Phenotyping clonal populations of glioma stem cell reveals a high degree of plasticity in response to changes of microenvironment

James A Innes,Andrew S Lowe,Raquel Fonseca,Natasha Aley,Tedani El-Hassan,Myrianni Constantinou,Joanne Lau,Ayad Eddaoudi,Silvia Marino,Sebastian Brandner

doi:10.1038/s41374-021-00695-2

Abstract

AbstractThe phenotype of glioma-initiating cells (GIC) is modulated by cell-intrinsic and cell-extrinsic factors. Phenotypic heterogeneity and plasticity of GIC is an important limitation to therapeutic approaches targeting cancer stem cells. Plasticity also presents a challenge to the identification, isolation, and propagation of purified cancer stem cells. Here we use a barcode labelling approach of GIC to generate clonal populations over a number of passages, in combination with phenotyping using the established stem cell markers CD133, CD15, CD44, and A2B5. Using two cell lines derived from isocitrate dehydrogenase (IDH)-wildtype glioblastoma, we identify a remarkable heterogeneity of the phenotypes between the cell lines. During passaging, clonal expansion manifests as the emergence of a limited number of barcoded clones and a decrease in the overall number of clones. Dual-labelled GIC are capable of forming traceable clonal populations which emerge after as few as two passages from mixed cultures and through analyses of similarity of relative proportions of 16 surface markers we were able to pinpoint the fate of such populations. By generating tumour organoids we observed a remarkable persistence of dominant clones but also a significant plasticity of stemness marker expression. Our study presents an experimental approach to simultaneously barcode and phenotype glioma-initiating cells to assess their functional properties, for example to screen newly established GIC for tumour-specific therapeutic vulnerabilities.The authors barcoded glioma-initiating cells (GIC) using combinations of virally encoded fluorophores. GIC show a strong tendency to form clonal populations over as few as two passages. Combined with stem cell marker phenotyping and computational analysis, they could trace the fate of such populations. This model presents an approach for rapid assessment of newly established GIC to assess tumour-specific vulnerabilities.

Highlights

Glioblastoma-initiating cells (GIC) can recreate aspects of inter and intra-tumour cancer stem cell (CSC) heterogeneity
Two cell lines derived from isocitrate dehydrogenase (IDH)- CD44 expression dominant but variably associated with A2B5 wildtype glioblastomas, designated G19 and G61, were used
The inherent plasticity of CSCs in adapting to these environmental cues renders them a diverse and dynamic target for therapeutic intervention[24], i.e., there are potential escape mechanisms from therapies that aim at populations of a certain differentiation state

Summary

Introduction

Glioblastoma-initiating cells (GIC) can recreate aspects of inter and intra-tumour cancer stem cell (CSC) heterogeneity. Advances in the understanding of phenotypic heterogeneity have challenged the definition of CSC differentiation as a unidirectional and irreversible hierarchy[3] Such plasticity is key to the understanding of pathogenesis of tumours, where phenotypic shift can occur during initiation, progression, and selection of clones during development of therapy resistance[4]. The identification and isolation of CSC populations to a high degree of purity using combinations of CSC markers remains a significant challenge due to their plasticity, and there are no established combinations of markers that can demonstrate a high degree of “CSC purity”[6] These difficulties could explain the variability between patient samples or the intrinsic adaptability of stem cells as a response to variations in experimental conditions

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Results

Conclusion