3D micro-addressable tissue models to understand spatiotemporal heterogeneity in transcriptional regulation

描述：3D微可寻址组织模型用于了解转录调控的时空异质性先进的体外细胞培养平台有可能揭示细胞应激反应和适应动力学的复杂转录和表观遗传调控，这是具有挑战性的或不可能在体内研究的，原因是动物模型固有的复杂性，无法通过实验操作绝大多数组织参数，以及缺乏高时空分辨率的测量技术。由于组织工程和生物材料的进步，这些平台进一步提供了对生理组织微环境的更接近，减少了在研究的早期阶段进行活体实验的需要。这些研究将在我们理解抗生素耐药性、个体化癌症药物以及开发有效和安全的干细胞疗法方面具有重要应用。然而，表型变化背后的复杂分子机制的说明一直非常有限，使工具能够研究这种过程的动力学在高时空分辨率将提供新的窗口，以前无法访问的生物学。虽然微制造策略已经使定义明确的异质模型组织成为可能，但对驻留在微尺度组织利基中的细胞进行广谱遗传或表观遗传分析是不可能的。我们的广泛假设是，微米级的时空异质性通过转录机制影响细胞的应激反应，扩大体外生理相关平台的能力将为理解这些动力学提供一个强大的、广谱的、高分辨率的工具。我们将通过追求两条协同途径朝着我们的目标努力：1)建立我们先前的微流体血管组织模型，以开发模拟脑瘤组织的模型，展示调节体内药物分布、代谢和化疗耐药发展的关键化学-机械-细胞特征；2)扩展我们通过染色质免疫沉淀(CHIP)分析转录水平调控的能力，我们已经在仅50个细胞上演示了这一点，以测量NF-kB在时空氧变化和细胞毒性应激反应之间的相互作用中所起的作用。该项目开发的能力将极大地增强3D细胞培养模型的实用性，并将提供在广泛背景下研究压力反应的转录机制。

DESCRIPTION: 3D micro-addressable tissue models to understand spatiotemporal heterogeneity in transcriptional regulation Advanced In vitro cell culture platforms have the potential to reveal the complex transcriptional and epigenetic regulation of cellular stress response and adaptation dynamics, which is challenging or impossible to study in vivo due to the inherent complexity of animal models, inability to experimentally manipulate the vast majority of tissue parameters, and a lack of high spatiotemporal resolution measurement techniques. Because of advances in tissue engineering and biomaterials, these platforms furthermore provide an ever-closer approximation of the physiological tissue microenvironment, reducing the need for in vivo experiments at earlier stages of research. Such studies will have important applications in our understanding of antibiotic resistance, personalized cancer medicine, and the development of effective and safe stem cell therapies. However, illustration of the complex molecular mechanisms behind the phenotype changes has been highly limited, and enabling tools to study the dynamics of such processes at high spatiotemporal resolution will provide new windows into previously inaccessible biology. While micro fabrication strategies have enabled well-defined heterogeneous model tissues, broad-spectrum genetic or epigenetic analysis of cells residing within micro scale tissue niches has not been possible. Our broad hypothesis is that spatiotemporal heterogeneity at micron scales impacts cellular stress response via transcriptional mechanisms, and that expanding the capabilities of physiologically relevant in vitro platforms will provide a powerful, broad spectrum, high-resolution tool to understand these dynamics. We will work towards our goals by pursuing two synergistic paths: 1) build on our prior microfluidic vascular tissue models to develop a brain tumor tissue mimic exhibiting the key chemo-mechano-cellular features regulating drug distribution, metabolism and chemoresistance development in vivo, and 2) extend our ability to analyze transcription level regulation via chromatin immunoprecipitation (ChIP), which we have already demonstrated on as few as 50 cells, to measure the role played by NF-kB in the interplay between spatiotemporal oxygen variations and cytotoxic stress response. The capabilities developed in this project will greatly enhance the utility of 3D cell culture models, and will provide access to the transcriptional machinery underlying stress response in a broad range of contexts.