Co-culture of Glutamatergic Neurons and Pediatric High-Grade Glioma Cells Into Microfluidic Devices to Assess Electrical Interactions

10.3791/62748 ◽  
2021 ◽  
Author(s):  
Quentin Fuchs ◽  
Aurélie Batut ◽  
Mélanie Gleyzes ◽  
Jessica Rontard ◽  
Louise Miny ◽  
...  
Keyword(s):  
Glioma Cells ◽  
Microfluidic Devices ◽  
High Grade Glioma ◽  
High Grade ◽  
Glutamatergic Neurons ◽  
Pediatric High Grade Glioma

scholarly journals PDTM-21. MULTI-MODAL PROFILING OF PEDIATRIC HIGH-GRADE GLIOMA SINGLE CELLS USING PATCH-SEQ

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi191-vi191
Author(s):  
Shawn Gillespie ◽  
Marlene Arzt ◽  
Pamelyn Woo ◽  
Michelle Monje
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Glioma Cells ◽  
High Grade Glioma ◽  
Neural Circuitry ◽  
Extracellular Potassium ◽  
High Grade ◽  
Transcriptional Profiles ◽  
Pediatric High Grade Glioma ◽  
Developmental Hierarchy

Abstract Pediatric and adult high-grade gliomas are characterized by extensive intra-tumoral transcriptional heterogeneity. When measured by single cell RNA sequencing, gliomas reveal themselves as continuums of stemness and differentiation programs with important implications for therapy, but to date this transcriptional information has not been directly linked to physiological behaviors of cells. Recent work from our group establishes the electrical integration of glioma cells into neural circuitry. One subpopulation of glioma cells participates in glutamatergic synaptic communication with neurons, and a distinct subpopulation of cells sense and respond to extracellular potassium flux of neuronal networks by an entirely distinct mechanism. Our data support a model in which both modes of electrical communication are critical to glioma growth, but current associations between the electrophysiological properties of a cell, its transcriptional profile and developmental state are correlational in nature. Patch-seq is needed to clarify the relationship between transcriptional profiles of quiescent/cycling stem-like cells and the observed electrophysiological behaviors. Put more simply, patch-seq will clarify where the synaptically-connected glioma cells exist along a developmental hierarchy. METHODS Here, we adapt a recently described technique called patch-seq to record the electrophysiological profiles of individual pediatric high-grade glioma cells by whole cell patch-clamp and subsequently isolate their mRNA for single cell sequencing by smart-seq2 and analysis using Seurat. In this way, we couple electrophysiological and transcriptomic profiles to unambiguously assign functional identities to cells with transcriptional profiles along a developmental hierarchy. RESULTS We report the successful adaptation of patch-seq for use with patient-derived diffuse intrinsic pontine glioma (DIPG) xenografts in acute brain slice preparations, enabling evaluation of single glioma cells integrated in intact neural circuitry. CONCLUSIONS Data synthesizing the electrophysiological and transcriptomic profiles of single glioma cells in the context of the developmental hierarchy will be presented.

scholarly journals P11.63 PI3K inhibition in conjunction with the ketogenic diet reduces growth and neuroinflammation in pediatric high-grade glioma

2019 ◽  
Vol 21 (Supplement_3) ◽  
pp. iii58-iii58
Author(s):  
E Noch ◽  
I Yim ◽  
L Cantley
Keyword(s):  
Oxidative Stress ◽  
Ketogenic Diet ◽  
Glucose Utilization ◽  
Glioma Cells ◽  
High Grade Glioma ◽  
Therapeutic Resistance ◽  
High Grade ◽  
Neuro Inflammation ◽  
Pediatric High Grade Glioma ◽  
Pro Inflammatory Cytokines

Abstract Background: Pediatric high-grade glioma remains a poorly treatable disease with high mortality.Therapeutic advances have lagged behind that of adult glioblastoma, due to small patient numbers, inappropriate generalization from adult tumor types, and unique biology. The use of targeted therapy has recently gained interest in this disease, but efficacy is limited by therapeutic resistance, often as a result of tumor heterogeneity. In the case of phosphatidylinositol 3-kinase (PI3K) inhibition, clinically relevant PI3K inhibitors represent a strong class of drugs for pediatric high-grade glioma, but their use is associated with insulin feedback that reactivates the PI3K pathway and drives therapeutic resistance. Here, we target insulin feedback that is the primary mechanism of PI3K inhibitor-related therapeutic resistance in pediatric high-grade glioma using the ketogenic diet. Materials and Methods: Patient-derived pediatric high-grade glioma stem cells were treated with vehicle or the pan-PI3K inhibitor, BKM-120, with glucose deprivation or phenformin to decrease glucose utilization. NSG mice containing patient-derived pediatric high-grade glioma xenografts were treated with vehicle or BKM-120 on a regular or ketogenic diet to determine whether reducing insulin feedback increases BKM-120 efficacy. To determine the effect of glioma cells on neuro-inflammation, we measured pro-inflammatory cytokines in glioma cells treated with BKM-120 and phenformin in comparison to vehicle-treated cells. We then applied conditioned medium from glioma cells treated with BKM-120 and phenformin to cortical neuronal cultures to measure oxidative stress and neuro-inflammation. Results: Pediatric high-grade glioma cells exhibited increased toxicity when exposed to BKM-120 with glucose deprivation or phenformin. Furthermore, mice with intracranial high-grade glioma xenografts survived longer when treated with BKM-120 on the ketogenic diet than with BKM-120 or the ketogenic diet alone. Phenformin reduced the production of pro-inflammatory cytokines by BKM-120-treated glioma cells. Cortical neurons treated with conditioned medium from BKM-120- and phenformin-treated glioma cells exhibited less oxidative stress than those treated with BKM-120 alone. Our results demonstrate that strategies to lower glucose utilization and insulin feedback increase efficacy of BKM-120 and decrease neuro-inflammation. Conclusions: We show that strategies to lower glucose utilization and insulin feedback increase efficacy of BKM-120. Furthermore, reducing insulin feedback decreases the production of pro-inflammatory cytokines in tumor cells and reduces oxidative stress in neurons treated with conditioned medium from BKM-120-treated glioma cells. By using the ketogenic diet to reduce glucose levels, this strategy may enhance efficacy of PI3K inhibitors in this patient population.

Abstract 3466: ALT-positive pediatric high grade glioma cells display chemosensitivity to ATR pathway inhibition

2017 ◽  
Author(s):  
Jacqueline A. Brosnan-Cashman ◽  
Anthony J. Rizzo ◽  
Ezgi Goger ◽  
Kaylar M. Myers ◽  
Reza Zarinshenas ◽  
...  
Keyword(s):  
Glioma Cells ◽  
High Grade Glioma ◽  
High Grade ◽  
Pathway Inhibition ◽  
Pediatric High Grade Glioma

scholarly journals EXTH-36. PI3K INHIBITION IN CONJUNCTION WITH THE KETOGENIC DIET REDUCES GROWTH AND NEUROINFLAMMATION IN PEDIATRIC HIGH-GRADE GLIOMA

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi89-vi90
Author(s):  
Evan Noch ◽  
Isaiah Yim ◽  
Lewis Cantley
Keyword(s):  
Ketogenic Diet ◽  
Cortical Neurons ◽  
Glioma Cells ◽  
High Grade Glioma ◽  
Therapeutic Resistance ◽  
High Grade ◽  
Pi3k Inhibitors ◽  
Pi3k Inhibition ◽  
Neuro Inflammation ◽  
Pediatric High Grade Glioma

Abstract Pediatric high-grade glioma remains a poorly treatable disease with high mortality. Targeted therapies have gained interest in this disease, but efficacy is limited by therapeutic resistance, often because of tumor heterogeneity. Phosphoinositide 3-kinase (PI3K) inhibitors represent a strong drug class for pediatric glioma, but their use is associated with insulin feedback that reactivates the PI3K pathway and drives therapeutic resistance. Here, we target insulin feedback that is the primary mechanism of PI3K inhibitor-related therapeutic resistance in pediatric high-grade glioma using the ketogenic diet. We treated patient-derived pediatric high-grade glioma stem cells with vehicle or the pan-PI3K inhibitor, BKM-120, in conjunction with phenformin to decrease glucose utilization. These cells exhibited 65% less proliferation when exposed to BKM-120 and phenformin. We treated NOD scid gamma (NSG) mice containing patient-derived pediatric high-grade glioma xenografts with vehicle or BKM-120 on a regular or ketogenic diet to determine whether reducing insulin feedback increases BKM-120 efficacy. Mice with intracranial glioma xenografts survived longer when treated with BKM-120 on the ketogenic diet than with BKM-120 or the ketogenic diet alone. We measured pro-inflammatory cytokines in glioma cells treated with BKM-120 and phenformin in comparison to vehicle-treated cells to determine their effect on neuro-inflammation. We also applied conditioned medium from glioma cells treated with BKM-120 and phenformin to cortical neurons to measure oxidative stress. We found that phenformin reduced the production of pro-inflammatory cytokines, including TNF-alpha, IFN-gamma, IL-1beta, and IL-6, by BKM-120-treated glioma cells. Cortical neurons treated with conditioned medium from BKM-120- and phenformin-treated glioma cells exhibited less oxidative stress than those treated with BKM-120 alone. Our results demonstrate that lowering glucose utilization and insulin feedback increases efficacy of PI3K inhibition and decreases neuro-inflammation. By using the ketogenic diet to reduce systemic glucose levels, this strategy may enhance efficacy and reduce morbidity of PI3K inhibitors in this population.

scholarly journals HG-10 * HYPOXIA PATHWAY DRIVEN BY HIF-1ALPHA ARE PREDOMINANTLY INVOLVED IN PEDIATRIC HIGH GRADE GLIOMA AND REPRESENT PROMISING THERAPEUTIC TARGETS

2015 ◽  
Vol 17 (suppl 3) ◽  
pp. iii12-iii12
Author(s):  
C. Lasthaus ◽  
M. Litzler ◽  
C. Bour ◽  
D. Guenot ◽  
N. Entz-Werle
Keyword(s):  
Therapeutic Targets ◽  
High Grade Glioma ◽  
High Grade ◽  
Pediatric High Grade Glioma

scholarly journals HGG-41. STRUCTURAL VARIANT DRIVERS IN PEDIATRIC HIGH-GRADE GLIOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii351-iii351
Author(s):  
Frank Dubois ◽  
Ofer Shapira ◽  
Noah Greenwald ◽  
Travis Zack ◽  
Jessica W Tsai ◽  
...  
Keyword(s):  
Copy Number ◽  
High Grade Glioma ◽  
High Grade ◽  
Structural Variants ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Effector Domains ◽  
Topologically Associating Domains ◽  
Genome Wide ◽  
Pediatric High Grade Glioma

Abstract BACKGROUND Driver single nucleotide variants (SNV) and somatic copy number aberrations (SCNA) of pediatric high-grade glioma (pHGGs), including Diffuse Midline Gliomas (DMGs) are characterized. However, structural variants (SVs) in pHGGs and the mechanisms through which they contribute to glioma formation have not been systematically analyzed genome-wide. METHODS Using SvABA for SVs as well as the latest pipelines for SCNAs and SNVs we analyzed whole-genome sequencing from 174 patients. This includes 60 previously unpublished samples, 43 of which are DMGs. Signature analysis allowed us to define pHGG groups with shared SV characteristics. Significantly recurring SV breakpoints and juxtapositions were identified with algorithms we recently developed and the findings were correlated with RNAseq and H3K27ac ChIPseq. RESULTS The SV characteristics in pHGG showed three groups defined by either complex, intermediate or simple signature activities. These associated with distinct combinations of known driver oncogenes. Our statistical analysis revealed recurring SVs in the topologically associating domains of MYCN, MYC, EGFR, PDGFRA & MET. These correlated with increased mRNA expression and amplification of H3K27ac peaks. Complex recurring amplifications showed characteristics of extrachromosomal amplicons and were enriched in coding SVs splitting protein regulatory from effector domains. Integrative analysis of all SCNAs, SNVs & SVs revealed patterns of characteristic combinations between potential drivers and signatures. This included two distinct groups of H3K27M DMGs with either complex or simple signatures and different combinations of associated variants. CONCLUSION Recurrent SVs associate with signatures shaped by an underlying process, which can lead to distinct mechanisms to activate the same oncogene.

Pediatric High-Grade Glioma Patterns of Failure by Molecular Subgroup

2020 ◽  
Vol 108 (3) ◽  
pp. e239-e240
Author(s):  
L.J. Sudmeier ◽  
T. Morgan ◽  
P. Mendoza ◽  
J. Switchenko ◽  
E. Schreibmann ◽  
...  
Keyword(s):  
High Grade Glioma ◽  
High Grade ◽  
Patterns Of Failure ◽  
Molecular Subgroup ◽  
Pediatric High Grade Glioma

scholarly journals HG-35MET-PET DELINEATES NON-CONTRAST ENHANCING TUMOR (nCET) REGIONS AT HIGH RISK FOR RECURRENCE IN PEDIATRIC HIGH GRADE GLIOMA

2016 ◽  
Vol 18 (suppl 3) ◽  
pp. iii55.2-iii55
Author(s):  
John T. Lucas ◽  
Nick Serrano ◽  
Hyun Kim ◽  
Yimei Li ◽  
Alberto Broniscer ◽  
...  
Keyword(s):  
High Risk ◽  
High Grade Glioma ◽  
High Grade ◽  
Pediatric High Grade Glioma
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