Molecular Analysis Using Liquid Crystal Technology

描述（由申请人提供）： 癌症是一个技术突破导致我们对疾病分子基础的理解取得重大进展的领域。关于关键信号传导蛋白在癌症中的作用的知识促使人们尝试开发针对信号传导靶点如表皮生长因子受体（EGFR）的抗癌剂。相当多的研究集中在拮抗EGFR的酪氨酸激酶活性上，并且最近已经鉴定出EGFR突变与激酶抑制剂治疗后的肿瘤缩小相关。然而，非小细胞肺癌（NSCLC）的临床试验显示，激酶拮抗剂如吉非替尼或厄洛替尼在癌症患者中的有效性水平高度可变。此外，关于这些抗癌剂在人类肿瘤中的确切作用机制知之甚少，主要是由于难以对有限的组织材料进行相关的分子测定。关于这些药物如何影响人类肿瘤中EGFR状态/信号传导的不确定性使得这些药物的临床开发极其困难。因此，本项目旨在进一步开发一种基于新型精密敏感工具的技术，该工具使用纳米结构表面和液晶（LC）来放大和成像分子相互作用。我们的初步数据表明，我们可以：a）制造纳米级形貌的表面，B）使用LC鉴定细胞制剂中的EGFR表达和磷酸化状态，以及c）检测暴露于EGFR拮抗剂后细胞提取物中EGFR磷酸化状态和激酶活性的抑制。我们的目标是完善，验证和实施这种方法，以允许对有限的临床标本进行高通量筛选，并最终使用这种技术来评估哪些肿瘤最有可能对EGFR拮抗剂产生反应。拟议的目标是：1）优化扭矩平衡方法，以利用基于LC的测定以高灵敏度、可重复性和定量的方式报告细胞制剂中EGFR的表达和磷酸化状态; 2）使用LC技术，评估和完善少量细胞足以以灵敏和定量的方式报告EGFR的表达、磷酸化状态和酪氨酸激酶活性的概念。预计这项研究将为癌症的研究、诊断和治疗提供一个强大而新颖的工具。

DESCRIPTION (provided by applicant): Cancer is an area where technological breakthroughs have resulted in major advances in our understanding of the molecular basis of disease. Knowledge concerning the role of key signaling proteins in cancer has prompted attempts to develop anti-cancer agents that are directed against signaling targets such as the epidermal growth factor receptor (EGFR). Considerable research has centered on antagonizing the tyrosine kinase activity of the EGFR, and EGFR mutations have recently been identified that correlate with tumor shrinkage following treatment with kinase inhibitors. However, clinical trials in non-small cell lung cancer (NSCLC) have revealed highly variable levels of effectiveness in cancer patients to kinase antagonists such as gefitinib or erlotinib. Moreover, little is known regarding the precise mechanisms of action of these anti-cancer agents in human tumors, primarily due to the difficulty in performing relevant molecular assays on limited tissue material. Uncertainty about how these agents affect EGFR status/signaling in human tumors has made clinical development of these drugs extremely difficult. Accordingly, the present project seeks to further develop a technology based on a new class of exquisitely sensitive tools that use nanostructured surfaces and liquid crystals (LCs) to amplify and image molecular interactions. Our preliminary data show that we can: a) fabricate surfaces with nanometer-scale topographies, b) identify EGFR expression and phosphorylation status in cell preparations using LCs, and c) detect inhibition of EGFR phosphorylation status and kinase activity in cell extracts following exposure to EGFR antagonists. Our goals are to refine, validate, and implement this methodology to allow high throughput screening on limited clinical specimens, and to ultimately use this technology to assess which tumors are most likely to respond to EGFR antagonists. The proposed Aims are: 1) Optimize the torque balance method for utilizing LC-based assays to report EGFR expression and phosphorylation status in cell preparations in a highly sensitive, reproducible and quantitative manner; 2) Using LC technology, evaluate and refine the concept that small numbers of cells are sufficient to report the expression, phosphorylation status and tyrosine kinase activity of the EGFR in a sensitive and quantitative fashion. It is anticipated that this research will provide a powerful and novel tool for the study, diagnosis, and treatment of cancer.