MOLECULAR PROFILING OF CELLS BY LASER SCANNING CYTOMETRY

这是一个垂直整合的项目，将推进激光扫描细胞术（LSC）的新兴技术，使其能够a）进行临床相关的组织分析研究，包括人类实体瘤中每个样本的50-100个荧光和免疫荧光测量，分组为每个细胞5-10个相关测量的组，每组约5，000个细胞，以及B）使用假设检验和/或探索性方法，能够对数据进行广泛的分析。 从市售仪器（CompuCyte Corp.，剑桥，MA）和先前针对淋巴组织开发的方法，在R-21阶段，我们将a）开发适合于上皮肿瘤的细胞制备方法，B）确定用于识别和勾画单个细胞轮廓以进行多参数分析的最佳初始测量（光散射相对于细胞角蛋白或微管蛋白），c）显影一种或多种染料组合以用作用于随后显影另外的染色板的模板，每个包含每个细胞4-6个相关的测量值，和d）确定可以用每个细胞最多5个另外的荧光探针再访问和再染色单个细胞的条件。 在R-33阶段，我们将a）开发一组核心的5-10个用于组织分析的免疫荧光板，每个板由每个细胞5-10个测量组成，再染色，使用在非交叉反应性和结合亲和力方面已经优化的抗体，B）开发软件，该软件将具有捕获、预处理、显示、分析、存储，并导出由相关、部分相关和不相关数据的混合物组成的混合数据集，c）通过添加额外的激光器和进行定制染料开发以增加每个细胞的潜在相关测量的数量来扩展CompuCyte仪器的能力，以及d）扩展与我们机构内的临床部门的交互，预计将设计具体的转化临床研究，以探索用于预后和治疗计划的组织分析，并在本资助期结束前启动此类研究。

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.