RNA-Seq sheds light on the lineage relationships between glioblastoma stem-like cells and other glioma cell types

Tumor-propagating glioblastoma (GBM) stem-like cells (GSCs) of the proneural and mesenchymal molecular subtypes have been described. However, it is unknown if these two GSC populations are sufficient to generate the spectrum of cellular heterogeneity observed in GBM. The lineage relationships and niche interactions of GSCs have not been fully elucidated.

UCSF researchers performed single-cell RNA-sequencing (scRNA-seq) and matched exome sequencing of human GBMs (12 patients; >37,000 cells) to identify recurrent hierarchies of GSCs and their progeny. They mapped sequenced cells to tumor-anatomical structures and identify microenvironment interactions using reference atlases and quantitative immunohistochemistry. They found that all GSCs can be described by a single axis of variation, ranging from proneural to mesenchymal. Increasing mesenchymal GSC (mGSC) content, but not proneural GSC (pGSC) content, correlates with significantly inferior survival. All clonal expressed mutations are found in the GSC populations, with a greater representation of mutations found in mGSCs. While pGSCs upregulate markers of cell-cycle progression, mGSCs are largely quiescent and overexpress cytokines mediating the chemotaxis of myeloid-derived suppressor cells. The researchers found mGSCs enriched in hypoxic regions while pGSCs are enriched in the tumor’s invasive edge. They show that varying proportions of mGSCs, pGSCs, their progeny and stromal/immune cells are sufficient to explain the genetic and phenotypic heterogeneity observed in GBM. This study sheds light on a long-standing debate regarding the lineage relationships between GSCs and other glioma cell types.

Single-cell sequencing reveals a single axis of variation in GBM cellular phenotypes

rna-seq

(A) A t-SNE plot of 32,151 10X cells from 6 patients. Cells are colored by the presence (red) or absence (black) of clonal CNVs. (B) (Top-left) Expression of GBM-enriched genes in IDH1-wildtype human GBMs from TCGA (n=144) and non-malignant human brain from Gtex (n=200). (Top-right) Average expression (+-SEM) of GBM marker-genes in cells classified as neoplastic (black) and non-neoplastic (grey). (Bottom) Heatmap of the 50 most specific genes (Wilcoxon rank-sum test) in clusters of non-neoplastic  cells.  (C)  (Top)  PCA  of  25,899  10X  neoplastic  cells.  Density  curves  of  a  Gaussian  mixture  model  fit  to  PC1  sample  scores  are  in  gray. (Bottom) Distributions of cells from each patient along PC1. (D) Differentially expressed genes between PCA clusters (abs. log2 fold-change>1 and adj. p  <0.001  in  red).  (E)  Fractions  of cycling cells,  ***  :  Fisher  p<0.001.  (F-G) Expression  of top-loading genes from PC1  (F)  and PC2  (G)  in  single cells. Cells are sorted by PC1/2 sample score resp.

Muller S, Di Lullo E, Bhaduri A, Alvarado B, Yagnik G, Kohanbash G, Aghi M, Diaz A. (2018) A draft single-cell atlas of human glioblastoma reveals a single axis of phenotype in tumor-propagating cells. bioRXiv [Epub ahead of print]. [abstract]

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