Spatially-resolved proteome mapping of senescent cells and their tissue microenvironment at single-cell resolution

单细胞分辨率下衰老细胞及其组织微环境的空间分辨蛋白质组图谱

• 批准号: 10552842
• 负责人: Ljiljana Pasa-Tolic
• 金额: $ 47.5万
• 依托单位: BATTELLE PACIFIC NORTHWEST LABORATORIES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-05 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10552842
• 关键词: Address; Aging; Atlases; Biological Markers; Blood capillaries; Breast; C57BL/6 Mouse; Cell Aging; Cell Cycle Arrest; Cell Separation; Cells; Collaborations; Collection; Coupled; Degenerative polyarthritis; Detection; Disease; Dorsal; Fluorescence; Formalin; Freezing; Goals; Heterogeneity; Human; Imaging technology; Immunofluorescence Immunologic; Immunohistochemistry; Internal Ribosome Entry Site; Label; Laboratories; Link; Liver; Lung; Malignant Neoplasms; Mammary Gland Parenchyma; Maps; Mass Spectrum Analysis; Measurement; Methods; Microfluidics; Molecular; Mus; Non-Insulin-Dependent Diabetes Mellitus; Normal Cell; Organ; Paraffin Embedding; Phase; Phenotype; Physiological Processes; Preparation; Procollagen; Proteins; Proteome; Proteomics; Reagent; Reproducibility; Research Personnel; Resolution; Resources; Robot; Role; Sampling; Site; Skin; Skin Tissue; Stains; Stress; Structure of parenchyma of lung; System; Technology; Therapeutic; Tissue Embedding; Tissues; base; candidate marker; cell type; comparative; design; draining lymph node; dysbiosis; epigenome; human disease; human tissue; improved; in vivo; insight; laser capture microdissection; multiple omics; nanoDroplet; novel; overexpression; promoter; protein biomarkers; protein profiling; senescence; success; targeted treatment; tissue mapping; transcriptome

PROJECT SUMMARY/ABSTRACT Cellular senescence is a permanent state of cell cycle arrest induced by many different stresses. Although senescent cells (SNCs) have been demonstrated with beneficial roles in normal physiological processes, they are increasingly recognized as the key determinants of many aging-related diseases, such as cancer, osteoarthritis, and type 2 diabetes. The SNCs and senescence-associated secretory phenotype (SASP) are found to be highly heterogeneous and vary in different types of cells and tissue regions. Currently, there are no “universal” biomarkers for identifying the SNCs in vivo. The first step towards advancing our understanding of cellular senescence and developing SNC-targeting therapy approaches is to comprehensively characterize cellular senescence in various human tissues. Mass spectrometry (MS)-based spatial proteomics can provide direct insights into cellular heterogeneity and reveal novel protein markers. However, current spatial proteomics technologies are limited by their poor spatial resolution and low analysis throughput. The overall objective of this project is to significantly advance our microfluidics-based spatial proteomics platform, termed laser capture microdissection coupled with nanodroplet processing in one-pot for trace samples (LCM-nanoPOTS), and apply this technology to map SNCs and their SASP in different mouse and human tissues. In the UG3 phase, we will establish a high-throughput and robust single-cell isolation system and couple it with nanoPOTS-MS. We will modify the Zeiss LCM system to enable reliable single-cell isolation and collection by designing and assembling a robot-addressable capillary sampling probe. Next, we will deploy the spatial single-cell proteomics platform for mapping of SNCs and their SASP in mouse skin tissue. We will develop a streamlined workflow to identify SNCs from FASST mouse skins. Immunohistochemistry or immunofluorescence will be used to validate novel protein marker candidates. In the UH3 phase, we will significantly enhance proteome coverage and analysis throughput of the nanoPOTS-LC-MS platform. We aim to achieve a throughput of >300 samples/day and a proteome coverage of >1500 proteins. The nanoPOTS sample preparation will be optimized for formalin-fixed paraffin- embedded (FFPE) tissues for broad applications of spatial proteomics technology. Next, we will demonstrate the improved platform in different tissue types, including mouse liver, skin draining lymph nodes, as well as human liver, breast, and lung tissues. We will establish collaborations with different TMCs to characterize SNCs and SASP using spatial proteomics in various human tissues and contribute to multiomics mapping of the tissues. Statement of Impact: The capability to generate unbiased and comprehensive proteome maps at single-cell resolution will enable the discovery of SNC protein markers across different cell types and organs, and advance our understanding of the impact of SNCs on tissue microenvironment.

项目摘要/摘要 细胞衰老是由多种不同的应激引起的细胞周期停滞的永久性状态。虽然 衰老细胞(SNCs)已被证明在正常的生理过程中具有有益的作用，它们 越来越多地被认为是许多与衰老有关的疾病的关键决定因素，如癌症， 骨性关节炎和2型糖尿病。SNCs和衰老相关分泌表型(SASP)是 发现高度异质性，并在不同类型的细胞和组织区域中有所不同。目前，没有 在活体内鉴定SNCs的“通用”生物标志物。推进我们对……理解的第一步 细胞衰老和发展的SNC靶向治疗方法是全面表征 人体各种组织中的细胞衰老。基于质谱仪(MS)的空间蛋白质组学可以提供 直接洞察细胞的异质性，揭示新的蛋白质标记。然而，目前空间蛋白质组学 技术受到空间分辨率低和分析吞吐量低的限制。这样做的总体目标是 该项目将显著推进我们基于微流体的空间蛋白质组学平台，称为激光捕获 微分离-纳米液滴一锅处理痕量样品(LCM-NanPOTS)及其应用 这项技术可以在不同的小鼠和人类组织中定位SNCs及其SASP。在UG3阶段，我们将 建立高通量和健壮的单细胞分离系统，并将其与纳米POTS-MS偶联。我们会 改装蔡司LCM系统，通过设计和组装实现可靠的单细胞分离和收集 一种机器人可寻址的毛细取样探头。接下来，我们将部署空间单细胞蛋白质组学平台 小鼠皮肤组织中SNCs及其SASP的定位。我们将开发一个简化的工作流程来识别SNC 从FASST老鼠皮中提取。将使用免疫组织化学或免疫荧光来验证新的蛋白质 应聘者。在UH3阶段，我们将显著提高蛋白质组覆盖率和分析吞吐量 纳米POTS-LC-MS平台。我们的目标是实现每天300个样品和一个蛋白质组的生产能力 覆盖&gt；1500蛋白质。纳米POTS样品的制备将针对福尔马林固定石蜡进行优化- 包埋组织(FFPE)为空间蛋白质组学技术的广泛应用提供了基础。接下来，我们将演示 不同组织类型的改进平台，包括小鼠肝脏、皮肤引流淋巴结节以及人类 肝脏、乳房和肺组织。我们将与不同的TMC建立合作，以表征SNC和 SASP在人类各种组织中使用空间蛋白质组学，并有助于组织的多组学图谱绘制。 影响陈述：在单细胞上生成无偏见和全面的蛋白质组图谱的能力 解决方案将使跨不同细胞类型和器官的SNC蛋白质标记的发现成为可能，并推动 我们对SNCs对组织微环境影响的理解。