Cell Ploidy Modulates Nucleolar Size via Dosage of Nucleolar Organizing Regions

Abstract Abnormal nucleolar morphology is a pathognomonic histologic feature of cancer cells. Nucleoli are biomolecular condensates and cytogenetic entities that form around nucleolar organizing regions (NORs), located on the short arms of acrocentric chromosomes. Given that aneuploidy is pervasive in human cancers and can involve NOR-bearing chromosomes, we investigated how aneuploidy-driven changes in NOR dosage impact nucleolar morphology. We used reversine, a spindle assembly checkpoint inhibitor, to induce chromosomal instability and complex aneuploidy in DLD1 colorectal cancer cells (near-euploid) and normal human colon organoids (colonoids, euploid). High resolution 3D imaging and quantitative analyses of reversine-treated cells showed overall doubling of total nucleolar volume per nucleus. Reversine-associated nucleolar enlargement occurred independently of global protein synthesis, and thus was not solely driven by increases in biosynthetic demand. The volume of individual nucleoli showed a direct linear correlation with the number of associated NORs, as revealed by 3D co-localization with NOR-specific FISH probes. These findings establish NOR dosage as a determinant of nucleolar morphology and metric of genomic instability, providing a new paradigm by which aneuploidy can modulate spatial chromosomal constraints and nuclear architecture in cancer. Significance statement Cancer cells often show enlarged and abnormal nucleoli. We show that aneuploidy, a common feature of cancer, can directly reshape nucleolar morphology by altering the copy number of acrocentric chromosomal regions around which nucleoli form, NORs. Using high-resolution 3D imaging in cell lines and patient-derived colonoids, we find that nucleolar number and volume directly correlate with NOR number, independent of overall protein synthesis. Individual nucleolar volume scales with the number of 3D NOR associations, and prominent single nucleoli arise when NORs accumulate. These results provide evidence that aneuploidy can lead to aberrant nucleolar morphologies seen in many human cancers.