Tracking genome evolution in single cell clones reveals the rates and features of copy number alterations generated by ongoing chromosomal instability in cancer

Cancer genomes exhibit extensive chromosomal alterations caused by ongoing Chromosomal Instability (CIN). The ensuing cell-cell heterogeneity facilitates evolution and cancer cell plasticity that can drive therapy resistance, yet cancer CIN driver mechanisms remain essentially uncharacterised. This lack of knowledge presents an untapped opportunity to target vulnerabilities associated with ongoing CIN for therapy. Existing methods to investigate the cellular mechanisms responsible for CIN rely on laborious functional assays, or inference from genomic alteration patterns from sequencing data. Current bulk sequencing derived copy number alteration pattern signatures lack the cell-cell resolution that would reveal recent genomic alterations caused by CIN. Large-scale single cell sequencing of cancer cell populations is now emerging. However, it is not known whether the effects of selection still obscure the spectrum of genomic alterations caused by recent CIN. To address this, we employed a single-cell whole-genome sequencing (scWGS) clonal outgrowth technique, that allows us to track the real-time evolution of cancer genomes at the single-cell level. Single cancer cells surprisingly re-establish heterogeneity that matches their parental population within ∼22 generations. By comparing the features of copy number alterations at different evolutionary timepoints we reveal that some alteration types are likely under negative selection and are thus only apparent in the most recent cell divisions, and not in the parental population. In one cell line we identify a particular chromosome subject to recurrent chromosomal deletions, and validated that this chromosome wasinvolved frequently in mis-segregation events during anaphase using fluorescenceIn-Situhybridisation.