSFORMATION IN GLIOMAS
Authors: Comba Andrea , J Dunn Patrick , E Argento Anna , Kadiyala Padma , Motsch Sebastien , Kahana Alon , E Kish Phillip , Castro Maria , Lowenstein Pedro 
Published: November 2019

Abstract
Mesenchymal gliomas are the most aggressive tumors that carry the worst prognosis. The origins of mesenchymal cells within brain tumors, remains poorly understood. They could originate either from invading mesenchymal cells, from perivascular smooth muscle actin+ cells, or from a mesenchymal transformation of tumor cells. Identifying the origin and function of mesenchymal cells within gliomas is essential as these cells contribute to increased glioma aggressiveness and tumor progression. In this study we used human biopsies and implantable and genetically engineered mouse models (GEMM) of GBM to study tumor mesenchymal transformation. GBM implantable models were used to analyze the molecular landscape by laser microdissection followed by RNA-Seq and bioinformatics analysis. Time lapse confocal imagining was implemented to analyze GBM cells dynamics. Our results indicate the existence of a complex intratumoral and peritumoral dynamic organization of glioma cells (i.e., Oncostreams). Multicellular structures of elongated cells compatible with mesenchymal differentiation. These structures play important roles in intratumoral movements, peritumoral invasion of normal brain, and overall glioma progression. We also show that oncostreams are molecularly distinct and display increased expression of mesenchymal genes such as Col1a1. Knocking down of Col1a1 in a GEMM of aggressive gliomas reduced tumor progression and significantly increased animal survival. Histological examination confirmed absence of Col1a1, and absence of morphologically identifiable oncostreams. Our results show that tumor cells, especially within oncostreams, display a fibroblastic-like morphology and express proteins typical of mesenchymal cells. The knockout of Col1a1 from tumoral cells eliminated oncostreams from tumors and delayed tumor progression. These data suggest that tumor cells expressing mesenchymal genes regulate the organization of mesenchymal multicellular structures, and determine glioma progression. We propose that inhibiting mesenchymal transformation of glioma cells will assist in the treatment of glioblastoma.
TMIC-60. COMPREHENSIVE SPATIAL CHARACTERIZATION OF IMMUNE CELLS IN THE CNS BRAIN TUMOR MICROENVIRONMENT
Authors: Kassab Cynthia , Zamler Daniel , Gupta Pravesh , Srinivasan Visish , Rao Ganesh , Prabhu Sujit , Huse Jason , Fuller Greg , Bruner Janet , Langford Lauren , McCutcheon Ian , Ferguson Sherise , Weinberg Jeffrey , Wistuba Ignacio , Lang Frederick , Bhat Krishna , Heimberger Amy 
Published: November 2019

Abstract
Previous immune profiling in brain tumors has mostly focused on the high-density tumor areas, and as such, little is known about the nature and types of immunological responses that occur across the tumor landscape, including at the tumor-central nervous system (CNS) interface. En bloc resections of glioblastomas (n=10) and CNS lung metastases (n=10) were oriented on slides as whole mount wedges spanning three anatomical areas including the invasive edge, tumor region, and necrotic core. Tumor segmentation was performed and regional differences were immunologically analyzed for 770 immune genes using the NanoString nCounter System with CIBERSORT analysis to delineate immune gene signatures. The analysis was validated using multiplex immunohistochemistry (IHC). The top upregulated immune genes in the GBM necrotic core were associated with macrophages, including the CD163 lineage marker, chemotactic factors (such as CCL18 and SAA1), and the phagocytosis stimulatory factors (such as IL-8 and MARCO). The necrotic core downregulates GBM antigens (such as IL13RA2 and MAGEB2), markers of dendritic cells (such as LILRA4), and immune stimulatory processes including MHC, IFN, IL-12, TNF, and ICOS expression. In direct contrast, the infiltrating edge of the GBM relative to the tumor is enriched with stimulators for NK cytotoxicity (i.e., CD244, the fractalkine receptor for immune cells), chemokines for thymocytes and dendritic cells, and immune stimulatory IL-12 receptors. Glioblastoma has rare focal isolated areas of CD3 T-cell reactivity within the tumor. Similar to GBM, the necrotic center of lung metastases is enriched in immune suppressive macrophages, as reflected by CD163 IHC staining and arginase expression; however, they are more frequently infiltrated with M1 macrophages. Yet the majority of lung cancers are more diffusely infiltrated with CD3 T cells, especially at the infiltrating edge. In general, we noted distinct inter- and intratumoral immune gene signatures, with macrophages dominating the brain tumors, especially the necrotic core.
TMIC-64. THE ROLE OF TUMOR-DERIVED GRANULOCYTE MACROPHAGE COLONY STIMULATING FACTOR (GM-CSF/CSF2) IN REGULATION OF MICROGLIA-DEPENDENT INVASION IN GLIOMAS
Authors: Poleszak Katarzyna , Pasierbinska Maria , Ellert-Miklaszewska Aleksandra , Wojtaś Bartosz , Wojnicki Kamil , Kaminska Bozena 
Published: November 2019

Abstract
Brain resident immune cells (microglia) and peripheral macrophages accumulate in malignant gliomas and constitute for 30–50% of the tumor mass. These immune cells are polarized by factors released by glioma and become the pro-invasive, immunosuppressive cells that support tumor progression. We have previously found that tumor-derived granulocyte macrophage colony stimulating factor (GM-CSF/CSF2) is a crucial factor controlling accumulation of microglia and macrophages in murine gliomas. The analysis of TCGA dataset revealed overexpression of the CSF2 gene (encoding GM-CSF) in a set of mesenchymal glioblastomas and its association with high immune gene expression. To study the role of GM-CSF in microglia-stimulated glioma invasion, we used a co-culture system, which mimics microglia interactions with tumor cells. We silenced the expression of CSF2 in glioma cells and found reduced microglia-dependent invasion of glioma cells. To translate those results into clinically relevant setting, we designed and tested humanized short peptides interfering with binding of GM-CSF to its receptor. Selected peptide effectively inhibited binding of GM-CSF to its receptor as demonstrated with different methods. We selected the non-cytotoxic peptides that potently blocked microglia-dependent glioma invasion in cell co-cultures. Blocking GM-CSF-receptor signaling pathway with a neutralizing antibody against a GM-CSF receptor also inhibited microglia-dependent invasion of glioma cells. Altogether, our results demonstrate that glioma-derived GM-CSF supports pro-tumorigenic polarization of microglia turning them into cells that facilitate glioma growth and shape the immune microenvironment. This work was supported by statutory budget of Nencki Institute of Experimental Biology and grant PBS3/B7/19/2015 from The National Centre for Research and Development, Poland.
TMIC-65. GLOBAL PROFILING OF IMMUNE RESPONSE OF MICROGLIA AND MACROPHAGES IN PRECLINICAL MODEL OF GLIOBLASTOMA.
Authors: Walentynowicz Kacper , Ochocka Natalia , Segit Paweł , Mieczkowski Jakub , Wojtaś Bartosz , Gielniewski Bartłomiej , Kostyra Kacper , Baluszek Szymon , Kostkiewicz Bogusław , Kaminska Bozena 
Published: November 2019

Abstract
Glioblastoma (GBM) is the most aggressive primary brain tumor showing the poorest prognosis. Despite extensive research, no significant improvement in glioma therapy. GBM microenvironment has been shown to contribute to the aggressiveness of the tumor and influences overall survival. Tumor associated microglia and macrophages (TAMs) contribute to the biggest portion of tumor infiltrative cellular component, and may drive the immune response within the tumor. Their influence in the preclinical studies has not been fully described. Reliable animal models are crucial to facilitate translational research and drug discovery. In the present study, we employed animal xenograft model, where human GBM cells were stereotactically implanted into the mouse brain. Early and late stage responses of TAMs were studied using RNA-seq. Epigenetic changes within the TAMs were assessed using Assay for Transposase Accessible Chromatin sequencing (ATAC-seq) and Chromatin Immunoprecipitation sequencing (ChIP-seq) for four histone modifications (active transcription H3K4me3, H3K27ac, and repressed H3K9me3, H3K27me3) to further identify molecular alterations responsible for transcriptomic profiles. To facilitate translational discovery and heterogenous population of infiltrative cells, we isolated human CD11b cells from GBM patients and performed single cell RNA-seq (scRNAseq) using 10X Genomics platform. Moreover, we compared profiles of CD11b cells in the xenograft model with syngeneic immunocompetent model to further characterize commonly used models. Preliminary analysis shows critical differences between the models. Gain of global activation of genes has not been identified in the xenograft model. Differences in critical pathways in naïve microglia were identified, between the two studied models. Such global and extensive analysis allows us to compare preclinical models of GBM and clinical samples, and identify common pathways that can serve as potential therapeutic targets. This study was supported by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 665735 (Bio4Med) and National Science Centre grant 2017/27/B/NZ3/01605.
TMOD-02. A MODEL OF THE INITIATION AND GENOMIC LANDSCAPE OF GLIOBLASTOMA (GBM)
Authors: Bohm Alexandra , DePetro Jessica , Binding Carmen , Grisdale Cameron , Ware Matthaeus , Lawn Sam , Chahley Nick , Bukhari Shazreh , Chen Cindy , Gerber Amanda , Thomas Kaitlin , Pedersen Haley , Omairi Hiba , Blough Michael , Gregory Cairncross J 
Published: November 2019

Abstract
IDH-wildtype GBM is the most common variant of this cancer and occurs in older adults. Unfortunately patients’ tumors are either inherently resistant to standard treatment, which includes radio- and chemo-therapy, or acquire resistance during the therapeutic process. Additionally, although effective in other cancers, targeted therapies have yielded disappointing results in GBM, perhaps because the fully developed disease has significant cellular and molecular heterogeneity, allowing the tumour to adapt to treatments. Better insight into managing GBM might result from a detailed knowledge of its initiating events, which have not yet been elucidated. With this in mind, we developed a mouse model of GBM in which the earliest stages can be studied. This ex vivo model recreates GBM by culturing subventricular zone (SVZ) cells, the putative ‘cell of origin’ of GBM in platelet-derived growth factor A (PDGFA). Under this condition SVZ cells from p53 null mice transform, becoming exogenous growth factor independent and tumorigenic in immune-competent mice. In contrast, wildtype SVZ cells do not proliferate in PDGFA and null cells in EGF/FGF do not transform. To discover why p53 null SVZ cells uniquely transform in PDGFA, we performed array comparative genomic hybridization (aCGH) on cells before and after transformation in PDGFA and whole genome sequencing (WGS) on transformed cells and tumours generated from PDGFA-transformed cells. aCGH and WGS revealed that the genomic landscape of transformed cells displayed a striking similarity to that observed in primary human GBM. Specifically, these studies showed that chromosomal alterations are a hallmark of culturing SVZ cells in PDGFA, an intriguing finding considering GBM is also characterized by a specific landscape of copy number alterations. This model may resemble the pathogenesis of human GBM and be leveraged to investigate the early stages of tumorigenesis, further leading to the development of preventative strategies and novel therapeutics.
TMOD-10. PRE-CLINICAL IMAGING MODEL OF GLIOBLASTOMA PSEUDOPROGRESSION USING DUAL FERUMOXYTOL AND GADOLINIUM CONTRAST ENHANCED MAGNETIC RESONANCE IMAGING
Authors: Barajas Ramon , Woltjer Randall , Han Seunggu , Muldoon Leslie , Neuwelt Edward 
Published: November 2019

Abstract

BACKGROUND
Temozolomide-based chemoradiotherapy (CRT) mediated neuroinflammation (pseudoprogression; Psp) within glioblastoma (GBM) is manifested by progressive gadolinium enhancement on magnetic resonance imaging (Gd-MRI)1. Unfortunately, Gd-MRI cannot reliably differentiate Psp from growing GBM. Macrophage infiltration is a prominent component of Psp, and can be noninvasively assessed by 24 hour delayed ferumoxytol-enhanced MRI (Fe-MRI)2. We have demonstrated that dual Fe- to Gd-contrast mismatch ratio may be a specific metric for identifying Psp3. The purpose of our study was to develop and characterize a preclinical model that specifically replicates the imaging context of macrophage mediated Psp.

METHODS
Athymic rats bearing intracerebral patient derived xenografts (GBM-39 and GSC-827) or U-87 GBM cells were randomized to; 1) untreated control (N= 5), 2) CRT only (irradiation [5 Gy] and temozolomide [20 mg/kg, PO] (N=4), or 3) CRT with Amphotericin B (Amp B) (0.2 mg/kg, IP QD x 3–7 days) (N=8)4. Three day post-therapeutic Fe- (ferumoxytol, 25 mg/kg IV) and Gd-MRI was performed as well as 7 day post-therapy Gd-MRI (N=7). Semi-automated enhancement segmentation and volume calculation was performed with Horos software5. Significance was evaluated using Students T-test with unequal variances.

RESULTS
The ratio of Fe- to Gd-enhancement volume 3 days after therapy was found to be significantly elevated within the CRT with Amp B group (1.7 ± 0.53) when compared to the CRT alone (0.62 ± 0.11) and control groups (0.43 ± 0.11); P< 0.01. Seven days after therapy, Gd-enhancement volume was significantly decreased within the CRT with Amp B group (0.87 ± 0.13) when compared to CRT alone (0.38 ± 0.04; P< 0.01), confirming treatment efficacy.

CONCLUSION
Preliminary results suggest that CRT combined with Amp B in this rat model replicates the imaging characteristics of human Psp and may result in improved efficacy3. Fe-MRI enhancement may capture the efficacious macrophage modulatory effects of Amp B.
TMOD-17. A SWINE MODEL OF GLIOBLASTOMA INDUCED BY SOMATIC GENE MODIFICATION
Authors: Tschida Barbara , Duerre Dylan , Taisto Mandy , Solin Staci , Watson Adrienne 
Published: November 2019

Abstract
Glioblastoma (GBM) is the most common and malignant primary brain tumor. New, effective treatments for GBM are needed since the standard of care universally fails to cure patients. A major hurdle for GBM therapeutic development is the lack of relevant large animal models with high translational value for preclinical studies. To overcome this, we are developing a model of GBM in outbred, immune-proficient swine which have comparable brain size and anatomy to humans. GBM tumors will be driven by somatic alterations to major signaling pathways frequently altered in human GBM and will express a secreted reporter for tumor growth detectable in the peripheral blood. These alterations and reporter expression cassettes will be induced by delivering transposons and CRISPR/Cas9 gene-editing tools to the subventricular brain cells of live swine by stereotactic injections. We have developed and deployed these gene-editing tools, demonstrating their efficacy in swine cells. We have identified stereotactic coordinates to reproducibly target the lateral ventricles of neonatal swine brains and have demonstrated successful plasmid delivery to cells at these coordinates. Stable, persistent transposon integration and clonal expansion of modified cells has been confirmed by sequencing transposon-genomic DNA junctions in brain tissue six-months post-injection. To induce GBM-like tumors, we have introduced gene delivery and gene editing reagents to alter six major human GBM-associated signaling pathways in a cohort of swine. Resulting tumors will be examined molecularly to detect the pathway-associated transposons and CRISPR/Cas9-induced mutations in tumor tissue and determine the resemblance to human GBM. This somatic cell gene-modification platform will be adaptable, allowing on-demand inclusion of preclinical study-relevant alterations in GBM tumors, and will allow the rapid development tumors histologically and genetically similar to human GBM, which will be valuable for use in pre-clinical therapeutic studies, imaging studies using human clinical grade equipment, and surgical technique development.
TMOD-25. GLIOBLASTOMA ORGANOIDS: A MODEL SYSTEM FOR PATIENT-SPECIFIC THERAPEUTIC TESTING
Authors: Salinas Ryan , Zhang Daniel , Jacob Fadi , Nguyen Phuong , Sheikh Saad , Prokop Stefan , Dorsey Jay , Nasrallah MacLean , Brem Steven , O’Rourke Donald , Ming Guo-li , Song Hongjun 
Published: November 2019

Abstract
Glioblastoma treatment options remain limited due to its aggressive and invasive nature. It is increasingly appreciated that molecular heterogeneity between tumors and within tumors likely contributes to the lack of therapeutic advances. To maintain the inherent heterogeneity of glioblastoma, we employed a novel method to rapidly culture glioblastoma organoids (GBOs) directly from neurosurgical resection. GBOs are routinely generated around two weeks following initial resection. Comprehensive histologic and sequencing analyses demonstrated similarity to primary tumors. Leveraging clinical molecular and sequencing data, selected GBOs were treated with radiation/temozolamide and targeted inhibitor therapies. The effect on proliferation was measured by the percentage of KI67+ cells and gene set enrichment (GSEA) analysis was performed to compare the pre-treated expression signature amongst responsive and non-responsive tumors. Treatment of organoids with radiation/temozolamide led to a decrease in the percentage of KI67+ cells in four of eight patient-derived organoid lines with some evidence of correlative radiographic response Gene sets associated with radiation response and TNF signaling were enriched in radiation/temozolamide sensitive GBOs. GBO response to EGFR inhibition via gefitinib treatment was specific to EGFR altered tumors, whose expression also enriched for EGF signaling pathway expression. Two GBOs had downstream NF1 mutations that responded to the MEK inhibitor trametinib. On GSEA, gene expression of NF1 mutated GBOs enriched for RAS signaling. One GBO line was found to have a PI3K mutation and responded dramatically to mTOR inhibition via everolimus. Dichotomous efficacy of MEK and mTOR inhibition was also noted by tumor-specific changes in GBO diameter following treatment. This novel culturing method of GBOs maintains intertumoral and intratumoral heterogeneity and allows for therapeutic testing within two weeks of neurosurgical resection. As clinical sequencing because increasingly prevalent, GBOs may become a valuable tool to functionally test mutation-specific treatment strategies in a patient-specific manner within a clinically relevant time frame.
TMOD-36. DEVELOPMENT OF A PATIENT-DERIVED ORTHOTOPIC XENOGRAFT GLIOBLASTOMA MODEL PHENOTYPICALLY REPRESENTATIVE OF THE CLINICAL FEATURES
Authors: Nagaraja Tavarekere , deCarvalho Ana , Griffith Brent , Brown Stephen , Divine George , Irtenkauf Susan , Knight Robert , Lee Ian , Panda Swayamprava , Cabral Glauber , Ewing James 
Published: November 2019

Abstract
In murine models, patient derived orthotopic xenografts (PDOXs) generated from neurosphere cultures of human glioblastomas (GBMs) are unmatched in representing the molecular heterogeneity of the disease. However, intra-axial tumors with blood-brain barrier (BBB) breakdown visible on magnetic resonance imaging (MRI) are rare in these models and, thus, phenotypic recapitulation of human tumors is typically absent. We hypothesized that this absence lay in the interaction between tumor and stroma and that manipulation of the tumor stromal composition might produce a consistent BBB breakdown. Thirty-two athymic nude rats were implanted intracerebrally with one of two neurosphere cell lines (HF2303 or HF3016) derived from the tumors of two GBM patients demonstrating post-gadolinium enhancement on MRI. Neurospheres (3x105 cells in 10μl) were co-injected with a preparation containing either 5μl Matrigel with high (M+) or low (M-) growth factors, or PBS (M0). When the lesion reached 4 to 5 mm in diameter on imaging, MRI with contrast was used to study BBB breakdown. Two animals with only extra-axial tumors were excluded from analysis, and the intra-axial tumors were graded for radiological evidence of BBB breakdown on a scale of 0 to 4, with 0 being no evidence, and 4 being florid enhancement. A two-way ordinal logistic regression model was fit with treatment factors (three levels: M0, M+ and M-) and cell line (two levels: HF2303 and HF3016). A Wilcoxon Rank Sum test analysis within each cell line demonstrated a significant BBB breakdown effect with Matrigel in HF3016 (N=16, p=0.004), but not for HF2303 (N=16, p=0.328). By producing phenotypic conditions closer to the human glioma pathology, a reliable BBB breakdown in PDOX models represents a significant advance in assessing experimental therapies. However, as demonstrated herein, the effect of stromal enhancement is cell-line dependent, and the biological correlates that accompany BBB breakdown still need to be described.