Validation of a single cell multiplexed in-situ biomarker analysis platform for p

描述(申请人提供)：GE Global Research的一个多学科团队(工程师、生物学家、图像分析师、统计学家)和范德比尔特大学的癌症/细胞生物学家将对福尔马林固定石蜡包埋(FFPE)正常和肿瘤结肠组织进行原位单细胞分析。到目前为止，在原位组织环境中对四个以上的蛋白质签名进行多路组合分析是不可能实现的。 GE Global Research在过去六年中开发了一种独特的方法，可以用60或更多的染料结合抗体反复对单个FFPE组织切片进行染色，而不会丢失这些抗原的信号。这使得高分辨率单细胞分割(四个或更多)所需的标记与标记相结合，以重新确定细胞表型和信号活动。已经开发了绘图软件来关联单个细胞内的特定表达模式，以产生对单个细胞表型的单细胞多维分析。该项目的总体目标是促进将这项技术从研究原型转化为世界各地多个实验室和医院普遍使用的系统。这种方法独特地允许单个细胞在自然组织环境中达到高水平，从而允许确定细胞之间的关联。这个项目将研究重要的生物学原理，如果没有这种分析单个细胞的多个标记物的高分辨率技术是不可能的。在第一项研究中，两个干细胞群体具有独特的增殖和信号传递能力，分别代表不同的可识别的 小鼠结肠中的细胞种群将通过先前建立的在该系统中起作用的标记来表征。在第二项研究中，这些细胞群在结肠癌中的变化将在小鼠模型中进行测试，这些模型通过基因靶向在干细胞中特异性地激活结肠癌。最后，这些研究将扩展到检查人类结肠癌中干细胞群体的性质，并将与疾病进展和复发相关的已知途径活动与这些细胞相关联。该项目包括增加新的技术，通过增加RNA分析(miRNA和信使核糖核酸FISH)来扩展平台以包括基因组特征。将对现有的自动化系统进行改进，以便更广泛地使用这项技术，并增加样品处理。这些研究的结果将使具有不同细胞成分的复杂细胞环境的定义方式发生范式转变，而不会丢失其他需要分散组织元素的单细胞分析方法中出现的信息。

DESCRIPTION (provided by applicant): A multidisciplinary team from GE Global Research (engineers, biologists, image analysts, statisticians) together with cancer/cell biologists from Vanderbilt University will validate in-situ single cell analysis of formalin fixed paraffin embedde (FFPE) normal and neoplastic colonic tissue. Multiplexed combinatorial analysis of more than four protein signatures within in situ tissue context has been, up till now, impossible to achieve. GE Global Research has developed over the past six years a unique method of iteratively staining individual FFPE tissue sections with 60 or more dye conjugated antibodies without loss of signal for these antigens. This has enabled markers needed for high-resolution single cell segmentation (four or more) to be combined with markers to re- fine cellular phenotype and signaling activities. Mapping software has been developed to correlate specific expression patterns within individual cells to produce single cell multidimensional analysis of individual cel phenotypes. The overall goal of the project is to foster translation of this technology from a research prototype into a system that is commonly used in multiple labs and hospitals around the world. This approach uniquely allows individual cell characterization to a high level within th native tissue context, thereby allowing associations between the cells to be determined. This project will examine important biological principles not possible without this high-resolution technique for analysis of multiple markers of individual cells. In the first study two stem cell populations with unique proliferative and signaling capacities that represent separate identifiable cell populations will be characterized in the mouse colon with markers previously established to work in this system. In the second, changes within these cell populations in colon cancer will be tested in mouse models that activate colon cancer specifically in the stem cells with genetic targeting. Finally, these studies will be extended to examine the nature of the stem cell population within human colon cancer and to correlate known pathway activities relevant to disease progression and recurrence to these cells. This project includes the addition of new technologies that expand the platform to include genomic characterization by adding RNA analysis (miRNA and mRNA FISH). Improvements will be made to an existing automated system to allow broader use of the technology and in- creased sample processing. The results of these studies will enable a paradigm shift in how complex cellular environments with diverse cellular elements are defined, without the loss of information that occurs in other single cell analysis methods that require dispersal of tissue elements.