Dynamic Imaging of EMT in the Breast Cancer Microenvironment

乳腺癌微环境中EMT的动态成像

基本信息

批准号：
9262882
负责人：
FRANK B GERTLER
金额：
$ 17.05万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-07-05 至 2017-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9262882
关键词：
3-Dimensional Alternative Splicing Animal Model Animals Architecture Back Biological Models Blood Circulation Breast Breast Cancer Cell Breast Cancer Model Breast Carcinoma Breast Epithelial Cells Breast cancer metastasis Caliber Cells Complex Confocal Microscopy Coupled Cues Cytometry Development Developmental Process Devices Disease Marker Distant Drug resistance Ear Engineering Epithelial Cells Event Exons Fatty acid glycerol esters Genes Goals Grant Human Image Implant Individual Intervention Label Lead Length MLPH gene Mammary Gland Parenchyma Mammary gland Measures Mesenchymal Methods Microscope Microscopic Miniaturization Modeling Molecular Molecular Target Monitor Mus Neoplasm Circulating Cells Neoplasm Metastasis Operative Surgical Procedures Optical reporter Optics Organ Pattern Performance Periodicity Phenotype Population Primary Neoplasm Process RNA Splicing Rattus Reporter Reporting Resistance Resolution Rodent Model Scanning Side Signal Transduction Site Testing Therapeutic Intervention Time Tissues Treatment Efficacy Tumor Cell Invasion Tumor Escape Tumor-Associated Vasculature Work base cancer cell cancer imaging cell motility design epithelial to mesenchymal transition fluorescence microscope imaging study implantable device implantation in vivo in vivo imaging intravital imaging malignant breast neoplasm metastatic process migration miniaturize molecular imaging mortality mouse model neoplastic cell next generation novel progression marker protein expression public health relevance response seal serial imaging therapy resistant tissue phantom tool tumor tumor microenvironment

项目摘要

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）：转移和对治疗的抵抗是乳腺癌最致命的方面，但是尚未完全了解导致癌细胞传播和抗性的肿瘤微环境中的状况。启动和维持这些过程的分子事件是作为肿瘤微型介绍中的罕见事件发生的，并揭示了微观环境中的这些早期，微妙的变化，将需要高分辨率，多参数的体内显微镜方法和记录器构建体，这些构建体可以表征会导致侵入，弹性，肠内和结肠的动态细胞事件。我们将监测带有光学记者的乳腺癌细胞，该细胞使用微型双轴共焦（DAC）荧光显微镜在培养物和两个动物模型中与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个标记，其新颖的结构使自由基的微型化有助于在啮齿动物模型中植入，以连续研究微环境。尽管这些设备用于体内成像的应用很广，但我们在这里着重于监视上皮对间质转变（EMT）和后级的关键步骤。 EMT是一个发育过程，其中有组织的上皮细胞转变为具有间质表型的分离的迁移细胞，并在转移过程中取决于关键步骤。我们已经确定了一组新型的与乳腺癌细胞EMT并MET相关的替代剪接事件。我们建议通过用可切换的双色荧光记者转导这些事件来设计这些事件，以报告这些事件，这些记者根据剪接模式指示EMT状态。当这些报告基细胞启动EMT或MET时，耦合的剪接事件将导致荧光蛋白表达的变化，从而使我们微型共聚焦显微镜评估微环境中的细胞状态。我们将测试以下假设：EMT剪接事件与三个目标的乳腺癌微环境相关并报告的关键早期步骤，从而导致乳腺癌微环境内部入侵和静脉内部：1）工程师乳腺肿瘤细胞以报告EMT/与Met相关的替代剪接事件报告，2）2）使用EMT/Met ember yember yemt andern hyment Synem in tage in noffer yemt/met comment throume throume throume throume throume tossext hument hymer andere tock tocking hymer tock tocking hyme nounder tock，并在鼠标中表达鼠标探索的探索方法显微镜和3）将DAC显微镜作为一种可植入的装置，以评估原位大鼠乳腺癌模型的肿瘤微环境中EMT/MET相关的剪接事件。这项工作将导致开发可植入的显微镜，用于串行，高分辨率，多参数成像和可切换的分子记者，这些记者揭示了活动物模型肿瘤微环境中动态过程。