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

项目摘要/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 这一点仍然鲜为人知，但可能涉及细胞内在因素的组合(例如， 细胞存活的能力在有丝分裂因子、营养物质可获得性或特定背景应激源的差异)和 外在效应(特定于组织的机械和生化线索)。获得对事件的分子洞察 涉及转移性殖民是具有挑战性的，因为这样的事件很少见，最初的殖民地很小， 潜在的殖民地点分布广泛。本TDU的重点是开发、验证 和传播用于深度多路复用组织成像的创新工具包；这些工具包将在#年开发 与我们的RTBS密切合作，并提供给更广泛的CCBIR财团。当成熟时，我们的方法 所描述的将使定量测量涉及~60个蛋白质或其他 保存的组织中亚细胞分辨率的生物分子。在目标1中，我们将组装一款自动驾驶 多尺度显微镜，利用组织清除方面的进步，全自动高速和高速扫描 分辨率光片荧光成像和计算机视觉，以识别转移的最早事件， 包括单个转移细胞对组织的定植。这台显微镜将有介观和 纳米成像模式。介观模块具有计算控制的放大倍率(0.63X至 6.3倍)，在2.1-21 mm的视场范围内提供约5-10微米的各向同性分辨率。纳米模块 在300微米视场内提供约330 Nm的各向同性分辨率。生物学特征(转移性 菌落)将用介观模块快速有效地识别并在高 使用纳米模块的分辨率。目标2将涉及物理和化学方面的发展 厚度足够(~200微米)的组织切片的加速60-plex循环免疫荧光分析 包括一个转移的群体及其组织生态位。厚片高复用化、高分辨率 然后，成像将与来自RTBS的CRISPR-Cas9工程细胞系相结合，以测试特定的 关于信号、分化和涉及转移的形态机制的假说。支持 空间转录图谱和组织蛋白质组学将有助于整合到更关注基因组的NCI 程序。AIM 3将开发一种全自动多技术显微镜，能够准确地描述 转移性异质性以一种统计稳健的方式。该仪器将结合深度各向同性分辨率 通过自动样品处理、贴标签、成像和分析，以高度多元化的方法进行成像。这 下一代显微镜将涉及几种可推广的全面剖析稀有物质的技术 转移和癌症启动中的事件，这是另一个罕见的事件。共同努力，我们的方法 预计将极大地促进我们对最不具特点和最具 实体瘤的致命性特征。

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.