Biomechanical regulation of cell extrusion and migration during metastasis

大多数成人肿瘤由紧密结合的上皮细胞组成，组织成连续的薄片。癌细胞从这些薄片中挤出是转移的重要初始步骤。以前认为，肿瘤细胞挤出是由上皮间质转化（EMT）驱动的，由此癌细胞失去上皮表型和与邻近细胞的粘附。然而，最近的证据表明一种更复杂的行为，不同的癌症亚型进行不同程度的EMT，在某些情况下，可能根本不需要EMT。此外，肿瘤细胞通常通过集体迁移传播，其中细胞群与相邻细胞之间的完整粘附一起迁移。然后，细胞集合体可以作为循环肿瘤细胞簇进入血液，由于它们的转移潜力比相同数量的单个循环细胞大50- 100倍，它们是“最可能的转移先兆”。内力（皮质张力）或外在生物力学力（细胞外环境）有助于a）细胞从上皮层挤出和B）个体相对于集体迁移。我们缺乏能够分离这些变量的生理相关模型，严重阻碍了我们研究它们在肿瘤细胞传播过程中的贡献和相互作用的能力。在这项工作中，我们的目标是使用新型的微制造设备来探索细胞-细胞接触，细胞-细胞界面张力和相邻组织施加的“挤压”力如何驱动肿瘤细胞挤出和分离。将双峰限制为几何形状（2D微图案化基底）对细胞间边界、皮质张力和细胞运动性具有显著影响。取决于张力水平，细胞显示出波状的弱连接和更快的迁移（圆形）或强连接和更少的运动性（三角形），类似于健康的上皮组织。这表明细胞皮层硬度和细胞内力学在影响粘附在一起或更快迁移的能力方面起着重要作用，我们假设（i）肿瘤细胞中可见的较低皮质张力通过削弱相邻细胞之间的凝聚力而增加肿瘤细胞的扩散，以及（ii）生物力学力和细胞-细胞&细胞-细胞之间的平衡，ECM粘附控制癌细胞作为个体与集群从良性肿瘤的分离和传播。作为癌发展的模型，我们将使用一组细胞：原代角质形成细胞，永生化角质形成细胞，以及来自患者的两组原发性肿瘤和转移性头颈癌细胞（可在Braga实验室获得;角质形成细胞衍生的肿瘤）。我们将设计新颖的平台，以提供机械应力的高度可控性。我们的目标是：设计下一代3D微孔阵列和微通道，以评估细胞几何形状和外部机械力对粘附特性和迁移的影响;定义细胞在不同转化阶段对内在皮质张力和外部机械力变化的反应;比较各种几何挑战中的致癌信号结果：该项目将加速我们对驱动肿瘤细胞挤出和运动的因素的理解，使我们能够设计新的抗转移策略来抑制肿瘤细胞侵袭。该项目将产生转移的机械力调节的全面知识，肿瘤进展的不同状态如何响应张力挑战，分子调节剂和筛选平台干扰该过程。

Most adult tumours are comprised of tightly bound epithelial cells organised into continuous sheets. The extrusion of cancer cells from these sheets is an important initial step in metastasis. It was previous believed that tumour cell extrusion is driven by epithelialmesenchymal transition (EMT) whereby cancer cells lose epithelial phenotypes and adhesions to neighbouring cells. However, recent evidence has suggested a more complex behaviour, where different cancer subtypes perform varying degrees of EMT and in certain cases, EMT may not be required at all. Furthermore, tumour cells often disseminate via collective migration where group of cells migrate together with intact adhesions among neighbours. Cell collectives can then enter the bloodstream as circulating tumour cell clusters, which are "the most likely harbingers of metastases" due to their 50-100X greater metastatic potential than equal numbers of individual circulating cells.We currently do not fully understand how cell-cell and cell-ECM adhesions, intrinsic forces (cortical tension) or extrinsic biomechanical forces (extracellular environment) contribute to the a) extrusion of cells from epithelial sheets and b) individual vs. collective migration. Our lack of physiologically-relevant models capable of isolating these variables severely hampers our ability to study their contributions and interactions during tumour cell dissemination. In this work, we aim to use novel microfabricated devices to explore how cell-cell contacts, cell-cell interfacial tension and "squeeze" forces applied by neighbouring tissues drive tumour cell extrusion and detachment. Confinement of doublets into geometric shapes (2D micropatterned substrates) has a dramatic influence on intercellular boundaries, cortical tension and cell motility. Depending on the tensional level, cells displayed undulated, weak junctions and migrate faster (circular shapes) or strong junctions and less motility (triangular shapes), resembling healthy epithelial tissues. This indicates an essential role of cell cortex stiffness and intracellular mechanics to influence the ability to stick together or to migrate faster, and that these can be controlled via geometric confinement.We hypothesize that (i) the lower cortical tension seen in tumour cells increases tumour cell dissemination by weakening cohesion among neighbours and (ii) biomechanical forces and the balance between cell-cell & cell-ECM adhesions control the detachment and dissemination of cancer cells as individual vs. clusters from benign tumours.As a model of carcinoma development, we will use a panel of cells: primary keratinocytes, immortalized keratinocytes, and two sets of primary tumour and metastatic head and neck carcinoma cells from patients (available in the Braga lab; keratinocyte-derived tumours). We will design novel platforms to provide high controllability of mechanical stress. We aim to: Design next generation 3D-microwell arrays and microchannels to evaluate the influence of cell geometry and external mechanical forces on adhesive properties and migration; Define the response of cells at various stages of transformation to variations in intrinsic cortical tension and external mechanical forces; Compare the oncogenic signalling in the various geometric challenges (3D cellular microwell) and cell detachment/motility as cohorts (microchannels).Outcomes: This project will accelerate our understanding of the factors that drive tumour cell extrusion and motility, enabling us to devise novel anti-metastatic strategies to inhibit tumour cell invasion. The project will generate comprehensive knowledge of mechanical force regulation of metastasis, how different states of tumour progression respond to tensional challenges, molecular regulators and screening platforms to interfere with the process.