Engineering extracellular matrix factories to study how the cellular microenvironment regulates gene expression

哺乳动物细胞和组织存在于复杂的三维（3D）微环境中，其中不同的信号分子和支持结构动态相互作用，形成复杂的网络或细胞外基质（ECM）。除了为细胞提供结构支持外，基质还传递环境信号，指导正常细胞行为的许多方面，包括形状，存活和迁移。我们发现在组织损伤时（Midwood et al. Nat Med 2009）和感染期间（Goh et al. JI 2010）ECM糖蛋白腱生蛋白-C的快速诱导能够在小鼠体内实现有效的宿主应答（Midwood et al. Nat Med 2009; Piccinini et al. Cell Reports 2012）。值得注意的是，我们发现生腱蛋白-C可以通过调节巨噬细胞microRNA表达来维持促炎细胞因子合成（Piccinini等人Cell Reports 2012）。因此，这种ECM蛋白创造了一种细胞微环境，通过调节microRNA水平，进而调节基因表达谱，深刻影响细胞表型和行为。然而，我们不知道生腱蛋白C是如何做到这一点的，也不知道这是否也发生在人类身上。在常规细胞培养系统中复制这些信号通常具有挑战性。此外，虽然人工创建的3D细胞培养模型在组织工程和干细胞研究中显示出了潜力，但它们未能密切模仿体内微环境。有些复制了矩阵的三维性，但缺乏生化线索;该项目的目的是设计生理相关的人类ECM工厂，其可用于定义ECM的特定组分，包括生腱蛋白-C，通过调节人类巨噬细胞和癌细胞的microRNA水平来影响基因表达谱。为此，CRISPR/Cas9技术将用于人类成纤维细胞系的基因组编辑，该细胞系将用于生成无细胞人类成纤维细胞衍生的3D基质模型。这些模型具有生物化学、生物药理学和功能特征，并用作培养感兴趣的细胞类型的底物，包括原代人单核细胞衍生的巨噬细胞。将通过实时PCR研究单个ECM分子耗竭对候选靶基因表达（例如早期反应炎性microRNA及其靶标）的影响。如果这些模型得到验证，也有可能进行RNA测序，以全面识别受基质影响的基因和途径。应急计划包括稳定的shRNA或siRNA敲低以耗尽候选ECM分子。总的来说，该项目开发了一种生物技术工具，用于彻底解剖来自ECM的个体、人类微环境信号在基因表达的转录后调控中的作用，为宿主对感染的反应和肿瘤微环境的研究提供了新的机会。

Mammalian cells and tissues exist in complex three-dimensional (3D) microenvironments, in which diverse signalling molecules and support structures dynamically interact forming a complex network or extracellular matrix (ECM). Beyond providing structural support to cells, the matrix conveys environmental signals that direct many aspects of normal cell behaviour, including shape, survival and migration. We found rapid induction of the ECM glycoprotein tenascin-C upon tissue damage (Midwood et al. Nat Med 2009) and during infection (Goh et al. JI 2010) that enables effective host responses in the mouse in vivo (Midwood et al. Nat Med 2009; Piccinini et al. Cell Reports 2012). Notably, we discovered that tenascin-C can sustain pro-inflammatory cytokine synthesis by regulating macrophage microRNA expression (Piccinini et al. Cell Reports 2012). Thus, this ECM protein creates a cellular microenvironment that profoundly influences cell phenotype and behaviour by regulating microRNA levels and, in turn, gene expression profiles. However, we do not know how tenascin-C does this and whether this occurs also in humans. Replicating these signals in conventional cell culture systems is often challenging. Moreover, while artificially-created 3D cell culture models have shown potential in tissue engineering and stem cell research, they fail to closely mimic the in-vivo-like microenvironment. Some reproduce the three dimensionality of the matrix, but lack the biochemical cues; others contain non-physiologically relevant microenvironmental signals or do not allow studying the contribution of individual components to the cellular process of interest.The aim of this project is to engineer physiologically relevant human ECM factories that can be used to define whether specific components of the ECM, including tenascin-C, impact gene expression profiles by regulating microRNA levels of human macrophages and cancer cells. For this, CRISPR/Cas9 technology will be employed for genome editing of human fibroblast cell lines, which will be used to generate cell-free human fibroblast-derived 3D matrix models. These models with be biochemically, biophysically and functionally characterized, and used as substrate for the culture of cell types of interest, including primary human monocyte-derived macrophages. Effects of individual ECM molecule depletion on candidate target gene expression (e.g. early response inflammatory microRNAs and their targets) will be investigated by real-time PCR. If these models are validated, there is also the possibility to perform RNA sequencing to globally identify genes and pathways that are affected by the matrix. Contingency plans include stable shRNA or siRNA knockdown to deplete candidate ECM molecules. Collectively, this project develops a biotechnology tool for thoroughly dissecting the role of individual, human microenvironmental signals from the ECM in the posttranscriptional regulation of gene expression, presenting new opportunities for research on host responses to infection, and tumour microenvironments.