High-Throughput Screening Under Static or Dynamic Hypoxia

摘要 在许多人类肿瘤中缺氧的存在是广泛观察到的负面预后指标。 动物模型和体外系统的实验室实验与临床观察一致 表明缺氧是癌症进展的主要驱动力。由于缺氧难以控制， 在动物模型中进行测量，体外方法为详细的分子分析提供了最好的机会。 氧对癌症发生、发展的影响及分析。然而，临床肿瘤显示出 氧气水平的范围与一些地区是慢性缺氧，而其他地区的经验时间- 依赖性变化，即循环缺氧。这进一步使缺氧反应的系统分析复杂化 因为实际上存在可以探测的无限数量的静态和循环缺氧模式。到目前为止， 循环缺氧的研究通常限于探测单一的、任意选择的循环模式， 与临床无关。为了便于筛选慢性和动态变化的缺氧，我们 我建议开发一种技术，可以提供一系列的缺氧水平，无论是静态或循环，每个 一排标准多孔组织培养板。这可以监测分子适应，细胞生长 和药物反应。初步研究将 在与当前多通道兼容的标准96孔板格式上演示该技术 移液器、读板器和实验室协议，允许立即采用任何肿瘤实验室。 相同的原理可用于任何多孔板，以帮助评估氧对细胞生长的影响 以及生存力、肿瘤进展、生物标志物识别和治疗反应。没有电流 技术，允许这些类型的调查在一个标准的高通量格式。这项技术 解决了RFA中至少两个感兴趣的领域，因为它将有助于（i）阐明基本机制 潜在的癌症起始和进展，以及（ii）促进/加速药物发现的过程。

ABSTRACT The presence of hypoxia in a many human tumors is a widely observed negative prognostic indicator. Laboratory experiments in animal models and in vitro systems are consistent with the clinical observations showing that hypoxia is a major driving force in cancer progression. Since hypoxia is difficult to control and measure in animal models, in vitro approaches offer the best opportunity for detailed molecular analysis of the influence of oxygen on cancer initiation, progression and analysis. However, clinical tumors display a range of oxygen levels with some regions that are chronically hypoxic whereas other regions experience time- dependent changes, i.e. cycling hypoxia. This further complicates systematic analysis of hypoxic responses as there are literally an infinite number of static and cycling hypoxia patterns that can be probed. To date, studies of cycling hypoxia usually are limited to probing a single, arbitrarily chosen cycling pattern that may not be clinically relevant. In order to facilitate screening of both chronic and dynamic changes in hypoxia, we propose to develop a technology that can deliver a range of hypoxia levels, either static or cycling, to each row of a standard multiwell tissue culture plate. This allows monitoring of molecular adaptation, cell growth and drug response under a range of oxygen concentrations simultaneously. The initial studies will demonstrate the technology on a standard 96-well plate format that is compatible with current multichannel pipettors, plate readers and laboratory protocols that allows immediate adoption to any oncology laboratory. The same principles can be used in any multiwell plate to aid in assessing the effect of oxygen on cell growth and viability, tumor progression, biomarker identification and therapy response. There is no current technology that allows these types of investigations in a standard high-throughput format. This technology addresses at least two areas of interest in the RFA as it will aid in (i) the elucidation of the basic mechanisms underlying cancer initiation and progression and (ii) facilitate/accelerate the processes of drug discovery.