In Vitro Modeling of Mechanics in Cancer Metastasis

In addition to a multitude of genetic and biochemical alterations, abnormal morphological, structural, and mechanical changes in cells and their extracellular environment are key features of tumor invasion and metastasis. Furthermore, it is now evident that mechanical cues alongside biochemical signals contribute to critical steps of cancer initiation, progression, and spread. Despite its importance, it is very challenging to study mechanics of different steps of metastasis in the clinic or even in animal models. While considerable progress has been made in developing advanced in vitro models for studying genetic and biological aspects of cancer, less attention has been paid to models that can capture both biological and mechanical factors realistically. This is mainly due to lack of appropriate models and measurement tools. After introducing the central role of mechanics in cancer metastasis, we provide an outlook on the emergence of novel in vitro assays and their combination with advanced measurement technologies to probe and recapitulate mechanics in conditions more relevant to the metastatic disease

3D In Vitro Models for Investigating the Role of Stiffness in Cancer Invasion

BACKGROUND: Tumorigenesis is attributed to the interactions of cancer cells with the tumor microenvironment through both biochemical cues and physical stimuli. Increased matrix deposition and realignment of the collagen fibers are detected by cancer cells, inducing epithelial-to-mesenchymal transition, which in turn stimulates cell motility and invasiveness. METHODS: This review provides an overview of current research on the role of the physical microenvironment in cancer invasion. This was achieved by using a systematic approach and providing meta-analyses. Particular focus was placed on in vitro three-dimensional models of epithelial cancers. We investigated questions such as the effect of matrix stiffening, activation of stromal cells, and identified potential advances in mechano-based therapies. RESULTS: Meta-analysis revealed that 64% of studies report cancer invasion promotion as stiffness increases, while 36% report the opposite. Experimental approaches and data interpretations were varied, each affecting the invasion of cancer differently. Examples are the experimental timeframes used (24 h to 21 days), the type of polymer used (24 types), and choice of cell line (33 cell lines). The stiffness of the 3D matrices varied from 0.5 to 300 kPa and 19% of these matrices' stiffness were outside commonly accepted physiological range. 100% of the studies outside biological stiffness range (above 20 kPa) report that stiffness does not promote cancer invasion. CONCLUSIONS: Taking this analysis into account, we inform on the type of experimental approaches that could be the most relevant and provide what would be a standardized protocol and reporting strategy

Poroelastic osmoregulation of living cell volume

Cells maintain their volume through fine intracellular osmolarity regulation. Osmotic challenges drive fluid into or out of cells causing swelling or shrinkage, respectively. The dynamics of cell volume changes depending on the rheology of the cellular constituents and on how fast the fluid permeates through the membrane and cytoplasm. We investigated whether and how poroelasticity can describe volume dynamics in response to osmotic shocks. We exposed cells to osmotic perturbations and used defocusing epifluorescence microscopy on membrane-attached fluorescent nanospheres to track volume dynamics with high spatiotemporal resolution. We found that a poroelastic model that considers both geometrical and pressurization rates captures fluid-cytoskeleton interactions, which are rate-limiting factors in controlling volume changes at short timescales. Linking cellular responses to osmotic shocks and cell mechanics through poroelasticity can predict the cell state in health, disease, or in response to novel therapeutics.Peer ReviewedPostprint (published version

Mechanical response of neural cells to physiologically relevant stiffness gradients

Understanding the influence of the mechanical environment on neurite behavior is crucial in the development of peripheral nerve repair solutions, and could help tissue engineers to direct and guide regeneration. In this study, a new protocol to fabricate physiologically relevant hydrogel substrates with controlled mechanical cues is proposed. These hydrogels allow the analysis of the relative effects of both the absolute stiffness value and the local stiffness gradient on neural cell behavior, particularly for low stiffness values (1–2 kPa). NG108‐15 neural cell behavior is studied using well‐characterized collagen gradient substrates with stiffness values ranging from 1 to 10 kPa and gradient slopes of either 0.84 or 7.9 kPa mm^{-1}. It is found that cell orientation is influenced by specific combinations of stiffness value and stiffness gradient. The results highlight the importance of considering the type of hydrogel as well as both the absolute value of the stiffness and the steepness of its gradient, thus introducing a new framework for the development of tissue engineered scaffolds and the study of substrate stiffness

High-Strouhal-number pulsatile flow in a curved pipe

The high-Strouhal-number pulsatile flow in a curved pipe is studied numerically. A general force analysis is developed for the bend force, where the new contribution from flow acceleration is identified and analysed. The mechanisms of secondary flow production are studied by extending Hawthorne's (Proc. R. Soc. Lond. A, vol. 206, issue 1086, 1951, pp. 374–387) model to account for viscous effects and applied to assess the distinct contributions from an inviscid stretching and no-slip condition. A detailed comparison is made between the numerical simulations and models for a pipe flow characterised by a volume flux Q=UbA|sinΩpt| (where Ub is the maximum bulk velocity, Ωp is the angular frequency and A is the pipe cross-sectional area). For high-Reynolds-number (Reb) and high-Strouhal-number (St), the bend force predictions are in good agreement with the numerical results over a wide range of bend curvature (Rc/D; where Rc is the bend radius of curvature and D is the pipe diameter) owing to the influence of the streamwise flow acceleration on the pressure field. At high-St, the streamwise vorticity (secondary flow) distribution is steady and close to the low-St case, which is explained using a linear secondary flow model

Acoustics and vibrations in a complex piping network with pump startup–shutdown transients

Pump dynamic operational conditions result in extreme transient events that can enhance the response of piping networks. The predominant transients during rapid startup and shutdown are mainly studied for centrifugal pumps and are scarce for reciprocating pumps. Our study extends the conventional steady-state analysis to include the effect of reciprocating pump dynamic loading on pulsatile flow-induced acoustics and vibrations in a complex piping network. The forced response resulting from acoustical–structural coupling is assessed by utilising the one-dimensional multiphysics piping acoustic model and beam structural model. The network responses to pulsatile flows during dynamic pump loading (rapid start-up-shutdown events) are compared to the responses due to pulsatile flows during steady-state pump loading. With pump startup–shutdowns operations accompanied by pulsatile flows, the network response is the result of the combination of the transient and steady-state characteristics of plane acoustic waves and structural vibrations. The dynamic pump loading excites the fundamental, low-frequency acoustic eigenmode that causes transient loading of pipeline similar to reservoir–pipe–valve (RPV) systems

Biofabrication of vasculature in microphysiological models of bone

Bone contains a dense network of blood vessels that are essential to its homoeostasis, endocrine function, mineral metabolism and regenerative functions. In addition, bone vasculature is implicated in a number of prominent skeletal diseases, and bone has high affinity for metastatic cancers. Despite vasculature being an integral part of bone physiology and pathophysiology, it is often ignored or oversimplified in in vitro bone models. However, 3D physiologically relevant vasculature can now be engineered in vitro, with microphysiological systems (MPS) increasingly being used as platforms for engineering this physiologically relevant vasculature. In recent years, vascularised models of bone in MPSs systems have been reported in the literature, representing the beginning of a possible technological step change in how bone is modelled in vitro. Vascularised bone MPSs is a subfield of bone research in its nascency, however given the impact of MPSs has had in in vitro organ modelling, and the crucial role of vasculature to bone physiology, these systems stand to have a substantial impact on bone research. However, engineering vasculature within the specific design restraints of the bone niche is significantly challenging given the different requirements for engineering bone and vasculature. With this in mind, this paper aims to serve as technical guidance for the biofabrication of vascularised bone tissue within MPS devices. We first discuss the key engineering and biological considerations for engineering more physiologically relevant vasculature in vitro within the specific design constraints of the bone niche. We next explore emerging applications of vascularised bone MPSs, and conclude with a discussion on the current status of vascularised bone MPS biofabrication and suggest directions for development of next generation vascularised bone MPSs

Time-dependent mechanics of living cells

Cells sense and generate both internal and external forces. They resist and transmit these forces to the cell interior or to other cells. Moreover a variety of cellular responses are excited and influenced by transducing mechanical stimulations into chemical signals that lead to changes in cellular behaviour. The cytoplasm represents the largest part of the cell by volume and hence its rheology sets the maximum rate at which any cellular shape change can occur. To date, the cytoplasm has generally been modelled as a single-phase viscoelastic material; however, recent experimental evidence suggests that its rheology can be described more effectively using a poroelastic formulation in which the cytoplasm is considered to be a biphasic system constituted of a porous elastic solid meshwork (cytoskeleton, organelles, macromolecules) bathing in an interstitial fluid (cytosol). In this framework, a single parameter, the poroelastic diffusion constant p D , sets cellular rheology scaling as ~ 2 / p D Ex m with E the elastic modulus, x the hydraulic pore size, and m the cytosolic viscosity. Though this poroelastic view of the cell is a conceptually attractive model, direct supporting evidence has been lacking. In this work, such evidence is presented and the concept of a poroelastic cell is validated to explain cellular rheology at physiologically relevant time-scales. In this work, the functional form of stress relaxation in response to rapid application of a localised force by atomic force microscopy microindentation is examined in detail and it is shown that at short time-scales cellular relaxations are poroelastic. Then, p D is measured in cells by fitting experimental stress relaxation curves to the theoretical model. VI Next, using indentation tests in conjunction with osmotic perturbations, the validity of the predicted scaling of p D with pore size is qualitatively verified. Using chemical and genetic perturbations, it is shown that cytoplasmic rheology depends strongly on the integrity of the actin cytoskeleton but not on microtubules or intermediate filaments. Finally, comparison of scaling of viscoelastic and poroelastic models suggests that shorttime scale viscoelasticity might be due to water redistribution within the cytoplasm and a simple scaling relating cytoplasmic viscosity to cellular microstructure is provided

Associated changes in stiffness of collagen scaffolds during osteoblast mineralisation and bone formation

OBJECTIVE: Engineering bone in 3D is important for both regenerative medicine purposes and for the development of accurate in vitro models of bone tissue. The changing material stiffness of bone tissue had not yet been monitored throughout the process of mineralisation and bone nodule formation by osteoblasts either during in vitro engineering or in development perspective. RESULTS: Within this short research note, stiffness changes (Young's modulus) during in vitro bone formation by primary osteoblasts in dense collagen scaffolds were monitored using atomic force microscopy. Data analysis revealed significant stiffening of 3D bone cultures at day 5 and 8 that was correlated with the onset of mineral deposition (p < 0.00005)

CNS cell distribution and axon orientation determine local spinal cord mechanical properties.

Mechanical signaling plays an important role in cell physiology and pathology. Many cell types, including neurons and glial cells, respond to the mechanical properties of their environment. Yet, for spinal cord tissue, data on tissue stiffness are sparse. To investigate the regional and direction-dependent mechanical properties of spinal cord tissue at a spatial resolution relevant to individual cells, we conducted atomic force microscopy (AFM) indentation and tensile measurements on acutely isolated mouse spinal cord tissue sectioned along the three major anatomical planes, and correlated local mechanical properties with the underlying cellular structures. Stiffness maps revealed that gray matter is significantly stiffer than white matter irrespective of directionality (transverse, coronal, and sagittal planes) and force direction (compression or tension) (K(g) = ∼ 130 P(a) vs. K(w) = ∼ 70 Pa); both matters stiffened with increasing strain. When all data were pooled for each plane, gray matter behaved like an isotropic material under compression; however, subregions of the gray matter were rather heterogeneous and anisotropic. For example, in sagittal sections the dorsal horn was significantly stiffer than the ventral horn. In contrast, white matter behaved transversely isotropic, with the elastic stiffness along the craniocaudal (i.e., longitudinal) axis being lower than perpendicular to it. The stiffness distributions we found under compression strongly correlated with the orientation of axons, the areas of cell nuclei, and cellular in plane proximity. Based on these morphological parameters, we developed a phenomenological model to estimate local mechanical properties of central nervous system (CNS) tissue. Our study may thus ultimately help predicting local tissue stiffness, and hence cell behavior in response to mechanical signaling under physiological and pathological conditions, purely based on histological data.The authors thank the CECAD Imaging Facility (and their staff members), Andreas Christ, Jochen Guck, Jolanta Kozlowski, Ryan MacDonald, Graham Sheridan, and Alex Winkel for helpful discussions and/or technical support. This work was supported by Köln Fortune Program/Faculty of Medicine, University of Cologne (Fellowship to D.E.K.), German National Academic Foundation (Scholarship to D.E.K.), Herchel Smith Foundation (Fellowship to E.M.), DAAD-PROMOS-Program (Scholarship to J.H.), Deutsche Forschungsgemeinschaft (grant KU2760/2-1 to S.K.), UK Medical Research Council (Career Development Award to K.F.), and the Human Frontier Science Program (Young Investigator Grant to K.F.).This is the final version of the article. It first appeared from Cell Press via http://dx.doi.org/10.1016/j.bpj.2015.03.03