Persister cells arise upon chronic treatment and constitute the origin of resistant cells. Understanding the biology and heterogeneity of persister cells is essential for the design of future treatment approaches. However, the study of persister cells has been strongly limited by our capacity to analyse small numbers of cells ex-vivo.

PC9 drug-tolerant/persister (DTP) cells were characterised at single cell resolution in vivo. To this end, PC9 cells were implanted in the flank of female mice. Once tumours acquired the desired volume, mice were orally administered osimertinib daily at a dose of 25 mg/kg for 14 days. Vehicle-treated samples were used as a reference. Tumours were then dissociated and processed for single-cell RNA-seq using the 10X Genomics reagents or for single-cell protein analysis using CyTOF.

Our results showed that the resulting osimertinib DTP population is transcriptionally heterogenous. The comparison of the different transcriptional states identified a putative target which was confirmed at protein level using CyTOF. Further in vivo studies in PC9 cells using an antibody-drug conjugate (ADCs) against the identified putative target in combination with osimertinib resulted in ablation of a specific subpopulation of DTPs.

Our single-cell RNA-seq analysis of osimertinib-treated tumours highlighted that it is essential to investigate therapeutic response at single-cell resolution. The understanding of cellular heterogeneity is key to better understand combinatorial treatments. In our preclinical model, a specific subpopulation of DTPs was eliminated by combinatorial treatment which resulted in reduction in the heterogeneity of the resulting DTP population and ultimately in delayed re-growth of tumours.