bluuebunny/crossref_metadata_2025 huggingface dataset:
TMIC-23. A SEQUENTIAL IMAGING STRATEGY TO STUDY ONCOLYTIC VIRUS INFILTRATION, REPLICATION AND TUMOR MICROENVIRONMENTAL PERTURBATIONS, EX VIVO
Authors: Smalley Munisha , Thyiagarajan Saravanan , Jain Misti , Best Douglas , Saha Suniti , Prasad Komal , Alonzo Emiliy , Hathaway Erin , Henderson Kira , Chiocca Antonio , Lawler Sean , Goldman Aaron
Published: November 2019

The use of oncolytic viruses (OV) as therapeutic agents for glioblastoma multiforme (GBM) is an emerging clinical focus. OV have the potential to reinvigorate the immune contexture in the tumor microenvironment (TME). However, current pre-clinical models are insufficient to study the immune effects of OV. Here, we describe an imaging approach using a human ex-vivo platform to study the effects of OV on tumor-associated immune cells in GBM. Using the rQNestin34.5v.1 herpes simplex virus (rQNestin) we treated human GBM ex-vivo, incorporating autologous patient-derived peripheral-blood mononucleated cells and whole tissue fragments. We treated GBM (n=10) with rQNestin, capable of expressing GFP, and profiled GFP expression as a proxy for replication. We analyzed serial sections of H&E, HSV-1, and multiplex immunohistochemistry (mIHC) to investigate the impact of rQNestin on the spatial context of the immune compartment. Cell Signaling Technology antibodies, CD3e (D7A6E™,ID:85061), CD68 (D4B9C,ID:76437), CD11c (D3V1E,49420), MHC-Class II ((HLA-DRB)LGII-612.14,NA) and Pan-keratin (C11,4545) were optimized for identifying intratumoral T-cells (CD3e+), macrophages (CD68) and dendritic cells (CD11c+MHCII+) in relation to the surrounding tissue architecture (pan cytokeratin). Using the serial-section imaging approach, we overlaid infiltration and replication of rQNestin. Additionally, strategic sectioning identified dynamic spatial alterations in the immune compartment, via visualization of the tumor-immune interface with indications of infiltration and replication. Quantitative pathology showed dynamic re-arrangement of immune cells - in spatial orientation, quantity and localization, relative to the tumor, under rQnestin pressure, ex-vivo. Here, we report that our ex-vivo human tumor model can be used to evaluate the effects of OV in the TME – not only viral replication but also the direct effect on the immune compartment. In particular, we can assess changes in immune subsets post-OV treatment in a 3-D manner, therefore providing greater insight into their spatial arrangement and distribution. These findings could develop our understanding of anti-tumor OV mechanisms.
TMIC-29. METABOLIC INTERACTIONS BETWEEN TREATMENT-RESISTANT SLOW-CYCLING CELLS AND THE IMMUNE SYSTEM IN HIGH-GRADE GLIOMA
Authors: Sayour Elias , Yang Changlin , Tian Guimei , Grippin Adam , Dajac Mariana , Nazareth Brian , Andrews Michael , Karachi Aida , Mendez-Gomez Hector , Stover Brian , Moore Ginger , Flores-Toro Joseph , Divita Bayli , Kresak Jesse , Rahman Maryam , Flores Catherine , Huang Jianping , Harrison Jeffrey , Mitchell Duane , Deleyrolle Loic
Published: November 2019

INTRODUCTION

Intratumoral heterogeneity is increasingly recognized as a determinant of therapy resistance and disease recurrence; this is exemplified by glioblastoma (GBM), one of the most lethal malignancies. We recently revealed that GBM contain cell subpopulations with distinct metabolic requirements, with fast-cycling cells (FCCs) harnessing aerobic glycolysis, and treatment-resistant slow-cycling cells (SCCs) preferentially engaging lipid metabolism. How the different tumor cell populations interact with immune cells and how this metabolic heterogeneity shapes the immune landscape in GBM has yet to be understood. OBJECTIVES: The objectives of this study are to understand the mechanisms of communication in the tumor microenvironment, specifically to characterize the metabolic interactions between SCCs (a therapeutically resistant population of cancer cells that drive disease progression and recurrence) and the immune compartment.

METHODS

The murine glioma cell line KR158 (derived from a Nf1;Trp53 mutant mouse) was used to establish the slow-cycling cell paradigm and metabolic heterogeneity in an immune-competent model of glioma.

RESULTS

Similar to what we observed in patient-derived specimens, mouse KR158-derived SCCs demonstrate tumorigenicity, treatment resistance and up-regulation of stemness programs and lipid metabolic pathways. We determined that tumor progression is regulated by the interaction of SCCs with the immune system and established that these cells are driving a pro-tumorigenic microenvironment via the recruitment of immune suppressive myeloid cells. Importantly, the immune microenvironment shaped by SCCs is marked by specific metabolic features showing enhanced lipid exchange capacities that we propose are exploited by SCCs to support their survival and functions.

CONCLUSION

Our study indicates that SCCs play a pivotal role in shifting the GBM milieu toward an immune regulatory phenotype but importantly reveals an unprecedented metabolic cooperation, which represents a novel therapeutic target to antagonize GBM.
TMIC-31. IMPACT OF IRON ON MACROPHAGE IMMUNE PHENOTYPE IN THE GLIOBLASTOMA TUMOR MICROENVIRONMENT
Authors: Shenoy Ganesh , Madhankumar Achuthamangalam , Slagle-Webb Becky , Mrowczynski Oliver , Schell Todd , Nesterova Darya , Lee Sang , Davalos Dimitrios , Berens Michael , Lathia Justin , Barnholtz-Sloan Jill , Rubin Joshua , Connor James
Published: November 2019

The tumor microenvironment (TME) in glioblastoma presents a significant hurdle to effective immunotherapies as it consists of an immunosuppressive niche that results in inhibition of anti-tumor immunity. Macrophages comprise a large portion of the glioblastoma TME as they are prolific secretors of immunosuppressive cytokines and can comprise of up to 30% of tumor volume. In addition to their crucial role in immune function, macrophages are important players in iron regulation owing to their ability to efficiently sequester and release iron. We studied the impact of iron status in macrophage anti-tumor immune functions and phenotypic plasticity using RAW264.7 macrophages and bone-marrow-derived macrophages (BMDMs). Using RAW264.7 cells, we found that the iron status of macrophages impacts their immune function by modulating expression of the co-stimulation membrane proteins CD80 and CD86 as well as the mannose receptor CD206. Interestingly, the iron-mediated immunomodulation was dependent on the formulation of iron with nanoparticle formulations such as ferumoxytol (Fe-NP) upregulating CD80, CD86, and decreasing CD206 while ferric ammonium citrate (FAC) downregulated CD80, CD86 and upregulated CD206. We analyzed expression of inflammatory cytokines in BMDMs with multiplex cytokine analysis and found that both FAC and Fe-NP increased expression of the chemotactic signals CXCL10, CCL1, CCL3, and CCL4. Additionally, we found that iron status impacts the ability of macrophages to repolarize from an inflammatory, immune-activating phenotype into an immunosuppressive phenotype upon exposure to glioblastoma tumor-conditioned media. We used qRT-PCR to examine gene expression of the tumor immunity-related genes TNFa, IL1B, NOS2, and IL10 and found that iron loaded RAW264.7 macrophages stimulated with lipopolysaccharide resisted repolarization into an immunosuppressive phenotype 24 hours after exposure to glioblastoma tumor-conditioned media. Our results suggest that understanding the link between iron status and immune function in the tumor microenvironment may be an important step in improving therapies against glioblastoma.
TMIC-33. INVESTIGATING S1PR3-MEDIATED GLIOBLASTOMA INVASION MECHANISMS USING 3D HYDROGEL - TISSUE CULTURE INSERT MODEL
Authors: Tate Kinsley , Chase Cornelison R , Bhargava Saloni , Munson Jennifer
Published: November 2019

Glioblastoma (GBM) is the most common malignant brain tumor and is characterized by its ability to invade into the surrounding microenvironment of the brain. The invasiveness of GBM makes this cancer extremely hard to treat, leading to a median patient survival of less than 16 months. Interactions between the tumor and surrounding tumor microenvironment (TME) play a key role in glioma invasion. Previous data from our xenograft mouse tumor implant model displays increased invasion in regions of fluid flow. Using this model, we identified an upregulation of sphingosine-1-phosphate receptor 3 (S1PR3) in the TME in regions of flow. We used a syngeneic GL261 mouse model and found S1PR3-/- mice display decreased flow-mediated glioma invasion in comparison to wild type mice. To further understand the individual contributions of the S1PR3-presenting cells in the TME, we have examined the role of S1PR3 in our in vitro system. This system is based on patient derived cellular ratios and incorporates collagen-hyaluronan hydrogels placed within 96 well tissue culture insert plates. The tunability of this model allows for interactions between various cell types and the impact of fluid flow on invasion to be examined. To examine the role of S1PR3 on invasion, TY52156 (an S1PR3 inhibitor) was applied to different cellular combinations including: G34 alone (a patient-specific cell line), +astrocytes, +microglia, +TME (astrocytes and microglia). A significant decrease in G34 flow stimulated invasion was observed with TY52156 but only in the presence of the TME or microglia alone. This data suggests that TY52156 thwarts the effects of flow and the microglia contributions to invasion. To further this work we plan to identify and evaluate other S1PR3-expressing cell types from mouse tumor implant samples using immunohistochemistry staining. This information will be used to determine further components that can be examined in our in vitro model.
TMIC-54. COMPARISON OF CELLULAR FEATURES AT AUTOPSY IN GLIOBLASTOMA PATIENTS WITH STANDARD TREATMENT OF CARE AND TUMOR TREATMENT FIELDS
Authors: Lowman Allison , Barrington Alexander , Cochran Elizabeth , Connelly Jennifer , Mueller Wade , LaViolette Peter
Published: November 2019

BACKGROUND

The FDA has approved the use of tumor treatment fields (TTFields) on patients with recurrent glioblastoma (GBM). A recent clinical trial showed that the use of TTFields along with standard treatment of care resulted in increased quality of life and survival over five years. TTFields work by delivering low intensity electric fields that disrupt GBM tumor cell division. This works to slow or even stop GBM tumor cells from dividing.

METHODS

Brain tissue samples were taken from thirteen GBM patients, diagnosed at autopsy. Three patients underwent standard clinical treatment and ten patients underwent TTField therapy in addition to the standard. Tissue samples were acquired from regions where enhancement was seen on the last clinical T1 plus contrast MRI prior to death. Samples were paraffin embedded and stained with hematoxylin and eosin (H&E) and Ki67. The slides were digitized, allowing analysis of the cellular features.

RESULTS

Using the H&E stained slides we compared cellular density between both groups. We found that there was not a noticeable difference in the nuclei density in areas of tumor between the groups. The percent of positive nuclei on the Ki67 stained slides was calculated and there was increased mitotically active cells per area of tissue in patients that did not undergo TTField therapy.

CONCLUSION

These findings suggest there is a difference in the cellular features in areas of tumor in patients treated with TTFields compared to those with standard treatment alone. Additional research needs to be done evaluating more patients.
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

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

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

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

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

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

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

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

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

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

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.
TMOD-39. EX-VIVO 3-DIMENSIONAL MODELS OF POST-SURGICAL RESIDUAL DISEASE IN HUMAN GLIOBLASTOMA
Authors: Rominiyi Ola , Myers Katie , Vanderlinden Aurelie , Dar Dawoud , Bagga Veejay , Gomez-Roman Natividad , Jellinek David , Carroll Thomas , Chalmers Anthony , Al-Tamimi Yahia , Collis Spencer
Published: November 2019

Translation of novel therapies for glioblastoma from laboratory to clinical trials has relied heavily on cells derived from within patient tumours. However, it is clear from over 40 years of limited improvement in survival rates, that the traditional paradigm of 2-dimensional in-vitro studies using these cells followed by implantation into mouse models poorly predicts the treatment response of glioblastoma cells left behind after surgery in patients. Additionally, recent increases in the number of new therapies requiring pre-clinical evaluation emphasise a need to apply disease models that faithfully and efficiently reproduce biological features of post-surgical residual glioblastoma in patients. Dependent on the anatomical considerations of individual cases, surgical resection occasionally provides an en-bloc specimen (e.g. partial lobectomy), which includes portions of infiltrated brain that would not otherwise be resected. Such specimens provide crucial material to model and understand residual (typically left behind) disease in glioblastoma. Using such specimens, we have developed a pragmatic methodology to liberate glioblastoma stem cells (GSC) from the tumour core and adjacent infiltrated brain to model resected and residual disease, respectively. Imperative considerations include careful patient selection and intraoperative clinical-academic collaboration, with immediate ex-vivo hemi-section and processing of suitable specimens. Initial biological characterisation of our resected and residual models reveals numerous divergent properties including morphological differences and elevated stem cell marker expression in residual models (p=0.0021). Importantly, although GSC within recently derived 2-dimensional residual disease models are frequently too migratory to reliably form discrete colonies, we are able to perform robust clonogenic survival studies using the same GSC within a novel customised 3-dimensional scaffold-based architecture (3.4-fold greater clonogenic potential, p=0.0010). In conclusion, we believe that our models provide a biologically relevant and cost-effective tool to investigate the nuances of residual glioblastoma and assess the potential of therapies to tackle disease normally left behind following surgery in patients.
TMOD-40. IN VIVO EVALUATION OF O6-METHYLGUANINE-DNA-METHYLTRANSFERASE (MGMT) PROMOTER METHYLATION STATUS FOR DE NOVO GLIOBLASTOMA PATIENTS USING DEEP LEARNING FEATURES
Authors: Rathore Saima , Nasrallah MacLean , Akbari Hamed , Shukla Gaurav , Bagley Stephen , Watt Christopher , Min Ha Sung , Mamourian Elizabeth , Sako Chiharu , Binder Zev , O’Rourke Donald , Davatzikos Christos
Published: November 2019

BACKGROUND

High expression of O6-methylguanine-DNA methyltransferase (MGMT) in glioblastoma is associated with resistance to temozolomide, as tumor cells lacking MGMT activity are significantly more sensitive to the cytotoxic effects of temozolomide. The MGMT promoter methylation status (MGMTpms) is typically determined as MGMT-methylated or MGMT-unmethylated. Some single-center studies have reported results ranging from 70–95% detection rates using MRI. We aim to further validate these findings using a multi-institutional data set. We hypothesize that transfer learning based features when integrated via machine learning may lead to non-invasive determination of MGMTpms.

METHODS

A total of 270 patients were included across the 3 institutions (Hospital of the University of Pennsylvania (HUP), Jefferson University Hospital (JUH); the TCIA). JUH and TCIA datasets comprised conventional modalities (T1,T2,T2-FLAIR,T1-Gd), whereas HUP dataset had additional modalities (DSC,DTI) as well. We used transfer learning and adapted a convolutional neural network (CNN) model pre-trained on 1.2 million 3-channel images of the ImageNet to extract deep learning features from the given images. A support vector machine multivariately integrated these features towards a non-invasive marker of MGMTpms.

RESULTS

The cross-validated accuracy of our MGMT marker in classifying the mutation status in individual patients was 86.95%, 81.56%, and 82.43%, respectively, in HUP, JUH, and TCIA. Our marker revealed MGMT-methylated tumors with lower neovascularization and cell density, when compared with MGMT-unmethylated tumors. MGMT-unmethylated tumors were found to be more lateralized to the right hemisphere, when compared with MGMT-methylated tumors.

CONCLUSION

Our findings suggest that transfer learning features when integrated via machine learning allow robust prediction of MGMTpms on mpMRI acquired within multiple institutions. The proposed non-invasive MGMT marker may contribute to (i) MGMTpms determination for patients with inadequate tissue/inoperable tumors, (ii) stratification of patients into clinical trials, (iii) patient selection for targeted therapy, and (iv) personalized treatment planning.
ANGI-03. PHARMACOLOGICAL TARGETING OF APELIN/APLNR SIGNALING BLUNTS THERAPY RESISTANCE TO VEGFA/VEGFR2 ANTI-ANGIOGENIC TREATMENT IN GLIOBLASTOMA
Authors: Kälin Roland , Mastrella Giorgia , Hou Mengzhuo , Li Min , Stoecklein Veit , Zdouc Nina , Volmar Marie , Miletic Hrvoje , Reinhard Sören , Herold-Mende Christel , Kleber Susanne , Eisenhut Katharina , Gargiulo Gaetano , Synowitz Michael , Vescovi Angelo , Harter Patrick , Penninger Josef , Wagner Ernst , Mittelbronn Michel , Bjerkvig Rolf , Hambardzumyan Dolores , Schüller Ulrich , Tonn Joerg-Christian , Radke Josefine , Glass Rainer
Published: November 2019

Anti-angiogenic therapy of glioblastoma with bevacizumab, a vascular endothelial growth factor-A (VEGFA) blocking antibody, may accelerate tumor cell invasion and induce alternative angiogenic pathways. We investigated the roles of the pro-angiogenic receptor APLNR and its cognate ligand apelin in VEGFA/VEGFR2 anti-angiogenic therapy against distinct subtypes of glioblastoma. In proneural glioblastoma, apelin levels were downregulated by VEGFA or VEGFR2 blockade by use of bevacizumab or ramucirumab, respectively. A central role for apelin/APLNR in controlling glioblastoma vascularization was corroborated in a serial implantation model of the angiogenic switch that occurs in human glioblastoma. Apelin and APLNR are broadly expressed in human glioblastoma, and knockdown or knockout of APLN in orthotopic models of proneural or classical glioblastoma subtypes massively reduced glioblastoma vascularization as compared with controls. What is more, direct infusion of the bioactive peptide apelin-13 was able to rescue this vascular loss-of-function phenotype, demonstrating the specific control of tumor vascularization by apelin/APLNR signaling. While high levels of apelin correlated with reduced tumor cell invasiveness, the reduction in apelin expression led to accelerated glioblastoma cell invasion. Analysis of stereotactic glioblastoma biopsies from patients as well as from in vitro and in vivo experiments revealed increased dissemination of APLNR-positive tumor cells when apelin levels were reduced. Most interestingly, application of apelin-F13A, a mutant APLNR ligand, blocked both tumor angiogenesis and glioblastoma cell invasion. Furthermore, co-targeting VEGFR2 and APLNR synergistically improved survival of mice bearing proneural glioblastoma. In summary, we show that apelin/APLNR signaling controls glioblastoma angiogenesis and invasion directly, and that both pathological features are blunted by apelin-F13A. We suggest that apelin-F13A can improve the efficiency and reduce the side effects of established anti-angiogenic treatments for distinct glioblastoma subtypes.
TMOD-41. ORTHOTOPIC PATIENT-DERIVED XENOGRAFT MODELS OF BRAIN METASTASIS: PLATFORMS FOR PRECISION ONCOLOGY AND UNDERSTANDING TUMOR BIOLOGY
Authors: Dankner Matthew , Savage Paul , Rose April , Lajoie Mathieu , Diaz Roberto , Park Morag , Watson Ian , Guiot Marie-Christine , Petrecca Kevin , Siegel Peter
Published: November 2019

RATIONALE

Brain metastasis (BrM) occurs in 10–20% of cancer patients and results in median survival times of less than 1 year. In order to accurately develop novel treatment strategies, there is an urgent need to establish animal models of BrM that resemble the human disease. OBJECTIVES: We sought to establish orthotopic patient-derived xenografts (PDX) models of BrM from diverse solid cancers to understand biological characteristics of BrM and to test novel drug candidates in their ability to treat BrM.

METHODS

35 PDXs were established by subcutaneous or mammary fat pad implantation and by intracranial injection. PDXs were validated by immunohistochemistry of routine pathological markers in patient specimens and matched primary and intracranial PDXs. PDXs were applied for precision medicine in class II BRAF mutant tumours treated with targeted therapy and in the pre-clinical development of a novel therapeutic agent, DZ-2384, which may have applications in treating BrM. They were also used to understand the underlying biology of leptomeningeal dissemination from parenchymal BrM.

RESULTS

PDXs reveal strong similarity to patient specimens by IHC staining. Class II BRAF mutant PDXs are optimally sensitive to BRAF and MEK inhibitors compared to either agent alone. DZ-2384 is effective in slowing the progression of breast cancer BrM. PDXs that most efficiently invade the leptomeninges display loss of E-Cadherin expression, suggesting the role of an epithelial-mesenchymal transition in this process.

CONCLUSIONS

PDXs of BrM represent important models that can be employed to test novel therapeutics and to improve understanding of molecular mechanisms engaged by BrM.
TMOD-42. PROSPECTIVE VALIDATION OF AN EX VIVO 3D ASSAY FOR PREDICTION OF TEMOZOLOMIDE RESPONSE IN GLIOBLASTOMA
Authors: Smith Ashley , Holmes Lillia , Shuford Stephen , Kanos Charles , Gardner Stephen , Hodge Philip , Lynn Michael , Edenfield Jeff , DesRochers Teresa
Published: November 2019

Standard treatment for newly diagnosed glioblastoma (GBM) is surgical resection followed by radiation with concurrent temozolomide (TMZ). For those who do not respond there are few clinical options, survival is low, and there no biomarkers to direct the decision of second-line treatment. We have developed a 3D cell culture assay to predict response to TMZ and 11 other potential therapies for GBM. Our assay uses GBM tissue obtained at surgical resection and the test’s 7-day turnaround maximizes clinical actionability of results. We performed a small, prospective study examining the ability of the assay to predict response to TMZ. Ten newly diagnosed GBM patients were enrolled over 12 months. Two patients were removed due to refusal of treatment, and three failed the assay (3/8, 62.5% success). The remaining five patients received radiation and concurrent TMZ following surgical resection. Using progression-free survival (PFS) measured from the completion of radiation therapy and setting 4 months as the cutoff for response, we were able to determine the ability of our assay to predict TMZ response. The assay correctly predicted all five patients (100%). The average PFS for the two predicted responders was 8 months compared to the three predicted non-responders at 3 months, p = 0.0634. In this limited dataset, MGMT methylation did not appear to play a role as two of three predicted non-responders were methylated along with one of two predicted responders. Average overall survival of the predicted TMZ responders was 13 months compared to 6 months for the predicted non-responders, p = 0.0634. This early data indicates the potential for this assay to inform clinicians of the best course of action for their patients. Further enrollment of patients into the 3D-PREDICT clinical trial will provide a larger dataset for better validation of the predictive value of this new assay.