Abstract 232: Qualitative Single-cell Assessment By Imaging Mass Cytometry (imc) Analysis Reveals Senescence-associated Stemness (sas) Induced By Erk5 S496 Phosphorylation In Atherosclerotic Plaque

The central role of ERK5 S496 phosphorylation in promoting senescence has been reported. Recently the conventional model of senescence as an event of cell cycle arrest has been challenged, given that senescence induced by various stresses reprograms cancer cells to acquire senescence-associated stemness (SAS), which allows them to escape senescence-induced cell cycle arrest with enhanced growth potential. To determine the role of ERK5 S496 phosphorylation in SAS, we utilized imaging mass cytometry (IMC) by using antibodies against various cell surface markers, senescence, and proliferation. We prepared tissue sections from the plaques in wild-type (WT) and ERK5 S496A knock-in (KI) mice, and the ablated plaque materials were evaluated by the CyTOF mass cytometer. We used the VISIOPHARM program, and single-cell features were computationally segmented using a watershed algorithm. Nine clusters were detected in WT (total 7903 cells) and ERK5 S496A KI (total 6166 cells) plaques. Single-cell analysis of p53 and Ki67 revealed three groups of the cells with different pattern of p53 and Ki67 expression (Fig.A). We calculated log scaled ratio of Ki67 and p53 of selected cells by log 10 (expression of Ki67/ expression of p53), and further calculated one-dimension density of this log scaled ratio to cluster cells into three groups by using cutoff of ration with log 10 (-1.01) and log 10 (-0.024) (Fig.B). Group 2 myeloid cells showed that p53 and Ki67 were co-expressed lineally, suggesting that this group of cells escaped from the growth-suppression effects of p53, which is also known as a senescence marker. Of note, the % of ERK5 S496A KI cells in group 2 were significantly lower than those in WT (Fig.C). Also, in vitro oxidized LDL remarkably increase β-gal + Ki67 + cells, which was significantly inhibited in ERK5 S496A KI macrophage compared to those in WT. Collectively, these data implicates ERK5 S496 phosphorylation in a unique role-regulation of SAS during atherogenesis.