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来设计癌症或宿主细胞，通过新的“外体Cre重组酶”结构不可逆转地标记附近的细胞。通过创造运输Cre的癌细胞，对Cre反应的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。这些类型的操作将使人们能够详细剖析塑造癌症扩散方向、维持休眠或控制癌症与免疫细胞相互作用的分子机制。外周小体是由癌细胞强有力地产生的纳米囊泡。将Cre定向到癌症外切体的特定标签将生成癌细胞在荧光报告动物体内转移过程中使用的可视(荧光)地图。这也将提供一种方法来分离与癌症接触的每个细胞，以便这些细胞的转录组可以与与癌症相似的细胞的转录组进行比较。这一策略有望为解剖肺癌转移过程中的微环境提供一种前所未有的工具。这项应用的具体目的是：1.创建和评估将荧光Cre-重组酶包装成外体以便运送到相邻细胞的工具。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，将红色荧光标记Cre与胞外体蛋白的特定运输结构域结合起来。外切体特异的Cre活性和转移将得到确认。绿色荧光蛋白报告细胞沿E-C癌细胞迁移路径的荧光转化将通过活细胞成像进行可视化和定量评估。目的2.启用并评价体内缺氧癌细胞Cre转移的生态位特异性活性。这一目标将展示Cre从癌细胞到旁观者细胞的微环境特异性转移。在血管内皮生长因子启动子6、7的控制下，荧光素酶表达的Lewis肺癌细胞(LL2/Luc-M38)将稳定地转染红色荧光外体Cre。静脉接种同基因Cre报告基因小鼠后的转移将通过全动物成像显示。然后，体外多光子共聚焦显微镜将测量候选缺氧区和转移轨迹上宿主细胞向绿色荧光的转化。处死前注射匹莫硝唑将用于验证候选区域的缺氧情况。将测试捕获和分析Cre靶向肿瘤周围细胞的能力。影响：此工具 将允许强大的成像和细胞数据收集，毫不含糊地描绘在所需微环境中发生在体内的癌症/旁观者细胞相互作用。预计它将创造一个史无前例的历史记录，记录每一个接触过癌细胞的细胞。这一记录可以阐明微环境中发生的影响转移的功能变化，以及这些变化如何与癌症扩散路线的变化相关联。 公共卫生相关性：癌细胞需要附近正常细胞的合作才能生存。关于正常细胞在肿瘤首次出现时如何协作支持它们的一些细节是已知的，然而，对于旁观者细胞是否或如何在转移过程中调节癌症的轨迹，人们知之甚少。这在一定程度上是因为癌细胞在转移过程中所走的确切路线无法可视化。该项目的目标之一是建立一种新的工具，使癌症在通过人体时接触到的每个细胞都将发出不可逆转的光。这将创建轨迹的可视(荧光)地图 转移过程中使用的癌细胞。此外，它还提供了一种分离接触癌症的每个细胞的方法，以便将这些细胞的行为与没有接触肺癌细胞的类似细胞的行为进行比较。这一策略可以揭示与转移性癌症对话中涉及的细胞因子，从而可以解剖和定向病理对话。

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. PUBLIC HEALTH RELEVANCE: Cancer cells require the collaboration of nearby normal cells in order to survive. Some details are known about how normal cells collaborate to support tumors when they first appear, however, little is known about whether or how bystander cells regulate the trajectory of cancer during the metastatic process. This is partly because the exact route that a cancer cell takes during metastasis cannot be visualized. One of the aims of this project is to establish a new tool so that every cell that cancer touches during its transit throug the body will glow irreversibly. This will create a visual (fluorescent) map of the trajectory that the cancer cells used during metastasis. Moreover, it provides a means to isolate each cell that contacted the cancer so that the behavior of those cells can be compared with that of similar cells that did not contact lung cancer cells. This strategy can uncover the cellular actors involve in dialogue with metastasizing cancer so that the pathological dialogue can be dissected and targeted.