Single-Cell Measurement of Lipid Signaling in Colorectal Cancer

描述（由申请人提供）：对能够评估来自疾病模型和患者的结直肠肿瘤中脂质信号传导的测定的需求日益增长。这种需求是由针对异常信号转导途径的分子靶向疗法的出现产生的，特别是涉及磷酸肌醇-3激酶（PI 3 K）的那些，现在已知其在癌症的起始和进展中起重要作用。测量失调的脂质信号传导蛋白的酶活性的能力对于指导治疗选择以及评估个体患者的治疗功效将是极其有利的。提出了一个跨学科的合作研究计划，以创建所需的仪器和化学工具，直接评估磷脂酰肌醇4，5-二磷酸（PI（4，5）P2）的代谢PI 3 K和相关酶在活细胞从结直肠癌细胞系和患者来源的转移性结直肠癌。研究人员将开发和优化微电泳平台，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。将设计、合成多种荧光PI（4，5）P2类似物，并筛选它们报告具有和不具有药理学抑制的PI 3 Ki细胞的细胞内活性的适用性。将使用纯化的酶和细胞裂解物在体外进行初始筛选。最有希望的化合物将在模型结直肠癌细胞系中进行测试。最佳报告基因将用于测试临床相关假设，例如PI 3 K通路是否以双峰方式在细胞中活化，以及PI 3 K通路是否用于响应靶向抑制剂的细胞信号传导的动态重编程。最后，在来自结直肠癌患者的转移性肿瘤的单个原代细胞中，在药物治疗之前和之后分析PI 3 K和其他PI（4，5）P2代谢酶的生物化学活性。所提出的技术将为与新兴的个性化医疗领域相关的临床检测提供新的方法。

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.