Targeting, Quantifying, and Isolating Heterogeneous Populations of Senescent Cells from Tissues via Cell Surface and Secreted Proteomes

National Institute On Aging

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Abstract

In regards to the objective to identify the secretome of senescent cells, substantial methodological and research progress has been made in identifying novel SASP proteins and refining the list of SASP biomarker candidates based on human cohorts studies. Over the last two years we have developed novel and physiologically relevant SASP profiles in monocytes for the first time. Applying a novel nanoparticle-based approach, we were able to achieve deep proteomic coverage in serum-supplemented secretome, which were then prioritized as biomarker candidates in humans. Proteomic profiling of senescent monocytes yielded a largely distinct list of senescence-associated proteins from earlier studies. To this end, we have collaborated closely with Dr. Keenan Walker to identify a panel of senescent monocyte proteins found to increase in circulation during aging in participants from the Baltimore Longitudinal Study on Aging and InCHIANTI study. Our analysis has found that a subset of proteins are predictive of aging and obesity-associated traits in these populations. More importantly, we have found a subset of this panel is predictive of key age-related clinical outcomes in humans. A subset of this protein panel in blood also strongly predict metabolic parameters such as obesity, BMI, LDLs, HDLs, and blood glucose levels. Our results suggest that senescent monocyte protein signatures are predictive of key age-related outcomes. Toward our second objective, we have collected early data on the cell-surface proteome (surfaceome) of senescent cells, from which we will identify and prioritize the most specific surfaceome candidates for targeting senescent cells. We have expanded and further validated our list of senescent cell surface markers by exploring multiple cell types including monocytes, vascular endothelial cells, and preadipocytes. We have validated several monocyte and fibroblast surfaceome markers with flow cytometry. Additionally, we have developed a rigorous computational pipeline to identify and prioritize the most likely cell surface proteins from cell surface proteomic datasets. To validate senescent markers in vivo in a tissue type that matches the cell culture experiments used for the initial discovery of the surfaceome, exploring human tissues for signs of senescent surfaceome markers in vivo. To validate cell surface markers in monocytes, our colleagues at the BLSA are sharing monocyte tissues from young and aged individuals. These specimens will be probed for candidate surfaceome proteins by immunofluorescence. Additionally, to validate the pre-adipocytes surfaceome, we are exploring fat tissues from a now completed clinical study, a completed IRB proposal originally initiated with Dr. Steven Cunningham (Ascension Saint Agnes Hospital, Baltimore). We collected omental and subcutaneous fat for the validation of senescence in young, old and obese individuals in vivo. Our efforts to quantify senescence burden and novel markers with histology in these tissues are ongoing.