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

项目总结/摘要 细胞衰老是由多种不同的应激诱导的细胞周期停滞的永久状态。虽然 衰老细胞（SNCs）已被证明在正常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， 骨关节炎和2型糖尿病。SNCs和衰老相关分泌表型（SASP）是 被发现是高度异质性的，并且在不同类型的细胞和组织区域中变化。目前没有 这些生物标志物是用于体内鉴定SNC的“通用”生物标志物。推进我们对知识的理解的第一步 细胞衰老和开发SNC靶向治疗方法是全面表征 各种人体组织中的细胞衰老。基于质谱（MS）的空间蛋白质组学可以提供 直接洞察细胞异质性和揭示新的蛋白质标记。然而，目前的空间蛋白质组学 这些技术受限于其差的空间分辨率和低的分析通量。本报告的总体目标 一个项目是显着推进我们的基于微流体的空间蛋白质组学平台，称为激光捕获 显微切割与纳米液滴处理结合在一锅微量样品（LCM-nanoPOTS），并应用 该技术用于绘制不同小鼠和人体组织中的SNCs及其SASP。在UG 3阶段， 建立一个高通量和稳健的单细胞分离系统，并将其与nanoPOTS-MS耦合。我们将 修改蔡司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管取样探针。接下来，我们将部署空间单细胞蛋白质组学平台， SNCs及其SASP在小鼠皮肤组织中的定位。我们将制定一个简化的工作流程，以确定SNCs FASST老鼠皮免疫组织化学或免疫荧光将用于验证新蛋白 候选标记。在UH 3阶段，我们将显著提高蛋白质组覆盖率和分析通量 nanoPOTS-LC-MS平台。我们的目标是实现>300个样品/天的通量和蛋白质组 覆盖>1500种蛋白质。nanoPOTS样品制备将针对福尔马林固定石蜡进行优化- 空间蛋白质组学技术的广泛应用。接下来，我们将演示 在不同的组织类型，包括小鼠肝脏，皮肤引流淋巴结，以及人类， 肝脏乳腺和肺组织我们将与不同的TMC建立合作，以表征SNCs， SASP在各种人体组织中使用空间蛋白质组学，并有助于组织的多组学作图。 影响声明：在单细胞水平上生成无偏倚和全面的蛋白质组图谱的能力 分辨率将能够发现不同细胞类型和器官的SNC蛋白标记物，并推进 我们对SNCs对组织微环境影响的理解。

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.