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

项目摘要/摘要： 二维(2D)组织培养模型是高度简化的癌症模型，无法捕获 体内发现的复杂性和异质性。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劲的活性迹象，但使体外测试中的一些 预见性最差。三维(3D)球体培养模型最近已经发展成为一座桥梁-- 并为组装更复杂的癌症相关组织提供了手段 微环境。尽管这些3D模型正在被工业界和学术界采用， 它们有局限性，并且受到以下因素的阻碍：吞吐量低、缺乏一致性、成本高以及需要 临床验证。斯克里普斯研究所分子筛选中心(SRIMSC)与 N3D生物科学公司，Greiner Bio-One USA Inc.，斯克里普斯研究部的Derek Duckett博士 分子治疗公司和冷泉港实验室(CSHL)医学博士、博士David Tuveson 建立了一项战略合作，以推动一项名为3D磁性生物打印的新技术的发展。磁性 3D生物打印通过利用n3D的核心技术纳米穿梭解决了这些关键问题 利用磁力使细胞悬浮和聚集，以产生球体/有机体。最终的结局 产品价格实惠；经HTS认证的384和1536微版格式，支持快速/一致 为包括原发肿瘤细胞系在内的多种细胞类型生产3D球体。最终目标是 使用筛选自动化加速3D球体培养，提高成本效益并实现快速药物 测试，如FDA批准的药物重新配方/再利用研究。这项技术的进步将 目标1：验证目前的384孔板纳米球技术 用于自动化兼容性的HTS设施。KRAS胰腺癌细胞三维模型与二维模型的比较 由Tuveson博士提供的模型。目的2：验证n3D球体技术在药物检测中的有效性 选择细胞毒性药物，NCI批准的肿瘤学药物集和斯克里普斯FDA批准的药物收集。CC50 2D和3D格式的数据，即产生50%细胞毒性的浓度将与 出版的文学作品。目标3：N3D生物科学公司将生产一种先进的1536孔板纳米穿梭驱动器 兼容HTS和药物发现工作。SRIMSC将评估和实施更高密度的格式 对于药物发现实用程序，最终将在一个包含~15万种化合物的大型库中进行测试，以 证明HTS已做好准备。目的4：将n3d球体技术应用于患者衍生 原代多形性胶质母细胞瘤(GBM)来源的细胞，最终目的是评估其在原发癌症中的作用 细胞研究。目的5：对n3d球体技术在胰腺原位肿瘤的体内治疗进行评价。 在临床前研究中的作用和应用。最终目标是转让和实施这项技术，并 全球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.