Imaging mechanisms of metastatic tumor formation in situ

项目摘要 为了响应RFA的细胞癌症生物学成像计划，我们提出了一个计划，重点是 成像和分子探测驱动新转移性肿瘤形成的细胞生物学事件。 具体来说，我们将解决两个问题：1）细胞内在和细胞内的变化的交叉点是如何产生的？ 与膜接头蛋白Caveolin-1表达变化相关的外源性信号影响了细胞膜的生物学行为。 儿童肉瘤的转移倾向（研究试验床1号单元）？2)细胞内毒素的作用是什么 和细胞外在的变化，脂质代谢对黑色素瘤转移模式（研究试验床单位2）？ 这两个问题的答案取决于技术，以捕捉分子，代谢和形态 单个转移细胞在远处定居时的状态：在技术开发单元-1中，我们 将开发一个多模式，多尺度的实时成像平台，以调查交叉的影响 跨生物体的微环境变化和细胞内在异质性对转移扩散的影响。的 该平台将利用斑马鱼胚胎精致的光学和生理特性来“观看” 一旦形成人类肿瘤异种移植物的细胞扩散到多个远端部位， 肿瘤的该显微镜将允许在高通量筛选模式和观察模式之间无缝切换。 在一个实验中，数十到数百个胚胎的转移模式和高分辨率成像模式 在XYZ中具有300 nm的全各向同性分辨率，可以对分子，代谢， 出现的转移性小生境内的单个细胞的形态学和增殖/存活状态。在 技术发展组-2我们会发展一个多尺度影像平台，以超光谱技术进行研究。 分析整个小鼠转移细胞的分子、代谢、形态和功能状态 机关该平台将利用先进的组织清除，全自动高速和高分辨率 光片荧光成像和计算机视觉，以集成介观成像模式， 采用纳米级的各向同性分辨率，采集高达20 x 20 x 20 mm的体积 成像模式提供300 nm XYZ分辨率，在器官中的任何地方都可以看到300微米的视野。 因此，可以快速识别生物特征，并立即以高亚细胞分辨率进行询问。 然后，我们将开发物理和化学加速的60重循环免疫荧光测定， 全面表征定殖细胞的分子、代谢和结构状态及其 在厚（~200微米）组织切片中的转移灶周围。为了准确描述转移性 异质性，整个系统，包括样品处理，标记和成像，将完全自动化， 以高通量的方式操作。我们使用此系统的目标是实现对 在100个完整组织样本中的异质细胞转移细胞行为。这些平台将共同 为原位癌细胞生物学的新时代产生多功能成像工具。

Project Summary In response to the RFA for a Cellular Cancer Biology Imaging Program we propose a program focused on imaging and molecularly probing the cell biological events that drive the formation of new metastatic tumors. Specifically, we will address two questions: 1) How does the intersection of shifts in cell-intrinsic and cell- extrinsic signals associated with shifts in expression of the membrane adaptor protein Caveolin-1 affect the metastatic propensity of pediatric sarcoma (Research Testbed Unit 1)? 2) What are the effects of cell-intrinsic and cell-extrinsic variation in lipid metabolism on melanoma metastasis patterns (Research Testbed Unit 2)? Answers to both questions depend on technology to capture the molecular, metabolic, and morphological states of individual metastatic cells as they colonize the distant site: In the Technology Development Unit-1 we will develop a multi-modal, multi-scale live imaging platform to investigate the effects of intersecting microenvironmental variation across an organism and cell intrinsic heterogeneity on metastatic spreading. The platform will leverage the exquisite optical and physiological properties of the zebrafish embryos to ‘watch’ at once how cells form human tumor xenografts spread to multiple distant sites where they form metastatic tumors. The microscope will allow seamless switching between a high-throughput screening mode observing the metastatic patterns in tens to hundreds of embryos in one experiment and a high-resolution imaging mode with fully isotropic resolution of 300 nm in XYZ that allows detailed analysis of the molecular, metabolic, morphologic, and proliferation/survival states of individual cells within an emerging metastatic niche. In the Technology Development Unit-2 we will develop a multi-scale imaging platform to investigate by hyper-spectral analysis the molecular, metabolic, morphological, and functional states of metastatic cells across entire mouse organs. The platform will leverage advances in tissue clearing, fully automated high-speed and high-resolution light-sheet fluorescence imaging, and computer vision, to integrate a mesoscopic imaging mode for fast acquisition of volumes of up to 20 x 20 x 20 mm at a ~5-10 micron isotropic resolution with a nanoscopic imaging mode providing 300 nm XYZ-resolution throughout a 300 micron field of view anywhere in the organ. Biological features can thus be rapidly identified and immediately interrogated with high subcellular resolution. We will then develop physically and chemically accelerated 60-plex cyclic immunofluorescence assays to comprehensively characterize the molecular, metabolic and architectural states of colonizing cells and their surroundings in the metastatic niche in thick (~200 microns) tissue sections. To accurately describe metastatic heterogeneity, the entire system, including sample handling, labeling, and imaging, will be fully automated and operated in a high-throughput fashion. Our goal with this system is to enable comprehensive profiling of heterogeneous cell metastatic cell behavior in 100’s of intact tissue specimens. Together, these platforms will generate versatile imaging tools for a new era of in situ cancer cell biology.