High-throughput on-chip platform for interrogation of mitochondrial membrane potential and apoptosis

用于检测线粒体膜电位和细胞凋亡的高通量芯片平台

基本信息

批准号：
9223679
负责人：
PETER J. BURKE
金额：
$ 41.51万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-02-01 至 2019-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9223679
关键词：
Antineoplastic Agents Apoptosis Apoptosis Inhibitor Apoptotic Area BCL2 gene BH3 peptide Biological Assay Biological Markers Calcium Cancer Biology Cancer Patient Cell Count Cell Death Cell Death Signaling Process Cell Proliferation Cells Cellular biology Chemicals Chemotherapy-Oncologic Procedure Clinical Trials Cultured Cells DNA Damage Defect Digitonin Effectiveness Electrodes Electron Transport Electrophysiology (science)Engineering Environment Etoposide Family Fluorescence Fluorescence Microscopy Frequencies Helper-Inducer T-Lymphocyte Human Individual Induction of Apoptosis Ion Channel Life Lipid Bilayers Location Malignant Neoplasms Mediating Membrane Membrane Potentials Metabolic Metabolism Methods Mitochondria Mitochondrial Membrane Protein Modeling Mus Muscle Fibers Mutation Myoblasts Nanotubes Oxidative Stress Pathway interactions Patients Peptides Permeability Pharmaceutical Preparations Pharmacology Primary Neoplasm Process Progression-Free Survivals Proteins Reader Regulation Relapse Research Resistance Signal Transduction Stem cells Survival Rate TP53 gene Techniques Technology Tissues Tumor Cell Line Tumor Tissue Work apoptosis deregulation base cancer stem cell cell growth chemotherapy clinical diagnostics cytokine experience high throughput screening high throughput technology human embryonic stem cell inhibitor/antagonist instrumentation mitochondrial membrane mitochondrial metabolism mouse model mutant nanochannel nanofluidic neoplastic cell novel therapeutics personalized medicine pluripotency public health relevance response screening statistics stem cell biology stem cell differentiation tool tumor metabolism tumor progression uncontrolled cell growth

项目摘要

DESCRIPTION (provided by applicant): In the proposed studies, we aim to develop and validate technology to allow for high-throughput assays of inhibitors and inducers of mitochondrial depolarization, the point of no return for apoptosis. As apoptosis is a key unregulated pathway in tumor progression, its study and ultimately control is of paramount important in cancer biology. Preliminary studies by some of us have shown a correlation between patient cancer progression-free survival rate and the response of mitochondria (depolarization of the inner membrane, permeabilization of the outer membrane) to pro-apoptotic BH3 peptides in digitonin permeabilized patient primary tumor cells. We also have shown that the patients whose mitochondria are more easily depolarized with BH3 peptides respond better to chemotherapy. In proof of concept work, we demonstrated an on-chip nanofluidic technology that was able to trap individual, living mitochondria isolated from cell and tissues, and to interrogate the membrane potential using potential sensitive fluorescence probes of these individual mitochondria in response various chemical environments, including substrates and inhibitors of the electron transport chain, as well as calcium challenges which resulted in mitochondrial flickering and depolarization. In addition, our initial work demonstrated in preliminary studies the electrical sensing of the opening and closing of individual ion channels in lipid bilayers using nanotube electrodes. In the proposed studies we will develop and validate this technology to sense the opening and closing of individual ion channels in mitoplasts and mitochondria, study their statistics and timing, and to develop a platform to enable, ultimately, high throughput assays of the electrophysiology of the mitochondrial electron transport chain and membrane depolarization at the single ion channel level. The technology will be transformative in three ways: First, it will validate a qualitatively new assay for study of apoptosis. Deregulation of apoptosis is well known as one of 6 "hallmarks" of cancer, however, methods to study mitochondrial depolarization have been lacking. Second, it will allow for a qualitatively new way to study electrophysiology at the single ion channel level. This will allow unprecedented studies of timing, location, and statistics of the mitochondrial membrane flickering and depolarization. Finally, the overall technology will enable new studies of mitochondrial metabolism. Cancer metabolism is one of the 2 new "hallmarks" added last year to cancer, and this instrumentation will enable the understanding of one important component of metabolic flux (namely, membrane potential). Because our high throughput technology will enable assays of thousands of individual mitochondria from small numbers of cells (even a single cell), it will enable an advance in instrumentation to study the interrelationships between metabolism, stem cells, and cancer biology.

描述（由适用提供）：在拟议的研究中，我们旨在开发和验证技术，以允许对线粒体沉积的抑制剂和影响的高通量评估，这是凋亡的回报点。由于凋亡是肿瘤进展中的关键不受管制的途径，因此其研究和最终控制在癌症生物学中至关重要。我们中的一些人的初步研究表明，患者癌症进展的无生存率与线粒体（内膜去极化，外膜的透化）与促凋亡的BH3 Pepperides在digitonin pepere蛋白通透性患者原主肿瘤中的反应之间存在相关性。我们还表明，通过BH3 Pepperides更容易部署线粒体的患者对化学疗法的反应更好。在概念验证工作中，我们展示了一种芯片纳米流体技术，该技术能够捕获个体，从细胞中分离出来的线粒体和组织，并使用这些单个线粒体的潜在敏感荧光项目在各种化学环境中（包括电子传输链的底物和抑制剂）以及钙挑战，以及导致线粒体闪烁和沉积的钙挑战。此外，我们最初的工作证明了在初步研究中在脂质双层中使用纳米管电极。在拟议的研究中，我们将开发和验证这项技术，以感知在线粒体和线粒体中的单个离子通道的开放和关闭，研究其统计数据和时机，并开发一个平台，以实现线粒体传输链链和膜型层级级别的单位型电动机电脑生理学的高吞吐量刺客。该技术将以三种方式进行变革：首先，它将验证质细胞增多症研究的定性评估。凋亡的放松管制是癌症的6个“标志”之一，但是，研究线粒体沉积的方法一直缺乏。其次，它将允许一种在单个离子通道水平上研究电生理学的新方法。这将允许对线粒体膜闪烁和去极化的定时，位置和统计的前所未有的研究。最后，整体技术将使线粒体代谢的新研究。癌症代谢是去年添加到癌症的两个新“标志”之一，该仪器将能够理解代谢通量的一个重要组成部分（即膜电位）。由于我们的高通量技术将从少量细胞（甚至是单个细胞）中实现数千个单个线粒体的测定，因此它将能够在仪器中进行进步，以研究代谢，干细胞和癌症生物学之间的相互关系。