Exosomal Recombinase-a tool to dissect metastasis and the cancer microenvironment

外泌体重组酶——剖析转移和癌症微环境的工具

• 批准号: 8703642
• 负责人: RICHARD A STEINMAN
• 金额: $ 22.74万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-11 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8703642
• 关键词: Anatomy; Animals; Behavior; Biological; Cancer Control; Cell Communication; Cell Line; Cells; Coculture Techniques; Collaborations; Collection; Confocal Microscopy; Data; Dissection; Effectiveness; Endothelial Cells; Engineering; Eukaryotic Cell; Fibroblasts; Fluorescence; Gene Expression Profile; Genetic Recombination; Hypoxia; Image; Immune; In Vitro; Incubated; Injection of therapeutic agent; Intravenous; Label; Lewis Lung Carcinoma; Life; Literature; Luciferases; Malignant Epithelial Cell; Malignant Neoplasms; Malignant neoplasm of lung; Maps; Measures; Molecular; Mus; Neoplasm Metastasis; Normal Cell; Parasites; Pathway interactions; Peptide Signal Sequences; Pimonidazole; Proteins; Reporter; Reporting; Research; Route; Shapes; Signal Pathway; Signal Transduction; Sorting - Cell Movement; Technology; Testing; Touch sensation; Toxoplasma; Vascular Endothelial Growth Factors; Visual; Work; cancer cell; cellular imaging; chemokine; expression vector; flotillin; fusion gene; in vivo; interest; macrophage; metastatic process; monoclonal antibody LL2; nanovesicle; novel; promoter; recombinase; tool; trafficking; tumor; tumor microenvironment; vector control; whole animal imaging

DESCRIPTION (provided by applicant): We propose a tool to engineer cancer or host cells to irreversibly mark nearby cells by means of novel "exosomal Cre recombinase" constructs. By creating cancer cells that transport Cre, the proximal microenvironment in Cre-responsive animals can be analyzed or manipulated to an extent well beyond our current capabilities. These sorts of manipulations would enable the detailed dissection of molecular mechanisms that shape the directionality of cancer spread, sustain dormancy or control cancer-immune cell interactions. Exosomes are nanovesicles that are robustly produced by cancer cells. Specific tags directing Cre to cancer exosomes will generate a visual (fluorescent) map of the trajectory that the cancer cells used during metastasis in fluorescent reporter animals. This will also provide a means to isolate each cell that contacted the cancer so that the transcriptome of those cells can be compared with that of similar cells that were na¿ve to the cancer. This strategy is expected to offer an unprecedented tool for the dissection of the lung cancer microenvironment that is engaged during the metastatic process. Specific Aims of this application are to: 1. Create and evaluate tools that package fluorescent Cre-recombinase into exosomes for delivery to adjacent cells. This aim will establish the optimal construct for Cre transfer between cancer and surrounding normal cells. We will construct fusion genes that combine Cre, a red fluorescent marker, and specific trafficking domains of exosomal proteins. Exosome-specific Cre activity and transfer will be confirmed. Fluorescent conversion of GFP-reporter cells along the path of migrating e-C cancer cells will be visualized through live cell imaging and quantitatively evaluated. Aim 2. Enable and evaluate niche-specific activity of Cre-transfer by hypoxic cancer cells in vivo. This aim will demonstrate microenvironment-specific transfer of Cre from cancer to bystander cells. Luciferase-expressing Lewis lung carcinoma cells (LL2/luc- M38) will be stably transfected with red fluorescent exosomal Cre under control of the VEGF promoter 6, 7. Metastases following intravenous inoculation of syngeneic Cre-reporter mice will be visualized using whole animal imaging. Ex vivo multiphoton confocal microscopy will then measure host cell conversion to green fluorescence at candidate hypoxic regions and along metastatic tracks. Pimonidazole injection prior to sacrifice will be used to validate hypoxia in candidate regions. The ability to capture and analyze Cre-targeted peritumoral cells will be tested. Impact: This tool will allow robust collection of imaging and cellular data that unambiguously delineate cancer/bystander cell interactions that occurred in vivo in desired microenvironments. It is expected to create an unprecedented historical record of each cell that touched a cancer cell. This record could illuminate functional changes that occur in the microenvironment that impact metastases, and how these changes are associated with alteration in the route of spread of cancer.

描述（由适用提供）：我们提出了一种工具来设计癌症或宿主细胞，以通过新颖的“外泌体CRE重组酶”构建体不可逆地标记。通过创建运输CRE的癌细胞，可以分析或操纵CRE响应动物的近端微环境，远远超出我们当前的能力。这类操作将使能够详细的分子机制详细解剖，从而塑造癌症扩散，休眠或控制癌症免疫细胞相互作用的方向性。外泌体是由癌细胞牢固产生的纳米膜。将CRE引导到癌症外泌体的特定标签将产生视觉（荧光）图的曲线图（荧光）图，该轨迹在荧光记者动物中转移时使用的癌细胞使用的轨迹。这还将提供一种分离接触癌症的细胞的方法，以便可以将这些细胞的转录组与与癌症相似的细胞的转录组进行比较。预计该策略将为转移过程中参与的肺癌微环境解剖提供空前的工具。该应用程序的具体目的是：1。创建和评估工具，将荧光CRE聚合酶包装到外泌体中以递送到相邻细胞中。该目标将建立癌症和周围正常细胞之间CRE转移的最佳结构。我们将构建结合CRE，红色荧光标记物和外泌体蛋白的特定运输域的融合基因。外泌体特异性的CRE活性和转移将得到确认。 GFP-Reporter细胞沿迁移E-C癌细胞的荧光转化将通过活细胞成像观察并进行定量评估。 AIM 2。启用并评估低氧癌细胞在体内Cre-Transfer的小众特异性活性。该目标将表明CRE从癌症转移到旁观者细胞的微环境特异性转移。表达荧光素酶表达的Lewis肺癌细胞（LL2/Luc-M38）将稳定地用红色荧光外泌体CRE转换，在VEGF启动子6、7中，将使用整个动物进行静脉接种静脉接种后的静脉接种静脉接种后的转移酶。然后体内多光子共聚焦显微镜将测量候选低氧区域和转移轨道的宿主细胞转化为绿色荧光。在牺牲前注射甲莫尼唑唑将用于验证候选区域的缺氧。将测试捕获和分析Cre靶向的周围细胞的能力。影响：此工具 将允许在所需的微环境中明确描绘出体内发生的癌症/旁观者细胞相互作用的明确描绘癌症/旁观者细胞相互作用的成像和细胞数据。预计将创建一个触及癌细胞的每个细胞的前所未有的历史记录。该记录可能会阐明影响转移酶的微环境中发生的功能变化，以及这些变化与癌症传播途径的改变有关。