Podosomes in space: macrophage migration and matrix degradation in 2D and 3D settings

Abstract

Migration of macrophages is a key process for a variety of physiological functions, such as pathogen clearance or tissue homeostasis. However, it can also be part of pathological scenarios, as in the case of tumor-associated macrophages. This review presents an overview of the different migration modes macrophages can adopt, depending on the physical and chemical properties of specific environments, and the constraints they impose upon cells. We discuss the importance of these environmental and also of cellular parameters, as well as their relative impact on macrophage migration and on the formation of matrix-lytic podosomes in 2D and 3D. Moreover, we present an overview of routinely used and also newly developed assays for the study of macrophage migration in both 2D and 3D contexts, their respective advantages and limitations, and also their potential to reliably mimic in vivo situations.

Keywords: MMP; cell migration; extracellular matrix; macrophages; podosomes; proteases.

Figure 1. Macrophage migration in 2D and 3D. (A) In vitro, on 2D surfaces, human macrophages adopt a rounded, flat cell shape and follow a classical five-step model of cell migration. Adhesion to the substrate is marked by red dots. (B) In vivo, macrophages are confronted with both 2D (B, bottom) and 3D (B, middle and top) situations. For example, during vascularization, macrophages can be found on vascular junctions attached to a 2D endothelial wall. Depending on the extracellular environment, macrophages migrating through interstitial space can adopt a rounded cell shape and use the amoeboid, non-proteolytic migration mode (B top) or a prolonged protrusion-rich morphology and use mesenchymal, proteolysis-dependent migration (B middle).

Figure 2. Macrophage podosomes in 2D and 3D. Schematic overview over podosome structure and ultrastructure in human macrophages attached to a 2D surface (left) or embedded in a 3D matrix (right). In 2D, macrophages form numerous dot-like podosomes (red dots) on the substrate-attached cell site. Podosome components are organized in core, ring, and cap structures. Reconstructed z-stacks of confocal micrographs of a single podosome show typical members of the different substructures (F-actin, vinculin, and supervillin) (adapted with permission from Bhuwania et al.³²). In dense 3D environments, human macrophages form 3D podosomes at protrusions. So far, no substructures have been described, and components of the core and ring of 2D podosomes mostly co-localize (see confocal micrographs of F-actin and vinculin; adapted with permission from van Goethem et al.⁵⁷). The table lists several parameters typical of podosomes in 2D and 3D, respectively.

Figure 3. Mesenchymal migration involves distinct proteases and nuclear constriction. (A) SEM micrographs (with permission from Renaud Poincloux, Tri Imaging Toulouse France; bars: 1 µm) of native collagen I and pepsin-extracted gelled collagen I show similar architecture and schematic presentation of distinct inter-fiber connectivity. (B) The percentage of macrophages migrating through native collagen I in the absence or presence of Ly-mix (lysosomal protease inhibitors) and/or GM6001 (MMP inhibitor), or Y27632 (ROCK inhibitor) was quantified. Macrophage migration through native collagen is not inhibited by Y27632 and is abolished by GM6001, indicating that cells use the mesenchymal migration mode, but not the amoeboid migration. hMDMs isolated from four independent healthy donors (n = 4). Statistics: two-tailed unpaired Student t test; **P < 0.005; n.s. P = 0.165. Macrophages penetrating matrices were examined by scanning electron microscopy (for methods, see van Goethem et al.¹⁷). Note the presence of holes made by cells (purple) in the matrix of native collagen I and pepsin-extracted gelled collagen I (arrows). Representative pictures are shown. Bars: 10 µm. (C) Reduced nuclear circularity, a signature of mesenchymal migration. Galleries showing confocal micrographs of macrophage nuclei stained with DAPI 72 h post-seeding (bars: 1 µm). Macrophages on 2D surfaces (upper left) mostly adopt the “fried egg” morphology, and nuclei are flattened, as shown by the higher nuclear area compared with nuclear area of cells in 3D matrices (upper middle and right). 3D matrices were prepared using pepsin-treated fibrillar collagen I. In gelled collagen I (upper right), cells use the mesenchymal migration mode and display a higher deformation of the nucleus compared with cells in fibrillar collagen, which use the amoeboid mode (upper middle). Both nuclear area (lower left graph) and nuclear circularity (lower right graph) were measured. Statistics: two-tailed unpaired Student t test. ***P < 0.0001, **P = 0.0056. Nuclear area and circularity were measured in a total of 25 (for fibrillar collagen) and 40 (for gelled collagen and 2D) macrophages from each time three independent donors.

Figure 4. Assays for the study of macrophage migration in 2D. In vitro, migration of macrophages on 2D surfaces can be analyzed by monitoring (A) the random movement of spontaneously migrating cells, (B) the directional movement of cells along a chemoattractant gradient (yellow; e.g., macrophage-colony stimulating factor), or (C and D) the directional movement of cells into a gap, which can be generated by the application of (C) a wound (red dotted line; based on a scratch by a pipette tip or needle) or (D) the removal of a silicon stopper. Directions of possible cell movement are indicated in boxes below. In all applications, cells can be imaged over time by standard microscopy methods and tracked/quantified with imaging software.

Figure 5. Migration assays for macrophages on 2D surfaces embedded in 3D matrices. Several assays enable the study of macrophage migration through 3D matrix (such as Matrigel^TM or type I collagen). However, cells still mostly adhere to 2D surfaces within these assays. (A) Standard Boyden chamber, with a thin layer of matrix added on the filter and within the pores, (B) gap closure assay (circular invasion assay), with a thick layer of matrix added on top, (C) chemotaxis chamber, with cells the seeded in 3D matrix. In Boyden/Chemotaxis chambers, cells migrate along a chemoattractant gradient (yellow), whereas in gap closure assays, cells migrate into the generated gap without additional stimulation. Directions of possible cell movement are indicated in boxes below. In all applications, cells can be imaged by standard microscopy methods. Note that Boyden chambers do not allow monitoring over time, as gap closure or chemotaxis chamber assays do, but enable easy access to the cells (e.g., for staining procedures).

Figure 6. Migration assays for macrophages in 3D. Macrophage infiltration into or migration through 3D matrices can be studied by vertical invasion, spherical invasion, or spheroid gel invasion assays. (A) In a vertical invasion assay, cells are placed on top of a thick matrix. Cells start to infiltrate into the gel (top right, red dots) and can be distinguished from non-invasive cells (black dots), which remain on the gel surface. The proportion of migrating cells, their morphology, and the distance of migration can be quantified over time from bright field microscopy image stacks. Cells can also be imaged by confocal microscopy, as shown by the z-stack reconstruction (bottom right) of human macrophages overexpressing creating a tunnel…white line; overexpressed mCherry-Lifeact (red) is used to stain F-actin. Bar: 20 µm (adapted, with permission, from van Goethem et al. and Guiet et al.⁶). (B) Spherical invasion assay (SIA). Cells are embedded in a dense plug of type I collagen, which is surrounded by slightly less dense collagen I, containing a chemoattractant. Cells invade into the surrounding matrix and can be monitored by confocal microscopy over time (right panel, bright field images of human macrophages, time of image acquisition after start of experiment is indicated; bar: 10 µm [adapted, with permission from Wiesner et al.⁶²]). The number of infiltrating cells, as well as distance from the plug border, can be quantified. (C) Spheroid gel invasion assay. Macrophages co-cultured for 3 d with spheroids of cancer cells (diameter: 0.5 mm) penetrate into spheroids. Spheroids infiltrated by macrophages are subsequently embedded in Matrigel^TM. Both macrophages and tumor cells leave the spheroid and invade the surrounding matrix. Cells are imaged by multi-photon microscopy (merged micrographs of human macrophages stained with cell tracker [red]; bar: 10 µm). Number of cells moving out of the spheroid (red triangles; bottom scheme) and area of cell invasion (green dotted line) can be quantified (adapted with permission from Guiet et al.⁶). Schemes at the bottom indicate direction of cell movement within each assay.