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。对关键信号蛋白在癌症中作用的了解促使人们尝试开发针对信号靶点的抗癌药物，如表皮生长因子受体(EGFR)。相当多的研究集中在拮抗EGFR的酪氨酸激酶活性，最近发现EGFR突变与用激酶抑制剂治疗后的肿瘤缩小有关。然而，非小细胞肺癌(NSCLC)的临床试验显示，癌症患者对吉非替尼或埃洛替尼等激酶拮抗剂的有效性水平差异很大。此外，人们对这些抗癌剂在人类肿瘤中的确切作用机制知之甚少，主要是因为在有限的组织材料上进行相关的分子分析是困难的。这些药物在人类肿瘤中如何影响EGFR状态/信号的不确定性使这些药物的临床开发变得极其困难。因此，本项目寻求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)制造具有纳米级形貌的表面，b)使用LCS鉴定细胞制剂中EGFR的表达和磷酸化状态，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。我们的目标是改进、验证和实施这一方法，以便在有限的临床样本上进行高通量筛查，并最终使用这项技术来评估哪些肿瘤最有可能对EGFR拮抗剂产生反应。建议的目标是：1)优化扭矩平衡方法，利用基于LC的检测方法，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，评估和完善这样一个概念，即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。预计这项研究将为癌症的研究、诊断和治疗提供一个强大的新工具。

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.