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P300-mediated adaptive conversion of glioma stem cells to vascular-like cells in response to therapeutic stress promotes tumor recurrence [RNA-seq_sorted GEC GPC]

ABSTRACT: Purpose: The goal of the study is to determine the transcriptional changes associated with non-radiated cells, radiated cells, and sorted non-radiated, and radiation-induced endothelial and pericyte-like cells to establish the identity of the vascular-like cells

ORGANISM(S): Homo sapiens

PROVIDER: GSE207729 | GEO | 2022/09/09

REPOSITORIES: GEO

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Project description:Purpose: The goal of the study is to determine the phenotypic and functional states of glioma cells induce in response to a standard glioblastoma therapy, ionizing radiation. Methods: We performed single-cell RNA-sequencing on a patient-derived gliomasphere line isolated from a primary glioblastoma tumor. Cells were exposed to a single dose of radiation ( 8 Gy) for 2- and 7-days to elaborate their gene expression profile. Single cell suspensions and cDNA library preparation was done using 10x Genomics Chromium single cell reagent kit v3 according to manufacturer's protocol. cDNA samples were sequenced on 1 lane of NovaSeq 6000 S2 flowcell. Reads were mapped to HumanGRCh38 genome using Cell Ranger v.3.0.2. More than 600 million reads were obtained for each sample. Average number of genes detected was 5875 (SE±357). Confident read mapping rates were 81.2-87.4% with over 86.8% of reads in cells. Seurat package v3.1.1 was used for integrated analysis by Canonical correlation analysis method with 75 dimensions . PCA was used for dimensional reduction, and Cell clustering was performed using shared nearest neighbor method. Each cell cluster was annotated by a combination of the following methods 1) canonical marker expression 2) Gene Set Enrichment Analysis (GSEA) of cluster specific markers determined by Find Markers function in Seurat and 3) Co-expression modules obtained by Louvain community detection clustering method. Trajectory analysis was done with monocle 3 (version 0.2.1.3) by converting Seurat object to monocle. Results: We identified 14 distinct clusters in all samples, and found dynamic alterations in frequency and size of clusters between non-radiated and radiated cells. Louvian community detection and co-expressed gene module analysis revealed that clusters expressing mitotic and DNA repair modules were diminshed, whereas clusters expressing stemness, mesenchymal and vasculogenesis modules increased post-radiation. Conclusion: Radiation therapy induces diverse functional states in glioma cells.

Project description:Pericytes have been implicated in regulation of inflammatory, reparative, fibrogenic and angiogenic responses in several different organs and pathologic conditions. Although the adult mammalian heart contains abundant pericytes, their fate and involvement in myocardial disease remains unknown. We used NG2Dsred;PDGFRaEGFP pericyte-fibroblast dual reporter mice and inducible NG2CreER mice to study the fate and phenotypic modulation of pericytes in a model of myocardial infarction. The transcriptomic profile of pericyte-derived fibroblasts was studied using PCR arrays. The transcriptomic profile of NG2 lineage cells (pericytes) was studied in control and infarcted hearts using single cell RNA-sequencing analysis. The role of TGF-b signaling in regulation of pericyte phenotype in vivo was investigated using pericyte-specific Tgfbr2 knockout mice. In vitro, the effects of TGF-b were studied in cultured human placental pericytes.In normal mouse hearts, NG2 and PDGFRa identified distinct non-overlapping populations of pericytes and fibroblasts respectively. Following myocardial infarction, a population of cells expressing both pericyte and fibroblast markers emerged. These cells expressed large amounts of extracellular matrix (ECM) genes. Lineage tracing demonstrated that in the infarcted region, a subpopulation of pericytes underwent fibroblast conversion. Single cell RNA-seq experiments demonstrated expansion and diversification of pericyte-derived cells in the infarct, associated with emergence of subpopulations exhibiting accentuated matrix gene synthesis. In vitro studies and the profile of pericyte-derived fibroblasts identified TGF-b as a potentially causative mediator in fibrogenic activation of infarct pericytes. However, pericyte-specific Tgfbr2 disruption had no significant effects on myofibroblast infiltration and collagen deposition in the infarct. Pericyte-specific TGF-b signaling was involved in vascular maturation, mediating formation of a mural cell coat investing infarct neovessels. These reparative effects of infarct pericytes protected the infarcted heart from dilative remodeling.

Project description:Background: Pericytes regulate vessel stabilization and function and their loss is associated with diseases such as diabetic retinopathy or cancer. Despite their physiological importance, pericyte function and molecular regulation during angiogenesis remain poorly understood. Methods: To decipher the transcriptomic programs of pericytes during angiogenesis, we crossed the Pdgfrb(BAC)-CreERT2 into the RiboTagflox/flox mice. Pericyte morphological changes were assessed in mural cell-specific R26-mTmG reporter mice, in which low doses of tamoxifen allowed labeling of single cell pericytes at high resolution. To study the role of phosphoinositide 3-kinase (PI3K) signaling in pericyte biology during angiogenesis, we used genetic mouse models which allow selective inactivation of PI3Kα and PI3Kβ isoforms and their negative regulator PTEN (phosphate and tensin homologue deleted on chromosome ten, PTEN) in mural cells. Results: At the onset of angiogenesis, pericytes exhibit molecular traits of cell proliferation and activated PI3K signaling, whereas during vascular remodeling pericytes upregulate genes involved in mature pericyte cell function, together with a remarkable decrease in PI3K signaling. Immature pericytes showed stellate shape and high proliferation, and mature pericytes were quiescent and elongated. Unexpectedly, we demonstrate that the PI3Kβ, but not PI3Kα, regulates pericyte proliferation and maturation during vessel formation. Genetic PI3Kβ inactivation in pericytes triggered early pericyte maturation. Conversely, unleashing PI3K signaling by means of PTEN deletion delayed pericyte maturation. Pericyte maturation was necessary to undergo vessel remodeling during angiogenesis. Conclusions: Our results identify new molecular and morphological traits associated to pericyte maturation and uncover PI3Kβ activity as a checkpoint to ensure appropriate vessel formation. In turn, our results may open new therapeutic opportunities to regulate angiogenesis in pathological processes through the manipulation of pericyte PI3Kβ activity.

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P300-mediated adaptive conversion of glioma stem cells to vascular-like cells in response to therapeutic stress promotes tumor recurrence­ [RNA-seq_sorted GEC GPC]

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P300-mediated adaptive conversion of glioma stem cells to vascular-like cells in response to therapeutic stress promotes tumor recurrence [RNA-seq_sorted GEC GPC]