Plasticity of extrachromosomal DNA segregation during drug adaptation.
Uneven segregation during mitosis is a striking feature of extrachromosomal DNA (ecDNA). Because ecDNA lacks a centromere, it is thought to segregate stochastically, generating intratumoral heterogeneity in genomic copy number. Drug treatment can readily change ecDNA copy number, enabling cells to acquire drug resistance, yet whether these changes reflect static selection of pre-existing clones or active reconfiguration under stress remains unresolved. To address this, we develop a high-throughput framework combining single-cell DNA sequencing with cellular barcoding for clonal tracking. Single-cell cloning reveals that not all clones exhibit identical segregation modes even under drug-free conditions. Under treatment, resistant populations do not simply arise from pre-existing clones with favorable ecDNA states; instead, some clones actively reconfigure their segregation behavior to generate resistant cells. Thus, although ecDNA generally segregates stochastically, it can undergo nonrandom, actively regulated segregation under drug stress, raising the possibility of therapeutically targeting ecDNA segregation mechanisms to counteract adaptive resistance.