Physical network regimes of 3D fibrillar collagen networks trigger invasive phenotypes of breast cancer cells.

The mechanical characteristics of the extracellular environment are known to significantly influence cancer cell behavior in vivo and in vitro. The structural complexity and viscoelastic dynamics of the extracellular matrix (ECM) pose significant challenges in understanding its impact on cancer cells. Herein, we report distinct regulatory signatures in the invasion of different breast cancer cell lines into three-dimensional (3D) fibrillar collagen networks, caused by systematic modifications of the physical network properties. By reconstituting collagen networks of thin fibrils, we demonstrate that such networks can display network strand flexibility akin to that of synthetic polymer networks, known to exhibit entropic rubber elasticity. This finding contrasts with the predominant description of the mechanics of fibrillar collagen networks by an enthalpic bending elasticity of rod-like fibrils. Mean-squared displacement analysis of free-standing fibrils confirmed a flexible fiber regime in networks of thin fibrils. Furthermore, collagen fibrils in both networks were softened by the adsorption of highly negatively charged sulfonated polymers and colloidal probe force measurements of network elastic modulus again proofed the occurrence of the two different physical network regimes. Our cell assays revealed that the cellular behavior (morphology, clustering, invasiveness, matrix metalloproteinase (MMP) activity) of the 'weakly invasive' MCF-7 and 'highly invasive' MDA-MB-231 breast cancer cell lines is distinctively affected by the physical (enthalpic/entropic) network regime, and cannot be explained by changes of the network elastic modulus, alone. These results highlight an essential pathway, albeit frequently overlooked, how the physical characteristics of fibrillar ECMs affect cellular behavior. Considering the coexistence of diverse physical network regimes of the ECM in vivo, our findings underscore their critical role of ECM's physical network regimes in tumor progression and other cell functions, and moreover emphasize the significance of 3D in vitro collagen network models for quantifying cell responses in both healthy and pathological states.

Development of a synthesis strategy for sulfamethoxazole derivatives and their coupling with hydrogel microparticles.

Sulfonamides were the first synthetic antibiotics broadly applied in veterinary and human medicine. Their increased use over the last few decades and limited technology to degrade them after entering the sewage system have led to their accumulation in the environment. A new hydrogel microparticle based biosensing application for sulfonamides is developed to overcome existing labour-intensive, and expensive detection methods to analyse and quantify their environmental distribution. This biosensing assay is based on the soft colloidal probe principle and requires microparticle functionalization strategies with target molecules. In this study, we developed a step-wise synthesis approach for sulfamethoxazole (SMX) derivatives in high yield, with SMX being one of the most ubiquitous sulfonamide antibiotics. After de novo synthesis of the SMX derivative, two coupling schemes to poly(ethylene glycol) (PEG) hydrogel microparticles bearing maleimide and thiol groups were investigated. In one approach, we coupled a cysteamine linker to a carboxyl group at the SMX derivative allowing for subsequent binding via the thiol-functionality to the maleimide groups of the microparticles in a mild, high-yielding thiol-ene "click" reaction. In a second approach, an additional 1,11-bis(maleimido)-3,6,9-trioxaundecane linker was coupled to the cysteamine to target the hydrolytically more stable thiol-groups of the microparticles. Successful PEG microparticle functionalization with the SMX derivatives was proven by IR spectroscopy and fluorescence microscopy. SMX-functionalized microparticles will be used in future applications for sulfonamide detection as well as for pull-down assays and screenings for new sulfomethoxazole binding targets.

Phenotype Switching of Breast Cancer Cells upon Matrix Interface Crossing.

Tumor cell growth, invasion, and metastasis are dependent on the tumor microenvironment. Many studies emphasize a correlation between the material characteristics of the tumor extracellular matrix (ECM) and the invasive properties of tumor cells and even a trigger of tumor aggressiveness. Herein, we report that the previously observed trigger of migration characteristics of MDA-MB-231 breast cancer cells during transmigration across interfaces of two differently porous matrices is strongly correlated with a persistent change in cell invasiveness and aggressiveness. Using an in vitro 3D model of fibrillar collagen-I matrices, we found an increase in migration directionality, strongly elongated morphology, higher proliferation, and an increase in aggressive markers in the genetic profile after cells crossed the interface from dense to open porous matrix microstructure. Moreover, our results indicate strong nuclear deformation and increased DNA damage during transmigration of the matrix interface as a possible trigger of the more aggressive phenotype. These findings suggest that distinct tissue interfaces or altered ECM conditions with differences in microstructure may instruct or even reprogram tumor cells toward more aggressive phenotypes in vivo. The biomedical relevance of our results is corroborated by additional findings that the transmigrated cells exhibit an increased resistance against a common breast cancer therapeutic.

Functional label-free assessment of fibroblast differentiation in 3D collagen-I-matrices using particle image velocimetry.

Fibroblasts are a diverse population of connective tissue cells that are a key component in physiological wound healing. Myofibroblasts are differentiated fibroblasts occurring in various physiological and pathological conditions, like in the healing of wounds or in the tumour microenvironment. They exhibit important functions compared to fibroblasts in terms of proliferation, protein secretion, and contractility. The gold standard to distinguish myofibroblasts is alpha-smooth muscle actin (αSMA) expression and its incorporation in stress fibres, which is only revealed by gene expression analysis and immunostaining. Here, we introduce an approach to functionally determine the myofibroblast status of live fibroblasts directly in in vitro cell culture by analysing their ability to contract the extracellular matrix around them without the need for labelling. It is based on particle image velocimetry algorithms applied to dynamic deformations of the extracellular matrix network structure imaged by phase contrast microscopy. Advanced image analysis allows us to distinguish between various differentiation stages of fibroblasts including the dynamic change over several days. We further apply machine learning classification to automatically evaluate different cell culture conditions. With this new method, we provide a versatile tool to functionally evaluate the dynamic process of fibroblast differentiation. It can be applied for in vitro screening studies in biomimetic 3D cell cultures with options to extend it to other cell systems with contractile phenotypes.

Stiffness Variation of 3D Collagen Networks by Surface Functionalization of Network Fibrils with Sulfonated Polymers.

Fibrillar collagen is the most prominent protein in the mammalian extracellular matrix. Therefore, it is also widely used for cell culture research and clinical therapy as a biomimetic 3D scaffold. Charged biopolymers, such as sulfated glycosaminoglycans, occur in vivo in close contact with collagen fibrils, affecting many functional properties such as mechanics and binding of growth factors. For in vitro application, the functions of sulfated biopolymer decorations of fibrillar collagen materials are hardly understood. Herein, we report new results on the stiffness dependence of 3D collagen I networks by surface functionalization of the network fibrils with synthetic sulfonated polymers, namely, poly(styrene sulfonate) (PSS) and poly(vinyl sulfonate) (PVS). A non-monotonic stiffness dependence on the amount of adsorbed polymer was found for both polymers. The stiffness dependence correlated to a transition from mono- to multilayer adsorption of sulfonated polymers on the fibrils, which was most prominent for PVS. PVS mono- and multilayers caused a network stiffness change by a factor of 0.3 and 2, respectively. A charge-dependent weakening of intrafibrillar salt bridges by the adsorbed sulfonated polymers leading to fibrillar softening is discussed as the mechanism for the stiffness decrease in the monolayer regime. In contrast, multilayer adsorption can be assumed to induce interfibrillar bridging and an increase in network stiffness. Our in vitro results have a strong implication on in vivo characteristics of fibrillar collagen I, as sulfated glycosaminoglycans frequently attach to collagen fibrils in various tissues, calling for an up to now overlooked impact on matrix and tendon mechanics.

3D Scaffold-Based Macrophage Fibroblast Coculture Model Reveals IL-10 Dependence of Wound Resolution Phase.

Persistent inflammation and impaired repair in dermal wound healing are frequently associated with cell-cell and cell-matrix miscommunication. A direct coculture model of primary human myofibroblasts (MyoFB) and M-CSF-differentiated macrophages (M-Mɸ) in fibrillar three-dimensional Collagen I (Coll I) matrices is developed to study intercellular interactions. The coculture experiments reveal the number of M-Mɸ regulated MyoFB dedifferentiation in a dose-dependent manner. The amount of MyoFB decreases in dependence of the number of cocultured M-Mɸ, even in the presence of MyoFB-inducing transforming growth factor ß1 (TGF-ß1 ). Gene expression analysis of matrix proteins (collagen I, collagen III, ED-A-fibronectin) confirms the results of an altered MyoFB phenotype. Additionally, M-Mɸ is shown to be the main source of secreted cytokine interleukin-10 (IL-10), which is suggested to affect MyoFB dedifferentiation. These findings indicate a paracrine impact of IL-10 secretion by M-Mɸ on the MyoFB differentiation status counteracting the TGF-ß1 -driven MyoFB activation. Hence, the in vitro coculture model simulates physiological situations during wound resolution and underlines the importance of paracrine IL-10 signals by M-Mɸ. In sum, the 3D Coll I-based matrices with a MyoFB-M-Mɸ coculture form a highly relevant biomimetic model of late stages of wound healing.

pH and Heat Resistance of the Major Celery Allergen Api g 1.

SCOPE: The major celery allergen Api g 1 is a member of the pathogenesis-related 10 class protein family. This study aims to investigate the impact of heat and pH on the native protein conformation required for Immunoglobulin E (IgE) recognition. METHODS AND RESULTS: Spectroscopic methods, MS and IgE-binding analyses are used to study the effects of pH and thermal treatment on Api g 1.0101. Heat processing results in a loss of the native protein fold via denaturation, oligomerization, and precipitation along with a subsequent reduction of IgE recognition. The induced effects and timescales are strongly pH dependent. While Api g 1 refolds partially into an IgE-binding conformation at physiological pH, acidic pH treatment leads to the formation of structurally heat-resistant, IgE-reactive oligomers. Thermal processing in the presence of a celery matrix or at pH conditions close to the isoelectric point (pI = 4.63) of Api g 1.0101 results in almost instant precipitation. CONCLUSION: This study demonstrates that Api g 1.0101 is not intrinsically susceptible to heat treatment in vitro. However, the pH and the celery matrix strongly influence the stability of Api g 1.0101 and might be the main reasons for the observed temperature lability of this important food allergen.