Finding Novel Pancreatic Cancer Oncogenes Using an Innovative 3D Culture System

使用创新的 3D 培养系统寻找新的胰腺癌癌基因

• 批准号: 8835927
• 负责人: Lindsey A Baker
• 金额: $ 5.42万
• 依托单位: COLD SPRING HARBOR LABORATORY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-02-01 至 2017-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8835927
• 关键词: Architecture; Area; Automobile Driving; Biochemical; Bioinformatics; Biological Assay; Biological Models; CabP2; Cancer Biology; Cancerous; Candidate Disease Gene; Cell Proliferation; Cell Separation; Cells; Clinical; Cultured Cells; Data; Data Set; Development; Dimensions; Dissection; Ductal; Ductal Epithelial Cell; Family; Fellowship; Flow Cytometry; Foundations; Funding; Gene Expression; Genes; Genetic; Genetic Engineering; Goals; Heterogeneity; Human; Laboratories; Laboratory Research; Learning; Lesion; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of pancreas; Methods; Modeling; Molecular Profiling; Mus; Oncogenes; Organoids; Pancreas; Pancreatic Ductal Adenocarcinoma; Pancreatic Intraepithelial Neoplasia; Pancreatic duct; Pathway interactions; Population; Positioning Attribute; Premalignant; Premalignant Cell; Proteins; RNA; RNA Sequence Analysis; RNA Sequences; Research; Research Personnel; Role; Sampling; Signaling Protein; Sorting - Cell Movement; Specimen; Stromal Cells; System; Techniques; Tissues; Training; Transplantation; Tumor Biology; Up-Regulation; cancer cell; in vivo; innovation; interest; knock-down; member; neoplastic; neoplastic cell; novel; overexpression; pancreatic cancer cells; pancreatic neoplasm; pancreatic tumorigenesis; programs; public health relevance; research study; single cell analysis; skills; therapy design; transcriptome sequencing; transcriptomics; tumor; tumor growth; tumorigenesis; tumorigenic

 DESCRIPTION (provided by applicant): I seek three years' funding to acquire the training needed to develop an independent research program examining cellular pathways that drive pancreatic ductal adenocarcinoma (PDA), with the ultimate aim of identifying new targets that could be used to develop treatments for this highly aggressive malignancy. I hypothesize that a subset of the gene expression differences between neoplastic and untransformed pancreatic cells represent cellular factors that function to promote PDA tumorigenesis, and the goal of this proposal is to define such factors. While previous studies have attempted to identify gene expression changes associated with PDA tumorigenesis, these studies suffered from a number of limitations, including the lack of a pure population of untransformed pancreatic ductal cells as a control and the presence of abundant stromal cells in pancreatic tumor specimens, making any gene expression changes identified in these samples difficult to attribute to neoplastic cells. In addition, the functional relevance of many genes identified through such studies to the development of pancreatic cancer has not been determined. Here, I propose to use an innovative three-dimensional organoid culture system that allows for the propagation of both normal and neoplastic pancreatic cells to define the cellular pathways driving PDA tumorigenesis. Specifically, I will first advance my training in bioinformatic approaches, which will be used to compare the gene expression profiles of normal, premalignant, and malignant pancreatic cells grown as organoids. Expression data will be integrated with other available datasets to define genes upregulated in neoplastic cells that are likely to promote tumorigenesis. In a preliminary study, I have identified a number of promising candidate genes upregulated in premalignant and malignant murine organoids, including S100a7a, which encodes a Ca+2-signaling protein previously found upregulated in human pancreatic lesions. I now plan to extend this analysis to identify genes upregulated in cancer cells derived from human pancreatic tumors and propagated as organoids. Second, I will learn flow cytometry and single cell analysis approaches, which will allow me to isolate normal and neoplastic pancreatic cells from primary tissue. Sorted cells will be used to confirm expression changes identified using the organoid system and to explore the cellular heterogeneity of those changes in vivo. Finally, I will develop the training to functionally assay tumorigenesis pathways, which will be used to determine the effect of promising candidate genes on tumorigenesis. I will genetically deplete or overexpress candidate genes of interest to look for changes in ductal organoid proliferation and survival. I will also determine whether candidate knockdown impairs tumor formation following orthotopic transplantation. Through the development of core competencies in bioinformatic, single cell, and cancer biology approaches, this fellowship will help me to elucidate new pathways that drive pancreatic tumorigenesis, and will place me in an excellent position to start my own independent research laboratory further dissecting these pathways.

 描述（由申请人提供）：我寻求三年的资金，以获得开发独立研究项目所需的培训，该项目检查驱动胰腺导管腺癌（PDA）的细胞通路，最终目的是确定可用于开发这种高度侵袭性恶性肿瘤治疗方法的新靶点。我假设，肿瘤和未转化的胰腺细胞之间的基因表达差异的一个子集代表的细胞因子，功能，以促进PDA肿瘤的发生，这个建议的目标是定义这样的因素。虽然以前的研究试图鉴定与PDA肿瘤发生相关的基因表达变化，但这些研究受到许多限制，包括缺乏纯的未转化胰腺导管细胞群， 对照和胰腺肿瘤标本中大量基质细胞的存在，使得在这些样品中鉴定的任何基因表达变化难以归因于肿瘤细胞。 此外，通过这些研究鉴定的许多基因与胰腺癌发展的功能相关性尚未确定。在这里，我建议使用一种创新的三维类器官培养系统，允许正常和肿瘤胰腺细胞的繁殖，以确定驱动PDA肿瘤发生的细胞途径。具体来说，我将首先推进我在生物信息学方法方面的培训，该方法将用于比较作为类器官生长的正常、癌前和恶性胰腺细胞的基因表达谱。表达数据将与其他可用的数据集整合，以确定可能促进肿瘤发生的肿瘤细胞中上调的基因。在一项初步研究中，我已经确定了一些有希望的候选基因在癌前和恶性小鼠类器官中上调，包括S100a7a，它编码一种以前在人类胰腺病变中发现上调的Ca+2信号蛋白。我现在计划扩展这项分析，以确定在人类胰腺肿瘤衍生的癌细胞中上调的基因，并作为类器官繁殖。其次，我将学习流式细胞术和单细胞分析方法，这将使我能够从原代组织中分离正常和肿瘤胰腺细胞。分选的细胞将用于确认使用类器官系统鉴定的表达变化，并探索这些变化在体内的细胞异质性。最后，我将开发功能性检测肿瘤发生途径的培训，这将用于确定有希望的候选基因对肿瘤发生的影响。我将从遗传学上耗尽或过度表达感兴趣的候选基因，以寻找导管类器官增殖和存活的变化。我还将确定候选基因敲减是否会损害原位移植后的肿瘤形成。通过生物信息学，单细胞和癌症生物学方法的核心竞争力的发展，这个奖学金将帮助我阐明驱动胰腺肿瘤发生的新途径，并将使我处于一个很好的位置，开始我自己的独立研究实验室进一步解剖这些途径。