PO-334 Identification of shared lineage programs across high-grade glioma subtypes by single-cell RNA-seq

Abstract BACKGROUND Pediatric treatment-induced high-grade glioma (TIHGG) is among the most severe late effects observed in childhood cancer survivors and is uniformly fatal. We previously showed that TIHGG are divergent from de novo pediatric high-grade glioma (pHGG) and cluster into two gene expression subgroups, one stemlike and the other inflammatory. Here we systematically compared TIHGG molecular profiles to pHGG and evaluated expression and single cell sequencing profiles in order to identify oncogenic mechanisms and the cellular basis for the observed TIHGG gene expression subgroups. MATERIALS/ METHODS 450/850K methylation and mutational signature analysis was conducted in 36 TIHGG samples. Resultant data were analyzed for the presence of chromothripsis, distinct molecular alterations, and mutational signatures in a subset of 10 samples with whole genome sequencing data. Five TIHGGs underwent single-cell RNA-Seq analysis (scRNAseq). RESULTS 26/36 TIHGG clustered with the pedRTK1 methylation class. TIHGG were characterized by an increased frequency of chromothripsis relative to pHGG (67% vs. 31%, p=0.036). FISH and WGS revealed frequent PDGFRA amplification secondary to enrichment in ecDNA. TIHGG were enriched for COSMIC mutational signatures 5 and 19 (p=0.0003) relative to pHGG. scRNAseq data showed that TIHGG tumors are composed of stem-like, neuronal, and inflammatory cell populations which may contribute to the previously described dominant expression profiles. CONCLUSIONS TIHGG represents a distinct molecular subtype of pHGG. Chromothripsis, leading to enriched expression of genes in extrachromosomal DNA, likely contribute to TIHGG oncogenesis. The dominant cell type (stem-like vs. inflammatory) may define the expression subgroup derived from bulk RNA-seq in heterogeneous tumors.

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IMMU-17. SINGLE-CELL RNA-SEQ REVEALS HIGH-GRADE GLIOMA ASSOCIATED MICROGLIA WITH PROINFLAMMATORY AND STEM-LIKE FEATURES SUPPORT TUMOR PROGRESSION

Neuro-Oncology ◽

10.1093/neuonc/noaa215.447 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii108-ii108

Author(s):

Hailong Liu ◽

Yongqiang Liu ◽

Janusz Franco-Barraza ◽

Xinguang Yu ◽

Shiyu Feng

Keyword(s):

Single Cell ◽

Tumor Cells ◽

Poor Response ◽

High Grade Glioma ◽

High Grade ◽

Rna Seq ◽

Wild Type ◽

Current Therapies ◽

Tgf Β1

Abstract Poor response of human glioblastoma to current therapies are influenced by tumor microenvironment. Although glioblastoma is recognized by large enrichment of microglia, characterization of diverse cell subsets and their functions remain challenging because of high heterogenicity. Here, we analyzed single-cell transcriptomics to comprehensively map the cell populations and determine the roles of microglia in IDH1/2 wild-type (IDH-wt) glioblastoma progression. Besides finding microglia were significantly enriched in IDH-wt glioblastoma compared to IDH1/2 mutant (IDH-mut) gliomas, we identified a unique high-grade glioma microglia (HGAM) subtype characterized by proinflammatory and stem-like features. In particular, HGAM’s pro-tumoral IL1β secretion is mediated via ApoE-induced activation of NLRP1 inflammasome. HGAM phagocytosed OPC-like malignant cells forming the neoplastic microglia, which presented the stem-like potential giving rise to activated microglia. Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation. Additionally, an intricated evaluation of glioma patients revealed that SETD2 mutation/low-expression correlated with adverse prognosis. Further analysis showed that SETD2 -dificient tumor cells presented hypersensitivity to HGAM-derived IL1β via epigenetic dysregulation of PHF6. Also, SETD2 -deficient tumor cells produced TGF-β1 contributing to microglia activation. Finally, targeting the TGF-β1/TβRI signaling impaired HGAM activation and tumor growth. Our studies identify a unique neoplastic microglia subpopulation and establish cellular basis of interactions with tumor cells important for disease progression.

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Single-Cell Transcriptomic Analysis Revealed a Critical Role of SPP1/CD44-Mediated Crosstalk Between Macrophages and Cancer Cells in Glioma

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.779319 ◽

2021 ◽

Vol 9 ◽

Author(s):

Cong He ◽

Luoyan Sheng ◽

Deshen Pan ◽

Shuai Jiang ◽

Li Ding ◽

...

Keyword(s):

Single Cell ◽

Tumor Heterogeneity ◽

Molecular Mechanisms ◽

Critical Role ◽

Cell Communication ◽

High Grade Glioma ◽

Sequencing Analysis ◽

High Grade ◽

Cellular Components

High-grade glioma is one of the most lethal human cancers characterized by extensive tumor heterogeneity. In order to identify cellular and molecular mechanisms that drive tumor heterogeneity of this lethal disease, we performed single-cell RNA sequencing analysis of one high-grade glioma. Accordingly, we analyzed the individual cellular components in the ecosystem of this tumor. We found that tumor-associated macrophages are predominant in the immune microenvironment. Furthermore, we identified five distinct subpopulations of tumor cells, including one cycling, two OPC/NPC-like and two MES-like cell subpopulations. Moreover, we revealed the evolutionary transition from the cycling to OPC/NPC-like and MES-like cells by trajectory analysis. Importantly, we found that SPP1/CD44 interaction plays a critical role in macrophage-mediated activation of MES-like cells by exploring the cell-cell communication among all cellular components in the tumor ecosystem. Finally, we showed that high expression levels of both SPP1 and CD44 correlate with an increased infiltration of macrophages and poor prognosis of glioma patients. Taken together, this study provided a single-cell atlas of one high-grade glioma and revealed a critical role of macrophage-mediated SPP1/CD44 signaling in glioma progression, indicating that the SPP1/CD44 axis is a potential target for glioma treatment.

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HGG-04. USE OF AN ADVANCED RNA-SEQ FUSION PIPELINE RESULTS IN THE TARGETED TREATMENT AND SUSTAINED CLINICAL RESPONSE OF CHILDREN WITH RECURRENT PEDIATRIC HIGH-GRADE GLIOMA

Neuro-Oncology ◽

10.1093/neuonc/noz036.098 ◽

2019 ◽

Vol 21 (Supplement_2) ◽

pp. ii87-ii87

Author(s):

Kallen Schwark ◽

Amy Bruzek ◽

Chandan Kumar-Sinha ◽

Sylvia Escolero ◽

Bernard Marini ◽

...

Keyword(s):

Clinical Response ◽

High Grade Glioma ◽

Targeted Treatment ◽

High Grade ◽

Rna Seq ◽

Pediatric High Grade Glioma

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Single-Cell Transcriptome Analysis of Lineage Diversity and Microenvironment in High-Grade Glioma

10.1101/250704 ◽

2018 ◽

Cited By ~ 1

Author(s):

Jinzhou Yuan ◽

Hanna Mendes Levitin ◽

Veronique Frattini ◽

Erin C. Bush ◽

Deborah M. Boyett ◽

...

Keyword(s):

Single Cell ◽

Glioma Cells ◽

High Grade Glioma ◽

Transformed Cells ◽

High Grade ◽

Tight Coupling ◽

Mesenchymal Tumors ◽

Neural Lineage ◽

Mesenchymal Transformation ◽

The Brain

ABSTRACTBackgroundDespite extensive molecular characterization, we lack a comprehensive understanding of lineage identity, differentiation, and proliferation in high-grade gliomas (HGGs). We sampled the cellular milieu of HGGs with massively-parallel single-cell RNA-Seq.ResultsWhile HGG cells can resemble glia or even immature neurons and form branched lineage structures, mesenchymal transformation results in unstructured populations. Glioma cells in a subset of mesenchymal tumors lose their neural lineage identity, express inflammatory genes, and co-exist with marked myeloid infiltration, reminiscent of molecular interactions between glioma and immune cells established in animal models. Additionally, we discovered a tight coupling between lineage resemblance and proliferation among malignantly transformed cells. Glioma cells that resemble oligodendrocyte progenitors, which proliferate in the brain, are often found in the cell cycle. Conversely, glioma cells that resemble astrocytes, neuroblasts, and oligodendrocytes, which are non-proliferative in the brain, are generally non-cycling in tumors.ConclusionsThese studies reveal a relationship between cellular identity and proliferation in HGG and distinct population structures that reflects the extent of neural and non-neural lineage resemblance among malignantly transformed cells.

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HGG-26. SINGLE-CELL RNA-SEQ OF PEDIATRIC HIGH-GRADE GLIOMAS IDENTIFIES COMMON ONCOGENIC PROCESSES AMONG DISTINCT TUMOR HISTOLOGIES

Neuro-Oncology ◽

10.1093/neuonc/noab090.090 ◽

2021 ◽

Vol 23 (Supplement_1) ◽

pp. i22-i22

Author(s):

John DeSisto ◽

Andrew Donson ◽

Rui Fu ◽

Bridget Sanford ◽

Kent Riemondy ◽

...

Keyword(s):

Dna Methylation ◽

Single Cell ◽

Immune Cells ◽

Immune Cell ◽

Treatment Modalities ◽

Patient Specific ◽

Cell Populations ◽

High Grade ◽

Methylation Data ◽

Rna Seq

Abstract Background Pediatric high-grade glioma (PHGG) is a deadly childhood brain tumor that responds poorly to treatment. PHGG comprises two major subtypes: cortical tumors with wild-type H3K27 and diffuse midline gliomas (DMG) that occur in the midline and have characteristic H3K27M mutations. Cortical PHGG is heterogeneous with multiple molecular subtypes. In order to identify underlying commonalities in cortical PHGG that might lead to better treatment modalities, we performed molecular profiling, including single-cell RNA-Seq (scRNA-Seq), on PHGG samples from Children’s Hospital Colorado. Methods Nineteen cortical PHGG tumor samples, one DMG and one normal margin sample obtained at biopsy were disaggregated to isolate viable cells. Fifteen were glioblastomas (GBM), including five with epithelioid and/or giant cell features and five radiation-induced glioblastomas (RIG). There were also four non-GBM PHGG. We performed scRNA-Seq using 10X Genomics v.3 library preparation to enable capture of infiltrating immune cells. We also performed bulk RNA-Seq and DNA methylation profiling. Results After eliminating patient-specific and cell-cycle effects, RIG, epithelioid GBM, and other GBM each formed identifiable subgroups in bulk RNA-Seq and scRNA-Seq datasets. In the scRNA-Seq data, clusters with cells from multiple tumor samples included a PDGFRA-positive population expressing oligodendrocyte progenitor markers, astrocytic, mesenchymal and stemlike populations, macrophage/monocyte immune cells, and a smaller T-cell population. Analyses of DNA methylation data showed PDGFRA and CDK4 amplification and CDKN2A deletion are common alterations among PHGG. Inferred copy number variation analysis of the single-cell data confirmed that individual tumors include populations that both include and lack the molecular alterations identified in the methylation data. RNA velocity studies to define tumor cells of origin and further analyses of the immune cell populations are underway. Conclusions Single-cell analysis of PHGG confirms a large degree of tumor heterogeneity but also shows that PHGG have stemlike, mesenchymal and immune cell populations with common characteristics.

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

Neuro-Oncology ◽

10.1093/neuonc/noz175.797 ◽

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.

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