p53 activity links chromosomal instability to pro-migratory extracellular vesicles

Abstract Chromosomal instability (CIN) promotes tumor evolution and metastasis while imposing cellular stress that can limit cell proliferation via activation of p53 and its downstream effector p21. However, how cells recover from this p53-mediated proliferation block while adopting a more malignant fate remains poorly understood. Here, using a p21–RFP reporter in p53-proficient NIH/3T3 cells, we show that acute CIN gives rise to two phenotypically distinct p21 cell populations with divergent recovery trajectories. p21-RFP LOW cells re-enter the cell cycle following a short cell-cycle arrest, while p21-RFP HIGH cells display a more persistent arrest that coincides with a senescence-like state. Longitudinal transcriptomic and extracellular vesicle (EV) proteomic analyses reveal that extracellular-matrix (ECM)- and adhesion-associated programs are initially suppressed following CIN but specifically reactivated in p21-RFP HIGH cells. The content of late-stage p21-RFP HIGH -derived EVs is enriched for ECM-, adhesion-, and cytoskeleton-associated proteins compared with that of p21-RFP LOW -derived EVs. Consequently, EVs derived from p21-RFP HIGH cells confer increased migration. Together, these findings reveal that CIN can yield distinct p21-expressing cell populations that display different cell-cycle recovery trajectories, EV makeup, and migration-associated paracrine activity.