Single-Cell Measurement of Lipid Signaling in Colorectal Cancer

描述（由申请人提供）：越来越需要能够从疾病模型和患者中评估结直肠肿瘤脂质信号的检测方法。这种需求是由于针对异常信号转导途径的分子靶向治疗的出现而产生的，特别是那些涉及磷酸肌醇-3激酶（PI3K）的分子靶向治疗，现在已知PI3K在癌症的发生和发展中起重要作用。测量失调的脂质信号蛋白的酶活性的能力对于指导治疗方案以及评估个体患者的治疗效果非常有利。一项跨学科合作研究计划被提议创建仪器和化学工具，以直接评估在结直肠癌细胞系和患者源性转移性结直肠癌活细胞中，PI3K和相关酶对磷脂酰肌醇4,5-二磷酸（PI(4,5)P2）的代谢。研究人员将开发和优化一个微电泳平台，用于使用荧光标记的脂质底物对单细胞样品进行生化分析。各种荧光PI(4,5)P2类似物将被设计、合成和筛选，以确定它们是否适合报告有或无药物抑制的PI3K i细胞内活性。初步筛选将在体外使用纯化酶和细胞裂解物进行。最有希望的化合物将在模型结肠直肠癌细胞系中进行测试。最佳报告因子将用于测试临床相关的假设，例如PI3K通路是否在细胞中以双峰方式激活，以及PI3K通路是否用于响应靶向抑制剂的细胞信号的动态重编程。最后，我们将在结直肠癌患者转移性肿瘤的单个原代细胞中分析药物治疗前后PI3K和其他PI(4,5) p2代谢酶的生化活性。提出的技术将提供一个新的方法，临床分析相关的个性化医疗的新兴领域。

DESCRIPTION (provided by applicant): There is a growing need for assays capable of assessing lipid signaling in colorectal tumors from disease models and patients. This need has been created by the advent of molecularly targeted therapies aimed at aberrant signal transduction pathways, particularly those involving phosphoinositide-3 kinase (PI3K), now known to play important roles in cancer initiation and progression. The ability to measure the enzymatic activity of dysregulated lipid signaling proteins would be extremely advantageous for directing treatment options as well as assessing treatment efficacy in individual patients. A collaborative interdisciplinary research program is proposed to create the instrumentation and chemical tools needed to directly assess the metabolism of phosphatidyl-inositol 4,5-bisphosphate (PI(4,5)P2) by PI3K and related enzymes in living cells from colorectal cancer cell lines and patient-derived metastatic colorectal cancer. The investigators will develop and optimize a microelectrophoresis platform for the biochemical analysis of single-cell samples using fluorescently labeled lipid substrates. A variety of fluorescent PI(4,5)P2 analogs will be designed, synthesized and screened for their suitability to report intracellular activity of PI3K i cells with and without pharmacologic inhibition. Initial screens will be performed in vitro using purified enzymes and cell lysates. The most promising compounds will then be tested in model colorectal carcinoma cell lines. The optimal reporter(s) will be employed to test clinically relevat hypotheses such as whether the PI3K pathway is activated in cells in a bimodal fashion and whether the PI3K pathway is used for dynamic reprogramming of cell signaling in response to targeted inhibitors. Finally, the biochemical activity of PI3K and other PI(4,5)P2-metabolizing enzymes will be profiled before and after pharmacologic therapy in single primary cells from metastatic tumors from colorectal cancer patients. The proposed technology will provide a new approach for clinical assays relevant to the emerging field of personalized medicine.