Dynamic Imaging of EMT in the Breast Cancer Microenvironment

乳腺癌微环境中EMT的动态成像

• 批准号: 8577999
• 负责人: CHRISTOPHER H CONTAG
• 金额: $ 66.88万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-05 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8577999
• 关键词: 3-Dimensional; Alternative Splicing; Animal Model; Animals; Architecture; Back; Biological Models; Blood Circulation; Breast; Breast Cancer Cell; Breast Carcinoma; Caliber; Cells; Complex; Confocal Microscopy; Coupled; Cues; Cytometry; Development; Developmental Process; Devices; Disease Progression; Distant; Drug resistance; Ear; Engineering; Epithelial Cells; Event; Exons; Fatty acid glycerol esters; Genes; Goals; Grant; Human; Image; Implant; Individual; Intervention; Label; Lead; Length; Life; Mammary Gland Parenchyma; Mammary Neoplasms; Mammary gland; Measures; Mesenchymal; Methods; Microscope; Microscopic; Miniaturization; Modeling; Molecular; Molecular Target; Monitor; Mus; Neoplasm Circulating Cells; Neoplasm Metastasis; Optical reporter; Optics; Organ; Pattern; Performance; Phenotype; Population; Primary Neoplasm; Process; RNA Splicing; Rattus; Reporter; Reporting; Resistance; Resolution; Rodent Model; Scanning; Side; Signal Transduction; Site; Testing; Therapeutic Intervention; Time; Tissues; Tumor Cell Invasion; Tumor-Associated Vasculature; Work; base; cancer cell; cell motility; design; epithelial to mesenchymal transition; fluorescence microscope; implantable device; implantation; in vivo; intravital imaging; malignant breast neoplasm; metastatic process; migration; miniaturize; molecular imaging; mortality; mouse model; neoplastic cell; next generation; novel; protein expression; public health relevance; response; seal; therapy resistant; tissue culture; tissue phantom; tool; tumor; tumor microenvironment

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.

描述（由申请人提供）：转移和对治疗的抗性是乳腺癌最致命的方面，但导致癌细胞扩散和抗性的肿瘤微环境中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，并且揭示微环境中的这些早期细微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，其可以表征导致远处组织的侵袭、内渗和定殖的动态细胞事件。我们将使用微型双轴共聚焦（DAC）荧光显微镜监测乳腺癌细胞窝藏光学记者，信号与文化中的转移过程相关的关键分子事件，并在两个动物模型。这种多光谱显微镜能够检测疾病进展的多个标志物，其新颖的结构使彻底的小型化有利于植入啮齿动物模型，以持续研究微环境。虽然这些设备在体内成像的应用是广泛的，我们在这里集中监测上皮间质转化（EMT）和回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，并且是转移过程中的关键步骤的基础。我们已经确定了一组新的选择性剪接事件，特别是与乳腺癌细胞EMT和MET。我们建议工程乳腺上皮细胞报告这些事件，通过转导他们与可切换的双色荧光报告，表明EMT状态的基础上剪接模式。当这些报告细胞启动EMT或MET时，偶联的剪接事件将导致荧光蛋白表达的变化，从而使我们的研究得以进行。 微型共聚焦显微镜来评估微环境内的细胞状态。我们将测试EMT剪接事件与乳腺癌微环境中导致侵袭和内渗的关键早期步骤相关的假设，并报告三个目标：1）工程化乳腺肿瘤细胞以报告EMT/MET相关的可变剪接事件，2）监测和验证EMT/使用现有的手持式DAC显微镜，在人乳腺组织外植体和异位小鼠模型系统内标记的乳腺癌细胞中MET报告基因表达，和3）使DAC显微镜适合作为可植入装置以评估原位大鼠乳腺癌模型的肿瘤微环境中的EMT/MET相关剪接事件。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报告，揭示活体动物模型肿瘤微环境内的动态过程。