High throughput optical coherence tomography (OCT)-based imaging platform for label-free, non-invasive characterization of 3D tumor spheroids.

项目摘要 90%以上的正在开发的药物在临床上由于缺乏疗效或意外的毒性而失败 审判临床开发后期的这种失败率在很大程度上是由于使用了过于简单的 体外细胞试验和体内动物模型，在药物治疗的各个阶段具有有限的预测价值， 的发现三维（3D）组织模型被期望提供新的生理和组织学模型。 药理学数据将更能预测临床中的药物疗效和毒性，因此将具有 在缩短时间表、降低成本、增加 药物发现的投资回报，并更有效地将新药带给更多的患者。虽然 高含量荧光成像，共焦和非共焦，在高分辨率成像中被大量用于此目的， 高通量筛选（HTS）实验室，荧光试剂的低渗透性和3D光散射 直径大于50 μm的肿瘤球体模型极大地限制了形态学的测量， 这是由于球体内部的生理学特性，并且需要对样品进行大量操作，包括固定、清除和分离。 和染色，这限制了其在大量化合物的HTS中的实际应用。最近我们 表明光学相干断层扫描（OCT）可以成像并获得形态和生理 在不假定其形状的情况下，可以获得尺寸超过1 mm的整个3D肿瘤球体的信息。此外，委员会认为， 我们开发了一种并行OCT成像技术， 最先进的商业OCT技术。在这个项目中，与Marc Ferrer博士的团队合作， 美国国立卫生研究院国家中心推进转化科学，我们计划：1）开发和优化一个无标签， 能够执行并行成像的非侵入式高通量OCT（HT-OCT）成像平台（16 通道）在384孔板上。我们预计整个盘子可以在不到5分钟的时间内扫描完毕， 包括阶段转换和数据存储所需的时间; 2）进行实时纵向研究， 单细胞和多细胞型肿瘤球体（SCTS和MCTS）的形态学和生理学， OCT系统;以及3）评价肿瘤药物的作用，其包括广泛的机制 从调节细胞信号通路的靶向治疗到标准化疗药物， 肿瘤球状体关于各种肿瘤球体模型的发展的3D和纵向信息将 成为同类中的第一个。成功完成这些开发和验证研究将建立一个标签- 免费的非侵入性HT-OCT成像平台，可用于精确定量分析3D 各种类型的肿瘤球体的形态和生理信息。技术发展于 该提议也将适用于任何其它3D天然和/或生物制造的组织模型，例如神经 球状体，正在开发用于再生医学和药物发现研究。

Project Summary More than 90% of the drugs being developed fail because of lack of efficacy or unexpected toxicity in clinical trials. This failure rate in the late stages of clinical development is in large part due to the use of overly simplistic in vitro cell assays, and in vivo animal models with limited predictive value during the various stages of drug discovery. Three-dimensional (3D) tissue models are expected to provide novel physiological and pharmacological data that will be more predictive of drug efficacy and toxicity in the clinic, and will therefore have a significant immediate and long-lasting impact in shortening of the timelines, reducing the costs, increasing the return on investment of drug discovery, and bringing new medicines to more patients more efficiently. Although high content fluorescence imaging, both confocal and non-confocal, is heavily used for this purpose in high throughput screening (HTS) laboratories, low penetration of fluorescent reagents and light scattering from 3D tumor spheroid models of a size of >50 μm diameter hugely limits the measurements of the morphology and physiology inside the spheroids, and requires significant manipulation of the samples, including fixation, clearing and staining, which limits its practical use for HTS of large collections of compounds. Recently, we have demonstrated that optical coherence tomography (OCT) can image and obtain morphological and physiological information of an entire 3D tumor spheroid over 1 mm in size without presumptions about its shape. Furthermore, we developed a parallel OCT imaging technology and achieved over 10-fold speed improvement compared to state-of-the-art commercial OCT technologies. In this program, in collaboration with Dr. Marc Ferrer’s group at the NIH National Center for Advancing Translational Sciences, we plan to: 1) Develop and optimize a label-free, non-invasive high throughput OCT (HT-OCT) imaging platform capable of performing parallel imaging (16 channels) on a 384-well plate. We expect that the entire plate can be scanned within less than 5 minutes, including time needed for stage translation and data storage; 2) Perform live, longitudinal studies to characterize the morphology and physiology of single- and multi-cell type tumor spheroids (SCTS and MCTS) using the HT- OCT system; and 3) Evaluate the effects of oncology drugs, which encompass a broad range of mechanisms from targeted therapies modulating cellular signaling pathways to standard of care chemotherapeutics, on 3D tumor spheroids. The 3D and longitudinal information about development of various tumor spheroid models will be the first of its kind. Successful completion of these development and validation studies will establish a label- free, non-invasive HT-OCT imaging platform that can be used to accurately and quantitatively analyze 3D morphological and physiological information of various types of tumor spheroids. The techniques developed in this proposal will also be applicable to any other 3D native and/or biofabricated tissue models, such as neural spheroids, being developed for regenerative medicine and drug discovery studies.