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（CSC），并表征其功能特性，以便进行靶向治疗。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-脊椎蛋白受体Lgr 5的表达定义了具有高效肿瘤引发能力的乳腺肿瘤干细胞群体;（B）在模型系统中放射治疗后，Lgr 5+细胞存活的剂量-反应曲线预测了肿瘤消除的剂量-反应曲线;和（c）Lgr 5+细胞在非消融性辐射后作为干细胞用于乳腺肿瘤再生。该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动物微辐照器，该微辐照器可向肿瘤提供均匀剂量的辐照，对周围组织的损伤可忽略不计。为了便于鉴定和分离Lgr 5+肿瘤细胞，将具有乳腺靶向癌基因的转基因小鼠与Lgr 5-报告菌株杂交。将通过流式细胞术从所得乳腺肿瘤中分选Lgr 5+细胞，并通过向受体动物注射有限细胞稀释液来测量其肿瘤引发能力。将Lgr 5+细胞与Lgr-和模拟分选的肿瘤上皮细胞进行比较，以确定大部分肿瘤起始能力是否与Lgr 5+细胞相关。由于Lgr 5是Wnt/β-连环蛋白途径的辅助受体，因此该蛋白质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自我更新的肿瘤球测定中进行测试。进一步的体内实验将测试Lgr 5+细胞是控制肿瘤对放射疗法的长期反应的CSC的假设。使用CT图像引导的小动物微辐照器向小鼠乳腺肿瘤递送单次高剂量，将在局部肿瘤控制的背景下研究辐照后Lgr 5+细胞消除的剂量-反应曲线。这将在体内检验肿瘤的消退取决于Lgr 5+细胞的消除的假设。这些细胞的治疗相关性的进一步证据将是 这是从谱系追踪实验确定的，该实验询问非消融性照射后肿瘤的再生长是否发生在Lgr 5 + CSC。如果获得预期的结果，他们将鉴定Lgr 5+细胞为CSC，其对于肿瘤发生的起始和放射治疗后的肿瘤存活/生长都至关重要。因此，我们期望鉴定出治疗相关的关键CSC群体。此外，由于Wnt信号传导抑制剂构成了研究药物的主要新类别，因此这些可能提供靶向Lgr 5 + CSC并使其易于消除的治疗手段。

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.