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。为此，我们与Keenan Walker博士密切合作，在巴尔的摩衰老纵向研究的参与者中发现了一组衰老单核细胞蛋白，这些蛋白在衰老过程中会增加循环。我们的分析发现，64种蛋白质的一个子集可以预测这一人群的衰老。更重要的是，我们发现这个小组的一个子集可以预测人类与年龄相关的关键临床结果，包括整体活动能力和身体健康，通过握力、行走速度、站立速度等来衡量。血液中该蛋白组的一个子集也强烈预测代谢参数，如肥胖、BMI、低密度脂蛋白、高密度脂蛋白和血糖水平。我们的研究结果表明，衰老的单核细胞蛋白特征可以预测关键的年龄相关结果。接下来，我们将在独立研究队列中验证这些预测。

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.