Single-cell analysis of subclonal heterogeneity in primary glioblastoma
Laura Richards1,2, Troy Ketela2, Neil Winegarden2, Fiona Couthino3, Michelle Kushida3, Peter Dirks3, Trevor J. Pugh1,2
1Department of Medical Biophysics, University of Toronto, Toronto, ON; 2Princess Margaret Cancer Centre, University Health Network, Toronto, ON; 3The Hospital for Sick Children, Toronto, ON
Glioblastomas (GBMs) exhibit high intratumoural heterogeneity and have been shown to contain a fraction of cells, termed brain tumour stem cells (BTSCs) that drive tumour growth and disease relapse. BTSCs can be isolated and expanded in culture from primary GBMs and retain self-renewal and proliferative capacity in vitro. However, the functional diversity within patient-derived BTSC cultures, as well as their relationship to the BTSC fraction in primary tumour tissues remains unknown.
As a pilot experiment, we profiled the transcriptomes of 1400 cells from two patient-derived BTSC cultures using a droplet-based single-cell RNA-seq technology (Drop-seq) that exploits 3’ end counting to assess gene expression. Dimensionality reduction (PCA, t-SNE) in combination with graph-based clustering identified 5 transcriptionally distinct clusters of cells across the two cultures. The cultures segregated independently, with the exception of a shared subpopulation of cells enriched for mitotic and cell-cycle DNA replication expression pathways. While this population may represent common cycling cells, we are investigating their role as a proliferative stem population. Other clusters not shared across the lines are enriched for glial cell development, kinetochore regulation and translational membrane targeting, highlighting the transcriptional diversity observed within the cultures.
To extend these findings, we are now profiling 6000 cells each from 7 matched bulk tumour and patient-derived BTSC cultures using the 10X Genomics Chromium platform. We expect these data will uncover the extent to which BTSC cultures functionally recapitulate the stem fraction in bulk primary tumours, as well as define networks of self-renewal, therapeutic resistance and targetable vulnerabilities.