Multiplexed Single-Cell Analysis of EGFR and HER2 Activity in Gastric Cancer Cell

描述（由申请人提供）：胃癌在全球范围内造成的死亡人数超过任何其他癌症，肺癌除外。虽然胃癌在西方国家的发病率相对较低，但该地区的患者很少出现在疾病的早期阶段。然而，高死亡率迫切需要改善治疗。分子靶向治疗如激酶抑制剂正在被广泛研究用于治疗胃癌。特别感兴趣的是抑制HER2和EGFR激酶的药物，这些激酶是在胃癌亚群中显示异常活性的人表皮生长因子受体（HER）家族的受体酪氨酸激酶。目前，FDA已批准使用HER2抑制剂曲妥珠单抗治疗某些胃癌，并且正在II期和III期临床试验中评估几种EGFR抑制剂与细胞毒性化疗联合治疗胃癌的疗效。随着这些靶向药物的出现，迫切需要准确确定患者是否适合抗HER治疗。这项工作提出了开发一种工具，解决了许多目前的诊断技术的局限性，直接测定异常活动的HER2和EGFR激酶在单细胞水平。HER2和EGFR激酶的高选择性肽底物将通过使用非天然侧链文库的肽支架的迭代组合优化来产生。 功能。将裂解负载荧光标记底物的单细胞，并使用单细胞分析系统进行分析，该系统由荧光显微镜和具有激光诱导荧光检测的高灵敏度毛细管电泳系统组成。该技术非常适合小型临床活检，将应用于胃癌模型和患者样本，以在单细胞水平上定量评估EGFR和HER2信号转导失调。

DESCRIPTION (provided by applicant): Gastric cancer causes more deaths worldwide than any other cancer, with the exception of lung cancer. Though the incidence of gastric cancer in Western nations is relatively low, patients in this region rarely present at an early stage of the disease. Consequent high mortality rates create a pressing need for improved treatment. Molecularly targeted therapies such as kinase inhibitors are under intensive investigation for treatment of gastric cancer. Of particular interest are drugs inhibiting HER2 and EGFR kinases, receptor tyrosine kinases of the human epidermal growth factor receptor (HER) family that demonstrate aberrant activity in subsets of gastric cancer. Currently, the FDA has approved the use of the HER2 inhibitor trastuzumab for the treatment of some gastric cancers, and several EGFR inhibitors are being evaluated in phase II and III clinical trials for efficacy in gastric caner therapy in combination with cytotoxic chemotherapy. With the advent of these targeted agents has arisen an urgent need to accurately determine patient eligibility for anti-HER treatment. This work proposes to develop a tool that addresses many of the limitations of current diagnostic technologies by directly assaying aberrant activity of HER2 and EGFR kinases at the single cell level. Highly selective peptide substrates of HER2 and EGFR kinase will be generated via iterative combinatorial optimization of a peptide scaffold using libraries of non-native side chain functionalities. Single cells loaded with fluorescently labeled substrates will be lysed and analyzed with a single-cell analysis system comprised of a fluorescence microscope and an exquisitely sensitive capillary electrophoresis system with laser-induced fluorescence detection. Ideally suited to small clinical biopsies, this technology will be applied both to gastric cancer models and to patient samples in order to quantitatively evaluate EGFR and HER2 signaling dysregulation at the single-cell level.