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，基于人类队列研究，在完善SASP生物标志物候选物列表方面取得了实质性进展。在与Luigi Ferrucci博士和Toshiko Tanaka博士的早期合作研究中，我们在巴尔的摩老龄化纵向研究中确定了与人类血浆中老龄化相关的衰老相关分泌表型（SASP）的一个子集。在过去的一年里，我们已经扩大了蛋白质组分析衰老单核细胞，这产生了很大程度上不同的列表从早期的研究衰老相关蛋白。为了测试单核细胞衰老特异性特征是否能预测人群的衰老、虚弱和疾病状态，我们继续与NIA人类队列研究合作。 对于我们的第二个目标，我们已经收集了关于衰老细胞的细胞表面蛋白质组（surfaceome）的早期数据，从中我们将确定并优先考虑针对衰老细胞的最具特异性的surfaceome候选者。为了鉴定最特异的表面蛋白，我们将我们在衰老成纤维细胞上鉴定的细胞表面蛋白与细胞表面蛋白图谱数据库进行了比较（Baush-Fluck等人，2015. PLOS One），并根据已知表达的细胞类型数量对候选细胞进行排名。为了扩展和验证我们的衰老细胞表面标志物列表，我们还使用称为糖细胞表面捕获的附加方法收集了衰老和非衰老细胞表面蛋白，该方法分离细胞表面N-连接的糖蛋白。在过去的一年中，我们优化了质谱采集方法，用于表面组研究的分析。我们还使用新的优化的糖细胞表面捕获方法在几种细胞类型中进行了初步的表面组研究：成纤维细胞，单核细胞，前脂肪细胞，血管平滑肌细胞和血管内皮细胞。我们现在正在对每种细胞类型的衰老细胞与非衰老细胞的细胞表面变化进行全面的定量比较。在早期的研究中，我们已经确定了4个候选的衰老特异性细胞表面蛋白在人体组织中进行验证。 为了在体内验证与用于表面组的最初发现的细胞培养实验相匹配的组织类型中的衰老标志物，我们已经开始合作以从人类获得单核细胞和脂肪组织。为了验证单核细胞中的细胞表面标志物，我们在BLSA的同事将分享来自年轻人和老年人的单核细胞组织。将通过免疫荧光法探测这些标本中的候选表面蛋白。此外，为了验证前脂肪细胞表面组，我们将获得脂肪组织。为此，我们与Steven Cunningham博士（Ascension Saint Agnes Hospital，巴尔的摩）一起提交了一份IRB提案，旨在收集网膜和皮下脂肪，用于在体内验证年轻、老年和肥胖个体的衰老。展望未来，我们将使用免疫细胞化学和流式细胞术在体内验证两种组织中的表面组标志物。

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.