Single-cell RNA sequencing reveals the impact of chromosomal instability on glioblastoma cancer stem cells

Yanding Zhao,Charles Gawad,Duane A Compton,Kristina M Godek,Chao Cheng,Robert Carter,Sivaraman Natarajan,Frederick S Varn

doi:10.1186/s12920-019-0532-5

Abstract

BackgroundIntra-tumor heterogeneity stems from genetic, epigenetic, functional, and environmental differences among tumor cells. A major source of genetic heterogeneity comes from DNA sequence differences and/or whole chromosome and focal copy number variations (CNVs). Whole chromosome CNVs are caused by chromosomal instability (CIN) that is defined by a persistently high rate of chromosome mis-segregation. Accordingly, CIN causes constantly changing karyotypes that result in extensive cell-to-cell genetic heterogeneity. How the genetic heterogeneity caused by CIN influences gene expression in individual cells remains unknown.MethodsWe performed single-cell RNA sequencing on a chromosomally unstable glioblastoma cancer stem cell (CSC) line and a control normal, diploid neural stem cell (NSC) line to investigate the impact of CNV due to CIN on gene expression. From the gene expression data, we computationally inferred large-scale CNVs in single cells. Also, we performed copy number adjusted differential gene expression analysis between NSCs and glioblastoma CSCs to identify copy number dependent and independent differentially expressed genes.ResultsHere, we demonstrate that gene expression across large genomic regions scales proportionally to whole chromosome copy number in chromosomally unstable CSCs. Also, we show that the differential expression of most genes between normal NSCs and glioblastoma CSCs is largely accounted for by copy number alterations. However, we identify 269 genes whose differential expression in glioblastoma CSCs relative to normal NSCs is independent of copy number. Moreover, a gene signature derived from the subset of genes that are differential expressed independent of copy number in glioblastoma CSCs correlates with tumor grade and is prognostic for patient survival.ConclusionsThese results demonstrate that CIN is directly responsible for gene expression changes and contributes to both genetic and transcriptional heterogeneity among glioblastoma CSCs. These results also demonstrate that the expression of some genes is buffered against changes in copy number, thus preserving some consistency in gene expression levels from cell-to-cell despite the continuous change in karyotype driven by CIN. Importantly, a gene signature derived from the subset of genes whose expression is buffered against copy number alterations correlates with tumor grade and is prognostic for patient survival that could facilitate patient diagnosis and treatment.

Highlights

Intra-tumor heterogeneity stems from genetic, epigenetic, functional, and environmental differences among tumor cells
Neural stem cells and glioblastoma cancer stem cells have distinct transcriptomes To investigate the relationship between gene expression levels and copy number variation (CNV) in chromosomally unstable cells, we performed single-cell RNA sequencing of chromosomally unstable GliNS2 glioblastoma CSCs and control normal, diploid CB660 neural stem cell (NSC) that were grown in identical serum-free culture conditions [9, 19, 20]
We performed principal component analysis (PCA), and similar to the unsupervised hierarchical clustering, we found that CB660 NSCs and GliNS2 CSCs separated into two distinct groups (Fig. 1b)

Summary

Introduction

Intra-tumor heterogeneity stems from genetic, epigenetic, functional, and environmental differences among tumor cells. Whole chromosome CNVs are caused by chromosomal instability (CIN) that is defined by a persistently high rate of chromosome mis-segregation. Intra-tumor heterogeneity stems from diverse populations of cells co-existing within the same tumor that have genetic, epigenetic, functional, and environmental differences [1,2,3]. An additional source of intra-tumor heterogeneity is genetic heterogeneity resulting from DNA sequence variation and/or whole chromosome and focal copy number variations (CNVs). CIN is a persistent and a high rate of chromosome mis-segregation that causes random chromosome losses and/or gains [7]. Both CSCs and non-CSCs display CIN [9]. Aneuploidy and CIN generate genetic diversity among tumor cells that contributes to therapeutic resistance and is correlated with poor patient prognosis [10,11,12]

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