Technical Development Unit 2: Intelligent Hyperspectral Imaging of Subcellular Molecular States at the Whole Organ Level

技术开发单元2：全器官水平亚细胞分子态智能高光谱成像

• 批准号: 10684861
• 负责人: Kevin Michael Dean
• 金额: $ 46.06万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-24 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10684861
• 关键词: 3-Dimensional; Acceleration; Address; Automation; Automobile Driving; Back; Biochemical; Biological; Biological Assay; Biosensor; CRISPR/Cas technology; Cell Line; Cells; Cellular Morphology; Chemicals; Chemistry; Communities; Computer Systems; Computer Vision Systems; Computer software; Computing Methodologies; Cues; Data Analyses; Development; Dimensions; Dyes; Electromagnetics; Engineering; Event; Feedback; Fostering; Frequencies; Goals; Heterogeneity; Image; Immunofluorescence Immunologic; In Situ; Intelligence; Interleukin-2; Intrinsic factor; Label; Laboratories; Light; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Measurement; Mechanics; Mediating; Metabolic; Methods; Microscope; Microscopy; Microtomy; Molecular; Molecular Analysis; Morphology; Neoplasm Metastasis; Nodule; Nutrient availability; Optics; Organ; Organism; Oxidation-Reduction; Performance; Periodicity; Physics; Preparation; Process; Proteins; Proteomics; Reagent; Refractive Indices; Resolution; Risk; Robotics; Sampling; Signal Transduction; Site; Slice; Solid Neoplasm; Solvents; Specimen; Speed; Technology; Testing; Thick; Thinness; Three-Dimensional Imaging; Tissue Preservation; Tissue imaging; Tissues; Transcript; Travel; Validation; automated analysis; cancer cell; cancer initiation; design; fluorescence imaging; high resolution imaging; imaging approach; imaging system; improved; innovation; insight; instrument; meter; multiplexed imaging; nano; next generation; programs; stressor; usability

PROJECT SUMMARY/ABSTRACT Cancers of different types preferentially metastasize to different tissues and specific sites in these tissues. Why this is true remains poorly understood but is likely to involve a combination of cell intrinsic factors (e.g., the ability of a cell to survive differences in mitogenic factors, nutrient availability, or context-specific stressors) and extrinsic effects (tissue-specific mechanical and biochemical cues). Gaining molecular insight into events involved in metastatic colonization is challenging, because such events are rare, colonies are initially small, and potential sites of colonization are widely distributed. The focus of this TDU is the development, validation and dissemination of innovative toolkits for deep multiplexed tissue imaging; these toolkits will be developed in close association with our RTBs and provided to the wider CCBIR consortium. When mature, the methods we described will enable quantitative measurement of molecular processes involving ~60 proteins or other biomolecules at subcellular resolution in a preserved tissue context. In Aim 1 we will assemble a self-driving multiscale microscope that leverages advances in tissue clearing, fully automated high-speed and high- resolution light-sheet fluorescence imaging, and computer vision, to identify the earliest events in metastasis, including the colonization of a tissue by a single metastatic cell. This microscope will have mesoscopic and nanoscopic imaging modes. The mesoscopic module has computationally controlled magnification (0.63X to 6.3X) and provides ~5-10 µm isotropic resolution throughout a 2.1-21mm field of view. The nanoscopic module provides ~330nm isotropic resolution throughout a 300 µm field of view. Biological features (metastatic colonies) will be rapidly and efficiently identified with the mesoscopic module and interrogated at high resolution using the nanoscopic module. Aim 2 will involve development of physically and chemically accelerated 60-plex cyclic immunofluorescence assays of tissue sections thick enough(~200 µm) to fully encompass a metastatic colony and its tissue niche. Thick section highly multiplexed and high-resolution imaging will then be combined with CRISPR-Cas9 engineered cell lines from the RTBs to test specific hypotheses about signaling, differentiation, and morphological mechanisms involved in metastasis. Support for spatial transcript profiling and tissue proteomics will aid with integration into more gnomically focused NCI programs. Aim 3 will develop a fully automated multi-technology microscope able to accurately describe metastatic heterogeneity in a statistically robust fashion. The instrument will combine deep isotropic resolution imaging with highly multiplexed methods via automated sample handling, labeling, imaging and analysis. This next-generation microscope will involve several generalizable technologies for comprehensively profiling rare events in metastasis and also cancer initiation, which is another rare event. Together, the approaches we describe are expected to substantially advance our understanding of one of the least characterized and most lethal features of solid tumors.

项目概要/摘要 不同类型的癌症优先转移到不同的组织和这些组织中的特定部位。为什么 这是真的，但我们仍然知之甚少，但很可能涉及细胞内在因素的组合（例如， 细胞在有丝分裂因子、营养可用性或特定环境压力因素的差异中生存的能力）和 外在效应（组织特异性机械和生化线索）。获得对事件的分子洞察 参与转移定植具有挑战性，因为此类事件很少见，集落最初很小， 潜在的定殖地点分布广泛。该 TDU 的重点是开发、验证 和传播用于深层多重组织成像的创新工具包；这些工具包将在 与我们的 RTB 密切联系，并提供给更广泛的 CCBIR 联盟。当成熟的时候，我们的方法 所描述的将能够定量测量涉及约 60 种蛋白质或其他蛋白质的分子过程 在保存的组织环境中以亚细胞分辨率观察生物分子。在目标 1 中，我们将组装一辆自动驾驶汽车 多尺度显微镜，利用组织透明化、全自动高速和高通量方面的进步 分辨率光片荧光成像和计算机视觉，以确定转移中的最早事件， 包括单个转移细胞在组织中的定殖。该显微镜将具有介观和 纳米级成像模式。介观模块具有计算控制的放大倍率（0.63X 至 6.3X），并在 2.1-21mm 视场范围内提供约 5-10 µm 各向同性分辨率。纳米级模块 在 300 µm 视场中提供约 330 nm 各向同性分辨率。生物学特征（转移性 菌落）将被介观模块快速有效地识别并在高温度下进行询问 使用纳米级模块的分辨率。目标 2 将涉及物理和化学的发展 对足够厚（~200 µm）的组织切片进行加速 60 重循环免疫荧光测定，以充分 包括转移集落及其组织生态位。厚切片高度多路复用和高分辨率 然后，成像将与来自 RTB 的 CRISPR-Cas9 工程细胞系相结合，以测试特定的 关于转移涉及的信号传导、分化和形态学机制的假设。支持 空间转录本分析和组织蛋白质组学将有助于整合到更注重基因学的 NCI 中 程序。 Aim 3 将开发一款全自动多技术显微镜，能够准确描述 以统计学上稳健的方式观察转移异质性。该仪器将结合深度各向同性分辨率 通过自动化样品处理、标记、成像和分析，采用高度多重方法进行成像。这 下一代显微镜将涉及多种通用技术，用于全面分析稀有物质 转移和癌症发生的事件，这是另一个罕见的事件。我们共同采取的方法 描述预计将大大促进我们对最不具有特征和最有特征的之一的理解 实体瘤的致死特征。