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: 8543689
• 负责人: RICHARD A STEINMAN
• 金额: $ 21.86万
• 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 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8543689
• 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

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（由申请人提供）：我们提出了一种通过新型“外泌体Cre重组酶”构建体来工程化癌症或宿主细胞以不可逆地标记附近细胞的工具。通过创造运输Cre的癌细胞，Cre反应动物的近端微环境可以被分析或操纵到远远超出我们目前能力的程度。这些操作将能够详细解剖形成癌症扩散方向的分子机制，维持休眠或控制癌症-免疫细胞相互作用。外泌体是由癌细胞稳健产生的纳米囊泡。将Cre引导至癌症外泌体的特异性标签将产生癌细胞在荧光报告动物中转移期间使用的轨迹的视觉（荧光）图。这也将提供一种方法来分离接触癌症的每个细胞，以便这些细胞的转录组可以与未接触癌症的类似细胞的转录组进行比较。这种策略有望为转移过程中参与的肺癌微环境的解剖提供前所未有的工具。本申请的具体目的是：1.创建和评估将荧光Cre重组酶包装到外泌体中以递送到相邻细胞的工具。这一目标将建立Cre在癌症和周围正常细胞之间转移的最佳构建体。我们将构建融合基因，结合联合收割机Cre，红色荧光标记，和外泌体蛋白的特定运输结构域。将确认外泌体特异性Cre活性和转移。GFP-报告细胞沿着迁移e-C癌细胞的路径的荧光转化将通过活细胞成像可视化并定量评价。目标2.通过体内缺氧癌细胞实现并评估Cre转移的小生境特异性活性。这一目标将证明Cre从癌症到旁观者细胞的微环境特异性转移。表达荧光素酶的刘易斯肺癌细胞（LL 2/luc-M38）将在VEGF启动子的控制下用红色荧光外泌体Cre稳定转染6，7。静脉内接种同基因Cre-reporter小鼠后的转移将使用整个动物成像可视化。然后，离体多光子共聚焦显微镜将测量候选缺氧区域和沿着转移轨迹的宿主细胞向绿色荧光的转化。在处死前注射匹莫硝唑将用于验证候选区域的缺氧。将测试捕获和分析Cre靶向的瘤周细胞的能力。影响：此工具 将允许稳健地收集成像和细胞数据，所述数据明确地描绘在期望的微环境中体内发生的癌症/旁观者细胞相互作用。预计它将创造一个前所未有的历史记录，记录每个接触癌细胞的细胞。这一记录可以阐明影响转移的微环境中发生的功能变化，以及这些变化如何与癌症扩散途径的改变相关。