Live Cell Interferometry for Quantifying Mass Transport in Cancer

描述(由申请人提供)：毛罗·费拉里(Mauro Ferrari)(1)将癌症描述为细胞内的多尺度运输问题，并指出，“细胞质内运输的物理学，如运输到不同的亚细胞位置……需要更深入的理解。”为此，需要新的技术来研究单细胞中的质量传输，以便1-回答关于癌症对细胞质内传输的影响的基本问题，以及2-设计新的治疗剂来攻击癌细胞内改变的质量传输机制。为了解决细胞质内运输的物理学问题，我提议全面开发一种新的方法，使通过细胞质的质量运输与特定标记蛋白质的运输联系起来的分析成为可能。我们的活细胞干涉测量系统已经以亚细胞分辨率实时跟踪数百个细胞的单细胞质量变化。我将增加同步高分辨率荧光功能，将质量传输与特定细胞成分的传输联系起来。这将使时间成为研究传统静态快照方法之外的大规模运输研究的基本第四维度。我将首先将这种新方法应用于HeLa、HEK293T和A549人类细胞系的质量运输和连接蛋白转位的研究，作为系统优化的模型和建立实用程序。在项目的第二阶段，我将使用活细胞干涉仪研究人类急性髓系白血病(AML)的小鼠初级模型中的质量传输，为这种方法在癌症中的应用提供翻译相关性。这将包括对Wiskott-Aldrich综合征蛋白WASP和N-WASP的沉默发夹RNA(ShRNA)敲除和EGFP标记的研究，这两种蛋白是细胞运输机械的已知组件，广泛运输，分别参与调节细胞质和细胞核内的肌动蛋白细胞骨架网络。我的目标是了解细胞骨架调节成分在自然和变化的环境条件下如何影响细胞内的质量运输，远远超过使用非定位肌动蛋白或微管解离剂的标准研究。最重要的是，我将沉浸在一个专注的、全面的指导培训计划中，重点是癌细胞生物学、技术开发和应用。该计划将包括癌症和细胞生物学方面的课程和研讨会，以及由个人和指导委员会提供的正式指导，这些指导来自癌症/细胞生物学和光学/显微镜、电子学和流体学工程学科的领先专家。这些严格的培训内容与我在斯坦福大学攻读博士期间获得的广泛的分子生物学和工程学课堂背景相辅相成。与一个有重点的跨学科研究项目一起，这个有组织的培训和指导计划将帮助我进入癌症细胞生物学的早期职业独立研究员职位，并在转化医学基础研究的靶向技术开发中得到强有力的应用。

DESCRIPTION (provided by applicant): Mauro Ferrari (1) describes cancer as a multi-scale transport problem inside cells and notes that, "the physics of transport within the cytoplasm, such as trafficking to different subcellular locations...requires much deeper understanding." Towards this end, new techniques are needed to study mass transport in single cells in order to 1- answer fundamental questions about the effects of cancer on transport within the cytoplasm, and 2- design novel therapeutic agents to attack altered mass transport mechanisms within cancer cells. In order to address the physics of transport within the cytoplasm, I propose to fully develop a novel approach that enables the analysis of mass transport through the cytoplasm linked to the trafficking of specifically tagged proteins. Our live cell interferometry system already tracks single cell mass changes in real time for hundreds of cells simultaneously with sub-cellular resolution. I will add simultaneous high-resolution fluorescence capabilities to link mass transport to the transport of specific cellular components. This will establish time as an essential 4th dimension for mass transport studies beyond traditional static snap-shot based approaches. I will apply this new approach to studies of mass transport and linked protein translocation in HeLa, HEK293T, and A549 human cell lines first as a model for system optimization and to establish utility. In the project second phase, I will use the live cell interferometer to study mass transport in a mouse primary model of human acute myeloid leukemia (AML) to provide translational relevance for this approach in cancer. This will include silencing hairpin RNA (shRNA) knockdown and eGFP-tagged studies of the Wiskott-Aldrich Syndrome proteins WASP and N-WASP, which are known components of the cellular transport machinery that traffic widely and are involved in regulation of the actin cytoskeleton network inside the cytoplasm and nucleus, respectively. My goal is to understand how cytoskeleton regulatory components affect, and are affected by, the transport of mass within cells under native and altered environmental conditions, far beyond standard studies that employ non-localized actin or microtubule dissociating agents. Most importantly, I will be immersed in a focused, comprehensive mentored training program with an emphasis on cancer cell biology, technology development, and applications. This program will include coursework and seminars in cancer and cell biology and formal mentoring, by individuals and a mentoring committee, from leading experts in cancer/cell biology and engineering disciplines in optics/microscopy, electronics and fluidics. These rigorous training components complement my extensive classroom background in molecular biology and engineering acquired during my Ph.D. studies at Stanford. Together with a focused, interdisciplinary research project, this structured training and mentored program will help propel me into an early career independent investigator position in cancer cell biology with powerful applications in targeted technology development for fundamental investigations in translational medicine.