Assessing the Transcriptional and Signaling Basis of Heterogeneity in the Epithelial-Mesenchymal Transition in Pancreatic Ductal Adenocarcinoma

项目总结 上皮-间充质转化(EMT)是一种发育过程，在 胰腺导管腺癌(PDAC)促进疾病进展和化疗耐药。PDAC肿瘤 细胞系通常含有异质混合的转化细胞，表现为上皮性或间充质 特征，使理解管理这一重要的 表型转换。观察到EMT可以由多种不同的生长因子启动，低 氧张力和基质介导的信号强烈表明，多个信号通路协同作用 驱动强大的EMT，并提高了EMT异质性仅由某些细胞的能力来解释的可能性 有力地激活协同驱动EMT的通路。另一种潜力，但不一定是相互的 对表型异质性的独家解释是，一些PDAC细胞准备接受EMT，因为 能够利用特定转录因子或信号通路的转录差异。基于 根据我们的初步数据，我们假设某些PDAC细胞在转录水平上已准备好接受EMT 而EMT的异质性进一步依赖于激酶调节信号在细胞间的变化 细胞群体内的过程。这里提出的工作的目标是通过以下方式检验这些假设 研究EMT异质性调控基础的定量系统生物学方法的发展 转录和激酶介导的信号传递过程。在目标1中，迭代免疫荧光成像 将开发管道，以收集不同处理的PDAC细胞种群的多路信号数据 EMT激动剂。在初步研究的基础上，我们建议测量七个不同信号的标记 路径节点和两个EMT标记，以创建包含针对数千个细胞测量的九个要素的数据集 每种实验条件。然后，我们将应用互信息数据科学方法来量化 识别协同驱动健壮EMT的信号通路。模型预测将使用以下工具进行测试 小分子抑制剂和siRNA介导的基因敲除。在目标2中，我们将使用遗传条形码 细胞群体中耐EMT或依从EMT谱系的转录转录图谱。单细胞RNA 将对EMT诱导前后的细胞测序数据进行分析，以确定 优先使PDAC细胞经历间充质转化。考生成绩单的相关性 为了解释EMT引爆，将通过击倒实验进行测试。在这项工作中开发的方法 将广泛适用于其他癌症环境下的EMT研究和替代类型的研究 表型转换。此外，具体的研究结果对小说组合的设计也有一定的指导意义 基于抑制EMT以促进对化疗的反应的PDAC的治疗。

PROJECT SUMMARY The epithelial-mesenchymal transition (EMT) is a developmental process that is aberrantly reactivated in pancreatic ductal adenocarcinoma (PDAC) to promote disease progression and chemoresistance. PDAC tumors and cell lines typically contain a heterogeneous mixture of transformed cells displaying epithelial or mesenchymal characteristics, complicating efforts to understand the regulatory mechanisms that govern this important phenotypic switching. The observation that EMT can be initiated by a variety of different growth factors, low oxygen tension, and matrix-mediated signaling strongly suggests that multiple signaling pathways cooperate to drive robust EMT and raises the possibility that EMT heterogeneity is explained by the ability of only some cells to activate robustly the pathways that cooperate to drive EMT. Another potential, but not necessarily mutually exclusive, explanation for phenotypic heterogeneity is that some PDAC cells are primed to undergo EMT due to transcriptional differences that enable utilization of specific transcription factors or signaling pathways. Based on our preliminary data, we hypothesize that certain PDAC cells are transcriptionally primed to undergo EMT and that EMT heterogeneity further depends upon cell-to-cell variations in kinase-regulated signaling processes within cell populations. The objective of the work proposed here is to test these hypotheses through the development of quantitative systems biology methods to study the basis of EMT heterogeneity regulation via transcriptional and kinase-mediated signaling processes. In Aim 1, an iterative immunofluorescence imaging pipeline will be developed to gather multiplexed signaling data on populations of PDAC cells treated with different EMT agonists. Based on preliminary studies, we propose to measure markers for seven distinct signaling pathway nodes and two EMT markers to create a dataset with nine features measured for thousands of cells for each experimental condition. We will then apply a mutual information data science approach for the quantitative identification of the signaling pathways that cooperate to drive robust EMT. Model predictions will be tested using small molecule inhibitors and siRNA-mediated knockdowns. In Aim 2, we will use genetic barcoding for the transcriptomic profiling of EMT-resistant or -compliant lineages within cell populations. Single-cell RNA sequencing data from cells before and after EMT induction will be analyzed to identify transcriptional states that preferentially enable PDAC cells to undergo the mesenchymal transition. The relevance of candidate transcripts for explaining EMT priming will be tested through knockdown experiments. The methods developed in this work will be broadly applicable to the study of EMT in other cancer settings and to the study of alternative types of phenotypic switching. Moreover, the specific results will have implications for the design of novel combination therapies for PDAC based on the objective of suppressing EMT to promote responsiveness to chemotherapy.