Cellular adaptation to cancer therapy along a resistance continuum [scRNA-seq: A375_PC9]

Advancements in precision oncology over the past decades have led to new therapeutic interventions, but the efficacy of such treatments is generally limited by an adaptive process that fosters drug resistance. In addition to genetic mutations, recent research has identified a role for non-genetic plasticity in transient drug tolerance and the acquisition of stable resistance. However, the dynamics of cell-state transitions that occur in the adaptation to cancer therapies remain unknown and require a systems-level longitudinal framework. Here we demonstrate that resistance develops through trajectories of cell-state transitions accompanied by a progressive increase in cell fitness, which we denote as the 'resistance continuum'. This cellular adaptation involves a stepwise assembly of gene expression programmes and epigenetically reinforced cell states underpinned by phenotypic plasticity, adaptation to stress and metabolic reprogramming. Our results support the notion that epithelial-to-mesenchymal transition or stemness programmes-often considered a proxy for phenotypic plasticity-enable adaptation, rather than a full resistance mechanism. Through systematic genetic perturbations, we identify the acquisition of metabolic dependencies, exposing vulnerabilities that can potentially be exploited therapeutically. The concept of the resistance continuum highlights the dynamic nature of cellular adaptation and calls for complementary therapies directed at the mechanisms underlying adaptive cell-state transitions. Drug-naïve A375 cells were treated in dose escalation from 30 nM to 4 uM of dabrafenib. Adapted cell populations (dabrafenib resistant) from the doses 30 nM, 60 nM, 125 nM, 250 nM, 500 nM, 1 uM, 2 uM and 4 uM (T003, T006, T0125, T0250, T0500, T1, T2 and T4) plus untreated control (C) were collected for single-cell sequencing using the 10X genomics platform. The same procedure was used to generate PC9 resistant cells to osimertinib. Drug-naïve PC9 cells were treated in dose escalation from 8 nM to 1.2 uM of osimertinib. Adapted cell populations (osimertinib resistant) from the doses 8 nM, 16 nM, 32 nM, 75 nM, 150 nM, 300 nM, 600 nM and 1.2 uM (T0008, T0016, T0032, T0075, T0150, T0300, T0600, T1.2) plus untreated control (C). An additional population of persister cells was generated by acute treatment with 1.2 uM (P1.2) for 9 days. Cells were collected for single-cell sequencing using the 10X genomics platform.