PROFILING SIGNALING ACTIVITY AND GENE EXPRESSION IN SINGLE, PANCREATIC ADENOCARCINOMA CELLS USING CE-RNA-SEQ

摘要 胰腺导管腺癌是一种极具破坏性的疾病，急需改进的治疗方法。 针对特定的信号通路。异常胰腺的分子生物学研究策略 组织和通知有针对性的治疗决定将对患者具有巨大价值 治疗。然而，由于对患者组织进行活组织检查，分子图谱极具挑战性。 是正常和恶性胰腺细胞的复杂混合物。此外，还有一个不断增长的 了解突变和基因表达本身与临床并不密切相关 回应。在目前的应用中，一个多学科研究团队提议开发一种 最先进的单细胞平台技术测量前哨的催化活性 KRAS通路中的激酶和通过RNA测序的基因表达。这个 调查人员将优化和验证微采样和微电泳法，以 分析单个细胞，同时克服细胞异质性和 样本量限制。KRAS-OUTLOW信号通路中激酶活性的新报道 将设计与单细胞毛细管相结合的新方法和仪器 高效捕获RNA的电泳法将成为先驱。人类肿瘤标本 维持在小鼠异种移植中将被检测以获得对肿瘤特性的独特见解 当前不可寻址。这项工作将直接将信使核糖核酸的产生与催化 患者来源的单个肿瘤细胞中的激酶活性。这项技术将使 诸如哪些激酶信号模式驱动经典表型与基础表型的问题 胰腺癌与单个肿瘤是否具有经典型和非典型性 基底型细胞。从实施这项技术中获得的数据和见解将 为临床化验提供了一种新的方法，有可能对 精准医学新兴领域的治疗策略。

Abstract Pancreatic ductal adenocarcinoma is a devastating disease in dire need of improved therapies targeted at specific signaling pathways. Strategies to molecularly profile aberrant pancreatic tissue and inform targeted therapeutic decisions would be of immense value in patient treatment. However, molecular profiling is extremely challenging since biopsied patient tissue is a complex mixture of normal and malignant pancreatic cells. Furthermore, there is a growing understanding that mutations and gene expression alone do not tightly correlate with clinical response. In the current application, a multidisciplinary research team proposes to develop a state-of-the-art, single-cell, platform technology to measure the catalytic activity of sentinel kinases within the KRAS pathway and gene expression through RNA sequencing. The investigators will optimize and validate microsampling and microelectrophoresis methods to assay single cells, simultaneously overcoming the challenges of cellular heterogeneity and sample-size limits. Novel reporters of kinase activity within KRAS-outflow signaling pathways will be designed and new methods and instrumentation combining single-cell capillary electrophoresis with efficient RNA capture will be pioneered. Human tumor samples maintained in murine xenografts will be assayed to gain unique insights into tumor properties not currently addressable. The work will directly link mRNA production with the catalytic activity of kinases in individual tumor cells derived from patients. The technology will enable questions such as which kinase signaling patterns drive the classical vs the basal phenotypes of pancreatic adenocarcinoma and whether a single tumor possesses a mixture of classical and basal-type cells. The data and the insights gained from implementation of this technology will provide a new approach for clinical assays with the potential for a profound impact on therapeutic strategies in the emerging field of precision medicine.