Dynamic Imaging of EMT in the Breast Cancer Microenvironment

描述（由申请人提供）：转移和对治疗的耐药性是乳腺癌最致命的方面，但导致癌细胞扩散和耐药性的肿瘤微环境内的条件尚未完全了解。启动和维持这些过程的分子事件在肿瘤微环境中作为罕见事件发生，并揭示微环境中的这些早期、微妙的变化将需要高分辨率、多参数体内显微镜方法和报告构建体，这些方法和报告构建体可以表征导致远处组织的侵袭、内渗和定植的动态细胞事件。我们将使用微型双轴共聚焦（DAC）荧光显微镜监测含有光学报告​​基因的乳腺癌细胞，这些光学报告基因发出与培养物和两种动物模型中的转移过程相关的关键分子事件。这种多光谱显微镜能够检测疾病进展的多种标志物，其新颖的结构实现了彻底的小型化，有利于植入啮齿动物模型中以持续研究微环境。尽管这些设备在体内成像的应用很广泛，但我们在此重点关注监测上皮间质转化 (EMT) 和 back-MET 的关键步骤。 EMT 是一个发育过程，其中有组织的上皮细胞转变为具有间充质表型的分离的迁移细胞，并且是转移过程中关键步骤的基础。我们已经确定了一组新的选择性剪接事件，特别与乳腺癌细胞 EMT 和 MET 相关。我们建议对乳腺上皮细胞进行改造，通过使用可切换的双色荧光报告基因转导乳腺上皮细胞来报告这些事件，该报告基因根据剪接模式指示 EMT 状态。当这些报告细胞启动 EMT 或 MET 时，耦合剪接事件将导致荧光蛋白表达的变化，从而使我们能够 微型共焦显微镜可评估微环境中的细胞状态。我们将测试以下假设：EMT 剪接事件与乳腺癌微环境中的侵袭和内渗相关的关键早期步骤相关，并报告导致乳腺癌微环境中侵袭和内渗的关键早期步骤，目的有三个：1) 改造乳腺肿瘤细胞以报告 EMT/MET 相关的选择性剪接事件，2) 使用现有手持式 DAC 显微镜监测和验证人乳腺组织外植体和异位小鼠模型系统中标记乳腺癌细胞中的 EMT/MET 报告基因表达，以及 3) 将 DAC 显微镜用作可植入设备来评估原位大鼠乳腺癌模型肿瘤微环境中 EMT/MET 相关剪接事件。这项工作将导致开发用于连续、高分辨率、多参数成像的植入式显微镜和可切换的分子报告基因，以揭示活体动物模型肿瘤微环境中的动态过程。

DESCRIPTION (provided by applicant): Metastasis and resistance to therapy are the most deadly aspects of breast cancer, and yet the conditions within the tumor microenvironment that lead to cancer cell dissemination and resistance are not fully understood. The molecular events that initiate and sustain these processes occur as rare events in the tumor microenvionment and revealing these early, subtle changes in the microenvironment will require high resolution, multiparametric in vivo microscopic approaches and reporter constructs that can characterize dynamic cellular events that lead to invasion, intravasation, and colonization of distant tissues. We will monitor breast cancer cells harboring optical reporters that signal key molecular events associated with the metastatic process in culture and in two animal models using a miniature dual-axis confocal (DAC) fluorescent microscope. This multispectral microscope is capable of detecting multiple markers of disease progression, and its novel architecture has enabled radical miniaturization conducive to implantation in rodent models for continuous study of the microenvironment. Although the applications for these devices for in vivo imaging are broad, we focus here on monitoring the critical steps of epithelial to mesenchymal transition (EMT) and back-MET. EMT is a developmental process in which organized epithelial cells transition into isolated, migratory cells with mesenchymal phenotypes, and underlies key steps in the metastatic process. We have identified a novel set of alternative splicing events associated specifically with breast cancer cell EMT and MET. We propose to engineer breast epithelial cells to report these events by transducing them with switchable bichromatic fluorescent reporters that indicate EMT status based on splicing patterns. As these reporter cells initiate EMT or MET, the coupled splicing events will result in a change in fluorescent protein expression, allowing our miniature confocal microscopes to assess cellular status within the microenvironment. We will test the hypothesis that EMT splicing events associate with, and report, key early steps leading to invasion and intravasation within the breast cancer microenvironment in three aims: 1) engineer breast tumor cells to report EMT/MET-associated alternative splicing events, 2) monitor and validate EMT/MET reporter expression in labeled breast cancer cells within human breast tissue explant and ectopic mouse model systems using an existing handheld DAC microscope, and 3) adapt the DAC microscope as an implantable device to evaluate EMT/MET-associated splicing events in the tumor microenvironment of an orthotopic rat mammary carcinoma model. This work will result in the development of implantable microscopes for serial, high resolution, multiparametric imaging, and switchable molecular reporters that reveal dynamic processes within the tumor microenvironment of living animal models.