Introduction: Cellular senescence is a stress response that causes an irreversible arrest of cell proliferation and development of a senescence-associated secretory phenotype. Pre-clinical mouse studies have demonstrated that targeted removal of senescent cells mitigates a variety of aging phenotypes. By current methods, senescence must be induced in culture to examine the characteristics of senescent human cells. However, a comprehensive understanding of the phenotype(s) of these cells in humans is lacking because they cannot, thus far, be isolated from tissues. Therefore, senescent cell-surface markers are needed to identify, isolate, characterize and ultimately target senescent cells for therapeutic interventions.
Methods: Senescent and non-senescent primary human fibroblasts (IMR90) were generated in cell culture by X-irradiation or mock irradiation. Cell-surface proteins were isolated using ‘Cell-Surface Capture’, an in-vivo biotinylation/affinity enrichment approach. Data-independent acquisitions (DIA) were performed on an orthogonal quadrupole time-of-flight TripleTOF 6600 mass spectrometer. Protein identification was performed with ProteinPilot and Spectronaut Pulsar. Secondary validation of surface proteins was performed using flow cytometry (BD LSR II).
Results: We identified 59 proteins significantly increased and 44 proteins significantly decreased on the surface of senescent compared to non-senescent human fibroblasts. Senescent cell-surface proteins were enriched for proteins in exosomal machinery, DNA damage response and cell death pathways. Three surface proteins – CALR, VCAM1, and CD44 – were independently validated by flow cytometry.
Conclusions: Our comprehensive proteomics approach has revealed novel, differentially expressed proteins for the cell-surface, exosomes, and the secretome of senescent cells. We will further focus on validating surface protein candidates and assessing their utility as biomarkers in order to isolate senescent cells from intact human tissues.