Imaging mechanisms of metastatic tumor formation in situ

项目摘要 为了响应细胞癌生物学成像计划的RFA，我们提出了一个专注于 成像和分子探测驱动新的转移性肿瘤形成的细胞生物学事件。 具体地说，我们将解决两个问题：1)细胞内在性和细胞内在性转移的交集是如何 与膜适配器蛋白Caveolin-1表达变化相关的外在信号影响 儿童肉瘤的转移倾向(研究试验床单元1)？2)细胞固有的影响是什么 黑色素瘤转移模式中脂肪代谢的细胞外源性变异(研究试验第二单元)？ 这两个问题的答案都取决于捕捉分子、代谢和形态的技术 单个转移细胞在远处定植时的状态：在技术发展单元-1中 将开发一个多模式、多尺度的实时成像平台来研究交叉点的影响 整个生物体的微环境变化和细胞在转移扩散上的内在异质性。这个 Platform将利用斑马鱼胚胎的精致光学和生理特性在 一旦细胞如何形成人类肿瘤，异种移植就会扩散到多个遥远的位置，在那里形成转移 肿瘤。该显微镜将允许在高通量筛选模式观察之间无缝切换 一次实验中数十至数百个胚胎的转移模式和高分辨率成像模式 XYZ的全各向同性分辨率为300 nm，允许详细分析分子、代谢、 新出现的转移生态位中单个细胞的形态和增殖/生存状态。在 技术开发单元-2我们将开发一个多尺度成像平台，用于高光谱调查 分析整个小鼠转移细胞的分子、代谢、形态和功能状态 器官。该平台将利用组织清理、全自动高速和高分辨率方面的进步 光片荧光成像，与计算机视觉相结合，实现了中观成像模式的快速 用纳米显微镜以~5-10微米的各向同性分辨率获取高达20x20x20 mm的体积 成像模式提供300纳米XYZ分辨率，覆盖器官内任何地方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-plex循环免疫荧光分析来 全面表征定植细胞的分子、代谢和构筑状态以及它们的 厚(约200微米)组织切片中转移灶的周围环境。准确地描述转移瘤 异构性，整个系统，包括样品处理、标签和成像，将完全自动化和 以高吞吐量的方式运行。我们使用该系统的目标是实现全面的分析 100‘S完整组织标本的异质性细胞转移行为。这些平台加在一起将 为原位癌细胞生物学的新时代生成多功能成像工具。

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.