Spheroid culture remodels mitosis and the proteome in tumor cells

Mitosis depends on precise spindle assembly and positioning, processes influenced by cell shape, size, and microenvironment. Most mechanistic insights into mitosis come from two-dimensional (2D) monolayer cultures, which lack the spatial constraints and extracellular matrix found in tissues, leaving the influence of the tissue environment on mitosis poorly understood. Here, we combine high-resolution imaging and quantitative proteomics to compare mitosis in three-dimensional (3D) multicellular spheroids, generated by magnetic levitation, with that in 2D monolayers. Using a non-transformed cell line and three cancer cell lines from breast, bone, and ovary, we show that 3D culture reshapes mitotic cells and their spindles. Tumor spheroids exhibited a prometaphase delay together with minor chromosome alignment defects, yet chromosome segregation remained largely accurate. Cells in spheroids were rounder, and their spindles were smaller, with increased multipolarity and defects in orientation and position, which varied by cell line. Proteomic profiling revealed broad downregulation of mitotic regulators in spheroids, including kinesins (KIF11, KIF4A), spindle checkpoint proteins, and APC/C components, accompanied by enrichment of metabolic and mitochondrial pathways. Together, our results reveal both shared and cell line-specific modes of mitotic restructuring and establish a framework that connects proteome state to mitotic architecture in 3D environments.