Estimation of cancer cell migration in biomimetic random/oriented collagen fiber microenvironments

Increasing data indicate that cancer cell migration is regulated by extracellular matrixes and their surrounding biochemical microenvironment,playing a crucial role in pathological processes such as tumor invasion and metastasis.However,conventional two-dimensional cell culture and animal models have limitations in studying the influence of tumor microenvironment on cancer cell migration.Fortunately,the further development of microfluidic technology has provided solutions for the study of such questions.We utilize microfluidic chip to build a random collagen fiber microenvironment(RFM)model and an oriented collagen fiber microenvironment(OFM)model that resemble early stage and late stage breast cancer microenvironments,respectively.By combining cell culture,biochemical concentration gradient construction,and microscopic imaging techniques,we investigate the impact of different collagen fiber biochemical microenvironments on the migration of breast cancer MDA-MB-231-RFP cells.The results show that MDA-MB-231-RFP cells migrate further in the OFM model compared to the RFM model,with significant differences observed.Furthermore,we establish concentration gradients of the anticancer drug paclitaxel in both the RFM and OFM models and find that paclitaxel significantly inhibits the migration of MDA-MB-231-RFP cells in the RFM model,with stronger inhibition on the high concentration side compared to the low concentration side.However,the inhibitory effect of paclitaxel on the migration of MDA-MB-231-RFP cells in the OFM model is weak.These findings suggest that the oriented collagen fiber microenvironment resembling the late-stage tumor microenvironment is more favorable for cancer cell migration and that the effectiveness of anticancer drugs is diminished.The RFM and OFM models constructed in this study not only provide a platform for studying the mechanism of cancer development,but also serve as a tool for the initial measurement of drug screening.

IL-17 induces NSCLC cell migration and invasion by elevating MMP19 gene transcription and expression through the interaction of p300-dependent STAT3-K631 acetylation and its Y705-phosphorylation

The cancer cell metastasis is a major death reason for patients with non-small cell lung cancer(NSCLC).Although researchers have disclosed that interleukin 17(IL-17)can increase matrix metalloproteinases(MMPs)induction causing NSCLC cell metastasis,the underlying mechanism remains unclear.In the study,we found that IL-17 receptor A(IL-17RA),p300,p-STAT3,Ack-STAT3,and MMP19 were up-regulated both in NSCLC tissues and NSCLC cells stimulated with IL-17.p300,STAT3 and MMP19 overexpression or knockdown could raise or reduce IL-17-induced p-STAT3,Ack-STAT3 and MMP19 level as well as the cell migration and invasion.Mechanism investigation revealed that STAT3 and p300 bound to the same region(−544 to−389 nt)of MMP19 promoter,and p300 could acetylate STAT3-K631 elevating STAT3 transcriptional activity,p-STAT3 or MMP19 expression and the cell mobility exposed to IL-17.Meanwhile,p300-mediated STAT3-K631 acetylation and its Y705-phosphorylation could interact,synergistically facilitating MMP19 gene transcription and enhancing cell migration and invasion.Besides,the animal experiments exhibited that the nude mice inoculated with NSCLC cells by silencing p300,STAT3 or MMP19 gene plus IL-17 treatment,the nodule number,and MMP19,Ack-STAT3,or p-STAT3 production in the lung metastatic nodules were all alleviated.Collectively,these outcomes uncover that IL-17-triggered NSCLC metastasis involves up-regulating MMP19 expression via the interaction of STAT3-K631 acetylation by p300 and its Y705-phosphorylation,which provides a new mechanistic insight and potential strategy for NSCLC metastasis and therapy.

Runx2 regulates peripheral nerve regeneration to promote Schwann cell migration and re-myelination

Runx2 is a major regulator of osteoblast differentiation and function;however,the role of Runx2 in peripheral nerve repair is unclea r.Here,we analyzed Runx2expression following injury and found that it was specifically up-regulated in Schwann cells.Furthermore,using Schwann cell-specific Runx2 knocko ut mice,we studied peripheral nerve development and regeneration and found that multiple steps in the regeneration process following sciatic nerve injury were Runx2-dependent.Changes observed in Runx2 knoc kout mice include increased prolife ration of Schwann cells,impaired Schwann cell migration and axonal regrowth,reduced re-myelination of axo ns,and a block in macrophage clearance in the late stage of regeneration.Taken together,our findings indicate that Runx2 is a key regulator of Schwann cell plasticity,and therefore peripheral nerve repair.Thus,our study shows that Runx2 plays a major role in Schwann cell migration,re-myelination,and peripheral nerve functional recovery following injury.

VISUALIZATION OF DYNAMIC ORGANIZATION OF CYTOSKELETON GELS IN LIVING CELLS BY HYBRID-SPM

We succeeded in performing of hybrid Scanning Probe Microscopy (hybrid-SPM) in which mechanical-SPM andfluorescence microscopy are combined. This technique is able to measure simultaneously mechanical properties anddistribution of cytoskeletons of living cells by using green fluorescent protein. We measured evolution of both local elasticityand distributions of actin stress fibers in an identical fibroblast living in physiological conditions. The SPM experimentsrevealed that stiffer lines develop in living cells, which correspond to actin stress fibers. The elasticity of the actin stressfibers is as high as 100 kPa. We discuss mechanical effects on the development of actin filament networks.

Cantharidin suppresses HCT116 colorectal carcinoma cell proliferation and migration by changing the cytoskeleton structure

Background:Cantharidin (CTD),a natural toxin produced from Chinese blister beetles,has extensive anti-tumor activity.The present study investigated the effect of CTD on a human colon cancer cell line to elucidate potential new insights regarding the mechanism(s) through which CTD exerts its anti-tumor effects.Materials and methods:The inhibitory effect of CTD on human colon cancer HCT116 cells was evaluated using the IncuCyte ZOOMTM analyzer.Apoptotic cells were detected by Annexin V-FITC/PI assay and cell cycle was evaluated with flow cytometry following propidium iodide staining.Alterations in F-actin microfilaments were analyzed by FITC-phalloidin staining and morphological changes were evaluated with a laser scanning confocal microscope.Cell migration assay was carried out to investigate the effects of CTD on migration of HCT116 cells in vitro.Results:CTD exhibited a significant growth inhibitory effect on HCT116 cells accompanied by an increase in G2/M phase cells,without a significant effect on apoptosis.CTD-treated cells also exhibited a dramatic collapse in their microfilament network and a significant reduction in cell adhesion.Conclusion:CTD inhibits growth by increasing G2/M phase cells and decreasing S phase cells,revealing that CTD exerts a significant growth inhibitory effect primarily through an inhibition of cell cycle progression (a cytostatic effect).Moreover,a negative effect on cell migration may also constitute a contributing factor to its anti-tumor potential.These findings suggest the potential use for developing CTD as a novel anti-cancer therapy that targets metastasis Giving full play to CTD may inhibit tumor transfer.

Experimental Study of Cell Migration and Functional Differentiation of Transplanted Neural Stem Cells Co-labeled with Superparamagnetic Iron Oxide and Brdu in an Ischemic Rat Model

Objective To explore the migration of transplanted neural stem cells co-labeled with superparamagnetic iron oxide （SPIO） and bromodeoxyuridine （Brdu） using the 4.7T MR system and to study the cell differentiation with immuno-histochemical method in ischemic rats. Methods Rat neural stem cells （NSCs） co-labelled with SPIO mediated by poly-L-lysine and bromodeoxyuridine （BrdU） were transplanted into the unaffected side of rat brain with middle cerebral artery occlusion （MCAO）. At weeks 1, 2, 3, 4, 5, and 6 after MCAO, migration of the labelled cells was monitored by MRI. At week 6 the rats were killed and their brain tissue was cut according to the migration site of transplanted ceils indicated by MRI and subjected to Prussian blue staining and immunohistochemical staining to observe the migration and differentiation of the transplanted NSCs. Results Three weeks after transplantation, the linear hypointensity area derived from the migration of labelled NSCs was observed by MRI in the corpus callosum adjacent to the injection site. Six weeks after the transplantation, the linear hypointensity area was moved toward the midline along the corpus callosum. MRI findings were confirmed by Prussian blue staining and immunohistochemical staining of the specimen at week 6 after the transplantation. Flourescence co-labelled immunohistochemical methods demonstrated that the transplanted NSCs could differentiate into astrocytes and neurons. Conclusion MRI can monitor the migration of SPIO-labelled NSCs after transplantation in a dynamical and non-invasive manner. NSCs transplanted into ischemic rats can differentiate into astrocytes and neurons during the process of migration.

Migratory properties of cultured olfactory ensheathing cells by single-cell migration assay

Olfactory ensheathing cells （OECs） are a unique type of glial cells that have axonal growth-promoting properties. OEC transplantation has emerged as a promising experimental therapy of axonal injuries and demyelinating diseases. However, some fundamental cellular properties of OECs remain unclear. In this study, we found that the distinct OEC subpopulations exhibited different migratory properties based on time-lapse imaging of single isolated cells, possibly due to their different cytoskeletal organizations. Moreover, OEC subpopulations displayed different attractive migratory responses to a gradient of lysophosphatidic acid （LPA） in single-cell migration assays. Finally, we found that OEC subpopulations transformed into each other spontaneously. Together, these results demonstrate, for the first time to our knowledge, that distinct OEC subpopulations display different migratory properties in vitro and provide new evidence to support the notion of OECs as a single cell type with malleable functional phenotypes.

TGF-β1-promoted epithelial-to-mesenchymal transformation and cell adhesion contribute to TGF-β1-enhanced cell migration in SMMC-7721 cells

Transforming growth factor-b 1 (TGF-β1), a multi-function polypeptide, is a double-edged sword in cancer. For some tumor cells, TGF-β1 is a potent growth inhibitor and apoptosis inducer. More commonly, TGF-β1 loses its growth-inhibitory and apoptosis-inducing effects, but stimulates the metastatic capacity of tumor cells. It is currently little known about TGF-β1-promoted cell migration in hepatocellular carcinoma (HCC) cells, let alone its mechanism. In this study, we found that TGF-β1 lost its tumor-suppressive effects, but significantly stimulated cell migration in SMMC-7721 human HCC cells. By FACS and Western blot analysis, we observed that TGF-β1 enhanced the expression of α5β1 integrin obviously, and subsequently stimulated cell adhesion onto fibronectin (Fn). Furthermore, we observed that TGF-β1 could also promote SMMC-7721 cells adhesion onto laminin (Ln). Our data also provided evidences that TGF-β1 induced epithelial-to-mesenchymal transformation (EMT) in SMMC-7721 cells. First, SMMC-7721 cells clearly switched to the spindle shape morphology after TGF-β1 treatment. Furthermore, TGF-β1 induced the down-regulation of E-cadherin and the nuclear translocation of β-catenin. These results indicated that TGF-β1-promoted cell adhesion and TGF-β1-induced epithelial-to-mesenchymal transformation might be both responsible for TGF-β1-enhanced cell migration.

Claudin 1 mediates tumor necrosis factor alpha-induced cell migration in human gastric cancer cells

AIM: To investigate the role of claudin 1 in the regulation of genes involved in cell migration and tumor necrosis factor alpha (TNF-α)-induced gene expression in human gastric adenocarcinoma cells.

Exploring the interconnectivity of biomimetic hierarchical porous Mg scaffolds for bone tissue engineering:Effects of pore size distribution on mechanical properties,degradation behavior and cell migration ability

Interconnectivity is the key characteristic of bone tissue engineering scaffold modulating cell migration,blood vessels invasion and transport of nutrient and waste.However,efforts and understanding of the interconnectivity of porous Mg is limited due to the diverse architectures of pore struts and pore size distribution of Mg scaffold systems.In this work,biomimetic hierarchical porous Mg scaffolds with tailored interconnectivity as well as pore size distribution were prepared by template replication of infiltration casting.Mg scaffold with better interconnectivity showed lower mechanical strength.Enlarging interconnected pores would enhance the interconnectivity of the whole scaffold and reduce the change of ion concentration,pH value and osmolality of the degradation microenvironment due to the lower specific surface area.Nevertheless,the degradation rates of five tested Mg scaffolds were no different because of the same geometry of strut unit.Direct cell culture and evaluation of cell density at both sides of four typical Mg scaffolds indicated that cell migration through hierarchical porous Mg scaffolds could be enhanced by not only bigger interconnected pore size but also larger main pore size.In summary,design of interconnectivity in terms of pore size distribution could regulate mechanical strength,microenvironment in cell culture condition and cell migration potential,and beyond that it shows great potential for personalized therapy which could facilitate the regeneration process.

Aquaporin 1 Facilitated Hepatocellular Carcinoma SMMC7221 Cell Migration Associated with Water Permeability

The authors investigated the regulation of human aquaporin I（hAQP1） and the involvement of aquaporin 1 （AQP 1） in the migration of human hepatocellular carcinoma SMMC-7221 cells using RNA intereference technology Firstly, two short hairpin RNA（shRNA） constructs in PBSU6 vector were reconstructed and their knockdown effects were identified in SMMC-7221 cells. Next, the involvement of endogenous hAQP1 in regulating the migration of SMMC-7221 cells was investigated via siRNA technology. HAQPI-shRNA can specifically inhibit AQP1 dependent osmotic water permeability. Meanwhile the migration of SMMC-7221 cells was inhibited remarkably after silencing AQP1 by performing transwell cell migration assay and in vitro wound healing assay. Furthermore, in the presence of an inhibitor HgCl2, the water permeability of the cell membrane was remarkably decreased, the expression of AQP1 was upregulated after HgCla treatment and the cell movement was decreased at the moment. Increased AQP1 cannot attenuate cell migration ability when cell membrane loses its water permeability function. This demonstrates that the cell migration was remarkably related to the transporting water function of cell membrane.

Effects of polar cortical cytoskeleton and unbalanced cortical surface tension on intercellular bridge thinning during cytokinesis

To probe the contributions of polar cortical cytoskeleton and the surface tension of daughter cells to intercellular bridge thinning dynamics during cytokinesis,we applied cytochalasin D（CD） or colchicine（COLC） in a highly localized manner to polar regions of dividing normal rat kidney（NRK） cells.We observed cellular morphological changes and analyzed the intercellular bridge thinning trajectories of dividing cells with different polar cortical characteristics.Global blebbistatin（BS） application was used to obtain cells losing active contractile force groups.Our results show that locally released CD or colchicine at the polar region caused inhibition of cytokinesis before ingression.Similar treatment at phases after ingression allowed completion of cytokinesis but dramatically influenced the trajectories of intercellular bridge thinning.Disturbing single polar cortical actin induced transformation of the intercellular bridge thinning process,and polar cortical tension controlled deformation time of intercellular bridges.Our study provides a feasible framework to induce and analyze the effects of local changes in mechanical properties of cellular components on single cellular cytokinesis.

Guided cell migration on a graded micropillar substrate

Cell migration is facilitated by the interaction of living cells and their local microenvironment.The local topography is one of the key factors regulating cell migration.Interaction between the surface topography and the cell behaviors is critical to understanding tissue development and regeneration.In this study,a dynamic mask photolithography technique has been utilized to fabricate a surface with graded micropillars.It has been demonstrated that the cells have been successfully guided to migrate from the sparse zone to the dense zone.The cell polarization angle has been characterized in both sparse zone and the dense zone.Compared to the dense zone,the cells in the sparse zone are more aligned along the direction of the micropillar spacing gradient,which enables the guided cell migration.Moreover,the effects of the micropillar spacing gradient,micropillar diameter,and micropillar height have been investigated in terms of the cell migration speed and cell spreading area.Finally,two issues significantly affecting the cell migration have been discussed:trapped cells between the micropillars and cell clusters.

Computational Study of Collective Cell Migration By Meshfree Method

The collective cell migration behavior on a substrate was studied using RKPM meshfree method.The cells were modeled as nematic liquid crystal with hyperelastic cell nucleus.The cell-substrate and cell-cell interactions were modeled by coarse-grained potential forces.Through this study,the pulling and pushing phenomenon during collective cell migration process was observed and it was found that the individual cell mobility significantly influenced the collective cell migratory behavior.More self-propelled cells are in the system along the same direction,the faster the collective group migrates toward coordinated direction.The parametric study on cell-cell adhesion strength indicated that as the adhesion strength increases,the collective cell migration speed increases.It also showed that the mechanical stress in leader cell is higher than stress in follower cells.

Targeting of RhoE inhibits epithelial-mesenchymal transition during colorectal cancer cell migration

Objective Despite microRNA （miR-200b） being proved to promote the proliferation of colorectal cancer （CRC） cells, the relationship between miR-200b and epithelial-mesenchymal transition （EMT） of CRC cells remains poorly understood. The aim of the study was to investigate the relationship between miR-200b and EMT during CRC cell migration. Methods The effect of miR-200b on EMT-associated markers E-cadherin and vimentin was evaluated by western blot in CRC cells （SW620 and HT-29） by treatment with miR-200b mimics and inhibitors. A lucifer- ase reporter assay was employed to detect downstream targets of miR-200b. Transwell migration assays were used to detect CRC cell migration. Results Westem blots revealed that treatment with miR-200b mimics led to up-regulation of E-cadherin and down-regulation of vimentin, metalloproteinase （MMP）-9, and MMP-2, whereas treatment with miR- 200b inhibitor exhibited opposite effects on expression of E-cadherin and vimentin. Luciferase reporter assays demonstrated that RhoE （RND3） was targeted by miR-200b. Two predicted target sites of miR-200b were present in the 3＇-UTR of RhoE. Predicted target site 1 was from nucleotides 1584 to 1591, and site 2 was from nucleotides 1729 to 1735. RhoE knockdown cell lines were also established to investigate the impact of RhoE and miR-200b on EMT and cell migration. RhoE knockdown enhanced the effect of miR- 200b mimics, up-regulating E-cadherin and down-regulating vimentin. RhoE knockdown also inhibited cell migration. Furthermore, miR-200b mimic treatment further promoted the inhibitory effect of RhoE knock- down on cell migration.

Effects of dexamethasone and HA1077 on actin cytoskeleton and β-catenin in cultured human trabecular meshwork cells

AIM：To investigate the effects of dexamethasone（DEX） and 1-（5-isoquinolinesulfonyl）-homopiperazine（HA1077） on actin cytoskeleton and β-catenin in cultured human trabecular meshwork（HTM） cells.METHODS： The HTM cells were separated from human eyeball and cultured in vitro.They were divided into control group,DEX（1×10^-6mol/L） group,HA1077（3×10^-5mol/L）group,and DEX（1×10^-6mol/L） and HA1077（3×10^-5mol/L）group.Actin cytoskeleton and β-catenin in HTM cells of the four groups were examined by immunofluorescence and Western blot analyses.RESULTS： In DEX group,there were reorganization of actin cytoskeleton and formation of cross linked actin networks（CLANs）,which were partially reversed in DEX and HA1077 group.DEX treatment also induced an increased expression of β-catenin,which was obviously reduced in DEX and HA1077 group.Meanwhile,the cultured HTM cells in HA1077 group had lower expression of β-catenin than that in the control group. CONCLUSION： Our results show that HA1077 can reverse the changes of actin organization and expression of β-catenin induced by DEX in cultured HTM cells,suggesting that HA1077 may play an important role in increasing outflow and reducing intraocular pressure.

Computational Investigation of Cell Migration Behavior in a Confluent Epithelial Monolayer

Cell migration plays a significant role in many biological activities,yet the physical mechanisms of cell migration are still not well understood.In this study,a continuum physics-based epithelial monolayer model including the intercellular interaction was employed to study the cell migration behavior in a confluent epithelial monolayer at constant cell density.The epithelial cell was modeled as isotropic elastic material.Through finite element simulation,the results revealed that themotile cellwas subjected to higher stress than the other jammed cells during the migration process.Cell stiffness was implied to play a significant role in epithelial cell migration behavior.Higher stiffness results in smaller displacement and lower migration speed.

LIM1863 is useful to explore collective cancer cell migration,and the group of heterogeneous cells undergoing collective migration behaves like a supracellular unit

Collective cancer cell migration(CCCM)and epithelial-to-mesenchymal transition(EMT)play key roles in metastasis.This study reports that the colorectal carcinoma cell line LIM1863 is useful for the study of CCCM and EMT.Methods:Hematoxylin and eosin staining,scanning electron microscopy,transmission electron microscopy,and western blot analysis were performed.Results:LIM1863 automatically grew as spheroids in suspension and had important typical epithelial properties,including several layers of cells arranged around a central lumen,apical-basal polarity,and types of cell-cell junctions.Treatment with a combination of both TGF beta 1 and TNF alpha induced definite and distinct EMT,a spheroid changing phenotype to form a monolayer high-confluent patch without lumen,without polarity.Spontaneous CCCM occurred in spheroids.Flat EMT cells adhered to the base of a dish,exhibited persistent movement as a cluster of cells,and then shed,resulting in a cluster.All cells from one cluster undergoing CCCM died.Otherwise,all cells undergoing EMT disappeared and almost all cells located in the cell reservoir survived and proliferated.Conclusion:LIM1863 is an excellent cell line to study CCCM and EMT.The group of heterogeneous cells undergoing CCCM behaves like a supracellular unit.

LINC complex independent perinuclear actin organization and cell migration

The link of the metazoan nucleus to the actin cytoskeleton is highly important for actin polymerization and migration of multiple cell types as well as for mechanotransduction and even affects the cellular transcriptome.Several mechanisms of organization of actin filaments next to the nuclear envelope have been identified.Among these mechanisms the most studied one is the Linker of nucleoskeleton and cytoskeleton(LINC)complex-dependent perinuclear actin organization.However,recently additional mechanisms have been identified:an Actin-related protein-2/3(Arp2/3)-dependent perinuclear actin polymerization during migration of dendritic cells and a perinuclear actin rim that is formed in response to external force application or migration cues.In parallel,there are also reports on cancer cells that migrate in a LINC complex independent manner and on cancers with reduced expression of the LINC complex components.Thus,suggesting that LINC complex independent migration may be associated with tumour formation.