GFP IMAGING FOR IN VIVO HIGH-THROUGHPUT DRUG SCREENING

本申请的目标是开发一种临床相关的， 体内高通量癌症药物筛选。 为了实现这一目标，我们将 进一步开发转移性癌症的小鼠模型， 荧光肿瘤，可以在外部成像，在非侵入性，快速， 实时方式适合高通量筛选抗肿瘤， 抗转移和抗血管生成剂。 作为迈向 为此，我们转导了绿色荧光蛋白（GFP）基因， 从生物发光水母Aequorea维多利亚，到一系列的50个人类 以及在移植后体内稳定表达GFP的啮齿类动物癌细胞系 合适的啮齿动物模型。 我们已经证明， 脑、肝和骨可以在外部、经皮全身成像 通过荧光定量。 在新的发展中，我们已经表明， 肺中表达GFP的肿瘤和转移可以在外部成像。 高通量模型的开发将具有以下特定目标： 1)确定与转移性癌症有关的器官，这些器官可以在外部进行治疗。 通过肿瘤GFP表达实时成像。 外部成像将是 与荧光肿瘤的直接成像相比; 2）测定和 改善外部成像的最大深度和最小肿瘤尺寸。 分辨率的改进将集中在信号/背景的增加上 1）表达超亮GFP的癌细胞克隆 将在体外选择，以增强尺寸和深度图像分辨率后， 选择性滤光片、可调谐激光和双光子 将使用成像以最大化深度和尺寸分辨率; 3）实时 肿瘤和转移性生长和血管生成和抑制 将对药物进行成像和定量，以证明使用 快速抗肿瘤、抗转移和抗血管生成药物的模型 屏幕 通过光学成像和标准品获得的剂量响应曲线 肿瘤测量将在结肠、胰腺、前列腺、黑色素瘤和 肺肿瘤模型。 拟定商业应用： 所有主要癌症的外部可成像、遗传荧光转移性小鼠模型 将开发用于快速筛选新型有效的抗肿瘤、抗转移 和抗血管生成剂。 目前抗癌新药体内试验的市场规模 随着基因组学、蛋白质组学和组合医学等新技术的发展， 化学合成和高通量体外筛选提供了许多新的 必须在体内进行测试和验证的电极导线。

The goal of this application is the development of a clinically-relevant in vivo high-throughput cancer drug screen. To achieve this goal, we will further develop mouse models of metastatic cancer with genetically fluorescent tumors that can be imaged externally in a non-invasive, rapid and real-time manner suitable for high-throughput screening of anti-tumor, anti-metastatic and anti-angiogenesis agents. As an important step toward this goal, we have transduced the green fluorescent protein (GFP) gene, cloned from the bioluminescent jellyfish Aequorea victoria, into a series of 50 human and rodent cancer cell lines that stably express GFP in vivo after transplantation to appropriate rodent models. We have already demonstrated that GFP tumors on the brain, liver, and bone can be externally, transcutaneously whole-body imaged quantitatively by fluorescence. In new developments, we have shown that GFP-expressing tumors and metastasis in the lung can be externally imaged. The high-throughput models will be developed with the following Specific Aims: 1) Determination of organs involved with metastatic cancer that can be externally imaged by tumor GFP expression in real-time. External imaging will be compared with direct imaging of the fluorescent tumors; 2) Determination and improvement of maximum depth and minimal tumor size for external imaging. Improvements in resolution will focus on increases in the signal/background ratio, as outlined below: 1) Ultra-bright GFP-expressing cancer cell clones will be selected in vitro to enhance size and depth image resolution after transplantation in vivo; 2) Selective filters, tunable laser and two-photon imaging to maximize depth and size resolution will be used; 3) Real-time tumor and metastatic growth and angiogenesis and inhibition by representative drugs will be imaged and quantified to demonstrate feasibility of use of the models for a rapid anti-tumor, anti-metastatic and anti-angiogenesis drug screen. Dose response curves obtained by optical imaging and by standard tumor measurements will be compared in colon, pancreas, prostate, melanoma and lung tumor models. PROPOSED COMMERCIAL APPLICATION: Externally-imageable, genetically-fluorescent metastatic mouse models of all major cancer types will be developed for rapid screening for novel effective anti-tumor, anti-metastatic and anti-angiogenic agents. The current market size for in vivo testing of new cancer drugs is rapidly expanding as new technologies of genomics, proteomics, and combinatorial chemical synthesis and high-throughput in vitro screening provide provide numerous new leads that must be tested and validated in vivo.