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)，并描述其功能特性，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，我们将检验以下假设：(A)R-pondin受体Lgr5的表达定义了具有高效致瘤能力的乳腺肿瘤干细胞群体；(B)在模型系统中进行放射治疗后，Lgr5+细胞存活的剂量-反应曲线预测了肿瘤消除；以及(C)Lgr5+细胞作为干细胞在非消融性照射后作为乳腺肿瘤再生的干细胞。这项研究将利用实验上容易处理的乳腺癌小鼠模型和一种最先进的小型动物微辐照器，这种微辐照器可以在对周围组织几乎不造成损害的情况下向肿瘤提供均匀剂量的辐射。为了便于Lgr5+肿瘤细胞的鉴定和分离，带有乳腺靶向癌基因的转基因小鼠将与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分离出来，并通过向受体动物注射有限细胞稀释度来测量它们的致瘤能力。Lgr5+细胞将与LGR-以及模拟分选的肿瘤上皮细胞进行比较，以确定大部分肿瘤启动能力是否与Lgr5+细胞有关。由于LGR5是Wnt/�-Catenin途径的辅助受体，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自我更新的肿瘤球体试验中得到验证。进一步的体内实验将验证Lgr5+细胞是控制肿瘤对放射治疗的长期反应的CSCs的假设。利用CT图像引导的小动物微照射器对小鼠乳腺肿瘤进行单次大剂量照射，在局部肿瘤对照的情况下，研究照射后Lgr5+细胞清除的剂量-效应曲线。这将在体内检验这样的假设，即肿瘤的消退取决于Lgr5+细胞的消除。这些细胞治疗相关性的进一步证据将是 由血统追踪实验确定，该实验询问非消融性照射后肿瘤的再生长是否发生在Lgr5+CSCs。如果获得了预期的结果，他们将确定Lgr5+细胞是对肿瘤形成的启动和放射治疗后肿瘤存活/生长至关重要的CSCs。因此，我们期望确定一个与治疗相关的关键CSCs群体。此外，由于Wnt信号抑制剂构成了一个主要的新的研究药物类别，这些药物可能提供一种靶向Lgr5+CSCs的治疗手段，使它们容易被消除。

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.