Unraveling the Molecular Mechanisms Underlying Spontaneous Multipolar Mitosis Through CIN‐seq

Multipolar mitosis, a hallmark of chromosomal instability (CIN), drives tumor heterogeneity and therapy resistance, yet remains difficult to study in live cells due to its rare and dynamic nature. To address this, we developed CIN-seq, a targeted single-cell multiomics method that enables large-scale profiling of rare CIN phenotypes and captures their temporally regulated gene expression. Applying CIN-seq, we investigated viable, spontaneous multipolar mitosis, an abnormal division process that cancer cells can survive. Genomic analysis revealed that this mitosis produces polyploid or chromosomally variable progeny, confirming its role in genomic instability. Aneuploidy of Chromosome 16 was linked to increased tripolar mitosis, a finding validated with CRISPR imaging. Transcriptomic analysis showed activation of the Rho GTPase cycle, which was associated with cytokinesis failure, while PTEN attenuation emerged as a key player of viable multipolar mitosis by promoting cell cycle progression and survival via BCL2L1. We also uncovered a novel link between this phenotype and degranulation-like stress responses, which may contribute to cancer cell adaptation to chromosomal instability. Overall, CIN-seq offers a powerful approach for studying rare, live CIN events at single-cell resolution and reveals new mechanisms by which cancer cells adapt to chromosomal instability.