Multiparameter Assay for Mechanistic Understanding of Carcinogens

描述（由申请人提供）：测定实验化合物的致癌活性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动，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难以用标准的高通量测定法来检测单个细胞的变化。解决这个问题的方法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，但由于样品制备过程漫长而复杂，从而降低了通量并显着增加了测定变异性，因此实施起来很困难。我们提出的解决方案是在一个样品中使用流式细胞术结合细胞条形码对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品中，以提供九种不同的细胞对致癌物的反应。此外，还可获得每个样品的细胞活力和细胞周期状态，从而实现化合物活性的多维分析。该系统受益于多路测量灵敏度的提高，同时保持了均匀高通量测定的精度。

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.