P15 - Fusion-Driven Shifts in Ploidy and CNV Landscapes in Triple-Negative Breast Cancer

Introduction: : Intratumor heterogeneity is characterized by cancer cell populations with diverse characteristics and variable responses to disease treatments. This is a driving force for the evolution of tumors, and impacts therapeutic response, adaptation, and resistance in many tumor types. Aneuploidy is one mechanism that contributes to cancer cell heterogeneity. Nearly 90% of solid tumors display alterations in ploidy as a result of mechanisms such as cell fusion or improper cell division including endoreplication, endomitosis, and failed cytokinesis. Cell fusion and resulting aneuploidy have been observed in many cancers including triple negative breast cancer (TNBC); however, a more comprehensive investigation into the relationship between fusion cells, ploidy, and the altered properties the fusion state may confer remains to be seen. Utilizing nuclear localized fluorescent populations, this study aimed to determine whether spontaneous cell fusion in TNBC leads to distinct genomic and phenotypic characteristics, including altered ploidy and cell responses that enhance adaptability or resistance to chemotherapeutics.

Materials and

Methods: : GFP- and mCherry-labeled H2B was stably expressed in the HCC1806 TNBC cell line via lentiviral transduction, enabling nuclear-localized fluorescent tracking. These populations were co-cultured for one week under standard culture conditions, allowing spontaneous fusion events to occur. Single-cell fusion and control clones were isolated using fluorescence-activated cell sorting (FACS) from multiple replicate co-cultures (Figure 1a). Clonal characterization included assessments of ploidy, growth rate, morphology, chemotherapeutic dose response, and copy number variation (CNV). Ploidy was analyzed through metaphase spread preparation and karyotyping. Growth rate and drug response were assessed via longitudinal imaging using an IncuCyte microscope, with and without chemotherapeutic perturbation. DNA was extracted for 1x whole-genome sequencing, and CNV profiles of fusion and control clones were analyzed.

Results, Conclusions, and Discussions:: Fusion clones exhibited significantly higher ploidy than controls, with an approximate doubling of chromosomal content. Figure 1b depicts the average ploidy of fusion clones which were around 4n while control clones remained around 2n (p= 5E-24). Additionally, ploidy analysis was repeated after culturing clones for approximately five weeks under standard culture conditions and patterns of increased ploidy persisted. Growth rates were significantly elevated and more variable among fusion clones. Morphologically, fusion clones displayed larger nuclear and cytoplasmic areas, but lower nuclear-to-cytoplasmic ratios—differences that were all statistically significant. Drug response profiling revealed increased variability in IC50 values among fusion clones across three chemotherapeutics commonly used in TNBC, with a trend toward higher resistance. CNV analysis identified multiple chromosomal regions with statistically significant copy number alterations distinguishing fusion from control clones (Figure 1c). Overrepresentation analysis was conducted on the sets of genes which were significantly different between fusion and control clones using g:Profiler. Notably, several gene sets related to DNA transcription were more prevalently expressed in fusion clones. Additionally, creating a heatmap of CNV profiles for fusion and control clones allowed for direct comparison between the two groups (Figure 1d).
These findings suggest that cell-cell fusion in TNBC contributes to both genomic and phenotypic heterogeneity. The observed differences in growth dynamics, morphology, CNV, and drug response support a model in which fusion promotes clonal diversification and potentially facilitates adaptation or resistance. Additionally, distinct morphological traits may serve as visual indicators of fusion within cellular populations.
This work advances our understanding of fusion-induced aneuploidy in TNBC and highlights its possible role in tumor evolution. Future studies will explore fusion dynamics within parental populations, examine the relationship between fusion and chemotherapeutic adaptation, and assess these phenomena in other cell models.

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