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 细胞感应是由许多不同应力引起的细胞周期停滞的永久状态。虽然 感觉细胞（SNC）在正常生理过程中具有有益作用，它们已证明它们 越来越多地被认为是许多与衰老有关的疾病（例如癌症）的关键决定者 骨关节炎和2型糖尿病。 SNC和相关的秘书表型（SASP）是 发现在不同类型的细胞和组织区域中是高度异质性的。目前，没有 “通用”生物标志物用于识别体内SNC。迈向我们理解的第一步 细胞感应和发展SNC靶向疗法的方法是全面表征 各种人体组织中的细胞感应。质谱（MS）的空间蛋白质组学可以提供 直接洞悉细胞异质性并揭示新的蛋白质标志物。但是，当前的空间蛋白质组学 技术受到其空间分辨率差和分析低吞吐量的限制。总体目标 项目将大大提高我们基于微流体的空间蛋白质组学平台，称为激光捕获 微分解与纳米光处理在一锅中的痕量样品（LCM纳米）中，并应用 这项技术可在不同的小鼠和人体组织中绘制SNC及其SASP。在UG3阶段，我们将 建立一个高通量和稳健的单细胞隔离系统，并将其与纳米摩托车搭配使用。我们将 修改Zeiss LCM系统以设计和组装启用可靠的单细胞隔离和收集 机器人 - 可调的毛细管采样探针。接下来，我们将部署空间单细胞蛋白质组学平台 SNC的映射及其在小鼠皮肤组织中的SASP。我们将开发一个简化的工作流程来识别SNC 来自Fasst鼠标皮。免疫组织化学或免疫荧光将用于验证新蛋白 标记候选人。在UH3阶段，我们将显着增强蛋白质组覆盖范围和分析吞吐量 Nanopots-LC-MS平台的图。我们的目标是获得> 300个样品/天的吞吐量和蛋白质组 覆盖> 1500蛋白。纳米机样品制剂将针对福尔马林固定石蜡进行优化 用于空间蛋白质组学技术广泛应用的嵌入式（FFPE）组织。接下来，我们将展示 改善了不同组织类型的平台，包括小鼠肝脏，皮肤排水淋巴结以及人类 肝，乳房和肺组织。我们将与不同的TMC建立合作，以表征SNC和 SASP使用各种人体组织中的空间蛋白质组学，并有助于组织的多样映射。 影响声明：在单细胞处生成公正和全面的蛋白质组图的能力 分辨率将使在不同的细胞类型和器官中发现SNC蛋白标记物，并提高 我们对SNC对组织微环境的影响的理解。