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

描述（由申请人提供）：组织高通量筛选平台。尽管我们对基因组学、蛋白质组学和基础医学中的细胞信号事件的了解不断增加，但有希望的药物样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都不能预测药物的整体疗效和安全性。尽管已知细胞和组织微环境的重要性，大多数用于高通量筛选（HTS）的基于细胞的系统都是在单层中培养的。乳腺肿瘤细胞的研究强调了在三维（3D）中培养细胞的重要性，细胞-细胞和基质相互作用。细胞环境和环境提供了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）可能是一种更具预测性的临床前模型。然而，技术和资金障碍阻碍了3D模型在当前HTS努力中实现其承诺。此外，3D培养物的有趣之处在于其固有的结构，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各不相同。因此，对于即插即用模块的新型3D培养系统，存在着真正的、未被满足的需求，这种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

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.