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

在我们的第一个目标中，在基于人类队列研究的SASP候选生物标志物列表的完善方面取得了实质性进展。在早期与dr。Luigi Ferrucci和Toshiko Tanaka，我们在巴尔的摩衰老纵向研究中发现了与人类血浆衰老相关的衰老相关分泌表型（SASP）的一个子集。在过去的一年里，我们已经将衰老的蛋白质组学分析扩展到单核细胞中，这已经产生了与早期研究截然不同的衰老相关蛋白质列表。为了测试单核细胞衰老特异性特征是否可以预测人类的衰老、虚弱和疾病状态，我们正在继续与NIA人类队列研究合作。

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 test whether monocyte-senescence specific signatures are predictive of aging, frailty, and disease status in human populations, we are continuing to collaborate with NIA human cohort studies. 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. Over the past year we have 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. 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. Going forward we will validate surfaceome markers in both tissue in vivo using immunocytochemistry and flow cytometry.