Biomechanical regulation of cell extrusion and migration during metastasis

大多数成人肿瘤是由紧密结合的上皮细胞组成的，这些细胞排列成连续的片状。癌细胞从这些薄片中排出是转移过程中重要的初始步骤。以前人们认为肿瘤细胞的挤出是由上皮间充质转化(EMT)驱动的，即癌细胞失去上皮表型和与邻近细胞的粘连。然而，最近的证据表明，一种更复杂的行为，不同癌症亚型进行不同程度的EMT，在某些情况下，EMT可能根本不需要。此外，肿瘤细胞通常通过集体迁移传播，其中一组细胞连同相邻细胞之间完整的粘连一起迁移。然后，细胞群可以作为循环的肿瘤细胞团进入血流，因为它们的转移潜力是相同数量的单个循环细胞的50-100倍。目前我们还不完全了解细胞-细胞和细胞-ECM的粘连、内在力(皮质张力)或外部生物机械力(细胞外环境)如何导致细胞从上皮片挤出，以及b)个体与集体迁移。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扩散过程中的作用和相互作用的能力。在这项工作中，我们的目标是使用新的微制造设备来探索细胞-细胞接触、细胞-细胞界面张力和邻近组织施加的“挤压”力如何驱动肿瘤细胞挤压和分离。将二倍体限制为几何形状(2D微图案衬底)对细胞间边界、皮质张力和细胞运动性有显著影响。根据拉伸程度的不同，细胞表现为波状、弱连接，迁移速度较快(圆形)，或连接较强、运动性较差(三角形)，类似于健康的上皮组织。这表明细胞皮质僵硬和细胞内机制在影响粘连或更快迁移的能力方面起着至关重要的作用，并且可以通过几何约束来控制。我们假设(I)在肿瘤细胞中看到的较低的皮质张力通过削弱相邻细胞之间的凝聚力来增加肿瘤细胞的扩散，以及(Ii)生物机械力和细胞-细胞-细胞间黏附之间的平衡控制着癌细胞作为个体与良性肿瘤集群的分离和扩散。作为癌症发展的模型，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，和两组来自患者的原发肿瘤和转移性头颈癌细胞(可在布拉加实验室获得；角质形成细胞来源的肿瘤)。我们将设计新的平台，提供高度可控的机械应力。我们的目标是：设计新一代3D微孔阵列和微通道，以评估细胞几何和外部机械力对粘附性和迁移的影响；确定处于不同转化阶段的细胞对内在皮质张力和外部机械力变化的反应；比较不同几何挑战(3D细胞微孔)和细胞分离/运动(微通道)中的致癌信号。结果：该项目将促进我们对驱动肿瘤细胞挤出和移动的因素的理解，使我们能够设计出新的抗转移策略来抑制肿瘤细胞的侵袭。该项目将产生关于转移的机械力调节、肿瘤进展的不同状态如何响应拉伸挑战、分子调节器和干扰这一过程的筛查平台的全面知识。

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.