Single-cell Transcriptomics reveals multi-step adaptations to endocrine therapy

Resistant tumours are thought to arise from the action of Darwinian selection on intratumoral genetic heterogeneity. However, clonal selection is incompatible with the late recurrence often characterising luminal breast cancers treated with endocrine therapy (ET), suggesting a more complex interplay between genetic and non-genetic factors. In the present study, we dissect the contributions of clonal genetic diversity and transcriptional plasticity during the early and late phases of ET at single-cell resolution. Using single-cell RNA-sequencing and imaging we disentangle the transcriptional variability of plastic cells and define a rare sub-population of pre-adapted (PA) cells which undergoes further transcriptomic reprogramming and copy number changes to acquire full resistance. PA cells show reduced oestrogen receptor a activity but increased features of quiescence and migration. We find evidence for sub-clonal expression of this PA signature in primary tumours and for dominant expression in clustered circulating tumour cells. We propose a multi-step model for ET resistance development and advocate the use of stage-specific biomarkers. Overall design: Single-cell RNA-seq libraries from cell lines or FACS-sorted subpopulations, generated using 10x Chromium and subjected to high-throughput sequencing.