MOLECULAR PROFILING OF CELLS BY LASER SCANNING CYTOMETRY

这是一个垂直整合的项目，将推动新兴的激光扫描细胞仪(LSC)技术，从而：a)能够进行临床相关的组织图谱研究，包括对人体实体肿瘤的每个样本进行50-100个荧光和免疫荧光测量，分成每组约5,000个细胞中每个细胞5-10个相关测量的面板，以及，b)使用假设检验和/或探索性方法对数据进行广泛分析。从一台商用仪器(马萨诸塞州坎布里奇的CompuCyte公司)和以前为淋巴组织开发的多色方案开始，在R-21阶段，我们将a)开发适合于上皮性肿瘤的细胞准备方法，b)确定用于识别和绘制多参数分析的单个细胞的最佳初始测量结果(光散射与细胞角蛋白或微管蛋白)，c)开发一种或多种染料组合作为后续开发额外多色板的模板，每个细胞包含4-6个相关的测量结果，以及d)确定在什么条件下单个细胞可以再次访问并用每个细胞最多5个额外的荧光探针进行染色。在R-33阶段，我们将a)开发一套核心的5-10个多色免疫荧光面板用于组织分析，每个面板由5-10个带有限制的细胞的测量组成，使用针对非交叉反应和结合亲和力进行优化的抗体，b)开发能够捕获、预处理、显示、分析、存储和导出由相关、部分相关和不相关数据混合组成的混合数据集的软件，c)通过添加额外的激光和进行定制染料开发来扩展计算细胞仪器的能力，以增加每个细胞的潜在相关测量的数量，以及d)扩大与我们机构内临床部门的互动，以期设计具体的转化性临床研究，以探索组织图谱对预后和治疗计划的影响，并在本赠款期限结束时启动此类研究。

This is a vertically integrated project that will advance the emerging technology of laser scanning cytometry (LSC) to the point that it will a) enable the performance of clinically relevant tissue profiling studies consisting of 50-100 fluorescent and immunofluorescent measurements per sample in human solid tumors, grouped in panels of 5-10 correlated measurements per cell on each of approximately 5,000 cells per panel, and, b) enable extensive analysis of the data, using hypothesis-testing and/or exploratory approaches. Starting with a commercially available instrument (CompuCyte Corp., Cambridge, MA) and multicolor protocols previously developed for lymphoid tissues, during the R-21 phase we will a) develop cell preparatory methods that are suitable for epithelial tumors, b) identify the best initial measurements for identifying and contouring individual cells for multiparameter analysis (light scatter vs cytokeratin, or tubulin), c) develop one or more dye combinations to serve as templates for subsequent development of additional multicolor panels, each encompassing 4-6 correlated measurements per cell, and d) determine the conditions under which individual cells can be revisited and restained with up to 5 additional fluorescent probes per cell. During the R-33 phase we will a) develop a core set of 5-10 multicolor immunofluorescent panels for tissue profiling, each consisting of 5-10 measurements per cell with restaining, using antibodies that have been optimized with respect to non-crossreactivity and binding affinity, b) develop software that will have the capability to capture, preprocess, display, analyze, store, and export mixed data sets consisting of mixtures of correlated, partially correlated, and uncorrelated data, c) extend the capabilities of the CompuCyte instrument by adding additional lasers, and doing custom dye development to increase the number of potential correlated measurements per cell, and d) expand interactions with clinical departments within our institution, in anticipation of devising specific translational clinical studies to explore tissue profiling for prognosis and treatment planning, and launching such studies by the time this grant period has been completed.