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

描述（由申请人提供）：我们提出了一种工具，通过新型的“外泌体Cre重组酶”构建来设计癌症或宿主细胞，使其不可逆地标记附近的细胞。通过制造运输Cre的癌细胞，可以分析或操纵Cre反应动物的近端微环境，其程度远远超出我们目前的能力。这类操作将使详细解剖形成癌症扩散方向、维持休眠或控制癌症免疫细胞相互作用的分子机制成为可能。外泌体是由癌细胞大量产生的纳米囊泡。在荧光报告动物中，将Cre导向癌症外泌体的特定标签将生成癌细胞在转移过程中使用的可视化（荧光）轨迹图。这也将提供一种分离每个与癌症接触的细胞的方法，以便将这些细胞的转录组与与癌症无关的类似细胞的转录组进行比较。该策略有望提供一种前所未有的工具，用于在转移过程中参与肺癌微环境的解剖。此应用程序的具体目的是：1。创建和评估将荧光cre -重组酶包装到外泌体中以传递到邻近细胞的工具。这一目标将建立Cre在癌细胞和周围正常细胞之间转移的最佳结构。我们将构建融合基因，结合红色荧光标记Cre和外泌体蛋白的特定运输结构域。外泌体特异性Cre活性和转移将被确认。gfp报告细胞沿迁移e-C癌细胞路径的荧光转化将通过活细胞成像可视化并定量评估。目标2。激活和评估体内缺氧癌细胞转移cre的利基特异性活性。这个目标将证明微环境特异性转移Cre从癌症到旁观者细胞。表达荧光素酶的Lewis肺癌细胞（LL2/luc- M38）将在VEGF启动子的控制下稳定地转染红色荧光外泌体Cre 6,7。静脉注射同源cre报告小鼠后的转移将使用全动物成像可视化。体外多光子共聚焦显微镜将测量宿主细胞在候选缺氧区域和转移路径上向绿色荧光的转化。牺牲前注射吡咪唑将用于验证候选区域的缺氧。捕获和分析cre靶向肿瘤周围细胞的能力将被测试。影响：这个工具

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.