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 our first objective, substantial progress has been made in refining the list of SASP biomarker candidates based on human cohorts studies. In early collaborative studies with Drs. Luigi Ferrucci and Toshiko Tanaka, we identified a subset of the senescence-associated secretory phenotype (SASP) that are associated with aging in human plasma in the Baltimore Longitudinal Study of Aging. Over the last year we have expanded proteomic profiling of senescence into monocytes, which has 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. Our analysis has found that a subset of 64 proteins are predictive of aging in this population. More importantly, we have found a subset of this panel is predictive of key age-related clinical outcomes in humans, including overall mobility and physical health, as measured by grip strength, walking speed, chair stand pace, etc. 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. Next, we will test these predictions in independent study cohorts for validation. 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. To identify the most specific surface proteins, we have compared the cell surface proteins we identified on senescent fibroblasts with the cell-surface protein atlas database (Baush-Fluck et al. 2015. PLOS One) and rank our candidates based on the number of cell-types they are known to be expressed in. To expand and validate our list of senescent cell surface markers, we have also collected senescent and non-senescent cell surface proteins using an additional method termed glyco-cell surface capture, which isolates cell surface N-linked glycoproteins. In the previous year we optimized mass spectrometry acquisisiton methods for the analysis of surfaceome studies. We have additionally performed pilot surfaceome studies using a new optimized glyco-cell surface capture approach in several cell types: fibroblasts, monocytes, pre-adipocytes, vascular smooth muscle cells, and vascular endothelial cells. We are now performing comprehensive quantitative comparison of cell surface changes in senescent versus non-senescent cells of each cell type. In early studies we have identified 4 candidate senescence-specific cell surface proteins for validation in human tissues. We have now identified and validated multiple cell surface protein candidates with flow cytometry - including CDCP1, CALR, F2R, VCAM1 and others. To validate senescent markers in vivo in a tissue type that matches the cell culture experiments used for the initial discovery of the surfaceome, we have initiated collaborations to obtain monocytes and adipose tissues from humans. To validate cell surface markers in monocytes, our colleagues at the BLSA will share 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 will obtain fat tissue. To this end we have submitted an IRB proposal with Dr. Steven Cunningham (Ascension Saint Agnes Hospital, Baltimore) to collect omental and subcutaneous fat for the validation of senescence in young, old and obese individuals in vivo. In the last year, the IRB proposal was approved and we have procured about 30 adipose tissues samples for validation and are optimizing antibodies for staining and validation of the presence of senescent cells in vivo.