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: 9003785
• 负责人: Lindsey A Baker
• 金额: $ 5.8万
• 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/9003785
• 关键词: Architecture; Area; Automobile Driving; Binding Proteins; Biochemical; Bioinformatics; Biological Assay; Biological Models; 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; 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; Research; Research Personnel; Role; Sampling; Signaling Protein; Sorting - Cell Movement; Specimen; Stromal Cells; System; Technical Expertise; Techniques; Tissues; Training; Transplantation; Tumor Biology; Up-Regulation; cancer cell; in vivo; innovation; interest; knock-down; member; mouse model; neoplastic; neoplastic cell; novel; overexpression; pancreatic cancer cells; pancreatic neoplasm; pancreatic tumorigenesis; programs; public health relevance; research study; single cell analysis; skills; therapy design; three dimensional cell culture; transcriptome sequencing; transcriptomics; tumor; tumor growth; tumor heterogeneity; 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(申请人提供)：我寻求三年的资金，以获得所需的培训，以开发一个独立的研究计划，检查导致胰腺导管腺癌(PDA)的细胞通路，最终目的是确定新的靶点，可以用来开发这种高度侵袭性的恶性肿瘤的治疗方法。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部分代表了促进PDA肿瘤发生的细胞因素，本提案的目标是定义这些因素。虽然以前的研究试图确定与pda肿瘤发生相关的基因表达变化，但这些研究受到一些限制，包括缺乏纯净的未转化胰腺导管细胞群体。 对照和胰腺肿瘤标本中大量间质细胞的存在，使得在这些样本中发现的任何基因表达变化很难归因于肿瘤细胞。 此外，通过这类研究确定的许多基因与胰腺癌发生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器官培养系统，允许正常和肿瘤胰腺细胞的增殖，以确定驱动PDA肿瘤发生的细胞途径。具体地说，我将首先提高我在生物信息学方法方面的培训，这将用于比较正常、癌前和恶性胰腺细胞作为类器官生长的基因表达谱。表达数据将与其他可用的数据集整合，以定义在肿瘤细胞中上调的基因，这些基因可能促进肿瘤的形成。在一项初步研究中，我已经确定了一些在癌前和恶性小鼠器官中上调的有希望的候选基因，包括S100a7a，它编码一种以前在人类胰腺病变中上调的Ca+2信号蛋白。我现在计划扩展这项分析，以确定在来源于人类胰腺肿瘤的癌细胞中上调的基因，并以有机类化合物的形式繁殖。其次，我将学习流式细胞术和单细胞分析方法，这将使我能够从原始组织中分离出正常和肿瘤的胰腺细胞。分选的细胞将被用来确认使用有机系统识别的表达变化，并探索这些变化在体内的细胞异质性。最后，我将开展对肿瘤发生途径进行功能性分析的培训，这将用于确定有希望的候选基因对肿瘤发生的影响。我将在基因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除是否会损害原位移植后的肿瘤形成。通过发展生物信息学、单细胞和癌症生物学方法的核心能力，该奖学金将帮助我阐明推动胰腺肿瘤发生的新途径，并将使我处于一个极好的位置，开始自己的独立研究实验室，进一步剖析这些途径。