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

描述(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，但有希望的类药物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的有效性和安全性。大多数用于高通量筛选(HTS)的基于细胞的系统是在单层培养的，尽管已知细胞和组织微环境的重要性。对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。细胞的背景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培养之所以有趣，很大程度上在于内在的结构，即内外细胞在生长、营养获取、缺氧和对药物的敏感性方面存在差异。因此，存在对即插即用模块中的新的3D培养系统的真正的、未得到满足的需求，该模块可以与多种细胞类型一起工作、具有成本效益，并且可以被任何人利用 HTS将改进他们的药物发现工作，特别是在寻找将转化为临床并在患者身上安全发挥作用的药物方面。我们认为，以多孔形式培养的多细胞球形细胞可以弥合这一差距。我们已经成功地将各种人类癌细胞系培养成符合HTS的96孔格式的3D球体。在适当的条件下，在3D和2D培养条件下的概念验证分析中，每孔可获得1个椭球形状，Z‘因子大于0.5。在我们的初步筛选中，已经确定了在2D单层培养的癌细胞中表现出与3D单层培养的癌细胞不同的抗肿瘤化合物。在这里，我们建议通过将人内皮细胞和成纤维细胞引入人类癌细胞来形成3D共培养，从而进一步提高我们模拟肿瘤微环境的能力。显然，肿瘤不是由一种细胞类型组成的，肿瘤的所有细胞对抗肿瘤治疗的整体反应都有贡献。此外，我们将放弃传统的细胞培养液配方和技术，转而在生理葡萄糖、谷氨酰胺和氧气浓度下培养细胞，以模拟人体。接下来，我们将开发3D共培养，使用正常的人类细胞作为药物安全性的模型。最后，我们将使用这些新模型执行概念验证HTS。用于HTS的上皮细胞、内皮细胞和基质细胞共培养以测试有效性或安全性的最终方案将在公共领域公布，任何线索都将使用高含量筛选进一步验证，以在其3D环境中可视化细胞。

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.