Dynamic Imaging of EMT in the Breast Cancer Microenvironment

描述（由申请人提供）：转移和对治疗的抵抗是乳腺癌最致命的方面，然而肿瘤微环境中导致癌细胞传播和抵抗的条件尚不完全清楚。启动和维持这些过程的分子事件在肿瘤微环境中是罕见的事件，揭示微环境中这些早期微妙的变化将需要高分辨率、多参数的体内显微镜方法和报告构建，这些方法可以表征导致远处组织入侵、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，在培养和两种动物模型中监测携带与转移过程相关的关键分子事件信号的乳腺癌细胞。这种多光谱显微镜能够检测疾病进展的多种标志物，其新颖的结构使其能够彻底小型化，有利于植入啮齿动物模型，以便连续研究微环境。尽管这些设备在体内成像的应用范围很广，但我们在这里关注的是监测上皮细胞向间质转化（EMT）和back-MET的关键步骤。EMT是一个发育过程，在这个过程中，有组织的上皮细胞转变为分离的、具有间充质表型的迁移细胞，是转移过程的关键步骤。我们已经确定了一组新的与乳腺癌细胞EMT和MET特异性相关的选择性剪接事件。我们建议通过使用可切换的双色荧光报告器（基于剪接模式指示EMT状态）来转导乳腺上皮细胞来报告这些事件。当这些报告细胞启动EMT或MET时，偶联剪接事件将导致荧光蛋白表达的变化，从而使我们的

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.