Characterizing Senescent Cell Heterogeneity by Surface Proteins: Single-Cell CITE-Seq

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ONGOING WORK AIM 1. To systematically identify cell surface proteins in multiple models of senescence. We have used an improved cell surface protein-labeling method designed for MS analysis and two models of cellular senescence: replicative senescence (RS) and ionizing radiation-induced senescence (IR-IS). Surface proteins were labeled by thiol-cleavable amine-reactive biotinylation, isolated by neutravidin agarose, and identified and quantified by MS analysis. We identified several proteins highly enriched on the surface of senescent cells RS (77) and IR-IS (144), while 58 were shared between the two comparisons. GO analysis of the 58 shared protein confirmed the enrichment of receptor and transmembrane proteins in the 58 shared proteins, including the highly enriched surface proteins CD44, CD54, and DPP4. These and other surface proteins will be targeted for Cite-Seq, senolytics and senomorphics. AIM 2. To use surface proteins in order to subclassify senescent cells using single cell-CITE-Seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing). Senescent cell heterogeneity represents a major challenge in targeting them efficiently by senotherapeutics. We and others have characterized the heterogeneity of the senescence transcriptome depending on the origin of the cell and the type of stress (Hernandez-Segura et al., 2017 and Casella et al., 2019). However, identifying shared surface proteins presents an excellent opportunity to subclassify senescent cells. We will use surface proteins from AIM 1 to identify senescent cell subtypes. Our preliminary analysis indicates that CD26 and CD106 mRNAs are highly expressed in bulk and single senescent cells compared to proliferating cells. Thus, we explored if these two surface proteins were highly expressed in distinct sub-populations of senescent cells. Interestingly, CD26- and CD106-positive cells may cluster in specific sub-populations of senescent cells. FUTURE PLANS: 1. To validate and functionally analyze senescence-associated surface proteins. 2. To use surface proteins to identify sub-populations of senescent cells. 3. To target surface proteins for senotherapeutics.