The mechanical rigidity of the extracellular matrix regulates the structure, motility, and proliferation of glioma cells

Glioblastoma multiforme (GBM) is a malignant astrocytoma of the central nervous system associated with a median survival time of 15 months, even with aggressive therapy. This rapid progression is due in part to diffuse infiltration of single tumor cells into the brain parenchyma, which is thought to involve aberrant interactions between tumor cells and the extracellular matrix (ECM). Here, we test the hypothesis that mechanical cues from the ECM contribute to key tumor cell properties relevant to invasion. We cultured a series of glioma cell lines (U373-MG, U87-MG, U251-MG, SNB19, C6) on fibronectin-coated polymeric ECM substrates of defined mechanical rigidity and investigated the role of ECM rigidity in regulating tumor cell structure, migration, and proliferation. On highly rigid ECMs, tumor cells spread extensively, form prominent stress fibers and mature focal adhesions, and migrate rapidly. As ECM rigidity is lowered to values comparable with normal brain tissue, tumor cells appear rounded and fail to productively migrate. Remarkably, cell proliferation is also strongly regulated by ECM rigidity, with cells dividing much more rapidly on rigid than on compliant ECMs. Pharmacologic inhibition of nonmuscle myosin II–based contractility blunts this rigidity-sensitivity and rescues cell motility on highly compliant substrates. Collectively, our results provide support for a novel model in which ECM rigidity provides a transformative, microenvironmental cue that acts through actomyosin contractility to regulate the invasive properties of GBM tumor cells.

Antibodies mediate formation of neutrophil extracellular traps in the middle ear and facilitate secondary pneumococcal otitis media

Otitis media (OM) (a middle ear infection) is a common childhood illness that can leave some children with permanent hearing loss. OM can arise following infection with a variety of different pathogens, including a coinfection with influenza A virus (IAV) and Streptococcus pneumoniae (the pneumococcus). We and others have demonstrated that coinfection with IAV facilitates the replication of pneumococci in the middle ear. Specifically, we used a mouse model of OM to show that IAV facilitates the outgrowth of S. pneumoniae in the middle ear by inducing middle ear inflammation. Here, we seek to understand how the host inflammatory response facilitates bacterial outgrowth in the middle ear. Using B cell-deficient infant mice, we show that antibodies play a crucial role in facilitating pneumococcal replication. We subsequently show that this is due to antibody-dependent neutrophil extracellular trap (NET) formation in the middle ear, which, instead of clearing the infection, allows the bacteria to replicate. We further demonstrate the importance of these NETs as a potential therapeutic target through the transtympanic administration of a DNase, which effectively reduces the bacterial load in the middle ear. Taken together, these data provide novel insight into how pneumococci are able to replicate in the middle ear cavity and induce disease.

Electrical imaging and fluid modeling of convective fingering in a shallow water-table aquifer

Unstable density-driven flow can lead to enhanced solute transport in groundwater. Only recently has the complex fingering pattern associated with free convection been documented in field settings. Electrical resistivity (ER) tomography has been used to capture a snapshot of convective instabilities at a single point in time, but a thorough transient analysis is still lacking in the literature. We present the results of a 2 year experimental study at a shallow aquifer in the United Arab Emirates that was designed to specifically explore the transient nature of free convection. ER tomography data documented the presence of convective fingers following a significant rainfall event. We demonstrate that the complex fingering pattern had completely disappeared a year after the rainfall event. The observation is supported by an analysis of the aquifer halite budget and hydrodynamic modeling of the transient character of the fingering instabilities. Modeling results show that the transient dynamics of the gravitational instabilities (their initial development, infiltration into the underlying lower-density groundwater, and subsequent decay) are in agreement with the timing observed in the time-lapse ER measurements. All experimental observations and modeling results are consistent with the hypothesis that a dense brine that infiltrated into the aquifer from a surficial source was the cause of free convection at this site, and that the finite nature of the dense brine source and dispersive mixing led to the decay of instabilities with time. This study highlights the importance of the transience of free convection phenomena and suggests that these processes are more rapid than was previously understood.

Fluid-structure interaction analysis by coupled FE-SPH model based on a novel searching algorithm

Fluid–Structure Interaction (FSI) problem is significant in science and engineering, which leads to challenges for computational mechanics. The coupled model of Finite Element and Smoothed Particle Hydrodynamics (FE-SPH) is a robust technique for simulation of FSI problems. However, two important steps of neighbor searching and contact searching in the coupled FE-SPH model are extremely time-consuming. Point-In-Box (PIB) searching algorithm has been developed by Swegle to improve the efficiency of searching. However, it has a shortcoming that efficiency of searching can be significantly affected by the distribution of points (nodes in FEM and particles in SPH). In this paper, in order to improve the efficiency of searching, a novel Striped-PIB (S-PIB) searching algorithm is proposed to overcome the shortcoming of PIB algorithm that caused by points distribution, and the two time-consuming steps of neighbor searching and contact searching are integrated into one searching step. The accuracy and efficiency of the newly developed searching algorithm is studied on by efficiency test and FSI problems. It has been found that the newly developed model can significantly improve the computational efficiency and it is believed to be a powerful tool for the FSI analysis.

Similarity solutions for flow and heat transfer of non-Newtonian fluid over a stretching surface

Similarity solutions are carried out for flow of power law non-Newtonian fluid film on unsteady stretching surface subjected to constant heat flux. Free convection heat transfer induces thermal boundary layer within a semi-infinite layer of Boussinesq fluid. The nonlinear coupled partial differential equations (PDE) governing the flow and the boundary conditions are converted to a system of ordinary differential equations (ODE) using two-parameter groups. This technique reduces the number of independent variables by two, and finally the obtained ordinary differential equations are solved numerically for the temperature and velocity using the shooting method. The thermal and velocity boundary layers are studied by the means of Prandtl number and non-Newtonian power index plotted in curves.

Myogel, a novel, basement membrane-rich, extracellular matrix derived from skeletal muscle, is highly adipogenic in vivo and in vitro

Background/Aims Biological and synthetic scaffolds play important roles in tissue engineering and are being developed towards human clinical applications. Based on previous work from our laboratory, we propose that extracellular matrices from skeletal muscle could be developed for adipose tissue engineering. Methods Extracellular matrices (Myogels) extracted from skeletal muscle of various species were assessed using biochemical assays including ELISA and Western blotting. Biofunctionality was assessed using an in vitro differentiation assay and a tissue engineering construct model in the rat. Results Myogels were successfully extracted from mice, rats, pigs and humans. Myogels contained significant levels of laminin α4- and α2-subunits and collagen I compared to Matrigel™, which contains laminin 1 (α1β1γ1) and collagen IV. Levels of growth factors such as fibroblast growth factor 2 were significantly higher than Matrigel, vascular endothelial growth factor-A levels were significantly lower and all other growth factors were comparable. Myogels reproducibly stimulated adipogenic differentiation of preadipocytes in vitro and the growth of adipose tissue in the rat. Conclusions We found Myogel induces adipocyte differentiation in vitroand shows strong adipogenic potential in vivo, inducing the growth of well-vascularised adipose tissue. Myogel offers an alternative for current support scaffolds in adipose tissue engineering, allowing the scaling up of animal models towards clinical adipose tissue engineering applications.

Heat transfer analysis of viscous incompressible fluid by combined natural convection and radiation in an open cavity

The effect of radiation on natural convection of Newtonian fluid contained in an open cavity is investigated in this study. The governing partial differential equations are solved numerically using the Alternate Direct Implicit method together with the Successive Over Relaxation method. The study is focused on studying the flow pattern and the convective and radiative heat transfer rates are studied for different values of radiation parameters namely, the optical thickness of the fluid, scattering albedo, and the Planck number. It was found that in the optically thin limit, an increase in the optical thickness of the fluid raises the temperature and radiation heat transfer of the fluid. However, a further increase in the optical thickness decreases the radiative heat transfer rate due to increase in the energy level of the fluid, which ultimately reduces the total heat transfer rate within the fluid.

Myoepithelial molecular markers in human breast carcinoma PMC42-LA cells are induced by extracellular matrix and stromal cells

The microenvironment plays a key role in the cellular differentiation of the two main cell lineages of the human breast, luminal epithelial, and myoepithelial. It is not clear, however, how the components of the microenvironment control the development of these cell lineages. To investigate how lineage development is regulated by 3-D culture and microenvironment components, we used the PMC42-LA human breast carcinoma cell line, which possesses stem cell characteristics. When cultured on a two-dimensional glass substrate, PMC42-LA cells formed a monolayer and expressed predominantly luminal epithelial markers, including cytokeratins 8, 18, and 19; E-cadherin; and sialomucin. The key myoepithelial-specific proteins α-smooth muscle actin and cytokeratin 14 were not expressed. When cultured within Engelbreth-Holm- Swarm sarcoma-derived basement membrane matrix (EHS matrix), PMC42-LA cells formed organoids in which the expression of luminal markers was reduced and the expression of other myoepithelial-specific markers (cytokeratin 17 and P-cadherin) was promoted. The presence of primary human mammary gland fibroblasts within the EHS matrix induced expression of the key myoepithelial-specific markers, α-smooth muscle actin and cytokeratin 14. Immortalized human skin fibroblasts were less effective in inducing expression of these key myoepithelial-specific markers. Confocal dual-labeling showed that individual cells expressed luminal or myoepithelial proteins, but not both. Conditioned medium from the mammary fibroblasts was equally effective in inducing myoepithelial marker expression. The results indicate that the myoepithelial lineage is promoted by the extracellular matrix, in conjunction with products secreted by breast-specific fibroblasts. Our results demonstrate a key role for the breast microenvironment in the regulation of breast lineage development.

Secreted products and extracellular matrix from testicular peritubular myoid cells influence androgen-binding protein secretion by Sertoli cells in culture

Metabolic cooperation mediated by secreted factors between Sertoli cells and peritubular myoid cells has been well documented. We have confirmed that factors secreted by peritubular myoid cells modulate androgen-binding protein (ABP) secretion by Sertoli cells and shown further that this can also be achieved with peritubular myoid cell extracellular matrix (ECM). While peritubular myoid cell ECM potentiated the stimulatory effect of dibutyryl cyclic AMP on Sertoli cell ABP secretion, secreted factors did not, suggesting that the two components influence Sertoli cells through distinct mechanisms. We also tested other factors and other cell lines for effects on ABP production by Sertoli cells. The addition of human plasma fibronectin or conditioned medium from the basement membrane-producing Englebreth-Holm- Swarm sarcoma also stimulated ABP secretion by Sertoli cells. Cocultures of epithelial Sertoli cells with the cells of mesenchymal origin, such as testicular peritubular myoid cells, embryonic skin fibroblasts, and bladder smooth muscle cells, significantly stimulated ABP secretion by Sertoli cells, but co-culture with the epithelial-derived Martin-Darby canine kidney cell line had no effect on Sertoli cell-secreted ABP levels. Our data further define the epithelial-mesenchymal cell interaction that exists between Sertoli cells and peritubular myoid cells in the mammalian testis.

The role of biological extracellular matrix scaffolds in vascularized three-dimensional tissue growth in vivo

An in vivo murine vascularized chamber model has been shown to generate spontaneous angiogenesis and new tissue formation. This experiment aimed to assess the effects of common biological scaffolds on tissue growth in this model. Either laminin-1, type I collagen, fibrin glue, hyaluronan, or sea sponge was inserted into silicone chambers containing the epigastric artery and vein, one end was sealed with adipose tissue and the other with bone wax, then incubated subcutaneously. After 2, 4, or 6 weeks, tissue from chambers containing collagen I, fibrin glue, hyaluronan, or no added scaffold (control) had small amounts of vascularized connective tissue. Chambers containing sea sponge had moderate connective tissue growth together with a mild "foreign body" inflammatory response. Chambers containing laminin-1, at a concentration 10-fold lower than its concentration in Matrigel™, resulted in a moderate adipogenic response. In summary, (1) biological hydrogels are resorbed and gradually replaced by vascularized connective tissue; (2) sponge-like matrices with large pores support connective tissue growth within the pores and become encapsulated with granulation tissue; (3) laminin-containing scaffolds facilitate adipogenesis. It is concluded that the nature and chemical composition of the scaffold exerts a significant influence on the amount and type of tissue generated in this in vivo chamber model.

ST7-mediated suppression of tumorigenicity of prostate cancer cells is characterized by remodeling of the extracellular matrix

Multiple lines of evidence have provided compelling evidence for the existence of a tumor suppressor gene (TSG) on chromosome 7q31.1. ST7 may be the target of this genetic instability but its designation as a TSG is controversial. In this study, we show that, functionally, ST7 behaves as a tumor suppressor in human cancer. ST7 suppressed growth of PC-3 prostate cancer cells inoculated subcutaneously into severe combined immunodeficient mice, and increased the latency of tumor detection from 13 days in control tumors to 23 days. Re-expression of ST7 was also associated with suppression of colony formation under anchorage-independent conditions in MDA-MB-231 breast cancer cells and ST7 mRNA expression was downregulated in 44% of primary breast cancers. Expression profiling of PC-3 cells revealed that ST7 predominantly induces changes in genes involved in re-modeling the extracellular matrix such as SPARC, IGFBP5 and several matrix metalloproteinases. These data indicate that ST7 may mediate tumor suppression through modification of the tumor microenvironment.

The influence of extracellular matrix on the generation of vascularized, engineered, transplantable tissue

In a recently described model for tissue engineering, an arteriovenous loop comprising the femoral artery and vein with interposed vein graft is fabricated in the groin of an adult male rat, placed inside a polycarbonate chamber, and incubated subcutaneously. New vascularized granulation tissue will generate on this loop for up to 12 weeks. In the study described in this paper three different extracellular matrices were investigated for their ability to accelerate the amount of tissue generated compared with a no-matrix control. Poly-D,L-lactic-co-glycolic acid (PLGA) produced the maximal weight of new tissue and vascularization and this peaked at two weeks, but regressed by four weeks. Matrigel was next best. It peaked at four weeks but by eight weeks it also had regressed. Fibrin (20 and 80 mg/ml), by contrast, did not integrate with the generating vascularized tissue and produced less weight and volume of tissue than controls without matrix. The limiting factors to growth appear to be the chamber size and the capacity of the neotissue to integrate with the matrix. Once the sides of the chamber are reached or tissue fails to integrate, encapsulation and regression follow. The intrinsic position of the blood supply within the neotissue has many advantages for tissue and organ engineering, such as ability to seed the construct with stem cells and microsurgically transfer new tissue to another site within the individual. In conclusion, this study has found that PLGA and Matrigel are the best matrices for the rapid growth of new vascularized tissue suitable for replantation or transplantation.

Alternating current electrohydrodynamics induced nanoshearing and fluid micromixing for specific capture of cancer cells

We report a new tuneable alternating current (ac) electrohydrodynamics (ac-EHD) force referred to as “nanoshearing” which involves fluid flow generated within a few nanometers of an electrode surface. This force can be externally tuned via manipulating the applied ac-EHD field strength. The ability to manipulate ac-EHD induced forces and concomitant fluid micromixing can enhance fluid transport within the capture domain of the channel (e.g., transport of analytes and hence increase target–sensor interactions). This also provides a new capability to preferentially select strongly bound analytes over onspecifically bound cells and molecules. To demonstrate the utility and versatility of nanoshearing phenomenon to specifically capture cancer cells, we present proof-of-concept data in lysed blood using two microfluidic devices containing a long array of asymmetric planar electrode pairs. Under the optimal experimental conditions, we achieved high capture efficiency (e.g., approximately 90%; %RSD=2, n=3) with a 10-fold reduction in nonspecific dsorption of non-target cells for the detection of whole cells expressing Human Epidermal Growth Factor Receptor 2 (HER2). We believe that our ac-EHD devices and the use of tuneable nanoshearing phenomenon may find relevance in a wide variety of biological and medical applications.