Regulation of PD-L1 expression by matrix stiffness in lung cancer cells

Expression of programmed death-ligand 1 (PD-L1) in tumor cells such as lung cancer cells plays an important role in mechanisms underlying evasion of an immune check point system. Lung cancer tissue with increased deposition of extracellular matrix is much stiffer than normal lung tissue. There is emerging evidence that the matrix stiffness of cancer tissue affects the phenotypes and properties of cancer cells. Nevertheless, the effects of substrate rigidity on expression of PD-L1 in lung cancer cells remain elusive. We evaluated the effects of substrate stiffness on PD-L1 expression in HCC827 lung adenocarcinoma cells by using polyacrylamide hydrogels with stiffnesses of 2 and 25?kPa. Expression of PD-L1 protein was higher on the stiffer substrates (25?kPa gel and plastic dish) than on the soft 2?kPa gel. PD-L1 expression was reduced by detachment of cells adhering to the substrate. Interferon-γ enhanced expression of PD-L1 protein cultured on stiff (25?kPa gel and plastic dishes) and soft (2?kPa gel) substrates and in the cell adhesion-free condition. As the stiffness of substrates increased, formation of actin stress fiber and cell growth were enhanced. Transfection of the cells with short interfering RNA for PD-L1 inhibited cell growth without affecting stress fiber formation. Treatment of the cells with cytochalasin D, an inhibitor of actin polymerization, significantly reduced PD-L1 protein levels. Taken together, a stiff substrate enhanced PD-L1 expression via actin-dependent mechanisms in lung cancer cells. It is suggested that stiffness as a tumor environment regulates PD-L1 expression, which leads to evasion of the immune system and tumor growth.     

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.     

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.     

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.     

The role of extracellular matrix stiffness in regulating cytoskeletal remodeling via vinculin in synthetic smooth muscle cells

Vinculin is a key player in sensing and responding to external mechanical cues such as extracellular matrix stiffness. Increased matrix stiffness is often associated with certain pathological conditions including hypertension induced cellular cytoskeleton changes in vascular smooth muscle (VSM) cells. However, little is known on how stiffness affects cytoskeletal remodeling via vinculin in VSM cells. Thus, we utilized matrices with elastic moduli that simulate vascular stiffness in different stages of hypertension to investigate how matrix stiffness regulates cell cytoskeleton via vinculin in synthetic VSM cells. Through selecting a suitable reference gene, we found that an increase in physiologically relevant extracellular matrix stiffness (2–50?kPa) downregulates vinculin gene expression but upregulates vinculin protein expression. This discrepancy, which was not observed previously for non-muscle cells, suggests that the vinculin-mediated mecahnotransduction mechanism in synthetic VSM cells may be more complex than those proposed for non-muscle cells. Also adding to previous findings, we found that VSM cell growth may be impeded by substrates that are either too soft or too rigid.     

The Effect of Substrate Stiffness,Thickness, and Cross-Linking Density on Osteogenic Cell Behavior

Osteogenic cells respond to mechanical changes in their environment by altering their spread area, morphology, and gene expression profile. In particular, the bulk modulus of the substrate, as well as its microstructure and thickness, can substantially alter the local stiffness experienced by the cell. Although bone tissue regeneration strategies involve culture of bone cells on various biomaterial scaffolds, which are often cross-linked to enhance their physical integrity, it is difficult to ascertain and compare the local stiffness experienced by cells cultured on different biomaterials. In this study, we seek to characterize the local stiffness at the cellular level for MC3T3-E1 cells plated on biomaterial substrates of varying modulus, thickness, and cross-linking concentration. Cells were cultured on flat and wedge-shaped gels made from polyacrylamide or cross-linked collagen. The cross-linking density of the collagen gels was varied to investigate the effect of fiber cross-linking in conjunction with substrate thickness. Cell spread area was used as a measure of osteogenic differentiation. Finite element simulations were used to examine the effects of fiber cross-linking and substrate thickness on the resistance of the gel to cellular forces, corresponding to the equivalent shear stiffness for the gel structure in the region directly surrounding the cell. The results of this study show that MC3T3 cells cultured on a soft fibrous substrate attain the same spread cell area as those cultured on a much higher modulus, but nonfibrous substrate. Finite element simulations predict that a dramatic increase in the equivalent shear stiffness of fibrous collagen gels occurs as cross-linking density is increased, with equivalent stiffness also increasing as gel thickness is decreased. These results provide an insight into the response of osteogenic cells to individual substrate parameters and have the potential to inform future bone tissue regeneration strategies that can optimize the equivalent stiffness experienced by a cell.     

Sensitivity of alveolar macrophages to substrate mechanical and adhesive properties

In order to understand the sensitivity of alveolar macrophages (AMs) to substrate properties, we have developed a new model of macrophages cultured on substrates of increasing Young's modulus: (i) a monolayer of alveolar epithelial cells representing the supple (approximately 0.1 kPa) physiological substrate, (ii) polyacrylamide gels with two concentrations of bis-acrylamide representing low and high intermediate stiffness (respectively 40 kPa and 160 kPa) and, (iii) a highly rigid surface of plastic or glass (respectively 3 MPa and 70 MPa), the two latter being or not functionalized with type I-collagen. The macrophage response was studied through their shape (characterized by 3D-reconstructions of F-actin structure) and their cytoskeletal stiffness (estimated by transient twisting of magnetic RGD-coated beads and corrected for actual bead immersion). Macrophage shape dramatically changed from rounded to flattened as substrate stiffness increased from soft ((i) and (ii)) to rigid (iii) substrates, indicating a net sensitivity of alveolar macrophages to substrate stiffness but without generating F-actin stress fibers. Macrophage stiffness was also increased by large substrate stiffness increase but this increase was not due to an increase in internal tension assessed by the negligible effect of a F-actin depolymerizing drug (cytochalasine D) on bead twisting. The mechanical sensitivity of AMs could be partly explained by an idealized numerical model describing how low cell height enhances the substrate-stiffness-dependence of the apparent (measured) AM stiffness. Altogether, these results suggest that macrophages are able to probe their physical environment but the mechanosensitive mechanism behind appears quite different from tissue cells, since it occurs at no significant cell-scale prestress, shape changes through minimal actin remodeling and finally an AMs stiffness not affected by the loss in F-actin integrity.     

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     

Collagen type V modulates fibroblast behavior dependent on substrate stiffness

Collagen type V is highly expressed during tissue development and wound repair, but its exact function remains unclear. Cell binding to collagen V affects various basic cell functions and increased collagen V levels alter the structural organization and the stiffness of the ECM. We studied the combined effects of collagen V and substrate stiffness on the morphology, focal adhesion formation, and actin organization of fibroblasts. We found that a hybrid collagen I/V coating impairs fibroblast spreading on soft substrates (<10 kPa), but not on stiffer substrates (68 kPa or glass). In sharp contrast, a pure collagen I coating does not impair cell spreading on soft substrates. The impairment of cell spreading by collagen V is accompanied by diffuse actin staining patterns and small focal adhesions. These observations suggest that collagen V plays an essential role in modifying cell behavior during development and remodeling, when very soft tissues are present.     

Effect of substrate stiffness on early mouse embryo development

It is becoming increasingly clear that cells are remarkably sensitive to the biophysical cues of their microenvironment and that these cues play a significant role in influencing their behaviors. In this study, we investigated whether the early pre-implantation embryo is sensitive to mechanical cues, i.e. the elasticity of the culture environment. To test this, we have developed a new embryo culture system where the mechanical properties of the embryonic environment can be precisely defined. The contemporary standard environment for embryo culture is the polystyrene petri dish (PD), which has a stiffness (1 GPa) that is six orders of magnitude greater than the uterine epithelium (1 kPa). To approximate more closely the mechanical aspects of the in vivo uterine environment we used polydimethyl-siloxane (PDMS) or fabricated 3D type I collagen gels (1 kPa stiffness, Col-1k group). Mouse embryo development on alternate substrates was compared to that seen on the petri dish; percent development, hatching frequency, and cell number were observed. Our results indicated that embryos are sensitive to the mechanical environment on which they are cultured. Embryos cultured on Col-1k showed a significantly greater frequency of development to 2-cell (68±15% vs. 59±18%), blastocyst (64±9.1% vs. 50±18%) and hatching blastocyst stages (54±25% vs. 21±16%) and an increase in the number of trophectodermal cell (TE,65±13 vs. 49±12 cells) compared to control embryos cultured in PD (mean±S.D.; p<.01). Embryos cultured on Col-1k and PD were transferred to recipient females and observed on embryonic day 12.5. Both groups had the same number of fetuses, however the placentas of the Col-1k fetuses were larger than controls, suggesting a continued effect of the preimplantation environment. In summary, characteristics of the preimplantation microenvironment affect pre- and post-implantation growth.     

Mechanical force affects expression of an in vitro metastasis-like phenotype in HCT-8 cells

Cancer deaths are primarily caused by metastases, not by the parent tumor. During metastasis, malignant cells detach from the parent tumor, and spread through the circulatory system to invade new tissues and organs. The physical-chemical mechanisms and parameters within the cellular microenvironment that initiate the onset of metastasis, however, are not understood. Here we show that human colon carcinoma (HCT-8) cells can exhibit a dissociative, metastasis-like phenotype (MLP) in vitro when cultured on substrates with appropriate mechanical stiffness. This rather remarkable phenotype is observed when HCT-8 cells are cultured on gels with intermediate-stiffness (physiologically relevant 21-47 kPa), but not on very soft (1 kPa) and very stiff (3.6 GPa) substrates. The cell-cell adhesion molecule E-Cadherin, a metastasis hallmark, decreases 4.73 ± 1.43 times on cell membranes in concert with disassociation. Both specific and nonspecific cell adhesion decrease once the cells have disassociated. After reculturing the disassociated cells on fresh substrates, they retain the disassociated phenotype regardless of substrate stiffness. Inducing E-Cadherin overexpression in MLP cells only partially reverses the MLP phenotype in a minority population of the dissociated cells. This important experiment reveals that E-Cadherin does not play a significant role in the upstream regulation of the mechanosensing cascade. Our results indicate, during culture on the appropriate mechanical microenvironment, HCT-8 cells undergo a stable cell-state transition with increased in vitro metastasis-like characteristics as compared to parent cells grown on standard, very stiff tissue culture dishes. Nuclear staining reveals that a large nuclear deformation (major/minor axis ratio, 2:5) occurs in HCT-8 cells when cells are cultured on polystyrene substrates, but it is markedly reduced (ratio, 1:3) in cells grown on 21 kPa substrates, suggesting the cells are experiencing different intracellular forces when grown on stiff as compared to soft substrates. Furthermore, MLP can be inhibited by blebbistatin, which inactivates myosin II activity and relaxes intracellular forces. This novel finding suggests that the onset of metastasis may, in part, be linked to the intracellular forces and the mechanical microenvironment of the tumor.     

Environmental Particulate (PM2.5) Augments Stiffness-Induced Alveolar Epithelial Cell Mechanoactivation of Transforming Growth Factor Beta

Dysfunctional pulmonary homeostasis and repair, including diseases such as pulmonary fibrosis (PF), chronic obstructive pulmonary disease (COPD), and tumorigenesis have been increasing over the past decade, a fact that heavily implicates environmental influences. Several investigations have suggested that in response to increased transforming growth factor - beta (TGFβ) signaling, the alveolar type II (ATII) epithelial cell undergoes phenotypic changes that may contribute to the complex pathobiology of PF. We have previously demonstrated that increased tissue stiffness associated with PF is a potent extracellular matrix (ECM) signal for epithelial cell activation of TGFβ. The work reported here explores the relationship between tissue stiffness and exposure to environmental stimuli in the activation of TGFβ. We hypothesized that exposure of ATII cells to fine particulate matter (PM2.5) will result in enhanced cell contractility, TGFβ activation, and subsequent changes to ATII cell phenotype. ATII cells were cultured on increasingly stiff substrates with or without addition of PM2.5. Exposure to PM2.5 resulted in increased activation of TGFβ, increased cell contractility, and elongation of ATII cells. Most notably, on 8 kPa substrates, a stiffness greater than normal but less than established fibrotic lung, addition of PM2.5 resulted in increased cortical cell stiffness, enhanced actin staining and cell elongation; a result not seen in the absence of PM2.5. Our work suggests that PM2.5 exposure additionally enhances the existing interaction between ECM stiffness and TGFβ that has been previously reported. Furthermore, we show that this additional enhancement is likely a consequence of intracellular reactive oxygen species (ROS) leading to increased TGFβ signaling events. These results highlight the importance of both the micromechanical and biochemical environment in lung disease initiation and suggest that individuals in early stages of lung remodeling during fibrosis may be more susceptible than healthy individuals when exposed to environmental injury adjuvants.     

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.     

The regulation of osteogenesis by ECM rigidity in MC3T3-E1 cells requires MAPK activation

Once thought to provide only structural support to tissues by acting as a scaffold to which cells bind, it is now widely recognized that the extracellular matrix (ECM) provides instructive signals that dictate cell behavior. Recently we demonstrated that mechanical cues intrinsic to the ECM directly regulate the behavior of pre-osteoblastic MC3T3-E1 cells. We hypothesized that one possible mechanism by which ECM compliance exerts its influence on osteogenesis is by modulating the mitogen-activated protein kinase (MAPK) pathway. To address this hypothesis, the differentiation of MC3T3-E1 cells cultured on poly(ethylene glycol) (PEG)-based model substrates with tunable mechanical properties was assessed. Alkaline phosphatase (ALP) levels at days 7 and 14 were found to be significantly higher in cells grown on stiffer substrates (423.9 kPa hydrogels and rigid tissue culture polystyrene (TCPS) control) than on a soft hydrogel (13.7 kPa). Osteocalcin (OCN) and bone sialoprotein (BSP) gene expression levels followed a similar trend. In parallel, MAPK activity was significantly higher in cells cultured on stiffer substrates at both time points. Inhibiting this activation pharmacologically, using PD98059, resulted in significantly lower ALP levels, OCN, and BSP gene expression levels on the hydrogels. Interestingly, the effectiveness of PD98059 was itself dependent on substrate stiffness, with marked inhibition of MAPK phosphorylation in cells grown on compliant hydrogels but insignificant reduction in cells grown on TCPS. Together, these data confirm a role for MAPK in the regulation of osteogenic differentiation by ECM compliance.     

Fibronectin accelerates the growth and differentiation of ameloblast lineage cells in vitro.

During tooth development, the growth and differentiation of ameloblast lineage (AL) cells are regulated by epithelial-mesenchymal interactions. To examine the dynamic effects of components of the basement membrane, which is the extracellular matrix (ECM) lying between the epithelium and mesenchyme, we prepared AL cells from the epithelial layer sheet of mandibular incisors of postnatal day 7 rats and cultured them on plates coated with type IV collagen, laminin-1, or fibronectin. The growth of AL cells was supported by type IV collagen and fibronectin but not by laminin-1 in comparison with that on type I collagen as a reference. Clustering and differentiation of AL cells were observed on all matrices examined. AL cells showed normal growth and differentiation at low cell density on fibronectin but not on type I collagen. Furthermore, the population of cytokeratin 14-positive cells on fibronectin was lower than that on other ECM components, suggesting that fibronectin may be a modulator to accelerate the differentiation of AL cells. After the cells had been cultured for 9 days on fibronectin, crystal-like structures were observed. These structures overlaid the cell clusters and were positive for von Kossa staining. These findings indicate that each matrix component has a regulative role in the proliferation and differentiation of AL cells and that fibronectin causes the greatest acceleration of AL cell differentiation.     

Integrins beta1, alpha6, and alpha3 contribute to mechanical strain-induced differentiation of fetal lung type II epithelial cells via distinct mechanisms

Mechanical forces regulate lung maturation in the fetus by promoting type II epithelial differentiation. However, the cell surface receptors that transduce these mechanical cues into cellular responses remain largely unknown. When distal lung type II epithelial cells isolated from embryonic day 19 rat fetuses were cultured on flexible plates coated with laminin, fibronectin, vitronectin, collagen, or elastin and exposed to a level of mechanical strain (5%) similar to that observed in utero, transmembrane signaling responses were induced under all conditions, as measured by ERK activation. However, mechanical stress maximally increased expression of the type II cell differentiation marker surfactant protein C when cells were cultured on laminin substrates. Strain-induced alveolar epithelial differentiation was inhibited by interfering with cell binding to laminin using soluble laminin peptides (IKVIV or YIGSR) or blocking antibodies against integrin beta1, alpha3, or alpha6. Additional studies were carried out with substrates coated directly with different nonactivating anti-integrin antibodies. Blocking integrin beta1 and alpha6 binding sites inhibited both cell adhesion and differentiation, whereas inhibition of alpha3 prevented differentiation without altering cell attachment. These data demonstrate that various integrins contribute to mechanical control of type II lung epithelial cell differentiation on laminin substrates. However, they may act via distinct mechanisms, including some that are independent of their cell anchoring role.     

An in vitro correlation of metastatic capacity, substrate rigidity, and ECM composition

The process of metastasis requires a metastatic cancer cell to invade a variety of micro-environments of variable stiffnesses. Unlike metastatic cells, normal cell function and viability is dependent on the stiffness of the environment and used as a cue to maintain cell health and proper tissue organization. In this study we have asked if metastatic cells can ignore the parameter of stiffness and if this ability is gradually acquired and if so, through what mechanism. Using a panel of mouse mammary tumor cells derived from the same parental tumor, but possessing different metastatic abilities, we cultured the cells on hard and soft substrates conjugated with collagen or fibronectin. Normal and non-metastatic tumor cells responded to changes in stiffness on fibronectin, but not collagen. However, the more metastatic cells ignored the change in stiffness on fibronectin-coated substrates. This lack of response on fibronectin correlated with a change in the expression level of the α3 integrin subunit, activation of the β1 subunit, and phosphorylation of FAKpY397. We conclude that through fibronectin, changes in the activation and tethering of the beta-1 integrin provides a mechanism for metastatic cells to disregard changes in compliance to survive and navigate in environments of different stiffness.     

Adaptation of aequorin functional assay to high throughput screening

AequoScreen, a cellular aequorin-based functional assay, has been optimized for luminescent high-throughput screening (HTS) of G protein-coupled receptor (GPCRs). AequoScreen is a homogeneous assay in which the cells are loaded with the apoaequorin cofactor coelenterazine, diluted in assay buffer, and injected into plates containing the samples to be tested. A flash of light is emitted following the calcium increase resulting from the activation of the GPCR by the sample. Here we have validated a new plate reader, the Hamamatsu Photonics FDSS6000, for HTS in 96- and 384-well plates with CHO-K1 cells stably coexpressing mitochondrial apoaequorin and different GPCRs (AequoScreen cell lines). The acquisition time, plate type, and cell number per well have been optimized to obtain concentration-response curves with 4000 cells/well in 384-well plates and a high signal:background ratio. The FDSS6000 and AequoScreen cell lines allow reading of twenty 96- or 384-well plates in 1 h with Z' values of 0.71 and 0.78, respectively. These results bring new insights to functional assays, and therefore reinforce the interest in aequorin-based assays in a HTS environment.