Hydrostatic pressure promotes migration and filamin-A activation in fibroblasts with increased p38 phosphorylation and TGF-ß production.

Fibroblast migration is closely regulated by the mechanical characteristics in surrounding microenvironment. While increased interstitial hydrostatic pressure (HP) is a hallmark in many pathological and physiological conditions, little is known about how the HP affects fibroblast motility. Using cell-culture chips with elevated HP conditions, we showed that 20 cmH2O HP significantly accelerated fibroblast migration. The HP-induced migration acceleration was dependent on the augmentation of transforming growth factor-ß1, and correlated with the activation of filamin A via the phosphorylation of p38 mitogen-activated protein kinase. Our results suggest that interstitial HP elevation associated with various pathological states could significantly regulate fibroblast migration.

Autophagy in fibroblasts induced by cigarette smoke extract promotes invasion in lung cancer cells.

We investigated the effects of cigarette smoke extract (CSE) on lung fibroblasts and found that the invasiveness of lung cancer cells was facilitated by the conditioned medium from CSE-treated fibroblasts. CSE induced autophagy in fibroblasts and increased the expression of autophagy-related proteins, including optineurin and Ras-related protein Rab1B. Afterward, the fibroblasts produced high levels of interleukin-8 (IL-8), which promoted cancer cell invasion. The inhibition of either optineurin or Rab1B abrogated a rise in microtubule-associated protein 1 light chain 3 ß and a decrease in p62 protein, as well as the production of IL-8, in CSE-treated fibroblasts. A three-dimensional invasion assay using cancer cell spheroids revealed that the invasion of cancer cells alone and the fibroblast-led cancer cell invasion were both enhanced by the conditioned media from CSE-treated fibroblasts. These results suggest that cigarette smoke may induce autophagy and IL-8 secretion in lung fibroblasts and modify the microenvironment to favor invasion of lung cancer cells.

Emerging Roles of Air Gases in Lipid Bilayers.

Recent studies indicate that changing the physical properties of lipid bilayers may profoundly change the function of membrane proteins. Here, the effects of dissolved nitrogen and oxygen molecules on the mechanical properties and stability of lipid bilayers are investigated using differential confocal microscopy, atomic force microscopy, and molecular dynamics simulations. All experiments evidence the presence of dissolved air gas in lipid bilayers prepared without gas control. The lipid bilayers in degassed solutions are softer and less stable than those in ambient solutions. High concentrations of nitrogen increase the bending moduli and stability of the lipid bilayers and impede phase separation in ternary lipid bilayers. The effect of oxygen is less prominent. Molecular dynamics simulations indicate that higher nitrogen affinity accounts for increased rigidity. These findings have fundamental and wide implications for phenomena related to lipid bilayers and cell membranes, including the origin of life.

Heparin co-factor II enhances cell motility and promotes metastasis in non-small cell lung cancer.

Using the Serial Analysis of Gene Expression (SAGE) database from the Cancer Genome Anatomy Project, we identified heparin co-factor II (HCII), which is over-expressed in non-small cell lung cancer (NSCLC). Here, we investigated the clinical significance of HCII and provided molecular evidence to support the suggestion that HCII could enhance cancer metastasis in NSCLC. We found that high HCII expression in tumour tissue was associated with increased cancer recurrence and shorter overall survival times in 75 clinically operable NSCLC patients. High pretreatment plasma concentration of HCII was associated with reduced overall survival in 57 consecutive NSCLC patients. We over-expressed and knocked down HCII expression in lung cancer cell lines and confirmed that HCII could promote cell motility, invasion ability and filopodium dynamics in NSCLC cells in vitro and increased metastatic colonization in an in vivo mouse model. Exogenous treatment of HCII promoted cancer cell migration, and this promigratory effect of HCII was independent of thrombin. We further showed that HCII could up-regulate cancer cell migration through the activation of PI3K, which acts upstream of Rac1 and Cdc42, and this effect could be blocked by heparin. We suggest that HCII is a novel metastasis enhancer and may be used as a prognostic predictor for heparin treatment in NSCLC.

Membrane roughness as a sensitive parameter reflecting the status of neuronal cells in response to chemical and nanoparticle treatments.

BACKGROUND: Cell membranes exhibit abundant types of responses to external stimulations. Intuitively, membrane topography should be sensitive to changes of physical or chemical factors in the microenvironment. We employed the non-interferometric wide-field optical profilometry (NIWOP) technique to quantify the membrane roughness of living neuroblastoma cells under various treatments that could change the mechanical properties of the cells. RESULTS: The membrane roughness was reduced as the neuroblastoma cell was treated with paclitaxel, which increases cellular stiffness by translocating microtubules toward the cell membranes. The treatment of positively charged gold nanoparticles (AuNPs) showed a similar effect. In contrast, the negatively charged AuNPs did not cause significant changes of the membrane roughness. We also checked the membrane roughness of fixed cells by using scanning electron microscopy (SEM) and confirmed that the membrane roughness could be regarded as a parameter reflecting cellular mechanical properties. Finally, we monitored the temporal variations of the membrane roughness under the treatment with a hypertonic solution (75 mM sucrose in the culture medium). The membrane roughness was increased within 1 h but returned to the original level after 2 h. CONCLUSIONS: The results in the present study suggest that the optical measurement on membrane roughness can be regarded as a label-free method to monitor the changes in cell mechanical properties or binding properties of nanoparticles on cell surface. Because the cells were left untouched during the measurement, further tests about cell viability or drug efficacy can be done on the same specimen. Membrane roughness could thus provide a quick screening for new chemical or physical treatments on neuronal cells.

Neurite growth induced by red light-caused intracellular reactive oxygen species production through cytochrome c oxidase activation.

The applications of red-light photobiomodulation (PBM) to enhance neurite growth have been proposed for many years. However, the detailed mechanisms require further studies. In the present work we used a focused red-light spot to illuminate the junction of the longest neurite and the soma of a neuroblastoma cell (N2a), and demonstrated enhanced neurite growth at 620 nm and 760 nm with adequate illumination energy fluences. In contrast, 680 nm light showed no effect on neurite growth. The neurite growth was accompanied with the increase of intracellular reactive oxygen species (ROS). Using Trolox to reduce the ROS level, this red light-induced neurite growth was hindered. Suppressing the activities of cytochrome c oxidase (CCO) by using either a small-molecule inhibitor or siRNA abrogated the red light-induced neurite growth. These results suggest that red light-induced ROS production through the activation of CCO could be beneficial for neurite growth.

A 3D culture system for evaluating the combined effects of cisplatin and anti-fibrotic drugs on the growth and invasion of lung cancer cells co-cultured with fibroblasts.

Fibrosis and fibroblast activation usually occur in the tissues surrounding a malignant tumor; therefore, anti-fibrotic drugs are used in addition to chemotherapy. A reliable technique for evaluating the combined effects of anti-fibrotic drugs and anticancer drugs would be beneficial for the development of an appropriate treatment strategy. In this study, we manufactured a three-dimensional (3D) co-culture system of fibroblasts and lung cancer cell spheroids in Matrigel supplemented with fibrin (fibrin/Matrigel) that simulated the tissue microenvironment around a solid tumor. We compared the efficacy of an anticancer drug (cisplatin) with or without pretreatments of two anti-fibrotic drugs, nintedanib and pirfenidone, on the growth and invasion of cancer cells co-cultured with fibroblasts. The results showed that the addition of nintedanib improved cisplatin's effects on suppressing the growth of cancer cell spheroids and the invasion of cancer cells. In contrast, pirfenidone did not enhance the anticancer activity of cisplatin. Nintedanib also showed higher efficacy than pirfenidone in reducing the expression of four genes in fibroblasts associated with cell adhesion, invasion, and extracellular matrix degradation. This study demonstrated that the 3D co-cultures in fibrin/Matrigel would be useful for assessing the effects of drug combinations on tumor growth and invasion.

Effects of the media conditioned by various macrophage subtypes derived from THP-1 cells on tunneling nanotube formation in pancreatic cancer cells.

BACKGROUND: Tunneling nanotubes (TNTs) are special membrane structures for intercellular communications. Vital cargoes (such as mitochondria) could be delivered from healthy cells to rescue damaged ones through TNTs. The TNTs could be utilized for the purpose of systematic delivery of therapeutic agents between cells. However, there are insufficient studies on the controlled enhancement of TNT formations. The purpose of this study is to understand how macrophages influence the TNT formation in cancer cells. RESULTS: Here we compared the capabilities of inducing TNTs in human pancreatic cancer cells (PANC-1) of the media conditioned by M0, M1 and M2 macrophages derived from THP-1 cells. The M0 and M1 macrophage conditioned media promoted TNT formation. Using a focused ion beam to cut through a TNT, we observed tunnel-like structures inside dense cytoskeletons with scanning electron microscopy. The TNT formation correlated with raised motility, invasion, and epithelial-mesenchymal transition in the PANC-1 cells. Mitochondria and lysosomes were also found to be transported in the TNTs. CONCLUSIONS: These results suggest that TNT formation could be one of the responses to the immune stress in pancreatic cancer cells caused by M0 and M1 macrophages. This finding is valuable for the development of macrophage-targeting cancer therapy.

Measurement of membrane rigidity on trapped unilamellar phospholipid vesicles by using differential confocal microscopy.

We use optical tweezers to trap a unilamellar phospholipid vesicle and measure the out-of-plane thermal fluctuations by using differential confocal microscopy. Bending moduli of the lipid membranes are calculated directly from the mean-square values of the fluctuation amplitudes. Owing to the refractive index contrast between the inner and outer solutions of the vesicle, optical tweezers trap the vesicle laterally and improve the reliability of the measured fluctuation amplitudes along the optical axis. Bending moduli of membranes in gel or fluid phases obtained by the combination of differential confocal microscopy and optical tweezers are close to those reported previously. We also obtain the bending modulus of sphingomyelin membranes in the gel phase, which was not reported previously.

Label-free quantification of asymmetric cancer-cell filopodium activities in a multi-gradient chip.

We use novel super-resolution bright-field optical microscopy to observe the filopodium activities of human lung cancer cells in a multi-gradient cell culture chip. Temporal variations of the filopodium numbers are measured without fluorescent labelling. By carefully designing the fluidic field inside the culture chip, we establish stable concentration gradients of the injected reagents. The reagents are injected via a separated central inlet, and the concentration gradients are different at different positions in the chip. The same chip can be used for both control and treated experiments. Using epidermal growth factor as the treatment, we verify that the protrusions of filopodia indicate the direction of concentration gradients experienced by a living cancer cell; while the treatment of bovine serum albumin shows no specific effect on the growth of filopodia. The combination of label-free, high-resolution optical microscopy and a micro cell culture chip establishes a convenient and versatile platform for dynamical cancer-cell analyses.

Wide-field optical nanoprofilometry using structured illumination.

We combine the differential height measurement concept with structured illumination microscopy to develop wide-field optical nanoprofilometry. Sub-diffraction-limit lateral resolution and axially sectioning imaging are achieved with structured illumination using a liquid-crystal spatial light modulator. As the sample surface is placed into the linear region of the sectioning axial response curve, the signal change owing to topographic variations provides nanometer depth sensitivity. The lateral resolution and the depth profiling accuracy are about 0.3 wavelengths and 6 nm, respectively. Depth profiling on solid-state specimens and label-free superresolution imaging of living cells are demonstrated.

Three-dimensional characterization of active membrane waves on living cells.

We measure the temporal evolution of three-dimensional membrane topography on living fibroblasts and characterize the propagation of membrane waves using a wide-field optical profiling technique. The measured membrane profiles are compared with the numerical results calculated by the active membrane model recently proposed by Shlomovitz and Gov. After the treatments of blebbistatin and latrunculin A separately, the membrane waves disappear and the membrane surfaces are flattened, verifying that the membrane waves are driven by the interactions between myosin II and actin polymerization.

Neurite regrowth stimulation by a red-light spot focused on the neuronal cell soma following blue light-induced retraction.

The interaction of light with biological tissues has been considered for various therapeutic applications. Light-induced neurite growth has the potential to be a clinically useful technique for neuron repair. However, most previous studies used either a large illumination area to accelerate overall neurite growth or employed a light spot to guide a growing neurite. It is not clear if optical stimulation can induce the regrowth of a retracted neurite. In the present work, we used blue light (wavelength: 473 nm) to cause neurite retraction, and we proved that using a red-light (wavelength: 650 nm) spot to illuminate the soma near the junction of the retracted neurite could induce neurite regrowth. As a comparison, we found that green light (wavelength 550 nm) had a 62% probability of inducing neurite regrowth, while red light had a 75% probability of inducing neurite regrowth at the same power level. Furthermore, the neurite regrowth length induced by red light was increased by the pre-treatment with inhibitors of myosin functions. We also observed actin propagation from the soma to the tip of the re-growing neurite following red-light stimulation of the soma. The red light-induced extension and regrowth were abrogated in the calcium-free medium. These results suggest that illumination with a red-light spot on the soma may trigger the regrowth of a neurite after the retraction caused by blue-light illumination.

Dynamics of cancer cell filopodia characterized by super-resolution bright-field optical microscopy.

We explore the dynamics of cancer cell filopodia of diameters around 200 nm by using super-resolution bright-field optical microscopy. The high contrast required by the super-resolution image-restoration process is from the nanometer topographic sensitivity of non-interferometric widefield optical profilometry, rather than fluorescence labeling. Because the image-acquisition rate of this bright-field system is 20 frames/min, fast cellular dynamics can be captured and then analyzed. We successfully observe the growth and activities of the filopodia of a CL1-0 lung cancer cell. In the culturing condition, we measure that the filopodia exhibit an average elongation rate of 90 nm/sec, and an average shrinkage rate of 75 nm/sec. With the treatment of epidermal growth factor, the elongation and shrinkage rates increase to 110 nm/sec and 100 nm/sec respectively. We also find that the treatment of epidermal growth factor raises the number of filopodia by nearly a factor of 2, which implies enhancement of cell motility.

Substrate properties modulate cell membrane roughness by way of actin filaments.

Cell membrane roughness has been proposed as a sensitive feature to reflect cellular physiological conditions. In order to know whether membrane roughness is associated with the substrate properties, we employed the non-interferometric wide-field optical profilometry (NIWOP) technique to measure the membrane roughness of living mouse embryonic fibroblasts with different conditions of the culture substrate. By controlling the surface density of fibronectin (FN) coated on the substrate, we found that cells exhibited higher membrane roughness as the FN density increased in company with larger focal adhesion (FA) sizes. The examination of membrane roughness was also confirmed with atomic force microscopy. Using reagents altering actin or microtubule cytoskeletons, we provided evidence that the dynamics of actin filaments rather than that of microtubules plays a crucial role for the regulation of membrane roughness. By changing the substrate rigidity, we further demonstrated that the cells seeded on compliant gels exhibited significantly lower membrane roughness and smaller FAs than the cells on rigid substrate. Taken together, our data suggest that the magnitude of membrane roughness is modulated by way of actin dynamics in cells responding to substrate properties.

The Affinity of Elongated Membrane-Tethered Ligands Determines Potency of T Cell Receptor Triggering.

T lymphocytes are important mediators of adoptive immunity but the mechanism of T cell receptor (TCR) triggering remains uncertain. The interspatial distance between engaged T cells and antigen-presenting cells (APCs) is believed to be important for topological rearrangement of membrane tyrosine phosphatases and initiation of TCR signaling. We investigated the relationship between ligand topology and affinity by generating a series of artificial APCs that express membrane-tethered anti-CD3 scFv with different affinities (OKT3, BC3, and 2C11) in addition to recombinant class I and II pMHC molecules. The dimensions of membrane-tethered anti-CD3 and pMHC molecules were progressively increased by insertion of different extracellular domains. In agreement with previous studies, elongation of pMHC molecules or low-affinity anti-CD3 scFv caused progressive loss of T cell activation. However, elongation of high-affinity ligands (BC3 and OKT3 scFv) did not abolish TCR phosphorylation and T cell activation. Mutation of key amino acids in OKT3 to reduce binding affinity to CD3 resulted in restoration of topological dependence on T cell activation. Our results show that high-affinity TCR ligands can effectively induce TCR triggering even at large interspatial distances between T cells and APCs.

Imaging live cells at high spatiotemporal resolution for lab-on-a-chip applications.

Conventional optical imaging techniques are limited by the diffraction limit and difficult-to-image biomolecular and sub-cellular processes in living specimens. Novel optical imaging techniques are constantly evolving with the desire to innovate an imaging tool that is capable of seeing sub-cellular processes in a biological system, especially in three dimensions (3D) over time, i.e. 4D imaging. For fluorescence imaging on live cells, the trade-offs among imaging depth, spatial resolution, temporal resolution and photo-damage are constrained based on the limited photons of the emitters. The fundamental solution to solve this dilemma is to enlarge the photon bank such as the development of photostable and bright fluorophores, leading to the innovation in optical imaging techniques such as super-resolution microscopy and light sheet microscopy. With the synergy of microfluidic technology that is capable of manipulating biological cells and controlling their microenvironments to mimic in vivo physiological environments, studies of sub-cellular processes in various biological systems can be simplified and investigated systematically. In this review, we provide an overview of current state-of-the-art super-resolution and 3D live cell imaging techniques and their lab-on-a-chip applications, and finally discuss future research trends in new and breakthrough research areas of live specimen 4D imaging in controlled 3D microenvironments.

Drug testing and flow cytometry analysis on a large number of uniform sized tumor spheroids using a microfluidic device.

Three-dimensional (3D) tumor spheroid possesses great potential as an in vitro model to improve predictive capacity for pre-clinical drug testing. In this paper, we combine advantages of flow cytometry and microfluidics to perform drug testing and analysis on a large number (5000) of uniform sized tumor spheroids. The spheroids are formed, cultured, and treated with drugs inside a microfluidic device. The spheroids can then be harvested from the device without tedious operation. Due to the ample cell numbers, the spheroids can be dissociated into single cells for flow cytometry analysis. Flow cytometry provides statistical information in single cell resolution that makes it feasible to better investigate drug functions on the cells in more in vivo-like 3D formation. In the experiments, human hepatocellular carcinoma cells (HepG2) are exploited to form tumor spheroids within the microfluidic device, and three anti-cancer drugs: Cisplatin, Resveratrol, and Tirapazamine (TPZ), and their combinations are tested on the tumor spheroids with two different sizes. The experimental results suggest the cell culture format (2D monolayer vs. 3D spheroid) and spheroid size play critical roles in drug responses, and also demonstrate the advantages of bridging the two techniques in pharmaceutical drug screening applications.

Membrane ripples of a living cell measured by non-interferometric widefield optical profilometry.

We measured the membrane topography and dynamics on a living fibroblast by using the non-interferometric widefield optical profilometry (NIWOP) technique. With a water-immersion objective of a 0.75 numerical aperture, our NIWOP system provides depth resolution about 20 nm. The imaging speed could be as high as 5 frames/min. We directly observed and profiled the inward propagation of membrane ripples near the cell edge. To verify if the membrane activity was driven by the underlying cytoskeleton, we changed the structure of the cell cortex while observing the membrane topography. After dissolving the actin cortex by cytochalasin D, we found that the propagation of the membrane ripples disappeared and the edge of the cell shank. The non-contact NIWOP technique does not affect the motility and viability of cells and therefore is suitable for the studies on cell physiology related to membrane motions.

Directional migration of cancer cells induced by a blue light intensity gradient.

We used a spatial light modulator to project an optical micropattern of 473 nm light with a quartic intensity gradient on a single lung cancer cell. We observed that the intracellular amounts of reactive oxygen species (ROS) of the cancer cells were proportional to the intensity of the blue light, and the blue light intensity gradients could drive directional cell migration. This optically induced directional cell migration was inhibited by a ROS scavenger in the culture medium in a dose-dependent manner. In contrast, the ROS levels in fibroblasts were saturated by the blue light at low intensity and therefore the fibroblasts did not exhibit directional migration in the intensity gradient.