Poster T10 - Defining the cellular behaviors that can predict multinucleation

Introduction: Multinucleated cells, which contain multiple nuclei within a shared cytoplasm, are particularly significant in cancer as they display resistance to chemotherapy and can produce aneuploid progeny. Multinucleation can result from failure of abscission, the final step of cytokinesis that is essential for cleaving the midbody and segregating the cytoplasm and organelles into two daughter cells. Features specific to the tumor microenvironment, including transforming growth factor beta (TGFβ), have been found to promote abscission failure. TGFβ activates the expression of Snail-family transcription factors and induces epithelial-mesenchymal transition. We previously found that in the presence of TGFβ, mammary epithelial cells cultured on stiff microenvironments overexpress the filament-forming protein septin6 and the midbody proteins anillin and Mklp1, which result in an increase in midbody persistence, abscission failure, and multinucleation. This study aims to define and uncover the phenotypic anomalies that precede cytokinesis failure, and that might be used to predict multinucleation and aneuploidy before they arise.

Materials and

Methods: Here, we used quantitative timelapse imaging analysis of MCF10A human mammary epithelial cells treated with and without TGFβ to compare the spatiotemporal dynamics of mother cells that give rise to multinucleated progeny with those that give rise to mononucleated progeny. We monitored several phenotypic parameters, including the duration of the cell cycle, nuclear morphology, cell motility, and metabolism. MCF10A cells were transfected with a FUCCI plasmid to distinguish between the different stages of the cell cycle; stained with a live nuclear dye to measure nuclear morphology and cell motility; and incubated with Mitoview 633 to measure mitochondrial membrane potential. Oxygen consumption and extracellular acidification rates were also measured using an Agilent Seahorse as a readout of cellular metabolism.

Results, Conclusions, and Discussions: We found that mothers of multinucleated progeny demonstrated irregular cell-cycle durations compared to those that gave rise to mononucleated progeny. Specifically, the former exhibited extended durations of both G1 and M phases. In addition, mothers of multinucleated progeny displayed nuclear pleomorphism with higher variability in their nuclear size. Although there were no differences in the mean values of nuclear areas and shape parameters between the groups, we observed significant differences in the variances of the transition rate, which is defined as the frequency at which a cell undergoes substantial changes in its nuclear area. Moreover, tracking cell trajectories over time showed that mothers of multinucleated progeny mainly exhibited persistent motion, suggesting enhanced directional movement. Finally, we found that treating cells with TGFβ decreased mitochondrial ATP production rates. Taken together, our data show higher variabilities in phenotypic parameters in mothers of multinucleated progeny, which suggests imprecise control over the molecular mechanisms that control homeostasis. These findings suggest the existence of a common pathway that controls aspects of cell-cycle duration, nuclear morphology, cell motility, metabolism, and cytokinesis. Given that TGFβ induces elevated expression of septin6, a protein integral to cytoskeletal interactions, we are currently investigating the role of septin6 in stabilizing cell and nuclear shape and regulating cell motility. This approach will enhance our understanding of how the tumor microenvironment disrupts these complex cellular processes and may identify novel targets for therapeutic intervention in cancer.

Acknowledgements (Optional): Work from the authors’ group was supported in part by grants from the National Institutes of Health (HL164861, HD099030, CA214292, HD111539), the National Science Foundation (2134935, and the Center for the Physics of Biological Function PHY-1734030), the National Cancer Institute (through Rutgers Cancer Institute of New Jersey 1T32CA257957), and a Faculty Scholars Award from the Howard Hughes Medical Institute.