Multiparameter Assay for Mechanistic Understanding of Carcinogens

描述(申请人提供)：实验化合物中致癌活性的测定是一个漫长而昂贵的过程，对许多核心行业产生负面影响，并可能威胁人类健康。需要更快、更可靠的方法来准确确定人类致癌活性，以快速识别环境问题，减少对动物模型的依赖。致癌物的多效性使它们很难用测量单个细胞变化的标准高通量分析来检测。这个问题的一个解决方案是同时监测细胞功能的几个方面。虽然这些方法具有适当的灵敏度，但由于漫长而复杂的样品制备过程降低了吞吐量，并显著增加了分析的可变性，因此它们的实施一直很困难。我们在这笔赠款中提出的解决方案是使用结合细胞条形码的流式细胞术对一个样本中的细胞反应进行多路测量。在这个系统中，多个生物传感器被编码在一个样本中，以提供细胞对致癌物反应的九种不同测量。此外，还可以获得每个样本的细胞活性和细胞周期状态，从而能够对化合物活性进行多维分析。该系统得益于多路测量灵敏度的提高，同时保持了均质高通量分析的精确度。 与公共卫生相关：化学致癌物引起细胞DNA和新陈代谢的复杂变化，使得使用单一终点分析难以识别致癌物。这里描述的平台使用已知的致癌信号通路的高含量、多路测量来改进临床和环境样本中致癌物的检测，并提供关于其作用机制的信息。

DESCRIPTION (provided by applicant): The determination of carcinogenic activity in experimental compounds is a long and expensive process that negatively impacts many core industries and can threaten human health. Faster, more reliable methods that can be used to accurately determine human oncogenic activity are needed to rapidly identify environmental problems and reduce the reliance on animal models. The pleiotropic effects of carcinogens have made them difficult to detect using standard high-throughput assays that measure a single cellular change. A solution to this problem is to monitor several aspects of cell function simultaneously. Although these methods have the appropriate sensitivity, their implementation has been difficult due to long and complicated sample preparation procedures which decrease the throughput and dramatically increase the assay variability. The solution we propose in this grant is the multiplexed measurement of cellular responses in one sample using flow cytometry combined with cellular barcoding. In this system, multiple biosensors are encoded in a single sample to provide nine distinct measures of cellular responses to carcinogens. In addition, cell viability and cell cycle status are obtained for each sample, enabling multi-dimensional analysis of compound activity. This system benefits from the increased sensitivity of multiplexed measurement while retaining the precision of a homogeneous high-throughput assay. PUBLIC HEALTH RELEVANCE: Chemical carcinogens induce complex changes in cellular DNA and metabolism, making it difficult to identify carcinogens using single end-point assays. The platform described here uses high-content, multiplexed measurements of known oncogenic signaling pathways to improve the detection of carcinogens in clinical and environmental samples and provide information about their mechanism of action.