High Content Analysis to Identify Biomarkers for Chemopreventive Drug Activity

描述(由申请人提供)： 尽管选择性雌激素受体调节剂(SERM)已经为雌激素受体(ER)阳性乳腺癌的成功化学预防奠定了基础，但迫切需要新的药物来预防ER阴性乳腺癌。合成雷克生类似物贝沙罗汀(Targretin，LGD1069)在临床前模型中预防乳腺癌，目前正在临床环境中进行研究。贝沙罗汀预防乳腺肿瘤的确切机制尚未确定；然而，该药物引起的个体表型变化可作为替代生物标记物，以模拟未来成功的化学预防药物的效果。贝沙罗汀调节的几个分子通路会聚在一起改变细胞周期，从而抑制细胞增殖。除了细胞生长，贝沙罗汀还调节新陈代谢的基因，包括参与甘油三酯合成的基因。此外，作为优先与RXR结合的配体，贝沙罗汀标志着RXR成为乳腺癌预防药物的潜在靶点。目前，将不同表型整合为变量的全面数据集几乎是不可用的，例如细胞周期、受体参与和单个细胞的代谢状态的变化。因此，我们建议对生物标志物进行高通量、多重、基于细胞的筛选，这些生物标志物容易定义与高活性和低毒性药物相关的功能和表型效应。在我们的研究中，我们将优化一种多重分析方法，以结合基于DNA含量和EDU掺入的细胞周期分析数据，以及RXRA的定量和细胞内定位以及中性脂标记，以响应正常乳腺上皮细胞中的贝沙罗汀。高通量荧光显微镜(HTM)将被用来同时定量核DNA含量、细胞形态、中性脂含量和分布的多个参数，以及在单个细胞水平上的核受体定位和活性，在大量细胞群中。我们研究的长期目标是定义新的生物标记物和细胞学表型，基于对经证明能有效预防乳腺癌的药物治疗的细胞的多参数细胞学测量。一组这些表型，细胞学指纹图谱可以应用于寻找具有更好的抗增殖作用和较低毒性的新化合物。这项研究的结果将阐明新的化学预防药物的潜在靶点，并有助于开发先进的诊断工具。这项技术的进一步发展可能会加快发现和开发新的癌症预防候选药物。

DESCRIPTION (provided by applicant): Although selective estrogen receptor modulators (SERMs) have set the stage for successful chemoprevention of estrogen receptor (ER) positive mammary tumors, novel agents are urgently needed to prevent ER-negative breast cancer. The synthetic rexinoid analogue bexarotene (Targretin, LGD1069) prevents breast cancer in preclinical models and is now studied in clinical settings. The exact mechanism by which bexarotene prevents mammary tumors has not been identified; however, individual phenotypic changes elicited by this drug could be used as surrogate biomarkers, to model the effects of future successful chemopreventive agents. Several of the molecular pathways modulated by bexarotene converge to alter cell cycle and thus suppress cell proliferation. In addition to cell growth, bexarotene also regulates genes governing metabolism, including genes involved in triglyceride synthesis. Furthermore, as a ligand preferentially binding to RXR, bexarotene marks RXR as potential target for breast cancer preventive agents. Currently, comprehensive datasets integrating different phenotypes as variables, such as changes in cell cycle, receptor engagement and metabolic status of individual cells is virtually unavailable. Therefore, we propose to perform high-throughput multiplex, cell-based screens for biomarkers that readily define functional and phenotypic effects linked to agents with high activity and low toxicity. In our study a multiplex assay will be optimized to incorporate cell cycle analysis data based on DNA content and EdU incorporation, along with quantitation and intracellular localization of RXRa and neutral lipid labeling, in response to bexarotene in normal mammary epithelial cells. High-throughput fluorescence microscopy (HTM) will be used to simultaneously quantitate multiple parameters of nuclear DNA content, cell morphology, neutral lipid content and distribution, and nuclear receptor localization and activity at the single cell level, in a large population of cells. The long term goal of our studies is to define novel biomarkers and cytological phenotypes based on multiparametric cytological measurements on cells treated with agents proven to effectively prevent breast cancer. A set of these phenotypes, a cytological fingerprint can be applied to search for new compounds with better anti-proliferative effects and less toxicity. The outcomes of this study will elucidate potential targets for new chemopreventive agents as well as help the development of advanced diagnostic tools. Further development of this technology may speed up the identification and development of novel drug candidates for the prevention of cancer.