Substrate Compliance versus Ligand Density in Cell on Gel Responses

Substrate stiffness is emerging as an important physical factor in the response of many cell types. In agreement with findings on other anchorage-dependent cell lineages, aortic smooth muscle cells are found to spread and organize their cytoskeleton and focal adhesions much more so on “rigid” glass or “stiff” gels than on “soft” gels. Whereas these cells generally show maximal spreading on intermediate collagen densities, the limited spreading on soft gels is surprisingly insensitive to adhesive ligand density. Bell-shaped cell spreading curves encompassing all substrates are modeled by simple functions that couple ligand density to substrate stiffness. Although smooth muscle cells spread minimally on soft gels regardless of collagen, GFP-actin gives a slight overexpression of total actin that can override the soft gel response and drive spreading; GFP and GFP-paxillin do not have the same effect. The GFP-actin cells invariably show an organized filamentous cytoskeleton and clearly indicate that the cytoskeleton is at least one structural node in a signaling network that can override spreading limits typically dictated by soft gels. Based on such results, we hypothesize a central structural role for the cytoskeleton in driving the membrane outward during spreading whereas adhesion reinforces the spreading.     

Substrate stiffness regulates apoptosis and the mRNA expression of extracellular matrix regulatory genes in the rat annular cells

Cells are subjected to static tension of different magnitudes when cultured on substrates with different stiffnesses. It has long been recognized that mechanical stress is an important modulator of the intervertebral disc degeneration. Here we studied the influence of substrate stiffness on cell morphology, apoptosis and extracellular matrix (ECM) metabolism of the rat annulus fibrosus (AF) cells which are known to be mechanosensitive cells. Polyacrylamide gel substrates with three different stiffnesses were prepared by varying the concentration of acrylamide and bisacrylamide, and the elastic modulus of the different gel substrates were measured with atomic force microscopy (AFM). First-passage rat annular cells were cultured on soft, intermediate, rigid substrates or plastics for 24 or 48 h. The percentages of apoptotic cells were detected by flow cytometry and caspase-3 activity, and morphologic changes were visualized by Hoechst 33258 staining and F-actin staining. In addition, the expression of ECM genes (Col1α1, Col2α1, aggrecan, MMP-3, MMP-13 and ADAMTS-5) were analyzed by RT-PCR. The three different substrates had elastic moduli varying between 1 ± 0.23 kPa (soft, 5% gel with 0.06% bis), 32 ± 2.89 kPa (intermediate, 10% gel with 0.13% bis) and 63 ± 3.45 kPa (rigid, 10% gel with 0.26% bis) with a thickness about 60-70 μm. Most of the rat AF cells appeared small and rounded, and lost most of their stress fibers when cultured on soft substrate. There was a significant increase in the percentage of apoptotic cells in the rat AF cells cultured on soft and intermediate substrates relative to those on plastic surface, with a parallel decrease in the area of cell spreading and nucleus. The AF cells grown on intermediate or rigid substrate had reduced expression of Col1α1, Col2α1 and aggrecan and enhanced expression of MMP-3, MMP-13, and ADAMTS-5 at 24 h or 48 h, respectively, relative to those cultured on plastic surface. Conversely, we observed an up-regulation of Col2α1 and aggrecan and no change in the gene expression of MMP-3, MMP-13, and ADAMTS-5 in AF cells on soft substrates. Rat AF cells are sensitive to substrate stiffness which can regulate the morphology, growth, apoptosis and ECM metabolism of rat AF cells, thus indicating the importance of substrate choice for cell transplantation and regeneration for the treatment of disc degeneration using tissue-engineering technique.     

Thickness sensing of hMSCs on collagen gel directs stem cell fate

Mechanically compliant substrate provides crucial biomechanical cues for multipotent stem cells to regulate cellular fates such as differentiation, proliferation and maintenance of their phenotype. Effective modulus of which cells sense is not only determined by intrinsic mechanical properties of the substrate, but also the thickness of substrate. From our study, it was found that interference from underlying rigid support at hundreds of microns away could induce significant cellular response. Human mesenchymal stem cells (hMSCs) were cultured on compliant biological gel, collagen type I, of different thickness but identical ECM composition and local stiffness. The cells sensed the thin gel (130 μm) as having a higher effective modulus than the thick gel (1440 μm) and this was reflected in their changes in morphology, actin fibers structure, proliferation and tissue specific gene expression. Commitment into neuronal lineage was observed on the thin gel only. Conversely, the thick gel (1440 μm) was found to act like a substrate with lower effective modulus that inhibited actin fiber polymerization. Stem cells on the thick substrate did not express tissue specific genes and remained at their quiescent state. This study highlighted the need to consider not only the local modulus but also the thickness of biopolymer gel coating during modulation of cellular responses.     

Changes in the stiffness of human mesenchymal stem cells with the progress of cell death as measured by atomic force microscopy

This note reports observations of the change of stiffness of human mesenchymal stem cells (hMSCs) with the progress of cell death as measured by AFM. hMSC with impaired membrane, dead and viable cells were labelled with Annexin V and Propidium Iodide after 24 h cold storage, followed by AFM measurement and Young's modulus of cells was derived. Viable hMSCs have a Young's modulus (E) in the range of 0.81–1.13 kPa and consistent measurement was observed when different measurement locations were chosen. E of cells with partially impaired membrane was 0.69±0.17 kPa or in the range of 2.04–4.74 kPa, depending upon the measurement locations. With the loss of membrane integrity, though there was no variation on measured E between different locations, a mixed picture of cell stiffness was observed as indicated by cells with E as low as 0.09±0.03 kPa, in a mid-range of 4.62±0.67 kPa, and the highest of up to 48.98±19.80 kPa. With the progress of cell death, the highest stiffness was noticed for cells showing a more granular appearance; also the lowest stiffness for cells with vacuole appearance. Findings from this study indicate that cell stiffness is significantly altered with the progress of cell death.     

Sdc1 negatively modulates carcinoma cell motility and invasion

During cancer progression, tumor cells eventually invade the surrounding collagen-rich extracellular matrix. Here we show that squamous cell carcinoma cells strongly adhere to Type I collagen substrates but display limited motility and invasion on collagen barriers. Further analysis revealed that in addition to the α2β1 integrin, a second collagen receptor was identified as Syndecan-1 (Sdc1), a cell surface heparan sulfate proteoglycan. We demonstrate that siRNA-mediated depletion of Sdc1 reduced adhesion efficiency to collagen I, whereas knockdown of Sdc4 was without effect. Importantly, silencing Sdc1 expression caused reduced focal adhesion plaque formation and enhanced cell spreading and motility on collagen I substrates, but did not alter cell motility on other ECM substrates. Sdc1 depletion ablated adhesion-induced RhoA activation. In contrast, Rac1 was strongly activated following Sdc1 knockdown, suggesting that Sdc1 may mediate the link between integrin-induced actin remodeling and motility. Taken together, these data substantiate the existence of a co-adhesion receptor system in tumor cells, whereby Sdc1 functions as a key regulator of cell motility and cell invasion by modulating RhoA and Rac activity. Downregulation of Sdc1 expression during carcinoma progression may represent a mechanism by which tumor cells become more invasive and metastatic.     

Fibroblast adaptation and stiffness matching to soft elastic substrates

Many cell types alter their morphology and gene expression profile when grown on chemically equivalent surfaces with different rigidities. One expectation of this change in morphology and composition is that the cell’s internal stiffness, governed by cytoskeletal assembly and production of internal stresses, will change as a function of substrate stiffness. Atomic force microscopy was used to measure the stiffness of fibroblasts grown on fibronectin-coated polyacrylamide gels of shear moduli varying between 500 and 40,000 Pa. Indentation measurements show that the cells’ elastic moduli were equal to, or slightly lower than, those of their substrates for a range of soft gels and reached a saturating value at a substrate rigidity of 20 kPa. The amount of cross-linked F-actin sedimenting at low centrifugal force also increased with substrate stiffness. Together with enhanced actin polymerization and cross-linking, active contraction of the cytoskeleton can also modulate stiffness by exploiting the nonlinear elasticity of semiflexible biopolymer networks. These results suggest that within a range of stiffness spanning that of soft tissues, fibroblasts tune their internal stiffness to match that of their substrate, and modulation of cellular stiffness by the rigidity of the environment may be a mechanism used to direct cell migration and wound repair.     

Mechanism-based inhibition of cancer metastasis with (−)-epigallocatechin gallate

Cell motility and cell stiffness are closely related to metastatic activity of cancer cells. (−)-Epigallocatechin gallate (EGCG) has been shown to inhibit spontaneous metastasis of melanoma cell line into the lungs of mice, so we studied the effects of EGCG on cell motility, cell stiffness, and expression of vimentin and Slug, which are molecular phenotypes of epithelial–mesenchymal transition (EMT). Treatments of human non-small cell lung cancer cell lines H1299 and Lu99 with 50 and 100 μM EGCG reduced cell motility to 67.5% and 43.7% in H1299, and 71.7% and 31.5% in Lu99, respectively in in vitro wound healing assay. Studies on cell stiffness using atomic force microscope (AFM) revealed that treatment with 50 μM EGCG increased Young’s modulus of H1299 from 1.24 to 2.25 kPa and that of Lu99 from 1.29 to 2.28 kPa, showing a 2-fold increase in cell stiffness, i.e. rigid elasticity of cell membrane. Furthermore, treatment with 50 μM EGCG inhibited high expression of vimentin and Slug in the cells at a leading edge of scratch. Methyl-β-cyclodextrin, a reagent to deplete cholesterol in plasma membrane, showed inhibition of EMT phenotypes similar that by EGCG, suggesting that EGCG induces inhibition of EMT phenotypes by alteration of membrane organization.     

Decorin modulates collagen I-stimulated, but not fibronectin-stimulated, migration of C2C12 myoblasts

Extracellular matrix factors, specifically fibronectin and collagen I, are essential for structural support during muscle regeneration. Decorin has been identified as an anti-fibrotic agent with binding sites located on both fibronectin and collagen I. Upon injury, activated myoblasts are required to migrate through the extracellular matrix factors deposited by the myofibroblasts to facilitate skeletal muscle regeneration. In this study we looked at the effects decorin on fibronectin- and collagen I-stimulated myoblast migration. Dose response studies demonstrated 10 μg/ml, 5 μg/ml and 25 μg/ml as the optimal stimulatory concentrations of decorin (1.2 fold increase), fibronectin (3.5 fold increase) and collagen I (2.4 fold increase), when compared with control respectively. A synergistic effect was identified when decorin and collagen I were added in combination; this effect was not evident when decorin was added with fibronectin. The effects of these factors on the ROCK signalling pathway were also analyzed. ROCK-2 was identified as the key Rho-activated kinase isoform involved in migration, due to its higher expression levels and localisation to focal points within migrating C2C12 myoblasts. Decorin and collagen I in combination stimulated an increase in the number of ROCK-2 localized focal points when compared with control, decorin and collagen I added separately. Fibronectin did not show any increase in ROCK-2 focal points when compared with control. These results show for the first time that decorin can modify collagen I-stimulated, but not fibronectin-stimulated myoblast migration in vitro. Furthermore, the synergistic, rather than additive, effect observed suggests a direct modification of collagen I signalling by decorin mediated, at least in part, by ROCK-2 rather than ROCK-1.     

Extracellular matrix effect on RhoA signaling modulation in vascular smooth muscle cells

Morphological adaptations of vascular smooth muscle cells (VSMC) to the mechanically active environment in which they reside, are mediated by direct interactions with the extracellular matrix (ECM) which induces physiological changes at the intracellular level. This study aimed to analyze the effects of the ECM on RhoA-induced mechanical signaling that controls actin organization and focal adhesion formation. VSMC were transfected with RhoA constructs (wild type, dominant negative or constitutively active) and plated on different ECM proteins used as substrate (fibronectin, collagen IV, collagen I, and laminin) or poly-l-lysine as control. Morphological changes of the VSMC were detected by fluorescence confocal microscopy and total internal reflection fluorescence (TIRF) microscopy, and were independently verified using adhesion assays and Western blot analysis. Our results showed that the ECM has an important role in cell spreading, adhesion and morphology with a direct effect on modulating RhoA signaling. RhoA activity significantly affected the stress fibers and focal adhesions reorganization, but in a context imposed by the ECM. Thus, RhoA activity modulation in VSMC induced an increased activation of stress fibers and FA formation at 5 h, while a significant inhibition was recorded at 24 h after plating on the different ECM. Our findings provide biophysical evidence that ECM modulates VSMC response to mechanical stimuli inducing intracellular biochemical signaling involved in cellular adaptation to the local microenvironment.     

Combining dynamic stretch and tunable stiffness to probe cell mechanobiology in vitro

Cells have the ability to actively sense their mechanical environment and respond to both substrate stiffness and stretch by altering their adhesion, proliferation, locomotion, morphology, and synthetic profile. In order to elucidate the interrelated effects of different mechanical stimuli on cell phenotype in vitro, we have developed a method for culturing mammalian cells in a two-dimensional environment at a wide range of combined levels of substrate stiffness and dynamic stretch. Polyacrylamide gels were covalently bonded to flexible silicone culture plates and coated with monomeric collagen for cell adhesion. Substrate stiffness was adjusted from relatively soft (G′ = 0.3 kPa) to stiff (G′ = 50 kPa) by altering the ratio of acrylamide to bis-acrylamide, and the silicone membranes were stretched over circular loading posts by applying vacuum pressure to impart near-uniform stretch, as confirmed by strain field analysis. As a demonstration of the system, porcine aortic valve interstitial cells (VIC) and human mesenchymal stem cells (hMSC) were plated on soft and stiff substrates either statically cultured or exposed to 10% equibiaxial or pure uniaxial stretch at 1Hz for 6 hours. In all cases, cell attachment and cell viability were high. On soft substrates, VICs cultured statically exhibit a small rounded morphology, significantly smaller than on stiff substrates (p<0.05). Following equibiaxial cyclic stretch, VICs spread to the extent of cells cultured on stiff substrates, but did not reorient in response to uniaxial stretch to the extent of cells stretched on stiff substrates. hMSCs exhibited a less pronounced response than VICs, likely due to a lower stiffness threshold for spreading on static gels. These preliminary data demonstrate that inhibition of spreading due to a lack of matrix stiffness surrounding a cell may be overcome by externally applied stretch suggesting similar mechanotransduction mechanisms for sensing stiffness and stretch.     

Collagen abundance in mechanically stimulated osteoblast cultures using near infrared microscopy

Collagen abundance in osteoblast cell cultures was determined using near infrared microscopy with chemical imaging (NIR-CI) with and without mechanical stimulation of the the cells. MC3T3-E1 mouse osteoblast cells seeded on a polycarbonate substrate were mechanically stimulated using static loads of 13.5 N, 27 N and 40 N applied to the substrates during 2, 4, 6 and 8 days of incubation. Results show that the cells increased their collagen production with 13.5 N and 27 N loads when compared to the control sample with a 27 N load resulting in a noteworthy increase (109%) in collagen production. The 40 N load on the other hand, resulted in an initial decrease in the collagen expression in the extracellular matrix, possibly as a result of cell death or inhibition of the protein secretion process followed by an increase in collagen after cell recovery and proliferation. Qualitative confirmation of these results was performed using confocal microscopy.     

Intrinsic Response Towards Physiologic Stiffness is Cell-Type Dependent

In the continuous search for better tissue engineering scaffolds it has become increasingly clear that the substrate properties dramatically affect cell responses. Here we compared cells from a physiologically stiff tissue, melanoma, to cells isolated from a physiologically soft tissue, brain. We measured the cell line responses to laminin immobilized onto glass or polyacrylamide hydrogels tuned to have a Young’s modulus ranging from 1 to 390?kPa. Single cells were analyzed for spreading area, shape, total actin content, actin-based morphological features and modification of immobilized laminin. Both cell types exhibited stiffness- and laminin concentration-dependent responses on polyacrylamide and glass. Melanoma cells exhibited very little spreading and were rounded on soft (1, 5, and 15?kPa) hydrogels while cells on stiff (40, 100, and 390?kPa) hydrogels were spread and had a polarized cell shape with large lamellipodia. On rigid glass surfaces, spreading and actin-based morphological features were not observed until laminin concentration was much higher. Similarly, increased microglia cell spreading and presence of actin-based structures were observed on stiff hydrogels. However, responses on rigid glass surfaces were much different. Microglia cells had large spreading areas and elongated shapes on glass compared to hydrogels even when immobilized laminin density was consistent on all gels. While cell spreading and shape varied with Young’s modulus of the hydrogel, the concentration of f-actin was constant. A decrease in laminin immunofluorescence was associated with melanoma and microglia cell spreading on glass with high coating concentration of laminin, indicating modification of immobilized laminin triggered by supraphysiologic stiffness and high ligand density. These results suggest that some cell lines are more sensitive to mechanical properties matching their native tissue environment while other cell lines may require stiffness and extracellular ligand density well above physiologic tissue before saturation in cell spreading, elongation and cytoskeletal re-organization are reached.     

Intrinsic mechanical properties of the extracellular matrix affect the behavior of pre-osteoblastic MC3T3-E1 cells

Mechanical cues present in the ECM have been hypothesized to provide instructive signals that dictate cell behavior. We probed this hypothesis in osteoblastic cells by culturing MC3T3-E1 cells on the surface of type I collagen-modified hydrogels with tunable mechanical properties and assessed their proliferation, migration, and differentiation. On gels functionalized with a low type I collagen density, MC3T3-E1 cells cultured on polystyrene proliferated twice as fast as those cultured on the softest substrate. Quantitative time-lapse video microscopic analysis revealed random motility speeds were significantly retarded on the softest substrate (0.25 ± 0.01 µm/min), in contrast to maximum speeds on polystyrene substrates (0.42 ± 0.04 µm/min). On gels functionalized with a high type I collagen density, migration speed exhibited a biphasic dependence on ECM compliance, with maximum speeds (0.34 ± 0.02 µm/min) observed on gels of intermediate stiffness, whereas minimum speeds (0.24 ± 0.03 µm/min) occurred on both the softest and most rigid (i.e., polystyrene) substrates. Immature focal contacts and a poorly organized actin cytoskeleton were observed in cells cultured on the softest substrates, whereas those on more rigid substrates assembled mature focal adhesions and robust actin stress fibers. In parallel, focal adhesion kinase (FAK) activity (assessed by detecting pY397-FAK) was influenced by compliance, with maximal activity occurring in cells cultured on polystyrene. Finally, mineral deposition by the MC3T3-E1 cells was also affected by ECM compliance, leading to the conclusion that altering ECM mechanical properties may influence a variety of MC3T3-E1 cell functions, and perhaps ultimately, their differentiated phenotype. bone; focal adhesion kinase; mechanotransduction; cytoskeleton; integrins     

Differential binding to dorsal and ventral cell surfaces of fibroblasts: effect on collagen phagocytosis

Matrix remodeling by phagocytic fibroblasts is essential for growth and development but the regulatory processes are undefined. We evaluated the impact of spreading on the binding step of collagen phagocytosis with a novel culture system that more closely replicates phagocytosis in vivo than previous models. 3T3 cells were plated on collagen-coated beads, thereby loading only ventral surfaces (adhesion with spreading), or were allowed to spread on collagen films and then loaded with beads on their dorsal surfaces (adhesion without spreading). Ventral surfaces bound three-fold more beads than dorsal surfaces which was accompanied by accelerated phagosomal maturation. Arp3 and cortactin, markers of the actin-associated spreading machinery, strongly accumulated around ventrally but not dorsally loaded beads, suggesting that spreading contributes to enhanced binding of ventral surfaces. Further, ventral surfaces exhibited two-fold more free alpha2beta1 integrins, the major collagen receptors. Notably, compared to cells spread on collagen substrates, spreading cells exhibited a three-fold higher alpha2beta1 mobile fraction which was correlated with limited engagement of ventral receptors by actin filaments. Thus integrin ligation by actin filaments regulates the mobility of collagen receptors which in turn mediates the enhanced binding of collagen beads on spreading surfaces.     

Comparing the mechanical influence of vinculin, focal adhesion kinase and p53 in mouse embryonic fibroblasts

Cytoskeletal reorganization is an ongoing process when cells adhere, move or invade extracellular substrates. The cellular force generation and transmission are determined by the intactness of the actomyosin-(focal adhesion complex)-integrin connection. We investigated the intracellular course of action in mouse embryonic fibroblasts deficient in the focal adhesion proteins vinculin and focal adhesion kinase (FAK) and the nuclear matrix protein p53 using magnetic tweezer and nanoparticle tracking techniques. Results show that the lack of these proteins decrease cellular stiffness and affect cell rheological behavior. The decrease in cellular binding strength was higher in FAK- to vinculin-deficient cells, whilst p53-deficient cells showed no effect compared to wildtype cells. The intracellular cytoskeletal activity was lowest in wildtype cells, but increased in the following order when cells lacked FAK+p53 > p53 > vinculin. In summary, cell mechanical processes are differently affected by the focal adhesion proteins vinculin and FAK than by the nuclear matrix protein, p53.     

Adhesion-mediated signal transduction in human articular chondrocytes: the influence of biomaterial chemistry and tenascin-C

Chondrocyte 'dedifferentiation' involves the switching of the cell phenotype to one that no longer secretes extracellular matrix found in normal cartilage and occurs frequently during chondrocyte expansion in culture. It is also characterized by the differential expression of receptors and intracellular proteins that are involved in signal transduction pathways, including those associated with cell shape and actin microfilament organization. The objective of this study was to examine the modulation of chondrocyte phenotype by cultivation on polymer substrates containing poly(ethylene glycol) (PEG). We observed differential arrangement of actin organization in articular chondrocytes, depending on PEG length. When cultivated on 300 g/mol PEG substrates at day 19, chondrocytes had lost intracellular markers characteristic of the differentiated phenotype, including type II collagen and protein kinase C (PKC). On these surfaces, chondrocytes also expressed focal adhesion and signaling proteins indicative of cell attachment, spreading, and FA turnover, including RhoA, focal adhesion kinase, and vinculin. The switch to a dedifferentiated chondrocyte phenotype correlated with integrin expression. Conversely, the expression of CD44 receptors coincided with chondrogenic characteristics, suggesting that binding via these receptors could play a role in maintaining the differentiated phenotype on such substrates. These effects can be similar to those of compounds that interfere in intracellular signaling pathways and can be utilized to engineer cellular response.     

Absence of Filamin A Prevents Cells from Responding to Stiffness Gradients on Gels Coated with Collagen but not Fibronectin

Cell types from many tissues respond to changes in substrate stiffness by actively remodeling their cytoskeletons to alter spread area or adhesion strength, and in some cases changing their own stiffness to match that of their substrate. These cell responses to substrate stiffness are linked to substrate-induced changes in the state, localization, and amount of numerous proteins, but detailed evidence for the requirement of specific proteins in these distinct forms of mechanical response are scarce. Here we use microfluidics techniques to produce gels with a gradient of stiffness to show the essential function of filamin A in cell responses to mechanical stimuli and dissociate cell spreading and stiffening by contrasting responses of a pair of human melanoma-derived cell lines that differ in expression of this actin cross-linking protein. M2 melanoma cells null for filamin A do not alter their adherent area in response to increased substrate stiffness when they link to the substrate only through collagen receptors, but change adherent area normally when bound through fibronectin receptors. In contrast, filamin A-replete A7 cells change adherent area on both substrates and respond more strongly to collagen I-coated gels than to fibronectin-coated gels. Strikingly, A7 cells alter their stiffness, as measured by atomic force microscopy, to match the elastic modulus of the substrate immediately adjacent to them on the gradient. M2 cells, in contrast, maintain a constant stiffness on all substrates that is as low as that of A7 cells on the softest gels examined (1000 Pa). Comparison of cell spreading and cell stiffening on the same gradient substrates shows that cell spreading is uncoupled from stiffening. At saturating collagen and fibronectin concentrations, adhesion of M2 cells is reduced compared to that of A7 cells to an extent approximately equal to the difference in adherent area. Filamin A appears to be essential for cell stiffening on collagen, but not for cell spreading on fibronectin. These results have implications for different models of cell protrusion and adhesion and identify a key role for filamin A in altering cellular stiffness that cannot be compensated for by other actin cross-linkers in vivo.     

Investigating the effect of cell substrate on cancer cell stiffness by optical tweezers

The mechanical properties of cells are influenced by their microenvironment. Here we report cell stiffness alteration by changing the cell substrate stiffness for isolated cells and cells in contact with other cells. Polydimethylsiloxane (PDMS) is used to prepare soft substrates with three different stiffness values (173, 88 and 17 kPa respectively). Breast cancer cells lines, namely HBL-100, MCF-7 and MDA-MB-231 with different level of aggressiveness are cultured on these substrates and their local elasticity is investigated by vertical indentation of the cell membrane. Our preliminary results show an unforeseen behavior of the MDA-MB-231 cells. When cultured on glass substrate as isolated cells, they are less stiff than the other two types of cells, in agreement with the general statement that more aggressive and metastatic cells are softer. However, when connected to other cells the stiffness of MDA-MB-231 cells becomes similar to the other two cell lines. Moreover, the stiffness of MDA-MB-231 cells cultured on soft PDMS substrates is significantly higher than the stiffness of the other cell types, demonstrating thus the strong influence of the environmental conditions on the mechanical properties of the cells.