BPS2026 – Homotypic fusion alters biophysical properties and cytoskeleton organization in TNBC cells

Cell-cell fusion, essential for processes such as fertilization and viral infection, enables mixing of cytoplasmic and nuclear contents. In cancer studies, fusion events involving cancerous and stromal or immune cells, can lead to epithelial-mesenchymal transition (EMT), enhanced migration, and drug resistance. Although heterotypic cancer cell fusion has been widely studied, the consequences of homotypic cancer cell fusion remain poorly understood. We investigated migratory and biophysical features of the fusion population generated from triple-negative breast cancer (TNBC) cells. Fusion cells were identified and isolated using the ClonMapper duo system (CMDuo) through reporter expression and fluorescence-associated index sequences paired with randomly generated nucleotide barcodes. In the wound-healing assay, fusion cells closed the gap area nearly five times more efficiently than control cells over 24 h. Gene set enrichment analysis demonstrated changes in cytoskeleton-related pathways involving actin and microtubules. Consistent with these transcriptomic signatures, immunofluorescence staining revealed altered cytoskeletal organization, with fusion cells displaying reduced alignment of the microtubule network. Morphological analysis showed that fusion cells were significantly larger and contained enlarged nuclei. They also had an increased cytoplasm-to-nucleus ratio and a more circular morphology relative to non-fused cells. Analysis of actin-rich protrusions indicated markedly longer filopodia in fusion cells, suggesting enhanced extracellular sensing and invasive potential. Together, these findings establish that homotypic fusion generates TNBC progeny with a distinct biophysical phenotype characterized by increased migratory ability and altered cytoskeletal organization.