Single-cell label-free identification of senescence by Raman microscopy and spatial genomics

项目摘要 衰老细胞的分子和细胞异质性仍然很差。的知识差距 主要是由于缺乏适当的技术来表征完整组织中的细胞状态、类型和电路。 因此，我们将需要新的技术来绘制衰老的多维参数， 在分子、细胞和形态学水平上以及在纵向时间框架上的组织环境。单个 细胞多组学和分子谱分析（例如，单细胞RNA-seq，单细胞ATAC-seq，单细胞 蛋白质组学、甲基化组学、代谢组学）为理解这些特性打开了新的窗口， 以前所未有的分辨率和规模研究细胞的调节、动力学和功能。然而，这些测定是 天生的破坏性。细胞需要被解离、固定或裂解以用于这些分子谱分析测定。拉曼 显微镜提供了一个独特的机会，全面报告的振动能级的分子 以亚细胞空间分辨率的无标记、非破坏性的方式。随着拉曼光谱的最新进展 显微镜，单细胞和空间多组学，以及机器学习，我们已经开发了“拉曼2 RNA”（R2 R）， 一个实验和计算框架，以推断活细胞中的单细胞表达谱，通过标签， 结合多模态数据集成和域的自由高光谱拉曼显微图像 翻译.在这项提案中，我们的目标是开发“SenNetRaman”，一个创新的实验和计算 通过无标记高光谱拉曼表征衰老细胞分子异质性的平台 显微镜、单细胞和空间基因组学以及机器学习。在UG 3阶段，我们的目标是开发 “SenNetRaman”用于表征肺组织中对应于年轻、自然衰老或应激的单细胞， 诱导的衰老状态从完善的小鼠模型。我们将开发一种高通量的拉曼 用于衰老细胞分子异质性的无标记表征的显微镜系统， 预测基因表达并对应于各种衰老细胞状态的拉曼信号/标志物， 类型在UH 3阶段，我们将展示“SenNetRaman”用于表征多个细胞中的衰老细胞。 衰老模型系统，包括来自已建立的人类衰老的人肺、脑和皮肤 组织绘图中心总的来说，“SenNetRaman”是一个模块化的通用框架， 单细胞多组学数据，用于构建人类衰老细胞的定量生物分子组织图谱。我们 该应用在通过利用成像的最新进展来研究衰老的方法中是创新的， 单细胞基因组学和机器学习。该项目的结果将有助于确定新的标记，并揭示 衰老的新生物学“SenNetRaman”建立在SenNet计划的基础上，可以随时适应 现有的NIH单细胞组织绘图工作，包括人类肿瘤图谱（HTAN），人类生物分子图谱， Atlas计划（HuBMAP）和人类细胞图谱（HCA）将改变未来的生物医学和临床研究。

PROJECT SUMMARY The molecular and cellular heterogeneity of senescent cells remains poorly characterized. The knowledge gap is mainly due to the lack of proper technology to characterize the cell states, types, and circuits in intact tissues. Thus, we will need novel technologies to map the multidimensional parameters of senescence across diverse tissue environments at molecular, cellular, and morphological levels and over longitudinal time frames. Single cell multi-omics and molecular profiling assays (e.g., single-cell RNA-seq, single-cell ATAC-seq, single-cell proteomics, methylomics, metabolomics) have opened new windows into understanding the properties, regulation, dynamics, and function of cells at unprecedented resolution and scale. However, these assays are inherently destructive. Cells need to be dissociated, fixed, or lysed for these molecular profiling assays. Raman microscopy offers a unique opportunity to comprehensively report on the vibrational energy levels of molecules in a label-free, nondestructive manner with subcellular spatial resolution. With recent advances in Raman microscopy, single-cell and spatial multi-omics, and machine learning, we have developed “Raman2RNA” (R2R), an experimental and computational framework to infer single-cell expression profiles in live cells through label- free hyperspectral Raman microscopy images combined with multi-modal data integration and domain translation. In this proposal, we aim to develop “SenNetRaman”, an innovative experimental and computational platform to character the molecular heterogeneity of senescent cells through label-free hyperspectral Raman microscopy, single cell and spatial genomics, and machine learning. In the UG3 phase, we aim to develop “SenNetRaman” for characterizing single cells in lung tissues corresponding to young, naturally aged or stress- induced senescence states from well-established mouse models. We will develop a high-throughput Raman microscopy system for label-free characterization of the molecular heterogeneity of senescent cells and identify Raman signals/markers predictive of gene expression and corresponding to various senescent cell states and types. In the UH3 phase, we will demonstrate “SenNetRaman” for characterizing senescent cells across multiple senescence model systems including human lungs, brains, and skins from an established human senescence tissue mapping center. Overall, “SenNetRaman” is a modular and universal framework to link imaging data with single-cell multi-omics data for building quantitative biomolecular tissue maps of human senescent cells. Our application is innovative in the approach to study senescence by leveraging the recent advances in imaging, single-cell genomics, and machine learning. The results of this project will help identify novel markers and reveal new biology of senescence. “SenNetRaman” builds upon the SenNet Initiative and can be readily adapted to existing NIH single-cell tissue mapping efforts, including the Human Tumor Atlas (HTAN), Human Biomolecular Atlas Program (HuBMAP), and Human Cell Atlas (HCA) that will transform future biomedical and clinical research.