High throughput screening in human 3D spheroids of epithelial, endothelial, and s

人类上皮细胞、内皮细胞和组织细胞 3D 球体的高通量筛选

• 批准号: 7936184
• 负责人: KRISTIINA VUORI
• 金额: $ 47.75万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7936184
• 关键词: Academia; Adherent Culture; Architecture; Biological Assay; Cancer cell line; Cancerous; Cell Culture Techniques; Cell Survival; Cells; Cellular Spheroids; Clinic; Clinical Trials; Coculture Techniques; Confocal Microscopy; Culture Media; Culture Techniques; Cultured Cells; Dimensions; Dose; Drug Formulations; Endothelial Cells; Engineering; Environment; Epithelial; Epithelial Cells; Evaluation; Event; Exhibits; Failure; Fibroblasts; Genomics; Glucose; Glutamine; Growth; Histology; Human; Human Cell Line; Human body; Hypoxia; Image; Industry; Knowledge; Libraries; Mammary Neoplasms; Medical; Miniaturization; Modeling; Nutrient; Oxygen; Patients; Pharmaceutical Preparations; Physiological; Play; Plug-in; Pre-Clinical Model; Proteomics; Protocols documentation; Public Domains; Publishing; Reagent; Safety; Science; Scientist; Screening procedure; Signal Transduction; Stromal Cells; System; Techniques; Tissue Model; Tissues; Translating; Tumor Tissue; Work; base; cancer cell; cell type; cost; density; drug candidate; drug discovery; drug efficacy; efficacy testing; high throughput screening; human tissue; improved; in vivo; interest; meetings; monolayer; neoplastic cell; prevent; response; tissue culture; tumor

DESCRIPTION (provided by applicant): Tissue High Throughput Screening Platforms. The failure of promising drug-like compounds to successfully complete clinical trials, despite our ever-increasing knowledge of genomics, proteomics, and cell signaling events in the basic medical sciences suggests that the preclinical models scientists are using in academia and industry alike are not predictive of the overall drug efficacy and safety. Most cell-based systems for high throughput screening (HTS) are cultured in a monolayer, despite the known importance of the cell and tissue microenvironment. Studies in breast tumor cells have highlighted the importance of culturing cells in 3-dimensions (3D), with cell-cell and stromal interactions. The cellular context and environment provide signals critical to a cell's fate. As such, it is recognized that 3D models (aka spheroids) may be a more predictive preclinical model. However, technical and financial hurdles prevent 3D models from meeting their promise in current HTS efforts. In addition, much of what makes 3D cultures interesting lies in the inherent architecture where the inner and outer cells vary with respect to growth, nutrient access, hypoxia, and sensitivity to drugs. Thus, there is a true, unmet need for new 3D culture systems in plug and play modules that can work with multiple cell types, is cost-effective, and can be utilized by anyone doing HTS to improve their drug discovery efforts, especially with respect to finding drugs that will translate to the clinic and act safely in patients. We propose that human cells cultured in a multi-well format as multicellular spheroids can bridge that gap. We have successfully cultured various human cancer cell lines as 3D spheroids in a 96-well format amenable for HTS. Under the appropriate conditions, 1 spheroid forms per well, and Z' factors of greater than 0.5 have been achieved in proof-of-concept assays which compared 3D to 2D culture conditions. In our preliminary screens, anti-tumor compounds have been identified which exhibit differential effects in the cancer cells cultured as 2D monolayers versus those in 3D. Here we propose to further our ability to model the tumor microenvironment by introducing human endothelial cells and fibroblasts to the human cancer cells to form 3D co-cultures. Clearly, tumors are not composed of one cell type, and all the cells of the tumor contribute to the overall response to anti-tumor therapy. In addition, we will step away from traditional cell culture medium formulations and techniques and instead culture the cells under physiological glucose, glutamine, and oxygen concentrations to mimic the human body. Next, we will develop 3D co-cultures using normal human cells as a model for drug safety. Lastly, we will perform a proof of concept HTS using these new models. The resulting protocols for the co-culture of epithelial, endothelial, and stromal cells for HTS to test efficacy or safety will be published in the public domain, and any leads will be further validated using high content screening to visualize the cells in their 3D context.

描述（由申请人提供）：组织高通量筛选平台。尽管我们对基础医学中的基因组学、蛋白质组学和细胞信号事件的了解不断增加，但有前景的类药化合物未能成功完成临床试验，这表明科学家在学术界和工业界使用的临床前模型并不能预测整体药物功效和安全性。尽管细胞和组织微环境的重要性众所周知，但大多数用于高通量筛选 (HTS) 的细胞系统都是在单层中培养的。对乳腺肿瘤细胞的研究强调了在 3 维 (3D) 中培养细胞以及细胞与细胞和基质相互作用的重要性。细胞背景和环境提供对细胞命运至关重要的信号。因此，人们认识到 3D 模型（又名球体）可能是更具预测性的临床前模型。然而，技术和财务障碍阻碍了 3D 模型在当前 HTS 工作中兑现其承诺。此外，3D 培养的有趣之处很大程度上在于其内在结构，其中内部和外部细胞在生长、营养获取、缺氧和对药物的敏感性方面存在差异。因此，对于即插即用模块中的新 3D 培养系统存在真正的、未满足的需求，该系统可以与多种细胞类型一起工作，具有成本效益，并且任何人都可以使用 HTS 改善其药物发现工作，特别是寻找可转化为临床并在患者中安全发挥作用的药物。我们建议以多孔形式培养的人类细胞作为多细胞球体可以弥补这一差距。我们已成功将各种人类癌细胞系培养为适合 HTS 的 96 孔格式的 3D 球体。在适当的条件下，在比较 3D 和 2D 培养条件的概念验证测定中，每孔形成 1 个球状体，并且 Z' 因子大于 0.5。在我们的初步筛选中，已鉴定出抗肿瘤化合物，它们对 2D 单层培养的癌细胞与 3D 单层培养的癌细胞表现出不同的作用。在这里，我们建议通过将人内皮细胞和成纤维细胞引入人癌细胞中形成 3D 共培养物，进一步增强我们对肿瘤微环境进行建模的能力。显然，肿瘤并非由一种细胞类型组成，肿瘤的所有细胞都有助于抗肿瘤治疗的整体反应。此外，我们将放弃传统的细胞培养基配方和技术，而是在生理葡萄糖、谷氨酰胺和氧气浓度下培养细胞，以模拟人体。接下来，我们将使用正常人体细胞作为药物安全模型来开发 3D 共培养物。最后，我们将使用这些新模型进行概念验证 HTS。由此产生的 HTS 共培养上皮细胞、内皮细胞和基质细胞以测试功效或安全性的方案将在公共领域发布，并且任何线索都将使用高内涵筛选进一步验证，以在 3D 背景下可视化细胞。