Clinical application of multispectral imaging flow cytometry

描述（由申请人提供）：细胞稳态和反应性变化依赖于细胞质和核质之间的信号转导。在癌症中，异常信号转导是常见的，针对这些信号级联的新疗法目前正在临床试验中进行评估，或者已经在治疗武器库中获得了一席之地。异常信号转导通常与特定信号中介的异常细胞内分布有关，因此它们的亚细胞定位可能被用作治疗反应的参数。传统上，分子的细胞内定位是通过分子技术或共聚焦显微镜下的免疫标记技术来研究的。这些方法的缺点是缺乏对目标细胞群的特异性或评估统计上可靠分析所需的大细胞群的能力。ImageStream 100多光谱成像流式细胞仪（IS 100）已开发出高分辨率的明场、暗场和荧光图像，以高达100个细胞/秒的速率制备悬浮细胞。IDEAS（tm）分析软件根据其图像对每个细胞的200多个形态和光度参数进行量化，包括测量探针亚细胞位置的参数。本应用的目的是评估多光谱成像流式细胞术的潜力，以评估靶细胞特异性，治疗诱导的特定信号通路中间体核细胞质分布的变化作为临床样品中的反应参数。拟议的研究最终将与急性髓性白血病（AML）患者的I期临床试验一起进行，该患者接受大环内酯雷帕霉素（西罗莫司）治疗，已知大环内酯雷帕霉素会影响核因子kb （NF-icB）的功能（和细胞定位）。在本申请的R21阶段，将优化系统硬件、软件和样品制备条件，以便使用细胞系模型系统高效准确地确定NF-icB易位事件。还将评估检测与NF-icB信号转导和雷帕霉素作用相关的其他蛋白质易位的适用性，如果成功，将在该应用的R33阶段测试其他假设。R33期将应用该技术分析患者样本，并将测试NF-icB的细胞分布可作为雷帕霉素治疗AML患者反应的决定因素的假设，还将测试theR21期产生的其他假设。通过高速率分析癌细胞图像，ImageStream细胞仪可以提供特定癌症治疗反应成功或失败的特定信息。拟议研究的目的是确定仪器硬件和分析软件的必要调整，以便在临床研究环境中准确有效地使用它。

DESCRIPTION (provided by applicant): Cellular homeostasis and reactive changes depend on signal transduction between the cytoplasm and nucleoplasm. In cancer, aberrant signal transduction is commonly observed and novel therapeutics targeting these signaling cascades are currently evaluated in clinical trials or have already secured a position in the therapeutic arsenal. Aberrant signal transduction is often associated with abnormal intracellular distribution of specific signaling intermediaries thus their sub-cellular localization could potentially be used as a parameter of treatment response. Traditionally, intracellular localization of molecules have been studied by molecular techniques or by immunological labeling techniques studied with (confocal) microscopy. These approaches have the disadvantage of lacking specificity for target cell populations or the ability to evaluate large cell populations required for statistically robust analyses. The ImageStream 100 multispectral imaging flow cytometer (IS 100) has been developed to produce high resolution brightfield, darkfield, and fluorescence images of cells prepared in suspension at rates up to 100 cells per second. The IDEAS(tm) analysis software quantifies over 200 morphometric and photometric parameters for each cell based on its imagery, including parameters that measure sub-cellular location of probes. The aim of this application is to evaluate the potential of multispectral imaging flow cytometry to evaluate target cell-specific, therapy-induced changes in nuclear-cytoplasmic distribution of specific signaling pathway intermediaries as response parameters in clinical samples. The proposed studies will ultimately be conducted in conjunction with a phase I clinical trial in patients with acute myeloid leukemia (AML) undergoing therapy with the macrolide rapamycin (sirolimus) which is known to affect the function (and cellular localization) of nuclear factor-KB (NF-icB). During the R21 phase of this application, system hardware, software and sample preparation conditions will be optimized to efficiently and accurately determine NF-icB translocation events using cell line model systems. Applicability of detecting other translocations of proteins associated with NF-icB signal transduction and rapamycin action, will also be assessed and if successful will lead to testing of additional hypotheses in the R33 phase of this application. The R33 phase will apply this technique in the analysis of patient samples and will test the hypothesis that cellular distribution of NF-icB can be used as a determinant of response in AML patients treated with rapamycin and will also test additional hypotheses generated in theR21 phase. Lay Summary: By analyzing images of cancer cells at a high rate the ImageStream cytometer can provide specific information on the success or failure of specific cancer treatment responses. The aim of the proposed studies is to determine necessary adjustments to this instrument's hardware and analysis software to accurately and efficiently use it in a clinical research setting.