High Content Analysis to Identify Biomarkers for Chemopreventive Drug Activity

描述（由申请人提供）： 虽然选择性雌激素受体调节剂（SERM）已经为成功的雌激素受体（ER）阳性乳腺肿瘤的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。合成的rexinoid类似物贝沙罗汀（Targretin，LGD 1069）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，这种药物引起的个体表型变化可用作替代生物标志物，以模拟未来成功的化学预防剂的作用。贝沙罗汀调节的几种分子途径会聚以改变细胞周期，从而抑制细胞增殖。除了细胞生长，贝沙罗汀还调节控制代谢的基因，包括参与甘油三酯合成的基因。此外，作为优先结合RXR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，整合不同表型作为变量的综合数据集，例如细胞周期的变化，受体参与和单个细胞的代谢状态几乎不可用。因此，我们建议进行高通量的多重，基于细胞的筛选生物标志物，容易定义功能和表型的影响与高活性和低毒性的代理。在我们的研究中，将优化多重测定，以纳入基于DNA含量和EdU掺入的细胞周期分析数据，沿着RXR α和中性脂质标记的定量和细胞内定位，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）将用于在大细胞群体中同时定量单个细胞水平的核DNA含量、细胞形态、中性脂质含量和分布以及核受体定位和活性的多个参数。我们研究的长期目标是基于对经证明可有效预防乳腺癌的药剂处理的细胞的多参数细胞学测量来定义新的生物标志物和细胞学表型。一组这些表型，细胞学指纹可以应用于寻找新的化合物具有更好的抗增殖效果和更低的毒性。这项研究的结果将阐明新的化学预防剂的潜在靶点，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用于预防癌症的新型候选药物的鉴定和开发。

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.