Background

Cells and the surrounding extracellular matrix (ECM) build functional entities that rely on dynamic adjustments of both, the matrix and the cells, to prevent disease. For years it was thought that the ECM solely provides structural support for embedded cells. However, recent research has highlighted the pivotal functions of the ECM beyond its scaffold function. Modifications of the ECM have been linked to disease progression, and particularly in the context of cancer, in metastasis initiation and subsequent links to clinical prognosis and patient survival.

On one hand, the ECM can create a barrier that impedes cell migration. Spatial confinement in the matrix provokes cell adaptations, such as cell body deformation, nuclear deformation and active ECM remodelling by matrix metalloproteinases (Bonnans et al., 2014; Jayo et al., 2016; Yamada and Sixt, 2019). Conversely, fibrous structures built by a network of collagen and matrix-associated proteins can support cell migration by providing a three-dimensional fibrillary physical scaffold to guide directed motility. ECM components, such as fibronectin provide anchor points for cell adhesion, which is important for mesenchymal migration modes. Cell migration in 2D has been extensively studied and the basic principles include repeated cycles of membrane protrusion at the leading edge, adhesion, F-actin retrograde flow, and actomyosin-driven cell retraction (Abercrombie, 1980, Yamada and Sixt, 2019). Cell migration in 3D is emerging to be a more complex process and cells have been reported to use mesenchymal, amoeboid, lobopodial and collective cell migration and can also interconvert between these forms depending on the mechanical properties of the microenvironment (van Helvert et al., 2018, Yamada and Sixt, 2019). Invasive cancer cells align along and attach to ECM tension fibres during migration and often leave behind tunnels due to MMP degradation at the front (Yamada and Sixt, 2019). Before the onset of locomotion, many migratory cells explore the ECM with actin-rich membrane protrusions, such as filopodia or lamellipodia, a prerequisite for successful navigation through 3D environments. This allows sensing of dynamic changes in the microenvironment and direct adaptation to the topography, stiffness and anchor points within the matrix (Leithner et al., 2016, Pfisterer et al., 2020).

Recent development of novel imaging tools and advanced microscopes with high resolution and frame rates allow the visualization of these processes in real-time with limited photo-toxicity. Various models have been used, including organotypic, cell-derived or 3D matrices, to investigate cell behaviour in a more physiological 3D environment. However, a detailed general 3D matrix protocol for broad applicability of cancer cell-ECM interaction studies has not been published, to our knowledge. We recently developed a 3D system with variable mechanical properties to monitor the phenotype of cancer cells and the role of the filopodia stabilizing protein fascin within this by live cell imaging (Pfisterer et al., 2020). Our system allows analysis of any protein of interest in living cells embedded into a collagen-fibronectin matrix. Further, we show how the matrix can be stiffened via non-enzymatic glycation to investigate the impact of matrix stiffening on cell or molecule behaviour. Finally, we provide a simple method for collagen staining with fluorescent dyes for simultaneous imaging of the matrix and the cells that allows to draw conclusions on reciprocal forces between the ECM and interacting cells and to predict correlations between cell morphology and behaviour and matrix deformation.