Collagen-based cell migration models in vitro and in vivo

Abstract

Fibrillar collagen is the most abundant extracellular matrix (ECM) constituent which maintains the structure of most interstitial tissues and organs, including skin, gut, and breast. Density and spatial alignments of the three-dimensional (3D) collagen architecture define mechanical tissue properties, i.e. stiffness and porosity, which guide or oppose cell migration and positioning in different contexts, such as morphogenesis, regeneration, immune response, and cancer progression. To reproduce interstitial cell movement in vitro with high in vivo fidelity, 3D collagen lattices are being reconstituted from extracted collagen monomers, resulting in the re-assembly of a fibrillar meshwork of defined porosity and stiffness. With a focus on tumor invasion studies, we here evaluate different in vitro collagen-based cell invasion models, employing either pepsinized or non-pepsinized collagen extracts, and compare their structure to connective tissue in vivo, including mouse dermis and mammary gland, chick chorioallantoic membrane (CAM), and human dermis. Using confocal reflection and two-photon-excited second harmonic generation (SHG) microscopy, we here show that, depending on the collagen source, in vitro models yield homogeneous fibrillar texture with a quite narrow range of pore size variation, whereas all in vivo scaffolds comprise a range from low- to high-density fibrillar networks and heterogeneous pore sizes within the same tissue. Future in-depth comparison of structure and physical properties between 3D ECM-based models in vitro and in vivo are mandatory to better understand the mechanisms and limits of interstitial cell movements in distinct tissue environments.

Figure 1

Structure of 3D fibrillar collagen reconstituted in vitro from different collagen sources. (A) Structure of collagen monomers containing telopeptide and respective pepsin cleavage site in both, solublized (acidic) and multimeric state. Aldehyde (CHO) moities are generated by lysyl oxidase (LOX) spontaneously assembling into aldimine cross-links (adapted from ). (B) Polymerized networks of pepsinized (telopeptide-free) or non-pepzinized (telopeptide-containing) collagens from different sources and suppliers (bovine skin, Nutacon, Leiden, The Netherlands; calf skin, IBFB, Leipzig, Germany; mouse tail, Lisa Coussens lab, San Francisco, USA; rat tail, Becton Dickinson). For all matrices, a final collagen concentration of 1.7 mg/ml and standard polymerization procedures were used, as described ( and protocols provided by suppliers). Top row, single xy scan; middle row, overlay covering 8 μm in depth; bottom row, single xz scan. Because of the matrix dimensions, collagen fibre orientation is mostly in parallel to the upper and lower cover slips, leading to longitudinal reflection in xy scans and dot-like cross-section like signal in xz scans. Arrowheads: single collagen fibres. Marked areas refer to Fig. 5. (C) Outlines of typical migrating cells in horizontal and vertical dimension in 1:1 ratio to collagen structures depicted in (A). The outline of the mesenchymal cell (ca. 50×10×10 μm and 75 μm² area in xz) is similar to many tumor cells from epithelial or mesenchymal origin, whereas trafficking leukocytes are smaller (dendritic cell, ca. 15×7×7 μm, 35 μm² area in xz; T cell, ca. 10×4×5, 15 μm² area in xz). Bars, 10 μm.

Fig. 2

Collagen spacing of 3D mouse connective tissue models in vivo. Intravital two-photon microscopy was performed in connective tissues of anaesthetized mice from different locations using different experimental procedures (in brackets): (A) cremaster muscle (open surgery) ^,, (B) skull (frontoparietal scalp; open surgery) ^,, (C, D) back skin dermis (DSFC) ^,, and (E) mammary fat pad (mammary imaging window)^,. (A) Mouse cremaster model. Asterisks, SHG-negative gaps. B) Mouse dermis of the head region. Asterisks indicate SHG-negative spaces presumably filled with vessel tubes, based on roundish morphology. (C) Mouse dermis of the back skin in papillary (superficial) and (D) deep reticular dermis. (E) Collagen-rich region of the mammary fat pad. (D, E) Fluorescent dextran (red) was used to counterstain perfused blood vessels and (after uptake) tissue-resident macrophages (asterisks) ^,. (E) Arrowheads, fat cells. (A–E) Xy images represent individual planes from z-stacks at different imaging depth (numbers in images, μm of depth). Dotted lines, location of xz and yz images shown in the left and lower panels, using the ImageJ software (version 1.41o). Small dotted rectangles in (B, C) show the sections used for quantification in Fig. 5. In some images, cell outlines from Fig. 1C are displayed in 1:1 ratio. Bars, 100 μm.

Figure 3

Fibrillar collagen density of the chick embryo CAM. A chicken embryo (at day 12) was injected i.v. with rhodamin-conjugated lens culinaris agglutinin (LCA), and the CAM was harvested after 20 min, fixed in Zn-formalin, and analyzed by MP microscopy. Collagen fibres as detected by SHG (green), ecto/ endodermal layer of epithelial cells (white arrowheads) and blood cells (located capillary plexus or extravasated; empty arrowheads) were detected by autofluorescence signal (blue-gray); blood vessels positive for rhodamin-LCA fluorescence (red, asterisks). Image processing was performed as described in Fig. 2. Bars, 100 μm.

Figure 4

Human dermis from different ex vivo sources. Two-photon-excited SHG (collagen, green) and autofluorescence (red) of 3D dermis from native back skin (A), abdomen (B), and cadaveric skin of unspecified body region (C) (AlloDerm). (A) Skin left-over from the tumor margin not needed for histopathological diagnosis was used as non-fixed whole-mount few hours post-surgery. (B) DED was obtained from abdominal skin corrections, cultivation for 2 weeks and fixation by paraformaldehyde. (C) AlloDerm was used as provided by the supplier (LifeCell Corporation, Branchburg, NJ, USA) . All samples were monitored from the open margin of the dermal side. Image processing was performed as described in Fig. 2. White arrowheads, collagen bundles; empty arrowheads, elastic fibres. Bars, 100 μm.

Figure 5

Structural and quantitative comparison of collagen scaffold spacing from different models. (A) Loose and dense collagen regions obtained from selected in vitro and in vivo tissues (all derived from Fig. 1 to 4, except breast tissue). Cell outlines (from Fig. 1C) are included at 1:1 ratio in xy and xz dimension. Collagen-free gaps and pores were used for digital image analysis and quantification, using Image J. (B) Quantification of pore areas and (C) distance between fibres. Data show the medians (line) and individual measurements from different tissue regions (symbols) compared to the cross-section area and diameters of mammalian cells during migration (compare Fig. 1C). Bars, 10 μm.