Mass Cytometry as a Tool for Investigating Senescence in Multiple Model Systems

Cellular senescence is a durable cell cycle arrest as a result of the finite proliferative capacity of cells. Senescence responds to both intrinsic and extrinsic cellular stresses, such as aging, mitochondrial dysfunction, irradiation, and chemotherapy. Here, we report on the use of mass cytometry (MC) to analyze multiple model systems and demonstrate MC as a platform for senescence analysis at the single-cell level. We demonstrate changes to p16 expression, cell cycling fraction, and histone tail modifications in several established senescent model systems and using isolated human T cells. In bone marrow mesenchymal stromal cells (BMSCs), we show increased p16 expression with subsequent passage as well as a reduction in cycling cells and open chromatin marks. In WI-38 cells, we demonstrate increased p16 expression with both culture-induced senescence and oxidative stress-induced senescence (OSIS). We also use Wanderlust, a trajectory analysis tool, to demonstrate how p16 expression changes with histone tail modifications and cell cycle proteins. Finally, we demonstrate that repetitive stimulation of human T cells with CD3/CD28 beads induces an exhausted phenotype with increased p16 expression. This p16-expressing population exhibited higher expression of exhaustion markers such as EOMES and TOX. This work demonstrates that MC is a useful platform for studying senescence at a single-cell protein level, and is capable of measuring multiple markers of senescence at once with high confidence, thereby improving our understanding of senescent pathways.