Atypical fusion among non-malignant cells: Unveiling the link between post-fusion-induced genome chaos and cancer initiation.

backgroundAtypical cell fusion events can disrupt the normal post-fusion process, initiating genome chaos through the merging of parental genomes accompanied by severe and complex gross structural changes. Consequently, unscheduled fusion events can accelerate tumorigenesis by fostering persistent genomic instability, serving as catalysts in the post-hybrid selection process and decisively contributing to the acquisition of novel and malignant properties. Understanding how hybrid cells survive after fusion and identifying specific transcriptional and phenotypic signatures that characterize hybrids across different fusion phases may provide valuable insights to counteract the evolution of cancer-initiating hybrids.

methodsTwo distinct populations of H2B-fluorescence-labeled M13SV1 breast epithelial cells were cocultured to yield H2B double-fluorescence-tagged hybrid clones derived from spontaneous cell fusion. Hybrid clones were picked and propagated individually to establish early- and late-passage hybrid populations. Hybrid clone and passage-dependent cell fate studies were conducted via live-cell imaging and high-resolution 3D imaging to capture aberrant mitotic outcomes. Hybrids were evaluated for ploidy status, cell cycle DNA damage, cell/nuclear morphology and transcriptomic profiling using a set of distinct methods, including flow cytometry (ploidy/DNA damage analysis), diverse cell metrics and image processing software (cell/nuclear scoring) and RNA sequencing (transcriptome profiling). Subsequently, EMT marker expression, migration/invasion, and colony- and sphere-forming capacities were investigated.

resultsNear-tetraploid hybrids undergo a hybrid clone-dependent ploidy reduction process upon long-term passaging, which is associated with cell shrinkage. Ploidy reduction was not coupled with substantial loss of DNA lesions but rather correlated with the degree of nuclear deformation. Hybrids were extremely chromosomally unstable, as evidenced by a high incidence of aberrant mitoses, nuclear pleomorphism, and elevated scores of micro- and multinucleation. Additionally, a subset of hybrids underwent ploidy reversal, characterized by endoreplication, cytokinesis failure or refusion, leading to the formation of exceptionally large or multinucleated cells. Transcriptome analysis revealed a differentially expressed gene profile of the hybrids associated with EMT, migratory aptitude and cancer-related traits, as validated by migration/invasion assays, Western Blots and colony/mammosphere assays.

conclusionCollectively, the data highlight the catastrophic potential of atypical fusion processes, wherein widespread and large-scale genomic changes can occur at a rapid macroevolutionary pace and potentiate the risk for subclonal heterogeneity and malignancy.