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.

Differential Smad2/3 linker phosphorylation is a crosstalk mechanism of Rho/ROCK and canonical TGF-ß3 signaling in tenogenic differentiation.

The transforming growth factor (TGF)-ß3 is a well-known inducer for tenogenic differentiation, signaling via the Smad2/3 pathway. Furthermore, other factors like extracellular matrix or mechanical force can induce tenogenic differentiation and possibly alter the response to TGF-ß3 by signaling via the Rho/ROCK pathway. The aim of this study was to investigate the interplay of Rho/ROCK and TGF-ß3/Smad signaling in tenogenic differentiation, with the Smad2/3 molecule hypothesized as a possible interface. Cultured as monolayers or on collagen I matrices, mesenchymal stromal cells (MSC) were treated with the ROCK inhibitor Y-27632 (10 µM), TGF-ß3 (10 ng/ml) or both combined. Control cells were cultured accordingly, without Y-27632 and/or without TGF-ß3. At different time points, MSC were analyzed by real-time RT-PCR, immunofluorescence, and Western blot. Cultivation of MSC on collagen matrices and ROCK inhibition supported tenogenic differentiation and fostered the effect of TGF-ß3. The phosphorylation of the linker region of Smad2 was reduced by cultivation on collagen matrices, but not by ROCK inhibition. The latter, however, led to increased phosphorylation of the linker region of Smad3. In conclusion, collagen matrices and the Rho/ROCK signaling pathway influence the TGF-ß3/Smad2/3 pathway by regulating different phosphorylation sites of the Smad linker region.

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.

Fibrillar biopolymer-based scaffolds to study macrophage-fibroblast crosstalk in wound repair.

Controlled wound healing requires a temporal and spatial coordination of cellular activities within the surrounding extracellular matrix (ECM). Disruption of cell-cell and cell-matrix communication results in defective repair, like chronic or fibrotic wounds. Activities of macrophages and fibroblasts crucially contribute to the fate of closing wounds. To investigate the influence of the ECM as an active part controlling cellular behavior, coculture models based on fibrillar 3D biopolymers such as collagen have already been successfully used. With well-defined biochemical and biophysical properties such 3D scaffolds enable in vitro studies on cellular processes including infiltration and differentiation in an in vivo like microenvironment. Further, paracrine and autocrine signaling as well as modulation of soluble mediator transport inside the ECM can be modeled using fibrillar 3D scaffolds. Herein, we review the usage of these scaffolds in in vitro coculture models allowing in-depth studies on the crosstalk between macrophages and fibroblasts during different stages of cutaneous wound healing. A more accurate mimicry of the various processes of cellular crosstalk at the different stages of wound healing will contribute to a better understanding of the impact of biochemical and biophysical environmental parameters and help to develop further strategies against diseases such as fibrosis.

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.

Biomimetic estrogen sensor based on soft colloidal probes.

An increasing number of reports substantiate the link between emerging estrogenic pollutants and a variety of adverse effects including developmental disorders, infertility, cancer and neurological disorders, threatening public health as well as environment. The detection of the diverse classes of estrogenic and antiestrogenic substances is still challenging due to analytics which needs to cover the whole range of compounds acting on estrogen receptors and the complex estrogen pathways. In this proof-of-concept study, we report a novel biomimetic detection scheme based on the specific recognition of estrogenic ligands by estrogen sulfotransferase 1E1 (SULT1E1), which acts as one of the key enzymes in estrogen homeostasis. SULT1E1 was site-specifically immobilized on transparent glass slides via a hexahistidine-tag in a multi-step procedure. Soft colloidal probes (SCPs) covalently functionalized with ligands of SULT1E1, namely estrone and estradiol 17-(ß-D-glucuronide), served as adhesion probes. The various functionalization steps were analyzed and optimized using epifluorescence, confocal laser scanning as well as reflection interference contrast microscopy (RICM). A competitive SCP binding assay probing the elastic SCP deformation driven by the specific interaction between SCPs and the SULT1E1 decorated glass slides was employed in conjunction with an optical readout by RICM and automated image analysis to detect estrogenic compounds by their inhibition of SCP adhesion. This sensing concept has demonstrated exceptional specificity for estrogenic steroid compounds compared to structurally related substance classes and provides promising options for multiplexed assays and incorporation of other proteins of the endocrine system to fully capture the whole ensemble of hormonally active substances.

Impact of binding mode of low-sulfated hyaluronan to 3D collagen matrices on its osteoinductive effect for human bone marrow stromal cells.

Synthetically sulfated hyaluronan derivatives were shown to facilitate osteogenic differentiation of human bone marrow stromal cells (hBMSC) by application in solution or incorporated in thin collagen-based coatings. In the presented study, using a biomimetic three-dimensional (3D) cell culture model based on fibrillary collagen I (3D Col matrix), we asked on the impact of binding mode of low sulfated hyaluronan (sHA) in terms of adsorptive and covalent binding on osteogenic differentiation of hBMSC. Both binding modes of sHA induced osteogenic differentiation. Although for adsorptive binding of sHA a strong intracellular uptake of sHA was observed, implicating an intracellular mode of action, covalent binding of sHA to the 3D matrix induced also intense osteoinductive effects pointing towards an extracellular mode of action of sHA in osteogenic differentiation. In summary, the results emphasize the relevance of fibrillary 3D Col matrices as a model to study hBMSC differentiation in vitro in a physiological-like environment and that sHA can display dose-dependent osteoinductive effects in dependence on presentation mode in cell culture scaffolds.

Construction of a 3D brain extracellular matrix model to study the interaction between microglia and T cells in co-culture.

Neurodegenerative disorders are characterised by the activation of brain-resident microglia cells and by the infiltration of peripheral T cells. However, their interplay in disease has not been clarified yet. It is difficult to investigate complex cellular dynamics in living animals, and simple two-dimensional (2D) cell culture models do not resemble the soft 3D structure of brain tissue. Therefore, we developed a biomimetic 3D in vitro culture system for co-cultivation of microglia and T cells. As the activation and/or migration of immune cells in the brain might be affected by components of the extracellular matrix, defined 3D fibrillar collagen I-based matrices were constructed and modified with hyaluronan and/or chondroitin sulphate, resembling aspects of brain extracellular matrix. Murine microglia and spleen-derived T cells were cultured alone or in co-culture on the constructed matrices. Microglia exhibited in vivo-like morphology and T cells showed enhanced survival when co-cultured with microglia or to a minor degree in the presence of glia-conditioned medium. The open and porous fibrillar structure of the matrix allowed for cell invasion and direct cell-cell interaction, with stronger invasion of T cells. Both cell types showed no dependence on the matrix modifications. Microglia could be activated on the matrices by lipopolysaccharide resulting in interleukin-6 and tumour necrosis factor-α secretion. The findings herein indicate that biomimetic 3D matrices allow for co-cultivation and activation of primary microglia and T cells and provide useful tools to study their interaction in vitro.

Fibronectin-functionalization of 3D collagen networks supports immune tolerance and inflammation suppression in human monocyte-derived macrophages.

The extracellular matrix (ECM) is dynamically reorganized during wound healing. Concomitantly, recruited monocytes differentiate into macrophages. However, the role of the wound's ECM during this transition remain to be fully understood. Fibronectin is a multifunctional glycoprotein present in early wound ECM with a potential immunomodulatory role during monocyte-to-macrophage differentiation. Hence, to investigate the impact of fibronectin during this differentiation step, 3D fibrillar collagen type I networks with or without fibronectin-functionalization were engineered with defined topology (fibril and pore diameter: 0.8 µm; 7 µm) and amount of adsorbed fibronectin (0.15 µg per µg collagen). Primary, human monocytes were then differentiated into macrophages inside these networks. The immunological imprinting of the resulting macrophages was monitored by means of the expression of FABP4, CLEC4E, SLC2A6, and SOD2 which discriminate naïve and tolerized macrophages, as well pro-inflammatory (M1) and anti-inflammatory (M2) macrophage polarization. The analyses indicate that fibronectin-functionalization of collagen I networks induces macrophage tolerance rather than M1 or M2 macrophage phenotypes. This finding was confirmed by release profiles of pro- and anti-inflammatory cytokines such as IL6, IL8, CXCL10, and IL10. Nevertheless, upon LPS challenge, immune suppression by fibronectin was overridden since these macrophages could then deploy an efficient immune response. Our results therefore provide new perspectives in biomaterial science of wound healing scaffolds and the design of instructive materials for human monocyte-derived cells.

Matrix Remodeling and Hyaluronan Production by Myofibroblasts and Cancer-Associated Fibroblasts in 3D Collagen Matrices.

The tumor microenvironment is a key modulator in cancer progression and has become a novel target in cancer therapy. An increase in hyaluronan (HA) accumulation and metabolism can be found in advancing tumor progression and are often associated with aggressive malignancy, drug resistance and poor prognosis. Wound-healing related myofibroblasts or activated cancer-associated fibroblasts (CAF) are assumed to be the major sources of HA. Both cell types are capable to synthesize new matrix components as well as reorganize the extracellular matrix. However, to which extent myofibroblasts and CAF perform these actions are still unclear. In this work, we investigated the matrix remodeling and HA production potential in normal human dermal fibroblasts (NHFB) and CAF in the absence and presence of transforming growth factor beta -1 (TGF-ß1), with TGF-ß1 being a major factor of regulating fibroblast differentiation. Three-dimensional (3D) collagen matrix was utilized to mimic the extracellular matrix of the tumor microenvironment. We found that CAF appeared to response insensitively towards TGF-ß1 in terms of cell proliferation and matrix remodeling when compared to NHFB. In regards of HA production, we found that both cell types were capable to produce matrix bound HA, rather than a soluble counterpart, in response to TGF-ß1. However, activated CAF demonstrated higher HA production when compared to myofibroblasts. The average molecular weight of produced HA was found in the range of 480 kDa for both cells. By analyzing gene expression of HA metabolizing enzymes, namely hyaluronan synthase (HAS1-3) and hyaluronidase (HYAL1-3) isoforms, we found expression of specific isoforms in dependence of TGF-ß1 present in both cells. In addition, HAS2 and HYAL1 are highly expressed in CAF, which might contribute to a higher production and degradation of HA in CAF matrix. Overall, our results suggested a distinct behavior of NHFB and CAF in 3D collagen matrices in the presence of TGF-ß1 in terms of matrix remodeling and HA production pointing to a specific impact on tumor modulation.

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.

Biomimetic tissue models reveal the role of hyaluronan in melanoma proliferation and invasion.

Interactions of hyaluronan (HA) and tumor and stromal cells are highly discussed as one of the major contributors in tumor progression and metastasis. The balance of HA in the tissue is highly regulated by two key enzyme classes; hyaluronan synthases (HAS) and hyaluronidases (HYAL). Current reports hint that the HA amount in the tissue is correlated with poor prognosis in melanoma, the most life-threatening skin tumor. In this work, we generated in vivo mouse models with low and high expression of Has2 and used the models for studying melanoma proliferation of the B78D14 melanoma cell line. We found that a strong reduction of HA amount in the skin was correlated to decreased tissue stiffness and a reduction in tumor weight. Since tumor cells have a direct contact to the HA in the tumor and at the stroma interface, we reconstituted different biomimetic in vitro models using fibroblasts derived from a mouse model to recapitulate melanoma cell behavior at the tumor boundary, namely, (i) decellularized fibroblast matrix (FbECM), (ii) fibroblast embedded into 3D collagen matrices (FbColl), and (iii) well-defined HA-functionalized 3D collagen matrices (HAColl). We found no considerable effect of high and low amounts of fibroblast-derived HA in the matrices on melanoma proliferation and invasion. However, HYAL1-treated FbECM and FbColl, as well as HAColl functionalized with low molecular weight HA (34 kDa) promoted proliferation and invasion of melanoma cells in a concentration dependent manner. Our results emphasize the molecular weight specific effects of HA in regulation of melanoma behavior and provide an alternative explanation for the in vivo observation of HA dependent tumor growth.

Surface Functionalization by Hydrophobin-EPSPS Fusion Protein Allows for the Fast and Simple Detection of Glyphosate.

Glyphosate, the most widely used pesticide worldwide, is under debate due to its potentially cancerogenic effects and harmful influence on biodiversity and environment. Therefore, the detection of glyphosate in water, food or environmental probes is of high interest. Currently detection of glyphosate usually requires specialized, costly instruments, is labor intensive and time consuming. Here we present a fast and simple method to detect glyphosate in the nanomolar range based on the surface immobilization of glyphosate's target enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) via fusion to the hydrophobin Ccg2 and determination of enzyme activity with a malachite green assay, which is a common photometric technique to measure inorganic phosphate (Pi). The assay demonstrates a new approach for a fast and simple detection of pesticides.

Fibril bending stiffness of 3D collagen matrices instructs spreading and clustering of invasive and non-invasive breast cancer cells.

Extracellular matrix stiffening of breast tissues has been clinically correlated with malignant transformation and poor prognosis. An increase of collagen fibril diameter and lysyl-oxidase mediated crosslinking has been observed in advanced tumor stages. Many current reports suggest that the local mechanical properties of single fibrillar components dominantly regulate cancer cell behavior. Here, we demonstrate by an independent control of fibril diameter and intrafibrillar crosslinking of three-dimensional (3D) collagen matrices that fibril bending stiffness instructs cell behavior of invasive and non-invasive breast cancer cells. Two types of collagen matrices with fibril diameter of either 650 nm or 800 nm at a similar pore size of 10 µm were reconstituted and further modified with the zero-length crosslinker 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide (EDC) at concentrations of 0, 20, 100 and 500 mM. This approach yields two sets of collagen matrices with overlapping variation of matrix elasticity. With these matrices we could prove the common assumption that matrix elasticity of collagen networks is bending dominated with a linear dependence on fibril bending stiffness. We derive that the measured variation of matrix elasticity is directly correlated to the variation of fibril bending stiffness, being independently controlled either by fibril diameter or by intrafibrillar crosslinking. We use these defined matrices to demonstrate that the adjustment of fibril bending stiffness allows to instruct the behavior of two different breast cancer cell lines, invasive MDA-MB-231 (human breast carcinoma) and non-invasive MCF-7 cells (human breast adenocarcinoma). Invasiveness and spreading of invasive MDA-MB-231 cells as well as clustering of non-invasive MCF-7 cells is thereby investigated over a broad parameter range. Our results demonstrate and quantify the direct dependence of cancer cell phenotypes on the matrix mechanical properties on the scale of single fibrils.

Biomimetic tumor microenvironments based on collagen matrices.

The extracellular matrix (ECM) establishes the basis for the cell's microenvironment providing structural and mechanical support to cells and tissues, and regulating multiple cell functions including gene expression, cell cycle, apoptosis, morphogenesis, and migration. Tumor development can be considered as a process where the cells are subjected to mutations. However, changes in the microenvironment of tumor cells can strongly impact the growth, invasion and survival of tumor cells. This specialized microenvironment is known to have an abundance of inflammatory cells and activated fibroblasts both expressing ECM components and growth factors that support the survival and proliferation of tumor cells in a paracrine fashion. Growing evidence points towards a key role of the ECM in the modulation of tumor progression and metastasis, even influencing therapeutic pharmacosensitivity. Accordingly, to better understand the mechanisms of tumor cell behavior, e.g. proliferation, invasion and survival, in dependence on microenvironmental cues as well as the cell-microenvironment interaction, it is necessary to engineer well-defined 3D matrices to closely mimic in vivo like microenvironments in a controlled in vitro setting. In that way the full repertoire of high-resolution, in-depth analytical technologies available for in vitro settings can be applied to reveal the underlying molecular mechanisms in an in vivo like ECM microenvironment. This review provides an overview of the current bioengineering techniques of defined biomimetic 3D models with a focus on naturally derived biopolymer components for the investigation of tumor cell behavior in vitro.

Quantitative label-free single cell tracking in 3D biomimetic matrices.

Live cell imaging enables an observation of cell behavior over a period of time and is a growing field in modern cell biology. Quantitative analysis of the spatio-temporal dynamics of heterogeneous cell populations in three-dimensional (3D) microenvironments contributes a better understanding of cell-cell and cell-matrix interactions for many biomedical questions of physiological and pathological processes. However, current live cell imaging and analysis techniques are frequently limited by non-physiological 2D settings. Furthermore, they often rely on cell labelling by fluorescent dyes or expression of fluorescent proteins to enhance contrast of cells, which frequently affects cell viability and behavior of cells. In this work, we present a quantitative, label-free 3D single cell tracking technique using standard bright-field microscopy and affordable computational resources for data analysis. We demonstrate the efficacy of the automated method by studying migratory behavior of a large number of primary human macrophages over long time periods of several days in a biomimetic 3D microenvironment. The new technology provides a highly affordable platform for long-term studies of single cell behavior in 3D settings with minimal cell manipulation and can be implemented for various studies regarding cell-matrix interactions, cell-cell interactions as well as drug screening platform for primary and heterogeneous cell populations.

Solvent-Assisted Micromolding of Biohybrid Hydrogels to Maintain Human Hematopoietic Stem and Progenitor Cells Ex Vivo.

Array-format cell-culture carriers providing tunable matrix cues are instrumental in current cell biology and bioengineering. A new solvent-assisted demolding approach for the fabrication of microcavity arrays with very small feature sizes down to single-cell level (3 µm) of very soft biohybrid glycosaminoglycan-poly(ethylene glycol) hydrogels (down to a shear modulus of 1 kPa) is reported. It is further shown that independent additional options of localized conjugation of adhesion ligand peptides, presentation of growth factors through complexation to gel-based glycosaminoglycans, and secondary gel deposition for 3D cell embedding enable a versatile customization of the hydrogel microcavity arrays for cell culture studies. As a proof of concept, cell-instructive hydrogel compartment arrays are used to analyze the response of human hematopoietic stem and progenitor cells to defined biomolecular and spatial cues.

Instructing Human Macrophage Polarization by Stiffness and Glycosaminoglycan Functionalization in 3D Collagen Networks.

Dynamic alterations of composition and mechanics of the extracellular matrix are suggested to modulate cellular behavior including plasticity of macrophages (MPhs) during wound healing. In this study, engineered 3D fibrillar matrices based on naturally occurring biopolymers (collagen I, glycosaminoglycans (GAGs)) are used to mimic matrix stiffening as well as modification by sulfated and nonsulfated GAGs at different stages of wound healing. Human MPhs are found to sensitively respond to these microenvironmental cues in terms of polarization toward proinflammatory or wound healing phenotypes over 6 days in vitro. MPhs exhibit a wound healing phenotype in stiffer matrices as determined by protein and gene expression of relevant cytokines (IL10, IL12, and TNFα). Presence of sulfated and nonsulfated GAGs inhibits this polarization effect. Furthermore, control experiments on 2D matrices stress the relevance of using stiffness-controlled 3D matrices, as MPhs show a reciprocal polarization behavior depending on GAG presence. Hence, the results indicate a strong influence of dimensionality, stiffness, and GAG presence of the biomaterial scaffold on MPh polarization and emphasize the need for matrices closely mimicking the 3D in vivo context with a variable stiffness and GAG composition in in vitro studies.

Molecular weight specific impact of soluble and immobilized hyaluronan on CD44 expressing melanoma cells in 3D collagen matrices.

Hyaluronan (HA) and its principal receptor CD44 are known to be involved in regulating tumor cell dissemination and metastasis. The direct correlation of CD44-HA interaction on proliferation and invasion of tumor cells in dependence on the molecular weight and the presentation form of HA is not fully understood because of lack of appropriate matrix models. To address this issue, we reconstituted 3D collagen (Coll I) matrices and functionalized them with HA of molecular weight of 30-50kDa (low molecular weight; LMW-HA) and 500-750kDa (high molecular weight; HMW-HA). A post-modification strategy was applied to covalently immobilize HA to reconstituted fibrillar Coll I matrices, resulting in a non-altered Coll I network microstructure and stable immobilization over days. Functionalized Coll I matrices were characterized regarding topological and mechanical characteristics as well as HA amount using confocal laser scanning microscopy, colloidal probe force spectroscopy and quantitative Alcian blue assay, respectively. To elucidate HA dependent tumor cell behavior, BRO melanoma cell lines with and without CD44 receptor expression were used for in vitro cell experiments. We demonstrated that only soluble LMW-HA promoted cell proliferation in a CD44 dependent manner, while HMW-HA and immobilized LMW-HA did not. Furthermore, an enhanced cell invasion was found only for immobilized LMW-HA. Both findings correlated with a very strong and specific adhesive interaction of LMW-HA and CD44+ cells quantified in single cell adhesion measurements using soft colloidal force spectroscopy. Overall, our results introduce an in vitro biomaterials model allowing to test presentation mode and molecular weight specificity of HA in a 3D fibrillar matrix thus mimicking important in vivo features of tumor microenvironments. STATEMENT OF SIGNIFICANCE: Molecular weight and presentation form (bound vs. soluble) of hyaluronan (HA) are intensively discussed as key regulators in tumor progression and inflammation. We introduce 3D fibrillar collagen matrices with defined microstructure and stiffness allowing the presentation of specific molecular weight forms of HA in soluble and bound manner. Mimicking in that way important in vivo features of tumor microenvironments, we found that only low molecular weight HA (LMW-HA) in soluble form promoted proliferation of a melanoma cell line (BRO), while it enhanced cell invasion in bound form. The molecular weight specificity of LMW-HA was verified to be CD44 receptor dependent and was correlated to adhesive ligand-receptor interactions in quantitative colloidal force spectroscopy at single cell level.