Clinical application of multispectral imaging flow cytometry

描述（由申请人提供）：细胞内稳态和反应性变化取决于细胞质和核质之间的信号转导。在癌症中，通常观察到异常信号转导，并且靶向这些信号级联的新型治疗剂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传导中间体的异常细胞内分布相关，因此它们的亚细胞定位可潜在地用作治疗反应的参数。传统上，分子的细胞内定位已经通过分子技术或免疫标记技术（共聚焦）显微镜研究。这些方法的缺点是缺乏对靶细胞群的特异性或缺乏评估统计学稳健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）已开发用于以高达每秒100个细胞的速率生成悬浮液中制备的细胞的高分辨率明场、暗场和荧光图像。IDEAS（tm）分析软件根据每个细胞的图像，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。本申请的目的是评价多光谱成像流式细胞术在评价靶细胞特异性、治疗诱导的特定信号传导途径中间体的核质分布变化（作为临床样本中的响应参数）方面的潜力。拟议的研究最终将与I期临床试验结合进行，该临床试验在接受大环内酯雷帕霉素（西罗莫司）治疗的急性髓性白血病（AML）患者中进行，已知雷帕霉素会影响核因子-KB（NF-κ B）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，以使用细胞系模型系统高效准确地测定NF-κ B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蛋白质的其他易位的适用性，如果成功，将导致在本申请的R33阶段测试其他假设。R33阶段将在患者样本分析中应用该技术，并将测试NF-icB的细胞分布可用作雷帕霉素治疗的AML患者的反应决定因素的假设，还将测试R21阶段产生的其他假设。概述：通过高速分析癌细胞图像，ImageStream细胞仪可以提供特定癌症治疗反应成功或失败的具体信息。拟议研究的目的是确定对该仪器的硬件和分析软件进行必要的调整，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.