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Bone metastasis is a leading cause of death in patients with breast cancer, but the underlying mechanisms are poorly understood. While much work focuses on the molecular and cellular events that drive breast cancer bone metastasis, it is mostly unclear what role bone extracellular matrix (ECM) properties play in this process. Bone ECM primarily consists of mineralized collagen fibrils, which are composed of non-stoichiometric carbonated apatite (HA) and collagen type I. Reduced bone mineral content is epidemiologically linked with increased risk of bone metastasis. Yet elucidating the potential functional impact of collagen mineralization on breast cancer cells has remained challenging because of a lack of model systems that allow studying tumor cell behavior as a function of physiological, intrafibrillar collagen mineralization. Here, we have developed cell culture substrates composed of mineralized collagen type I fibrils using a polymer-induced liquid-precursor (PILP) process. Intrafibrillar HA decreased breast cancer cell adhesion forces and accordingly reduced collagen fiber alignment relative to cells cultured on control collagen. The resulting mineral-mediated changes in collagen network characteristics and mechanosignaling correlated with increased cell motility, but inhibited directed migration of breast cancer cells. These results suggest that physiological mineralization of collagen fibrils reduces tumor cell adhesion with potential functional consequences on skeletal homing of disseminated tumor cells in early stages of breast cancer metastasis., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Polymer hydrogels can be readily modulated with regard to their physical properties and functionalized to recapitulate molecular cues of the extracellular matrix (ECM). However, they remain structurally different from the hierarchical supramolecular assemblies of natural ECM. Accordingly, we herein report a set of hydrogel composite materials made from starPEG-peptide conjugates, maleimide-functionalized heparin and collagen type I that combine semisynthetic and ECM-derived components. Collagen fibrillogenesis was controlled by temperature and collagen concentration to form collagen microstructures which were then homogeneously distributed within the 3D composite matrix during hydrogel formation. The collagen-laden hydrogel materials showed a heterogeneous local variation of the stiffness and adhesion ligand density. Composite gels functionalized with growth factors and cell adhesive peptides (RGDSP) supported the growth of embedded human umbilical cord vein endothelial cells (HUVECs) and induced the alignment of embedded bone marrow-derived human mesenchymal stem cells (MSCs) to the collagen microstructures in vitro. The introduced composite hydrogel material is concluded to faithfully mimic cell-instructive features of the ECM., Statement of Significance: Cell-instructive materials play an important role in the generation of both regenerative therapies and advanced tissue and disease models. For that purpose, biofunctional polymer hydrogels recapitulating molecular cues of the extracellular matrix (ECM) were successfully applied in various different studies. However, hydrogels generally lack the hierarchical supramolecular structure of natural ECM. We have therefore developed a hydrogel composite material made from starPEG-peptide conjugates, maleimide-functionalized heparin and collagen type I fibrils. The collagen-laden scaffolds showed a heterogeneous local variation in the stiffness of the material. The composite gels were successfully tested in culture experiments with human umbilical cord vein endothelial cells and bone marrow-derived human mesenchymal stem cells. It was concluded that the composite scaffold was able to faithfully mimic important cell-instructive features of the ECM., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Purpose: To use nanoscopically defined, two-dimensional matrices assembled from aligned collagen type I fibrils as a sheet substratum for in vitro cultivation of human corneal endothelial cells (HCECs). To assess the effect of matrix architecture on HCEC morphology and to characterize integrin-mediated HCEC-matrix interaction., Methods: Cell alignment and cell-matrix interactions of primary HCECs and three different immortalized HCEC populations on native and UV-cross-linked collagen type I matrices were examined by time-lapse microscopy. Specific integrin α(2)β(1) binding to the collagen matrix was demonstrated using a function-blocking α(2) antibody. Integrin α(2) subunit expression levels of the four HCEC populations were analyzed by Western blot analysis., Results: All HCEC populations aligned along the oriented collagen fibrils. Primary HCECs and, to a lesser extent, the other tested HCEC populations exerted high traction forces, leading to progressive matrix destruction. Cross-linking of the collagen matrices considerably increased matrix stability. Integrin subunit α(2) expression levels of the four cell types correlated with the degree of cell alignment and exertion of traction forces. In turn, blocking integrin subunit α(2) reduced cell alignment and prevented matrix destruction., Conclusions: HCECs align directionally along parallel arrays of collagen type I fibrils. The interactions of HCECs with collagen type I are primarily mediated by integrin α(2)β(1). Integrin subunit α(2) levels correlate with matrix contraction and subsequent destruction. Sustained cultivation of HCECs on ultrathin collagen matrices thus requires matrix cross-linking and moderate integrin α(2)β(1) expression levels.

Atomic force microscopy (AFM)-based single-cell force spectroscopy (SCFS) enables the quantitative study of cell adhesion under physiological conditions. SCFS probes adhesive interactions of single living cells with substrates such as extracellular matrix (ECM) proteins and other cells. Here we present a protocol to study integrin-mediated adhesion of HeLa cells to collagen type I using SCFS. We describe procedures for (i) functionalization of AFM cantilevers with the lectin concanavalin A and supports with collagen, (ii) cell handling and attachment to the AFM cantilever, (iii) measurement of adhesion forces and (iv) data analysis and interpretation. Although designed to measure HeLa cell adhesion to collagen, the protocol can be modified for other cell lines and ECM proteins. Compared with other SCFS assays (for example, optical tweezer, biomembrane force probe), AFM-based SCFS has a more versatile force detection range, and it can therefore be used to address a broader range of biological questions. The protocol can be completed in 2-3 d.

Galectins are a taxonomically widespread family of galactose-binding proteins of which galectin-3 is known to modulate cell adhesion. Using single cell force spectroscopy, the contribution of galectin-3 to the adhesion of Madin-Darby canine kidney (MDCK) cells to different extracellular matrix proteins was investigated. When adhering to collagen-I or -IV, some cells rapidly entered an enhanced adhesion state, marked by a significant increase in the force required for cell detachment. Galectin-3-depleted cells had an increased probability of entering the enhanced adhesion state. Adhesion enhancement was specific to integrin alpha(2)beta(1), as it was not observed when cells adhered to extracellular matrix substrates by other integrins. The adhesion phenotype of galectin-3-depleted cells was mimicked in a galactoside-deficient MDCK cell line and could be complemented by the addition of recombinant galectin-3. We propose that galectin-3 influences integrin alpha(2)beta(1)-mediated adhesion complex formation by altering receptor clustering.

We have characterized early steps of alpha(2)beta(1) integrin-mediated cell adhesion to a collagen type I matrix by using single-cell force spectroscopy. In agreement with the role of alpha(2)beta(1) as a collagen type I receptor, alpha(2)beta(1)-expressing Chinese hamster ovary (CHO)-A2 cells spread rapidly on the matrix, whereas alpha(2)beta(1)-negative CHO wild-type cells adhered poorly. Probing CHO-A2 cell detachment forces over a contact time range of 600 s revealed a nonlinear adhesion response. During the first 60 s, cell adhesion increased slowly, and forces associated with the smallest rupture events were consistent with the breakage of individual integrin-collagen bonds. Above 60 s, a fraction of cells rapidly switched into an activated adhesion state marked by up to 10-fold increased detachment forces. Elevated overall cell adhesion coincided with a rise of the smallest rupture forces above the value required to break a single-integrin-collagen bond, suggesting a change from single to cooperative receptor binding. Transition into the activated adhesion mode and the increase of the smallest rupture forces were both blocked by inhibitors of actomyosin contractility. We therefore propose a two-step mechanism for the establishment of alpha(2)beta(1)-mediated adhesion as weak initial, single-integrin-mediated binding events are superseded by strong adhesive interactions involving receptor cooperativity and actomyosin contractility.