Lgr5, mammary tumor stem cells, and radiation therapy

描述（由申请人提供）：乳腺癌研究的一个主要目标是鉴定癌症干细胞（CSCs），并表征其功能特性，以期进行靶向治疗。基于来自小肠和乳腺的令人信服的最新数据，我们将验证以下假设：(a) R-spondin受体Lgr5的表达定义了具有高效肿瘤启动能力的乳腺肿瘤干细胞群体；(b)在模型系统中放疗后，Lgr5+细胞存活的剂量反应谱预测肿瘤消除的剂量反应谱；(c) Lgr5+细胞作为干细胞参与非消融放疗后乳腺肿瘤再生。这项研究将利用实验上易于处理的乳腺癌小鼠模型和最先进的小型动物微辐射器，该微辐射器向肿瘤提供均匀剂量的辐射，对周围组织的损害可以忽略不计。为了方便Lgr5+肿瘤细胞的鉴定和分离，将带有乳腺靶向癌基因的转基因mic与Lgr5-报告菌株杂交。Lgr5+细胞将通过流式细胞术从产生的乳腺肿瘤中进行分类，并通过向受体动物注射限制细胞稀释剂来测量其肿瘤启动能力。将Lgr5+细胞与Lgr-细胞以及模拟分类的肿瘤上皮细胞进行比较，以确定肿瘤启动能力的大部分是否与Lgr5+细胞有关。由于Lgr5是Wnt/¿-catenin通路的辅助受体，该蛋白不仅可以作为干细胞标记物，还可以作为CSC特性的功能决定因素。这一概念将在体外CSC自我更新的肿瘤球试验中得到验证。进一步的体内实验将验证Lgr5+细胞是控制肿瘤对放射治疗长期反应的CSCs的假设。利用CT图像引导的小动物微辐照器对小鼠乳腺肿瘤进行单次高剂量照射，在局部肿瘤控制的背景下，研究照射后Lgr5+细胞消除的剂量-反应谱。这将在体内验证肿瘤的消灭依赖于Lgr5+细胞的消除的假设。这些细胞治疗相关性的进一步证据将被发现

DESCRIPTION (provided by applicant): A major goal of breast cancer research is the identification of cancer stem cells (CSCs), and characterization of their functional properties wit a view to targeted therapy. Based on compelling recent data from the small intestine and mammary gland, we will test the hypotheses (a) that expression of the R-spondin receptor Lgr5 defines a population of mammary tumor stem cells with highly efficient tumor- initiating capacity; (b) that, following radiotherapy in a model system, the dose-response profile of Lgr5+ cell survival predicts that of tumor elimination; and (c) that Lgr5+ cells act as stem cells for mammary tumor regeneration after non-ablative radiation. The research will exploit experimentally tractable mouse models of breast cancer and a state-of-the-art small animal microirradiator that delivers uniform doses of irradiation to tumors with negligible damage to surrounding tissue. To facilitate identification and isolation of Lgr5+ tumor cells, transgenic mic with a mammary-targeted oncogene will be crossed with Lgr5- reporter strains. Lgr5+ cells will be sorted from the resulting mammary tumors by flow cytometry and their tumor-initiating capacity measured by injecting recipient animals with limiting cell dilutions. Lgr5+ cells will be compared with Lgr-, and with mock-sorted tumor epithelial cells, to determine whether the bulk of tumor-initiating capacity is associated with Lgr5+ cells. Since Lgr5 is an accessory receptor for the Wnt/¿-catenin pathway, the protein may serve not only as a stem cell marker but also as a functional determinant of CSC properties. This notion will be tested in tumorsphere assays of CSC self-renewal in vitro. Further in vivo experiments will test the hypothesis that Lgr5+ cells are CSCs which govern the tumor's long-term response to radiation therapy. Using a CT image-guided small animal microirradiator to deliver single high doses to mouse mammary tumors, the dose-response profile of Lgr5+ cell elimination following irradiation will be studied in the contex of local tumor control. This will test the hypothesis in vivo that extinction of the tumor depends on elimination of Lgr5+ cells. Further evidence of the therapeutic relevance of these cells will be determined from lineage tracing experiments asking whether regrowth of tumors following non-ablative irradiation occurs from Lgr5+ CSCs. If the expected results are obtained, they will identify Lgr5+ cells as CSCs that are critical for both initiation of tumorigenesis and for tumor survival/growth following radiation treatment. Thus we expect to identify a therapeutically relevant key population of CSCs. Moreover, since Wnt signaling inhibitors constitute a major new category of investigational drugs, these may provide a therapeutic means of targeting Lgr5+ CSCs and rendering them susceptible to elimination.