CEGS: Microscale Life Sciences Center

描述(由申请人提供)：越来越明显的是，理解、预测和诊断疾病状态被原位细胞群体固有的异质性所混淆。细胞命运的这种变化可能是戏剧性的，例如，一个细胞活着，而相邻的细胞死亡。因此，为了了解疾病状态所涉及的基本途径，有必要在单个细胞水平上将先前存在的细胞状态与疾病过程中的细胞命运联系起来。华盛顿大学的微尺度生命科学中心(MLSC)致力于解决这一问题，通过开发用于高通量基因组水平和多参数单细胞分析的尖端微尺度技术，并将该技术应用于生物学和健康的基本问题。我们的愿景是直接在个体细胞水平上解决疾病状态的途径，在不断增加的复杂性水平上逐步转移到体内对疾病的理解。我们建议将MLSC技术创新应用于关注细胞增殖和细胞死亡之间的平衡的问题。美国前三大杀手癌症、心脏病和中风都与细胞决策过程的不平衡有关。由于生死决策中固有的细胞异质性，这个基本的细胞生物学问题是一个例子，对单个细胞的分析对于发展基因组学、细胞功能和疾病之间的联系是必不可少的。要研究的特定系统是小鼠巨噬细胞模型中的促炎细胞死亡(下垂)，以及肿瘤 巴雷特食道(BE)癌前病变模型进展。在每种情况下，特定细胞状态的诊断特征将通过测量生理参数(细胞周期、倍体、呼吸速率、膜电位)和基因组参数(单细胞蛋白质组学、qRT-PCR和转录组学的基因表达谱；晚期-PCR的杂合性缺失)参数来确定。然后，在实施挑战后，这些指标将通过相同的测量集合与细胞命运相关，例如，针对下垂的细胞死亡刺激或针对BE的易感风险因素挑战(酸反流)。最终，时间序列将被用来绘制出决定生死的路径。最后，这些信息将被用于 作为一个在单细胞水平上定义细胞间相互作用的平台，将关于疾病途径的信息推向更大的体内相关性。将开发新技术并将其集成到现有的MLSC活细胞分析盒系统中，以支持这些雄心勃勃的生物学目标，包括1)用于细胞放置的自动化系统、芯片外设备互连和具有用户友好界面的高通量数据分析；2)基于新检测平台的新型光学和电子传感器、新型染料和纳米线；以及3)用于单细胞qRT-PCR、Late-PCR的新微模块，包括单细胞焦测序、芯片上单细胞蛋白质组学和使用条形码纳米棒的单细胞转录组学。

DESCRIPTION (provided by applicant): Increasingly, it is becoming apparent that understanding, predicting, and diagnosing disease states is confounded by the inherent heterogeneity of in situ cell populations. This variation in cell fate can be dramatic, for instance, one cell living while an adjacent cell dies. Thus, in order to understand fundamental pathways involved in disease states, it is necessary to link preexisting cell state to cell fate in the disease process at the individual cell level. The Microscale Life Sciences Center (MLSC) at the University of Washington is focused on solving this problem, by developing cutting-edge microscale technology for high throughput genomic-level and multi-parameter single-cell analysis, and applying that technology to fundamental problems of biology and health. Our vision is to address pathways to disease states directly at the individual cell level, at increasing levels of complexity that progressively move to an in vivo understanding of disease. We propose to apply MLSC technological innovations to questions that focus on the balance between cell proliferation and cell death. The top three killers in the US, cancer, heart disease and stroke, all involve an imbalance in this cellular decision-making process. Because of intrinsic cellular heterogeneity in the live/die decision, this fundamental cellular biology problem is an example of one for which analysis of individual cells is essential for developing the link between genomics, cell function, and disease. The specific systems to be studied are proinflammatory cell death (pyroptosis) in a mouse macrophage model, and neoplastic progression in the Barrett's Esophagus (BE) precancerous model. In each case, diagnostic signatures for specific cell states will be determined by measuring both physiological (cell cycle, ploidy, respiration rate, membrane potential) and genomic (gene expression profiles by single-cell proteomics, qRT-PCR and transcriptomics; LOH by LATE-PCR) parameters. These will then be correlated with cell fate via the same sets of measurements after a challenge is administered, for instance, a cell death stimulus for pyroptosis or a predisposing risk factor challenge (acid reflux) for BE. Ultimately, time series will be taken to map out the pathways that underlie the live/die decision. Finally, this information will be used as a platform to define cell-cell interactions at the single-cell level, to move information on disease pathways towards greater in vivo relevance. New technology will be developed and integrated into the existing MLSC Living Cell Analysis cassette system to support these ambitious biological goals including 1) automated systems for cell placement, off-chip device interconnects, and high throughput data analysis with user friendly interfaces; 2) new optical and electronic sensors based on a new detection platform, new dyes and nanowires; and 3) new micromodules for single-cell qRT-PCR, LATE-PCR for LOH including single-cell pyrosequencing, on-chip single-cell proteomics, and single-cell transcriptomics using barcoded nanobeads.