Changes in Tissue Fluidity Predict Tumor Aggressiveness In Vivo.

Cancer progression is caused by genetic changes and associated with various alterations in cell properties, which also affect a tumor's mechanical state. While an increased stiffness has been well known for long for solid tumors, it has limited prognostic power. It is hypothesized that cancer progression is accompanied by tissue fluidization, where portions of the tissue can change position across different length scales. Supported by tabletop magnetic resonance elastography (MRE) on stroma mimicking collagen gels and microscopic analysis of live cells inside patient derived tumor explants, an overview is provided of how cancer associated mechanisms, including cellular unjamming, proliferation, microenvironment composition, and remodeling can alter a tissue's fluidity and stiffness. In vivo, state-of-the-art multifrequency MRE can distinguish tumors from their surrounding host tissue by their rheological fingerprints. Most importantly, a meta-analysis on the currently available clinical studies is conducted and universal trends are identified. The results and conclusions are condensed into a gedankenexperiment about how a tumor can grow and eventually metastasize into its environment from a physics perspective to deduce corresponding mechanical properties. Based on stiffness, fluidity, spatial heterogeneity, and texture of the tumor front a roadmap for a prognosis of a tumor's aggressiveness and metastatic potential is presented.

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.

Structural Breakdown of Collagen Type I Elastin Blend Polymerization.

Biopolymer blends are advantageous materials with novel properties that may show performances way beyond their individual constituents. Collagen elastin hybrid gels are a new representative of such materials as they employ elastin's thermo switching behavior in the physiological temperature regime. Although recent studies highlight the potential applications of such systems, little is known about the interaction of collagen and elastin fibers during polymerization. In fact, the final network structure is predetermined in the early and mostly arbitrary association of the fibers. We investigated type I collagen polymerized with bovine neck ligament elastin with up to 33.3 weight percent elastin and showed, by using a plate reader, zeta potential and laser scanning microscopy (LSM) experiments, that elastin fibers bind in a lateral manner to collagen fibers. Our plate reader experiments revealed an elastin concentration-dependent increase in the polymerization rate, although the rate increase was greatest at intermediate elastin concentrations. As elastin does not significantly change the structural metrics pore size, fiber thickness or 2D anisotropy of the final gel, we are confident to conclude that elastin is incorporated homogeneously into the collagen fibers.

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.

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.

Environmentally controlled magnetic nano-tweezer for living cells and extracellular matrices.

The magnetic tweezer technique has become a versatile tool for unfolding or folding of individual molecules, mainly DNA. In addition to single molecule analysis, the magnetic tweezer can be used to analyze the mechanical properties of cells and extracellular matrices. We have established a magnetic tweezer that is capable of measuring the linear and non-linear viscoelastic behavior of a wide range of soft matter in precisely controlled environmental conditions, such as temperature, CO2 and humidity. The magnetic tweezer presented in this study is suitable to detect specific differences in the mechanical properties of different cell lines, such as human breast cancer cells and mouse embryonic fibroblasts, as well as collagen matrices of distinct concentrations in the presence and absence of fibronectin crosslinks. The precise calibration and control mechanism employed in the presented magnetic tweezer setup provides the ability to apply physiological force up to 5 nN on 4.5 µm superparamagnetic beads coated with fibronectin and coupled to the cells or collagen matrices. These measurements reveal specific local linear and non-linear viscoelastic behavior of the investigated samples. The viscoelastic response of cells and collagen matrices to the force application is best described by a weak power law behavior. Our results demonstrate that the stress stiffening response and the fluidization of cells is cell type specific and varies largely between differently invasive and aggressive cancer cells. Finally, we showed that the viscoelastic behavior of collagen matrices with and without fibronectin crosslinks measured by the magnetic tweezer can be related to the microstructure of these matrices.

Effect of Nuclear Stiffness on Cell Mechanics and Migration of Human Breast Cancer Cells.

The migration and invasion of cancer cells through 3D confined extracellular matrices is coupled to cell mechanics and the mechanics of the extracellular matrix. Cell mechanics is mainly determined by both the mechanics of the largest organelle in the cell, the nucleus, and the cytoskeletal architecture of the cell. Hence, cytoskeletal and nuclear mechanics are the major contributors to cell mechanics. Among other factors, steric hindrances of the extracellular matrix confinement are supposed to affect nuclear mechanics and thus also influence cell mechanics. Therefore, we propose that the percentage of invasive cells and their invasion depths into loose and dense 3D extracellular matrices is regulated by both nuclear and cytoskeletal mechanics. In order to investigate the effect of both nuclear and cytoskeletal mechanics on the overall cell mechanics, we firstly altered nuclear mechanics by the chromatin de-condensing reagent Trichostatin A (TSA) and secondly altered cytoskeletal mechanics by addition of actin polymerization inhibitor Latrunculin A and the myosin inhibitor Blebbistatin. In fact, we found that TSA-treated MDA-MB-231 human breast cancer cells increased their invasion depth in dense 3D extracellular matrices, whereas the invasion depths in loose matrices were decreased. Similarly, the invasion depths of TSA-treated MCF-7 human breast cancer cells in dense matrices were significantly increased compared to loose matrices, where the invasion depths were decreased. These results are also valid in the presence of a matrix-metalloproteinase inhibitor GM6001. Using atomic force microscopy (AFM), we found that the nuclear stiffnesses of both MDA-MB-231 and MCF-7 breast cancer cells were pronouncedly higher than their cytoskeletal stiffness, whereas the stiffness of the nucleus of human mammary epithelial cells was decreased compared to their cytoskeleton. TSA treatment reduced cytoskeletal and nuclear stiffness of MCF-7 cells, as expected. However, a softening of the nucleus by TSA treatment may induce a stiffening of the cytoskeleton of MDA-MB-231 cells and subsequently an apparent stiffening of the nucleus. Inhibiting actin polymerization using Latrunculin A revealed a softer nucleus of MDA-MB-231 cells under TSA treatment. This indicates that the actin-dependent cytoskeletal stiffness seems to be influenced by the TSA-induced nuclear stiffness changes. Finally, the combined treatment with TSA and Latrunculin A further justifies the hypothesis of apparent nuclear stiffening, indicating that cytoskeletal mechanics seem to be regulated by nuclear mechanics.

Fast and reliable advanced two-step pore-size analysis of biomimetic 3D extracellular matrix scaffolds.

The tissue microenvironment is a major contributor to cellular functions, such as cell adhesion, migration and invasion. A critical physical parameter for determining the effect of the microenvironment on cellular functions is the average pore-size of filamentous scaffolds, such as 3D collagen fiber matrices, which are assembled by the polymerization of biopolymers. The scaffolds of these matrices can be analyzed easily by using state-of-the-art laser scanning confocal imaging. However, the generation of a quantitative estimate of the pore-size in a 3D microenvironment is not trivial. In this study, we present a reliable and fast analytical method, which relies on a two-step 3D pore-size analysis utilizing several state-of-the-art image analysis methods, such as total variation (TV) denoising and adaptive local thresholds, and another crucial parameter, such as pore-coverage. We propose an iterative approach of pore-size analysis to determine even the smallest and obscure pores in a collagen scaffold. Additionally, we propose a novel parameter, the pseudo-pore-size, which describes a virtual scaffold porosity. In order to validate the advanced two-step pore-size analysis different types of artificial collagens, such as a rat and bovine mixture with two different collagen concentrations have been utilized. Additionally, we compare a traditional approach with our method using an artificially generated network with predefined pore-size distributions. Indeed, our analytical method provides a precise, fast and parameter-free, user-independent and automatic analysis of 3D pore topology, such as pore-sizes and pore-coverage. Additionally, we are able to determine non-physiological network topologies by taking the pore-coverage as a goodness-of-fit parameter.

"All of a sudden, a lot less still makes it worthwhile to be alive:" Palliative care patients' views on life.

OBJECTIVE: We aimed to elucidate the views on life as narrated by patients in palliative care (PC) to find out what patients deem to be essential in their life, whether something has changed concerning their view of life in light of the disease, and whether interviewees would like to give others something to take with them. METHOD: Data were collected from narrative audio and video interviews with 18 inpatients in a specialized PC unit. Interviews were recorded and transcribed verbatim and analyzed using qualitative content analysis applying MAXQDA software. CDs and DVDs with recorded interviews were provided to patients. RESULT: Eighteen interviews were analyzed: 11 audio and 7 video recordings. The age range was 41-80 years. Patients reported on changes in their views on life. Despite it being a complex and painful process, patients still gave examples of benefits experienced during their illness trajectory. Patients identified resources and coping strategies such as meaningful contacts with close others and mindfulness. Shifts have occurred in terms of taking more time for themselves, enjoying the moment, being more calm, and spending more time with family and friends. What patients wanted to pass on to others was to pay attention to the needs of both the self and the others, shape your life individually, confront yourself early with issues of death and dying, and care for your fellow human beings. Patients and relatives valued the opportunity to keep their interview as a CD/DVD. SIGNIFICANCE OF RESULTS: Results support the idea that many people facing terminal illness continue to focus on living and remain within their biographies and the contexts of their lives, even if their functional status declines. Patients and relatives appreciated that interviews were provided as kind of a legacy. Yet, more robust research is needed to decide whether such interviews yield any therapeutic effect.