(7) Novel imaging devices for measurement and control of tumor microenvironments

项目摘要/摘要 了解细胞在肿瘤微环境中的身份、位置和功能对于 了解它们是如何支配肿瘤表型并促进扩散和转移的。一个 这方面的例子是三部分结构TMEM(用于转移的肿瘤微环境)，包括 巨噬细胞、内皮细胞和肿瘤细胞的并列，它们共同发挥作用，起着 转移扩散的通道。我们创造了新的成像技术，包括 1)嵌入微流体的可植入窗口，允许连续高分辨率单细胞显微镜 几天到几周的原发和转移部位，以及2)多模式图像对齐技术 将固定的、染色的组织切片彼此配准或与获取的活体成像电影配准。灯 嵌入微流控系统中的激活阀门将用于提供微环境改变 化学试剂(如模拟缺氧药物、化疗)和生物试剂(如功能阻断抗体) 用荧光抗体标记未标记的组织，所有这些都是在实时成像组织反应的同时进行的。 活体成像将用于对组织进行成像，并提供覆盖 整个组织的时间跨度从几秒钟到几天，甚至几周不等。最后，活动细胞将被 被捕获，要么通过化学吸引到微流体室，要么通过激光捕获微解剖 固定，用于进一步的表情特征分析。这些技术在小学和小学教育研究中的应用 继发性病变具有前所未有的能力，可以探测细胞的身份、位置、数量和功能。 构成了异质的微环境。此外，在体内测试影响靶向的药物 形成TMEM的细胞的分布、功能和动态，以及它们与之相关的细胞 相互作用，将使快速确定哪些药物具有最好的治疗潜力。这项工作将 确定1)缺氧、细胞外基质密度、免疫细胞密度和化疗如何启动和定义肿瘤 原发和继发部位的异质性；2)化疗和已知的TMEM阻滞剂 组装和功能影响原发和继发血管内扩散的动力学 3)细胞行为和表型与细胞身份和位置的关系。 这个项目中使用的技术是普遍适用的，并将允许分子鉴定， 肿瘤异质性在多种癌症和疾病模型中的定位和量化。

PROJECT SUMMARY/ABSTRACT Understanding the identity, location and function of cells in the tumor microenvironment is essential to understanding how they dominate tumor phenotype and contribute to dissemination and metastasis. An example of this is the tri-partite structure TMEM (for Tumor MicroEnvironment of Metastasis) consisting of the juxtaposition of a macrophage, an endothelial cell and a tumor cell which function together to act as the doorway for metastatic dissemination. We have created novel imaging technologies consisting of 1)implantable windows with embedded microfluidics that allow serial high-resolution, single-cell microscopy of the primary and metastatic sites over days to weeks, and 2)multi-modal image alignment technologies to register fixed, stained, tissue sections to each other or to the acquired intravital imaging movies. Light activated valves embedded in the microfluidic system will be used to deliver microenvironment-altering chemical (e.g. hypoxia mimetics, chemotherapy) and biological (e.g. function blocking antibodies) agents and label unmarked tissues with fluorescent antibodies, all while imaging the tissue response in real time. Intravital imaging will be used to image the tissue and provide single-cell resolution images that cover the entire tissue over time spans ranging from seconds, to days, and even weeks. Finally, motile cells will be captured, either by chemo-attraction to microfluidic chambers, or laser capture microdissection after fixation, for further expression profiling. Application of these technologies to the study of primary and secondary lesions an unprecedented ability to probe the identity, location, quantity and function of the cells composing the heterogeneous microenvironment. Further, testing, in vivo, the targeting agents affecting the distribution, function and dynamics of the cells forming TMEM, as well as and the cells with which they interact, will enable the rapid determination of those agents with best therapeutic potential. This work will determine how 1) hypoxia, ECM density, immune cell density and chemotherapy initiate and define tumor heterogeneity in the primary and secondary sites; 2) chemotherapy and known blockers of TMEM assembly and function affect the dynamics of intravasation and dissemination in the primary and secondary sites; and 3) the relationship between cellular behavior and phenotype with cellular identity and location. The techniques utilized in this project are generally applicable and will allow molecular identification, localization and quantification of tumor heterogeneity in many cancers and disease models.