Advanced Development and Validation of 3 Dimensional Spheroid Culture of Primary Cancer Cells using Nano3D Technology

项目总结/摘要： 二维（2D）组织培养模型是高度简化的癌症模型，其不能捕获肿瘤细胞。 复杂性和异质性。大约95%的抗癌新药最终在临床试验中失败 尽管在现有的体外临床前模型中有强有力的活性迹象，但使体外测试成为一些 最少预测。三维（3D）球体培养模型最近已经发展到桥接“内-内-外”。 并提供了用于组装更复杂的癌症相关组织的手段 微环境尽管这些3D模型正在被工业界和学术界所采用， 它们具有局限性，并且受到低吞吐量、缺乏一致性、高成本和需要 临床验证斯克里普斯研究所分子筛选中心（SRIMSC）与 n3D Biosciences Inc.，Greiner Bio-One USA Inc.，斯克里普斯研究所的德里克·杜克特博士 分子治疗学和大卫Tuveson博士，医学博士，博士。在冷泉港实验室（CSHL）， 创建了一个战略合作，以推进一种称为3D磁性生物打印的新技术。磁 3D生物打印通过利用n3 D的核心技术NanoShuttle来解决这些关键问题 使用磁力悬浮和聚集细胞以产生球状体/类器官。极限端 产品将是一种经济实惠的; HTS已验证的384和1536微孔板格式，支持快速/一致的 为包括原发性肿瘤细胞系在内的多种细胞类型产生3D球状体。最终目标是 使用筛选自动化加速3D球状体培养，提高成本效益，并允许快速药物 测试，如FDA批准的药物在重新配方/重新用途的研究。这项技术的发展将 目标1：在一个实验室中验证当前的384孔板纳米球技术， HTS设施用于自动化兼容性。将KRAS胰腺癌细胞的3D结果与2D模型进行比较 图维森博士提供的模型。目的2：验证n3 D球体技术用于药物检测， 选择细胞毒性药物，NCI批准的肿瘤药物集和Scripps FDA批准的药物集。CC50 将2D和3D格式的数据（即产生50%细胞毒性的浓度）与 出版的文献。目标3：n3 D Biosciences将生产先进的1536孔板NanoShuttle驱动器 与HTS和药物发现工作兼容。SRIMSC将评估和实施更高密度格式 用于药物发现实用程序，最终将在约15万种化合物的大型库中进行测试， 证明HTS准备就绪。目的4：n3 D球体技术将用于患者源性 原代多形性胶质母细胞瘤（GBM）衍生细胞，最终目标是评估其在原发性癌症中的效用 细胞研究目的5：n3 D球体技术将在胰腺原位肿瘤中进行体内评价 作用及其在临床前研究中的效用。最终目标是转让和实施这项技术， 癌症研究和早期药物发现的方法。

PROJECT SUMMARY/ABSTRACT: Two-dimensional (2D) tissue culture models are highly simplified cancer models unable to capture the complexity and heterogeneity found in-vivo. Around 95% of new anticancer drugs eventually fail in clinical trial despite robust indications of activity in existing in vitro pre-clinical models, making in vitro testing some of the least predictive. Three dimensional (3D) spheroid culture models have recently advanced to bridge the “in- vitro to in-vivo gap” and provide the means for assembling more complex cancer relevant tissue microenvironments. Although these 3D models are being adopted by industry and the academic community, they have limitations and are hampered by low throughput, lack of consistency, high costs and the need for clinical validation. The Scripps Research Institute Molecular Screening Center (SRIMSC) in partnership with n3D Biosciences Inc., Greiner Bio-One USA Inc., Dr. Derek Duckett at Scripps Research department of Molecular Therapeutics and Dr. David Tuveson, M.D, Ph.D. at Cold Spring Harbor Laboratory (CSHL), have created a strategic collaboration to advance a novel technology known as 3D magnetic bioprinting. Magnetic 3D bioprinting addresses the these critical issues by utilizing n3D's core technology known as the NanoShuttle to levitate and aggregate cells using magnetic forces to produce spheroids/organoids. The ultimate end product will be an affordable; HTS validated 384 and 1536 microplate format that supports rapid/consistent production of 3D spheroids for a wide array of cell types including primary tumor lines. The end goal is to accelerate 3D spheroid cultivation using screening automation, improve cost efficiency and allow for rapid drug testing such as FDA approved drugs in reformulation/repurposing studies. Advancement of this technology will be facilitated through the following: Aim 1: Validation of the current 384 well plate nanosphere technology in a HTS facility for automation compatibility. Compare 3D results to 2D models of KRAS pancreatic cancer cell models as provided by Dr. Tuveson. Aim 2: Validation of n3D spheroid technology for drug testing against select cytotoxic drugs, NCI approved oncology drug set and the Scripps FDA Approved drug collection. CC50 data, i.e. the concentration that produces 50% cellular cytotoxicity, in 2D and in 3D formats will be compared to published literature. Aim 3: n3D Biosciences will produce an advance 1536 well plate NanoShuttle driver compatible for HTS and drug discovery efforts. SRIMSC will evaluate and implement the higher density format for drug discovery utility which will culminate in its testing on a large library of ~150K compounds to demonstrate HTS readiness. Aim 4: The n3D spheroid technology will be employed against patient derived primary Glioblastoma Multiform (GBM) derived cells with the end goal of evaluating its utility in primary cancer cell research. Aim 5: The n3D spheroid technology will be evaluated in-vivo for pancreatic orthotopic tumor effect and its utility in preclinical research. The end goal is to transfer and implement this technology and methods worldwide for cancer research and early drug discovery.