Migration of breast cancer cells : understanding the roles of volume exclusion and cell-to-cell adhesion

We study MCF-7 breast cancer cell movement in a transwell apparatus. Various experimental conditions lead to a variety of monotone and nonmonotone responses which are difficult to interpret. We anticipate that the experimental results could be caused by cell-to-cell adhesion or volume exclusion. Without any modeling, it is impossible to understand the relative roles played by these two mechanisms. A lattice-based exclusion process random-walk model incorporating agent-to-agent adhesion is applied to the experimental system. Our combined experimental and modeling approach shows that a low value of cell-to-cell adhesion strength provides the best explanation of the experimental data suggesting that volume exclusion plays a more important role than cell-to-cell adhesion. This combined experimental and modeling study gives insight into the cell-level details and design of transwell assays.

Studies of bone cell adhesion and proliferation on ion implanted titanium discs

Argon ions were implanted on titanium discs to study its effect on bone cell adhesion and proli feration. Polished titanium discs were prepared and implanted with argon ions with different doses. Afterwards the samples were sterilized using UV light, inocu lated with human bone cells and incubated. Once fixed and rinsed, image analysis has been used to quantify the number of cells attached to the titanium discs. Cell proliferation tests were also conducted after a period of 120 hours. Cell adhesion was seen to be higher with ion im planted surface. SEM analysis has shown that the cells attached spread more on ion implanted surface. The numbers of cells attached were seen to be higher on implanted surfaces; they tend to occupy wider areas with healthier cells.

Stroma regulates increased epithelial lateral cell adhesion in 3D culture : a role for actin/cadherin dynamics

BACKGROUND: Cell shape and tissue architecture are controlled by changes to junctional proteins and the cytoskeleton. How tissues control the dynamics of adhesion and cytoskeletal tension is unclear. We have studied epithelial tissue architecture using 3D culture models and found that adult primary prostate epithelial cells grow into hollow acinus-like spheroids. Importantly, when co-cultured with stroma the epithelia show increased lateral cell adhesions. To investigate this mechanism further we aimed to: identify a cell line model to allow repeatable and robust experiments; determine whether or not epithelial adhesion molecules were affected by stromal culture; and determine which stromal signalling molecules may influence cell adhesion in 3D epithelial cell cultures. METHODOLOGY/PRINCIPAL FINDINGS: The prostate cell line, BPH-1, showed increased lateral cell adhesion in response to stroma, when grown as 3D spheroids. Electron microscopy showed that 9.4% of lateral membranes were within 20 nm of each other and that this increased to 54% in the presence of stroma, after 7 days in culture. Stromal signalling did not influence E-cadherin or desmosome RNA or protein expression, but increased E-cadherin/actin co-localisation on the basolateral membranes, and decreased paracellular permeability. Microarray analysis identified several growth factors and pathways that were differentially expressed in stroma in response to 3D epithelial culture. The upregulated growth factors TGFβ2, CXCL12 and FGF10 were selected for further analysis because of previous associations with morphology. Small molecule inhibition of TGFβ2 signalling but not of CXCL12 and FGF10 signalling led to a decrease in actin and E-cadherin co-localisation and increased paracellular permeability. CONCLUSIONS/SIGNIFICANCE: In 3D culture models, paracrine stromal signals increase epithelial cell adhesion via adhesion/cytoskeleton interactions and TGFβ2-dependent mechanisms may play a key role. These findings indicate a role for stroma in maintaining adult epithelial tissue morphology and integrity.

Cell adhesion molecule uvomorulin expression in human breast cancer cell lines : relationship to morphology and invasive capacities

Loss of cell-cell adhesion in carcinoma cells may be an important step in the acquisition of an invasive, metastatic phenotype. We have examined the expression of the epithelial-specific cell adhesion molecule uvomorulin (E-cadherin, cell-CAM 120/80, L-CAM) in human breast cancer cell lines. We find that fibroblastoid, highly invasive, vimentin-expressing breast cancer cell lines do not express uvomorulin. Of the more epithelial-appearing, less invasive, keratin-expressing breast cancer cell lines, some express uvomorulin, and some do not. We examined the morphologies of the cell lines in the reconstituted basement membrane matrix Matrigel and measured the ability of the cells to traverse a Matrigel-coated filter as in vitro models for detachment of carcinoma cells from neighboring cells and invasion through basement membrane into surrounding tissue. Colonies of uvomorulin-positive cells have a characteristic fused appearance in Matrigel, whereas uvomorulin-negative cells appear detached. Cells which are uvomorulin negative and vimentin positive have a stellate morphology in Matrigel. We show that uvomorulin is responsible for the fused colony morphology in Matrigel since treatment of uvomorulin-positive MCF-7 cells with an antibody to uvomorulin caused the cells to detach from one another but did not induce invasiveness in these cells, as measured by their ability to cross a Matrigel-coated polycarbonate filter in a modified Boyden chamber assay. Two uvomorulin-negative, vimentin-negative cell lines are also not highly invasive as measured by this assay. We suggest that loss of uvomorulin-mediated cell-cell adhesion may be one of many changes involved in the progression of a carcinoma cell to an invasive phenotype.

Bone sialoprotein supports breast cancer cell adhesion proliferation and migration through differential usage of the αvβ3 and αvβ5 integrins

Bone sialoprotein (BSP), a secreted glycoprotein found in bone matrix, has been implicated in the formation of mammary microcalcifications and osteotropic metastasis of human breast cancer (HBC). BSP possesses an integrin-binding RGD (Arg-Gly-Asp) domain, which may promote interactions between HBC cells and bone extracellular matrix. Purified BSP, recombinant human BSP fragments and BSP-derived RGD peptides are shown to elicit migratory, adhesive, and proliferative responses in the MDA-MB-231 HBC cell line. Recombinant BSP fragment analysis localized a significant component of these activities to the RGD domain of the protein, and synthetic RGD peptides with BSP flanking sequences (BSPRGD) also conferred these responses. The fibronectin-derived RGD counterpart, GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro), could not support these cellular responses, emphasizing specificity of the BSP configuration. Although most of the proliferative and adhesive responses could be attributed to RGD interactions, these interactions were only partly responsible for the migrational responses. Experiments with integrin-blocking antibodies demonstrated that BSP-RGD-induced migration utilizes the αvβ3 vitronectin receptor, whereas adhesion and proliferation responses were αvβ5-mediated. Using fluorescence activated cell sorting, we selected two separate subpopulations of MDA-MB-231 cells enriched for αvβ3 or αvβ5 respectively. Although some expression of the alternate αv integrin was still retained, the αvβ5-enriched MDA-MB-231 cells showed enhanced proliferative and adhesive responses, whereas the αvβ3-enriched subpopulation was suppressed for proliferation and adhesion, but showed enhanced migratory responses to BSP-RGD. In addition, similar analysis of two other HBC cell lines showed less marked, but similar RGD-dependent trends in adhesion and proliferation to the BSP fragments. Collectively, these data demonstrate BSP effects on proliferative, migratory, and adhesive functions in HBC cells and that the RGD-mediated component differentially employs αvβ3 and αvβ5 integrin receptors.

Tunable “Nano-Shearing” : A physical mechanism to displace nonspecific cell adhesion during rare cell detection

We report a tunable alternating current electrohydrodynamic (ac-EHD) force which drives lateran fluid motion within a few nanometers of an electrode surface. Because the magnitude of this fluid shear force can be tuned externally (e.g., via the application of an ac electric field), it provides a new capability to physically displace weakly (nonspecifically) bound cellular analytes. To demonstrate the utility of the tunable nanoshearing phenomenon, we present data on purpose-built microfluidic devices that employ ac-EHD force to remove nonspecific adsorption of molecular and cellular species. Here, we show that an ac-EHD device containing asymmetric planar and microtip electrode pairs resulted in a 4-fold reduction in nonspecific adsorption of blood cells and also captured breast cancer cells in blood, with high efficiency (approximately 87%) and specificity. We therefore feel that this new capability of externally tuning and manipulating fluid flow could have wide applications as an innovative approach to enhance the specific capture of rare cells such as cancer cells in blood.

Single-cell force spectroscopy, an emerging tool to quantify cell adhesion to biomaterials

Cell adhesion receptors play a central role in sensing and integrating signals provided by the cellular environment. Thus, understanding adhesive interactions at the cell-biomaterial interface is essential to improve the design of implants that should emulate certain characteristics of the cell's natural environment. Numerous cell adhesion assays have been developed; among these, atomic force microscopy-based single-cell force spectroscopy (AFM-SCFS) provides a versatile tool to quantify cell adhesion at physiological conditions. Here we discuss how AFM-SCFS can be used to quantify the adhesion of living cells to biomaterials and give examples of using AFM-SCFS in tissue engineering and regenerative medicine. We anticipate that in the near future, AFM-SCFS will be established in the biomaterial field as an important technique to quantify cell-biomaterial interactions and thereby will contribute to the optimization of implants, scaffolds, and medical devices.

Platelet endothelial cell adhesion molecule 1 (PECAM-1) and its interactions with glycosaminoglycans: 2. Biochemical analyses

Platelet endothelial cell adhesion molecule 1 (PECAM-1) (CD31), a member of the immunoglobulin (Ig) superfamily of cell adhesion molecules with six Ig-like domains, has a range of functions, notably its contributions to leukocyte extravasation during inflammation and in maintaining vascular endothelial integrity. Although PECAM-1 is known to mediate cell adhesion by homophilic binding via domain 1, a number of PECAM-1 heterophilic ligands have been proposed. Here, the possibility that heparin and heparan sulfate (HS) are ligands for PECAM-1 was reinvestigated. The extracellular domain of PECAM-1 was expressed first as a fusion protein with the Fc region of human IgG1 fused to domain 6 and second with an N-terminal Flag tag on domain 1 (Flag-PECAM-1). Both proteins bound heparin immobilized on a biosensor chip in surface plasmon resonance (SPR) binding experiments. Binding was pH-sensitive but is easily measured at slightly acidic pH. A series of PECAM-1 domain deletions, prepared in both expression systems, were tested for heparin binding. This revealed that the main heparin-binding site required both domains 2 and 3. Flag-PECAM-1 and a Flag protein containing domains 1-3 bound HS on melanoma cell surfaces, but a Flag protein containing domains 1-2 did not. Heparin oligosaccharides inhibited Flag-PECAM-1 from binding immobilized heparin, with certain structures having greater inhibitory activity than others. Molecular modeling similarly identified the junction of domains 2 and 3 as the heparin-binding site and further revealed the importance of the iduronic acid conformation for binding. PECAM-1 does bind heparin/HS but by a site that is distinct from that required for homophilic binding.

Platelet endothelial cell adhesion molecule 1 (PECAM-1) and its interactions with glycosaminoglycans: 1. Molecular modeling studies

Platelet endothelial cell adhesion molecule 1 (PECAM-1) has many functions, including its roles in leukocyte extravasation as part of the inflammatory response and in the maintenance of vascular integrity through its contribution to endothelial cell−cell adhesion. PECAM-1 has been shown to mediate cell−cell adhesion through homophilic binding events that involve interactions between domain 1 of PECAM-1 molecules on adjacent cells. However, various heterophilic ligands of PECAM-1 have also been proposed. The possible interaction of PECAM-1 with glycosaminoglycans (GAGs) is the focus of this study. The three-dimensional structure of the extracellular immunoglobulin (Ig) domains of PECAM-1 were constructed using homology modeling and threading methods. Potential heparin/heparan sulfate-binding sites were predicted on the basis of their amino acid consensus sequences and a comparison with known structures of sulfate-binding proteins. Heparin and other GAG fragments have been docked to investigate the structural determinants of their protein-binding specificity and selectivity. The modeling has predicted two regions in PECAM-1 that appear to bind heparin oligosaccharides. A high-affinity binding site was located in Ig domains 2 and 3, and evidence for a low-affinity site in Ig domains 5 and 6 was obtained. These GAG-binding regions were distinct from regions involved in PECAM-1 homophilic interactions.

The neuronal cell adhesion molecule ICAM-5

The neuronal cell adhesion molecule ICAM-5 ICAM-5 (telencephalin) belongs to the intercellular adhesion molecule (ICAM)-subgroup of the immunoglobulin superfamily (IgSF). ICAMs participate in leukocyte adhesion and adhesion-dependent functions in the central nervous system (CNS) through interacting with the leukocyte-specific b2 integrins. ICAM-5 is found in the mammalian forebrain, appears at the time of birth, and is located at the cell soma and neuronal dendrites. Recent studies also show that it is important for the regulation of immune functions in the brain and for the development and maturation of neuronal synapses. The clinical importance of ICAM-5 is still under investigation; it may have a role in the development of Alzheimer s disease (AD). In this study, the role of ICAM-5 in neuronal differentiation and its associations with a-actinin and N-methyl-D-aspartic acid (NMDA) receptors were examined. NMDA receptors (NMDARs) are known to be involved in many neuronal functions, including the passage of information from one neuron to another one, and thus it was thought important to study their role related to ICAM-5. The results suggested that ICAM-5 was able to induce dendritic outgrowth through homophilic adhesion (ICAM-5 monomer binds to another ICAM-5 monomer in the same or neighbouring cell), and the homophilic binding activity appeared to be regulated by monomer/multimer transition. Moreover, ICAM-5 binding to a-actinin was shown to be important for neuritic outgrowth. It was examined whether matrix metalloproteinases (MMPs) are the main enzymes involved in ICAM-5 ectodomain cleavage. The results showed that stimulation of NMDARs leads to MMP activation, cleavage of ICAM-5 and it is accompanied by dendritic spine maturation. These findings also indicated that ICAM-5 and NMDA receptor subunit 1 (NR1) compete for binding to a-actinin, and ICAM-5 may regulate the NR1 association with the actin cytoskeleton. Thus, it is concluded that ICAM-5 is a crucial cell adhesion molecule involved in the development of neuronal synapses, especially in the regulation of dendritic spine development, and its functions may also be involved with memory formation and learning.

Characterisation of cell adhesion to substrate materials and the resistance to enzymatic and mechanical cell-removal

Cell-implant adhesive strength is important for prostheses. In this paper, an investigation is described into the adhesion of bovine chondrocytes to Ti6Al4V-based substrates with different surface roughnesses and compositions. Cells were cultured for 2 or 5 days, to promote adhesion. The ease of cell removal was characterised, using both biochemical (trypsin) and mechanical (accelerated buoyancy and liquid flow) methods. Computational fluid dynamics (CFD) modelling has been used to estimate the shear forces applied to the cells by the liquid flow. A comparison is presented between the ease of cell detachment indicated using these methods, for the three surfaces investigated. © 2008 Materials Research Society.