Project description:High-grade gliomas are aggressive primary brain cancers with poor response to standard regimens, driven by immense heterogeneity. In isocitrate dehydrogenase (IDH) wild-type high-grade glioma (glioblastoma, GBM), increased intra-tumoral heterogeneity is associated with more aggressive disease. Recently, spatial technologies have emerged to dissect this complex heterogeneity within the tumor ecosystem by preserving cellular organization in situ. Here, we construct a high- resolution molecular landscape of GBM and IDH-mutant high-grade glioma patient samples to investigate the cellular subtypes and spatial communities that compose high-grade glioma using digital spatial profiling and spatial molecular imaging. This uncovered striking diversity of the tumor and immune microenvironment, that is embodied by the heterogeneity of the inferred copy- number alterations in the tumor. Reconstructing the tumor architecture revealed brain-intrinsic niches, composed of tumor cells reflecting brain cell types and microglia; and brain-extrinsic niches, populated by mesenchymal tumor cells and monocytes. We further reveal that cellular communication in these niches is underpinned by specific ligand-receptor pairs. This primary study reveals high levels of intra-tumoral heterogeneity in high-grade gliomas, associated with a diverse immune landscape within spatially localized regions.
Project description:Affymetrix GeneChip® Human Genome U133 Plus 2.0 Array profiling of 20 primary, multi-region high-grade glioma samples. Total RNA was extracted from primary high-grade glioma samples and hybridized to GeneChip® Human Genome U133 Plus 2.0 Array according to the manufacturer's instructions.
Project description:Single-cell RNA-Seq was conducted on 19 pediatric high-grade glioma patient samples to study tumor heterogeneity and tumor/immune cell interaction.
Project description:We perform bulk RNA sequencing, tumor/normal DNA sequencing, and spatial transcriptomics on an initial discovery cohort of eleven glioma samples (comprising oligodendrogliomas, astrocytomas, one diffuse midline glioma, and glioblastomas) to identify both commonalities and distinct characteristics within the tumor microenvironment. Six additional high-grade IDH wild-type, EGFR positive glioblastomas are analyzed to validate subclonal somatic aneuploidy and copy number alterations associated with extrachromosomal DNA double-minutes. As part of the standard spatial transcriptomic analysis, we develop an integrated analysis framework combining germline/tumor exomes, bulk RNA-seq, and spatial transcriptomics to identify loss of heterozygosity by a hidden Markov model for segmenting consecutively expressed SNPs. In the discovery cohort, we identify focally amplified ecDNA in four of the eleven cases, with subclonal tumor heterogeneity present in two EGFR-amplified grade IV glioblastomas. In a TP53-mutated glioblastoma, we detect a subclone with EGFR amplification on ecDNA coupled to chromosome 17 LOH. To evaluate the impact of spatial subclonal chromosomal alterations on the p53/EGFR axis, we examine six additional EGFR positive gliomas, identifying MDM2/MDM4 ecDNA subclones in two samples. The spatial DNA heterogeneity of EGFR and p53 inactivation underscores the role of ecDNA in enabling rapid oncogene amplification and enhancing tumor adaptability under selective pressure.
Project description:To reveal the spectrum of gene mutations in grade II/III gliomas, whole exome sequencing of 52 samples including 4 multi-regional and 10 multi-time points sampling cases and 291 SNP-array were performed. Our analysis revealed high degrees of temporal/spatial heterogeneity generated during tumor expansion and relapse.
Project description:Purpose: The most clearly established genetic hallmark in oligodendroglial tumors (OTs) is the combined loss of 1p/19q, a molecular alteration characteristic of tumors responding to therapy. Markers of tumoral progression have not yet been studied. Experimental Design: Novel markers of tumoral progression in OTs were sought through gene expression profiling analysis. Results: Unsupervised hierarchical cluster analysis classified OTs into two main groups associated with tumoral grade, independent of histological subtype and 1p/19q status. Differential gene expression analysis between low- and high-grade OTs revealed that only cell cycle-related genes were significantly upregulated in high-grade OTs. Among the deregulated genes, NDRG2 downregulation was detected in high-grade OTs with combined loss of 1p/19q. Expression analysis revealed low transcript levels of NDRG2 relative to non-tumoral brain tissue in 45% (9/20) of high-grade OTs. Furthermore, the low transcript levels of NDRG2 were significantly associated with a worse clinical outcome in patients. Transcript levels of NDRG2 were associated with promoter hypermethylation, which was detected in 38.4% (10/26) of high-grade OTs. The treatment of glioma cell lines T98 and LN18 with demethylating agents increased the mRNA expression levels of NDRG2 relative to the control cell line. Additionally, cell proliferation was significantly reduced and cell cycle was arrested in G1 phase after treatment with demethylating agents. Conclusions: Taken together, our results suggest that NDRG2 is a candidate tumor suppressor gene in OTs whose inactivation could be involved in tumoral progression and worse patient survival. The microarray study involved 28 glioma samples including oligodendrogliomas and oligoastrocytomas WHO grade II and III. Six non-tumoral brain tissues were used as controls, two of which were purchased from Stratagene (La Jolla, CA) and Clontech (Mountain View, CA). RNAs from several cell lines (Universal Human RNA, Stratagene, La Jolla, CA) was used as a standard reference in all hybridizations
