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.

项目摘要/摘要 不同类型的癌症优先转移到这些组织中的不同组织和特定部位。为什么 这是正确的理解，但可能涉及细胞内在因素的组合（例如， 细胞在有丝分裂因素，养分可用性或上下文特异性压力方面生存差异的能力）和 外部效应（组织特异性的机械和生化提示）。获得分子洞察事件 参与转移性定殖是具有挑战性的，因为这样的事件很少见，菌落最初很小， 定植的潜在位点被广泛分布。 TDU的重点是开发，验证 并传播用于深度多重组织成像的创新工具包；这些工具包将在 与我们的RTB密切相关，并提供给更广泛的CCBIR财团。成熟后，我们的方法 所描述的将对涉及约60种蛋白或其他的分子过程进行定量测量 在保存的组织环境下亚细胞分辨率处的生物分子。在目标1中，我们将组装自动驾驶 多尺度显微镜，该显微镜利用组织清除，全自动高速和高 - 分辨率灯页荧光成像和计算机视觉，以确定转移中最早的事件， 包括单个转移细胞对组织的定殖。该显微镜将具有介观，并且 纳米成像模式。介质模块具有计算控制的放大倍数（0.63倍至 6.3倍），并在2.1-21mm的视野中提供约5-10 µm的各向同性分辨率。纳米镜模块 在300 µm的视野中提供约330nm的各向同性分辨率。生物特征（转移性 菌落将通过介观模块迅速有效地识别，并在高处进行询问 使用纳米镜模块的分辨率。 AIM 2将涉及物理和化学的发展 组织切片的加速60-循环免疫荧光测定厚度足以完全（〜200 µm） 涵盖转移菌落及其组织生态位。厚截面高度多路复用和高分辨率 然后，成像将与RTB的CRISPR-CAS9工程细胞系结合，以测试特定 关于转移涉及的信号，分化和形态学机制的假设。支持 空间转录物分析和组织蛋白质组学将有助于整合到更注重的NCI中 程序。 AIM 3将开发出完全自动化的多技术显微镜，能够准确描述 以统计强大的方式转移异质性。该仪器将结合深度各向同性分辨率 通过自动样品处理，标记，成像和分析的高度多路复用方法成像。这 下一代显微镜将涉及几种可概括的技术，以全面分析稀有 转移和癌症倡议的事件，这是另一个罕见的事件。在一起，我们的方法 预期的描述会大大提高我们对最不具体和大多数特征的理解之一 实体瘤的致命特征。