Dynamic Imaging of EMT in the Breast Cancer Microenvironment

描述(申请人提供)：转移和耐药是乳腺癌最致命的方面，但肿瘤微环境中导致癌细胞扩散和耐药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作为罕见事件发生，揭示微环境中的这些早期、细微的变化将需要高分辨率、多参数的活体显微方法和报告结构，以表征导致远处组织侵袭、血管内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个动物模型中监测携带光学记者的乳腺癌细胞，这些光学记者向与转移过程相关的关键分子事件发出信号。这种多光谱显微镜能够检测疾病进展的多个标志物，其新颖的结构使其能够实现激进的微型化，有利于植入啮齿动物模型中，用于连续研究微环境。虽然这些设备在活体成像中的应用很广泛，但我们在这里重点监测上皮向间充质转化(EMT)和Back-Met的关键步骤。EMT是有组织的上皮细胞转变为具有间充质表型的孤立的迁移细胞的发育过程，是转移过程中的关键步骤。我们已经确定了一组与乳腺癌细胞EMT和MET相关的新的选择性剪接事件。我们建议设计乳腺上皮细胞来报告这些事件，方法是用可切换的双色荧光记者来转导这些细胞，这些双色荧光记者基于剪接模式指示EMT状态。当这些报告细胞启动EMT或MET时，耦合的剪接事件将导致荧光蛋白表达的变化，从而使我们的 微型共聚焦显微镜，用于评估微环境中的细胞状态。我们将从三个目标检验这一假设，即EMT剪接事件与乳腺癌微环境中的关键早期步骤相关并报告其在乳腺癌微环境中的侵袭和侵袭：1)设计乳腺肿瘤细胞以报告EMT/MET相关的选择性剪接事件；2)使用现有的手持DAC显微镜监测和验证EMT/MET报告基因在人乳腺组织外植体和异位小鼠模型系统中标记的乳腺癌细胞中的表达；以及3)将DAC显微镜作为植入式设备来评估EMT/MET相关的原位大鼠乳腺癌模型肿瘤微环境中的剪接事件。这项工作将导致可植入显微镜的开发，用于连续、高分辨率、多参数成像和可切换的分子记者，以揭示活体动物模型的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.