Magnetic tweezers in cell mechanics.

The chapter provides an overview of the applications of magnetic tweezers in living cells. It discusses the advantages and disadvantages of magnetic tweezers technology with a focus on individual magnetic tweezers configurations, such as electromagnetic tweezers. Solutions to the disadvantages identified are also outlined. The specific role of magnetic tweezers in the field of mechanobiology, such as mechanosensitivity, mechano-allostery and mechanotransduction are also emphasized. The specific usage of magnetic tweezers in mechanically probing cells via specific cell surface receptors, such as mechanosensitive channels is discussed and why mechanical probing has revealed the opening and closing of the channels. Finally, the future direction of magnetic tweezers is presented.

Phenotypic Heterogeneity, Bidirectionality, Universal Cues, Plasticity, Mechanics, and the Tumor Microenvironment Drive Cancer Metastasis.

Tumor diseases become a huge problem when they embark on a path that advances to malignancy, such as the process of metastasis. Cancer metastasis has been thoroughly investigated from a biological perspective in the past, whereas it has still been less explored from a physical perspective. Until now, the intraluminal pathway of cancer metastasis has received the most attention, while the interaction of cancer cells with macrophages has received little attention. Apart from the biochemical characteristics, tumor treatments also rely on the tumor microenvironment, which is recognized to be immunosuppressive and, as has recently been found, mechanically stimulates cancer cells and thus alters their functions. The review article highlights the interaction of cancer cells with other cells in the vascular metastatic route and discusses the impact of this intercellular interplay on the mechanical characteristics and subsequently on the functionality of cancer cells. For instance, macrophages can guide cancer cells on their intravascular route of cancer metastasis, whereby they can help to circumvent the adverse conditions within blood or lymphatic vessels. Macrophages induce microchannel tunneling that can possibly avoid mechanical forces during extra- and intravasation and reduce the forces within the vascular lumen due to vascular flow. The review article highlights the vascular route of cancer metastasis and discusses the key players in this traditional route. Moreover, the effects of flows during the process of metastasis are presented, and the effects of the microenvironment, such as mechanical influences, are characterized. Finally, the increased knowledge of cancer metastasis opens up new perspectives for cancer treatment.

Extracellular Matrix Cues Regulate Mechanosensing and Mechanotransduction of Cancer Cells.

Extracellular biophysical properties have particular implications for a wide spectrum of cellular behaviors and functions, including growth, motility, differentiation, apoptosis, gene expression, cell-matrix and cell-cell adhesion, and signal transduction including mechanotransduction. Cells not only react to unambiguously mechanical cues from the extracellular matrix (ECM), but can occasionally manipulate the mechanical features of the matrix in parallel with biological characteristics, thus interfering with downstream matrix-based cues in both physiological and pathological processes. Bidirectional interactions between cells and (bio)materials in vitro can alter cell phenotype and mechanotransduction, as well as ECM structure, intentionally or unintentionally. Interactions between cell and matrix mechanics in vivo are of particular importance in a variety of diseases, including primarily cancer. Stiffness values between normal and cancerous tissue can range between 500 Pa (soft) and 48 kPa (stiff), respectively. Even the shear flow can increase from 0.1-1 dyn/cm2 (normal tissue) to 1-10 dyn/cm2 (cancerous tissue). There are currently many new areas of activity in tumor research on various biological length scales, which are highlighted in this review. Moreover, the complexity of interactions between ECM and cancer cells is reduced to common features of different tumors and the characteristics are highlighted to identify the main pathways of interaction. This all contributes to the standardization of mechanotransduction models and approaches, which, ultimately, increases the understanding of the complex interaction. Finally, both the in vitro and in vivo effects of this mechanics-biology pairing have key insights and implications for clinical practice in tumor treatment and, consequently, clinical translation.

The role of ADAM8 in the mechanophenotype of MDA-MB-231 breast cancer cells in 3D extracellular matrices.

The majority of investigations of cancer cells have been performed in an oversimplified 2D in vitro environment. In the last decade there is a trend toward more sophisticated 3D in vitro cell culture model systems that can bridge the existing gap between 2D in vitro and in vivo experiments in the field of biophysical and cell biological cancer cell research. Here, we hypothesize that the bidirectional interplay between breast cancer cells and their tumor microenvironment is critical for the outcome of the disease. Thereby, the tissue remodeling processes evoked by cancer cells are important for cancer cell-driven mechanical probing of their matrix environment and on cancer cell adhesion and motility. When remodeling processes have been explored, the emphasis was placed on matrix metalloproteinases and rather not on a disintegrin and metalloproteases (ADAMs). However, the role of ADAM8 in cell mechanics regulating cellular motility in 3D collagen matrices is still unclear. Thus, in this study, we focus on the function of ADAM8 in matrix remodeling and migration of 3D extracellular matrix scaffolds. Therefore, human MDA-MB-231 breast carcinoma cells with ADAM8 knocked down, referred to as ADAM8-KD cells, as well as MDA-MB-231 scrambled control cells, referred to as ADAM8-Ctrl cells, have been used to examine their ability to interact with and migrate in dense extracellular 3D matrices. The fiber displacements, as the capacity of cells to deform the environmental 3D matrix scaffold, has been observed. ADAM8-KD cells displace collagen fibers more strongly than ADAM8-Ctrl cells. Moreover, ADAM8-KD cells migrated more numerous in 3D collagen matrices compared to ADAM8-Ctrl cells. The impairment of ADAM8 using the ADAM8 inhibitor BK-1361 led to significantly increased fiber displacements of ADAM8-Ctrl cells to the levels of ADAM8-KD cells. In contrast, the inhibitor had no effect on ADAM8-KD cells in terms of fiber displacements as well as on the quantitative characteristics of cell invasion of ADAM8-Ctrl cells, albeit the cells that were found in the matrix invaded considerably deeper. When matrix remodeling by cells is impaired through GM6001, a broad-band metalloproteinase inhibitor, the fiber displacements of both cell types increased. In fact, ADAM8 is known to degrade fibronectin in a direct and/or indirect manner. The supplementation of fibronectin before polymerization of the 3D collagen matrices caused an enhancement in fiber displacements as well as in cell invasion into fibronectin-collagen matrices of ADAM8-Ctrl cells, whereas the fiber displacements of ADAM8-KD cells did not change. However, fibrinogen and laminin supplementation induced an increase in fiber displacements of both cell types. Thus, the impact of fibronectin on selective increase in fiber displacement of ADAM8-Ctrl cells appears to be ADAM8-dependent. As a consequence, the presence of ADAM8 may provide an explanation for the longstanding controversial results of fibronectin enrichment on malignant progression of cancers such as breast cancer. Finally, ADAM8 is apparently essential for providing cell-driven fiber displacements of the extracellular matrix microenvironment, which fosters 3D motility in a fibronectin-rich environment. Contribution to the field. Currently, the role of ADAM8 has been explored in 2D or at maximum 2.5D in vitro cell culture motility assays. However, the mechanical characteristics of these two cell types have not been examined. In this study, the function of ADAM8 in breast cancer is refined by providing in vitro cell investigations in 3D collagen fiber matrices of various conditions. ADAM8 has been shown to be involved in the reduced generation of fiber displacements and in influencing breast cancer cell migration. However, especially in the presence of fibronectin in 3Dcollagen fiber matrices, the fiber displacements of ADAM8-Ctrl cells are increased.

The versatile roles of ADAM8 in cancer cell migration, mechanics, and extracellular matrix remodeling.

The posttranslational proteolytic cleavage is a unique and irreversible process that governs the function and half-life of numerous proteins. Thereby the role of the family of A disintegrin and metalloproteases (ADAMs) plays a leading part. A member of this family, ADAM8, has gained attention in regulating disorders, such as neurogenerative diseases, immune function and cancer, by attenuating the function of proteins nearby the extracellular membrane leaflet. This process of "ectodomain shedding" can alter the turnover rate of a number of transmembrane proteins that function in cell adhesion and receptor signal transduction. In the past, the major focus of research about ADAMs have been on neurogenerative diseases, such as Alzheimer, however, there seems to be evidence for a connection between ADAM8 and cancer. The role of ADAMs in the field of cancer research has gained recent attention, but it has been not yet been extensively addressed. Thus, this review article highlights the various roles of ADAM8 with particular emphasis on pathological conditions, such as cancer and malignant cancer progression. Here, the shedding function, direct and indirect matrix degradation, effects on cancer cell mobility and transmigration, and the interplay of ADAM8 with matrix-embedded neighboring cells are presented and discussed. Moreover, the most probable mechanical impact of ADAM8 on cancer cells and their matrix environment is addressed and debated. In summary, this review presents recent advances in substrates/ligands and functions of ADAM8 in its new role in cancer and its potential link to cell mechanical properties and discusses matrix mechanics modifying properties. A deeper comprehension of the regulatory mechanisms governing the expression, subcellular localization, and activity of ADAM8 is expected to reveal appropriate drug targets that will permit a more tailored and fine-tuned modification of its proteolytic activity in cancer development and metastasis.

Physical and biological advances in endothelial cell-based engineered co-culture model systems.

Scientific knowledge in the field of cell biology and mechanobiology heavily leans on cell-based in vitro experiments and models that favor the examination and comprehension of certain biological processes and occurrences across a variety of environments. Cell culture assays are an invaluable instrument for a vast spectrum of biomedical and biophysical investigations. The quality of experimental models in terms of simplicity, reproducibility, and combinability with other methods, and in particular the scale at which they depict cell fate in native tissues, is critical to advancing the knowledge of the comprehension of cell-cell and cell-matrix interactions in tissues and organs. Typically, in vitro models are centered on the experimental tinkering of mammalian cells, most often cultured as monolayers on planar, two-dimensional (2D) materials. Notwithstanding the significant advances and numerous findings that have been accomplished with flat biology models, their usefulness for generating further new biological understanding is constrained because the simple 2D setting does not reproduce the physiological response of cells in natural living tissues. In addition, the co-culture systems in a 2D stetting weakly mirror their natural environment of tissues and organs. Significant advances in 3D cell biology and matrix engineering have resulted in the creation and establishment of a new type of cell culture shapes that more accurately represents the in vivo microenvironment and allows cells and their interactions to be analyzed in a biomimetic approach. Contemporary biomedical and biophysical science has novel advances in technology that permit the design of more challenging and resilient in vitro models for tissue engineering, with a particular focus on scaffold- or hydrogel-based formats, organotypic cultures, and organs-on-chips, which cover the purposes of co-cultures. Even these complex systems must be kept as simplified as possible in order to grasp a particular section of physiology too very precisely. In particular, it is highly appreciated that they bridge the space between conventional animal research and human (patho)physiology. In this review, the recent progress in 3D biomimetic culturation is presented with a special focus on co-cultures, with an emphasis on the technological building blocks and endothelium-based co-culture models in cancer research that are available for the development of more physiologically relevant in vitro models of human tissues under normal and diseased conditions. Through applications and samples of various physiological and disease models, it is possible to identify the frontiers and future engagement issues that will have to be tackled to integrate synthetic biomimetic culture systems far more successfully into biomedical and biophysical investigations.

A Beginner's Guide to the Characterization of Hydrogel Microarchitecture for Cellular Applications.

The extracellular matrix (ECM) is a three-dimensional, acellular scaffold of living tissues. Incorporating the ECM into cell culture models is a goal of cell biology studies and requires biocompatible materials that can mimic the ECM. Among such materials are hydrogels: polymeric networks that derive most of their mass from water. With the tuning of their properties, these polymer networks can resemble living tissues. The microarchitectural properties of hydrogels, such as porosity, pore size, fiber length, and surface topology can determine cell plasticity. The adequate characterization of these parameters requires reliable and reproducible methods. However, most methods were historically standardized using other biological specimens, such as 2D cell cultures, biopsies, or even animal models. Therefore, their translation comes with technical limitations when applied to hydrogel-based cell culture systems. In our current work, we have reviewed the most common techniques employed in the characterization of hydrogel microarchitectures. Our review provides a concise description of the underlying principles of each method and summarizes the collective data obtained from cell-free and cell-loaded hydrogels. The advantages and limitations of each technique are discussed, and comparisons are made. The information presented in our current work will be of interest to researchers who employ hydrogels as platforms for cell culture, 3D bioprinting, and other fields within hydrogel-based research.

PINCH1 Promotes Fibroblast Migration in Extracellular Matrices and Influences Their Mechanophenotype.

Cell migration performs a critical function in numerous physiological processes, including tissue homeostasis or wound healing after tissue injury, as well as pathological processes that include malignant progression of cancer. The efficiency of cell migration and invasion appears to be based on the mechano-phenotype of the cytoskeleton. The properties of the cytoskeleton depend on internal cytoskeletal and external environmental factors. A reason for this are connections between the cell and its local matrix microenvironment, which are established by cell-matrix adhesion receptors. Upon activation, focal adhesion proteins such as PINCH1 are recruited to sites where focal adhesions form. PINCH1 specifically couples through interactions with ILK, which binds to cell matrix receptors and the actomyosin cytoskeleton. However, the role of PINCH1 in cell mechanics regulating cellular motility in 3D collagen matrices is still unclear. PINCH1 is thought to facilitate 3D motility by regulating cellular mechanical properties, such as stiffness. In this study, PINCH1 wild-type and knock-out cells were examined for their ability to migrate in dense extracellular 3D matrices. Indeed, PINCH1 wild-type cells migrated more numerously and deeper in 3D matrices, compared to knock-out cells. Moreover, cellular deformability was determined, e.g., elastic modulus (stiffness). PINCH1 knock-out cells are more deformable (compliable) than PINCH1 wild-type cells. Migration of both PINCH1-/- cells and PINCH1fl/fl cells was decreased by Latrunculin A inhibition of actin polymerization, suggesting that actin cytoskeletal differences are not responsible for the discrepancy in invasiveness of the two cell types. However, the mechanical phenotype of PINCH1-/- cells may be reflected by Latrunculin A treatment of PINCH1fl/fl cells, as they exhibit resembling deformability to untreated PINCH1-/- cells. Moreover, an apparent mismatch exists between the elongation of the long axis and the contraction of the short axis between PINCH1fl/fl cells and PINCH1-/- cells following Latrunculin A treatment. There is evidence of this indicating a shift in the proxy values for Poisson's ratio in PINCH1-/- cells compared with PINCH1fl/fl cells. This is probably attributable to modifications in cytoskeletal architecture. The non-muscle myosin II inhibitor Blebbistatin also reduced the cell invasiveness in 3D extracellular matrices but instead caused a stiffening of the cells. Finally, PINCH1 is apparently essential for providing cellular mechanical stiffness through the actin cytoskeleton, which regulates 3D motility.

Viscoelasticity, Like Forces, Plays a Role in Mechanotransduction.

Viscoelasticity and its alteration in time and space has turned out to act as a key element in fundamental biological processes in living systems, such as morphogenesis and motility. Based on experimental and theoretical findings it can be proposed that viscoelasticity of cells, spheroids and tissues seems to be a collective characteristic that demands macromolecular, intracellular component and intercellular interactions. A major challenge is to couple the alterations in the macroscopic structural or material characteristics of cells, spheroids and tissues, such as cell and tissue phase transitions, to the microscopic interferences of their elements. Therefore, the biophysical technologies need to be improved, advanced and connected to classical biological assays. In this review, the viscoelastic nature of cytoskeletal, extracellular and cellular networks is presented and discussed. Viscoelasticity is conceptualized as a major contributor to cell migration and invasion and it is discussed whether it can serve as a biomarker for the cells' migratory capacity in several biological contexts. It can be hypothesized that the statistical mechanics of intra- and extracellular networks may be applied in the future as a powerful tool to explore quantitatively the biomechanical foundation of viscoelasticity over a broad range of time and length scales. Finally, the importance of the cellular viscoelasticity is illustrated in identifying and characterizing multiple disorders, such as cancer, tissue injuries, acute or chronic inflammations or fibrotic diseases.

Viscoelasticity Acts as a Marker for Tumor Extracellular Matrix Characteristics.

Biological materials such as extracellular matrix scaffolds, cancer cells, and tissues are often assumed to respond elastically for simplicity; the viscoelastic response is quite commonly ignored. Extracellular matrix mechanics including the viscoelasticity has turned out to be a key feature of cellular behavior and the entire shape and function of healthy and diseased tissues, such as cancer. The interference of cells with their local microenvironment and the interaction among different cell types relies both on the mechanical phenotype of each involved element. However, there is still not yet clearly understood how viscoelasticity alters the functional phenotype of the tumor extracellular matrix environment. Especially the biophysical technologies are still under ongoing improvement and further development. In addition, the effect of matrix mechanics in the progression of cancer is the subject of discussion. Hence, the topic of this review is especially attractive to collect the existing endeavors to characterize the viscoelastic features of tumor extracellular matrices and to briefly highlight the present frontiers in cancer progression and escape of cancers from therapy. Finally, this review article illustrates the importance of the tumor extracellular matrix mechano-phenotype, including the phenomenon viscoelasticity in identifying, characterizing, and treating specific cancer types.

Cell mechanical properties of human breast carcinoma cells depend on temperature.

The knowledge of cell mechanics is required to understand cellular processes and functions, such as the movement of cells, and the development of tissue engineering in cancer therapy. Cell mechanical properties depend on a variety of factors, such as cellular environments, and may also rely on external factors, such as the ambient temperature. The impact of temperature on cell mechanics is not clearly understood. To explore the effect of temperature on cell mechanics, we employed magnetic tweezers to apply a force of 1 nN to 4.5 µm superparamagnetic beads. The beads were coated with fibronectin and coupled to human epithelial breast cancer cells, in particular MCF-7 and MDA-MB-231 cells. Cells were measured in a temperature range between 25 and 45 °C. The creep response of both cell types followed a weak power law. At all temperatures, the MDA-MB-231 cells were pronouncedly softer compared to the MCF-7 cells, whereas their fluidity was increased. However, with increasing temperature, the cells became significantly softer and more fluid. Since mechanical properties are manifested in the cell's cytoskeletal structure and the paramagnetic beads are coupled through cell surface receptors linked to cytoskeletal structures, such as actin and myosin filaments as well as microtubules, the cells were probed with pharmacological drugs impacting the actin filament polymerization, such as Latrunculin A, the myosin filaments, such as Blebbistatin, and the microtubules, such as Demecolcine, during the magnetic tweezer measurements in the specific temperature range. Irrespective of pharmacological interventions, the creep response of cells followed a weak power law at all temperatures. Inhibition of the actin polymerization resulted in increased softness in both cell types and decreased fluidity exclusively in MDA-MB-231 cells. Blebbistatin had an effect on the compliance of MDA-MB-231 cells at lower temperatures, which was minor on the compliance MCF-7 cells. Microtubule inhibition affected the fluidity of MCF-7 cells but did not have a significant effect on the compliance of MCF-7 and MDA-MB-231 cells. In summary, with increasing temperature, the cells became significant softer with specific differences between the investigated drugs and cell lines.

Inhomogeneities in 3D Collagen Matrices Impact Matrix Mechanics and Cancer Cell Migration.

Cell motility under physiological and pathological conditions including malignant progression of cancer and subsequent metastasis are founded on environmental confinements. During the last two decades, three-dimensional cell migration has been studied mostly by utilizing biomimetic extracellular matrix models. In the majority of these studies, the in vitro collagen scaffolds are usually assumed to be homogenous, as they consist commonly of one specific type of collagen, such as collagen type I, isolated from one species. These collagen matrices should resemble in vivo extracellular matrix scaffolds physiologically, however, mechanical phenotype and functional reliability have been addressed poorly due to certain limitations based on the assumption of homogeneity. How local variations of extracellular matrix structure impact matrix mechanics and cell migration is largely unknown. Here, we hypothesize that local inhomogeneities alter cell movement due to alterations in matrix mechanics, as they frequently occur in in vivo tissue scaffolds and were even changed in diseased tissues. To analyze the effect of structural inhomogeneities on cell migration, we used a mixture of rat tail and bovine dermal collagen type I as well as pure rat and pure bovine collagens at four different concentrations to assess three-dimensional scaffold inhomogeneities. Collagen type I from rat self-assembled to elongated fibrils, whereas bovine collagen tended to build node-shaped inhomogeneous scaffolds. We have shown that the elastic modulus determined with atomic force microscopy in combination with pore size analysis using confocal laser scanning microscopy revealed distinct inhomogeneities within collagen matrices. We hypothesized that elastic modulus and pore size govern cancer cell invasion in three-dimensional collagen matrices. In fact, invasiveness of three breast cancer cell types is altered due to matrix-type and concentration indicating that these two factors are crucial for cellular invasiveness. Our findings revealed that local matrix scaffold inhomogeneity is another crucial parameter to explain differences in cell migration, which not solely depended on pore size and stiffness of the collagen matrices. With these three distinct biophysical parameters, characterizing structure and mechanics of the studied collagen matrices, we were able to explain differences in the invasion behavior of the studied cancer cell lines in dependence of the used collagen model.