A PDMS Device Coupled with Culture Dish for In Vitro Cell Migration Assay

Cell migration and invasion are important factors during tumor progression and metastasis. Wound-healing assay and the Boyden chamber assay are efficient tools to investigate tumor development because both of them could be applied to measure cell migration rate. Therefore, a simple and integrated polydimethylsiloxane (PDMS) device was developed for cell migration assay, which could perform quantitative evaluation of cell migration behaviors, especially for the wound-healing assay. The integrated device was composed of three units, which included cell culture dish, PDMS chamber, and wound generation mold. The PDMS chamber was integrated with cell culture chamber and could perform six experiments under different conditions of stimuli simultaneously. To verify the function of this device, it was utilized to explore the tumor cell migration behaviors under different concentrations of fetal bovine serum (FBS) and transforming growth factor (TGF-β) at different time points. This device has the unique capability to create the “wound” area in parallel during cell migration assay and provides a simple and efficient platform for investigating cell migration assay in biomedical application.     

Rab5 in the regulation of cell motility and invasion

Cellular invasion requires careful regulation of the cell migration and apoptotic signaling cascades, allowing cell movement and survival of the emigrating populations. Components of the endosomal machinery are involved in these processes, and in particular the role of small GTPases of the Rab family has become appreciated. Rab5 is best known for its role in regulating the trafficking of early endosomes, however, it has recently been appreciated to associate with and regulate the routing of complexes containing integrins, the primary cellular receptors for the extracellular matrix. The association regulates the spatio temporal activation of signals of downstream growth factors and integrins. Rab proteins have also been linked to apoptosis mediated by cell surface death receptors, which elicit the activation of the death cascade via activation of caspase 8. Recently, the link between trafficking, apoptosis and cell migration was strengthened, as Rab5 was determined to work in conjunction with caspase 8 in promoting tumor cell motility and metastasis by regulating β1 integrin traffic. The capacity to connect and regulate these pathways identifies Rab5 as a key player in future studies of cell migration and tumor dissemination.     

Microfluidic three-dimensional biomimetic tumor model for studying breast cancer cell migration and invasion in the presence of interstitial flow

Cell migration and invasion are critical steps in cancer metastasis, which are the major cause of death in cancer patients. Tumor-associated macrophages(TAMs) and interstitial flow(IF) are two important biochemical and biomechanical cues in tumor microenvironment, play essential roles in tumor progression. However, their combined effects on tumor cell migration and invasion as well as molecular mechanism remains largely unknown. In this work, we developed a microfluidic-based 3 D breast cancer model by co-culturing tumor aggregates, macrophages, monocytes and endothelial cells within 3 D extracellular matrix in the presence of IF to study tumor cell migration and invasion. On the established platform, we can precisely control the parameters related to tumor microenvironment and observe cellular responses and interactions in real-time. When co-culture of U937 with human umbilical vein endothelial cells(HUVECs) or MDA-MB-231 cells and tri-culture of U937 with HUVECs and MDA-MB-231 cells, we found that mesenchymal-like MDA-MB-231 aggregates activated the monocytes to TAM-like phenotype macrophages. MDA-MB-231 cells and IF simultaneously enhanced the macrophages activation by the stimulation of colony-stimulating factor 1(CSF-1). The activated macrophages and IF further promoted vascular sprouting via vascular endothelial growth factor(VEGFα) signal and tumor cell invasion. This is the first attempt to study the interaction between macrophages and breast cancer cells under IF condition. Taken together, our results provide a new insight to reveal the important physiological and pathological processes of macrophages-tumor communication. Moreover, our established platform with a more mimetic 3 D breast cancer model has the potential for drug screening with more accurate results.     

Cell motility assays on tissue culture dishes via non-invasive confinement and release of cells

In vitro cell migration assays are useful for screening bioactive agents that regulate angiogenesis, tumor metastasis, would healing, and immune responses by effecting changes in the rate of cell migration. Here we have developed a noninvasive in vitro migration assay that operates through release of confluent groups of cells initially confined within patterns of cell-resistant polyelectrolyte. Cell-resistant patterns of polyelectrolyte, separating groups of confluent cells, are rendered cell adhesive by adsorption of a second, cell adhesive polyelectrolyte of opposite charge; thereby, resulting in migration of cells into the separating regions. By dynamically controlling cell-surface interactions through self-assembly of cell-adhesive and cell resistant polyelectrolytes, this method eliminates the need to mechanically wound cells, as is done in current cell migration assays. The utility of this technique in identifying molecules and mechanisms that regulate cell migration is demonstrated by its application as an assay for the effects of platelet derived growth factors, cytoskeleton disrupting agents, and Merlin overexpression, on the migration of NIH 3T3 fibroblasts.     

Co-culture of tumor spheroids and monocytes in a collagen matrix-embedded microfluidic device to study the migration of breast cancer cells

A 3D co-culture microfluidic device was developed to study the effects of ECM stiffness and TAMs on tumor cells migration.     

Characterization of the interaction between fibroblasts and tumor cells on a microfluidic co‐culture device

Fibroblasts and tumor cells have been involved in the process of cancer development, progression and therapy. Here, we present a simple microfluidic device which enables to study the interaction between fibroblasts and tumor cells by indirect contact co‐culture. The device is composed of multiple cell culture chambers which are connected by a parallel of cell migration regions, and it enables to realize different types of cells to communicate each other on the single device. In this work, human embryonic lung fibroblasts cells were observed to exhibit obvious migration towards tumor cells instead of normal epithelial cells on the co‐culture device. Moreover, transdifferentiation of human embryonic lung fibroblast cells was recognized by the specific expression of α‐smooth musle actin, indicating the effect of tumor cells on the behavior of fibroblasts. Furthermore, multiple types of cell co‐culture can be demonstrated on the single device which enables to mimic the complicated microenviroment in vivo. The device is simple and easy to operate, which enables to realize real‐time observation of cell migration after external stimulus. This microfluidic device allows for the characterization of various cellular events on a single device sequentially, faciliating the better understanding of interaction between heterotypic cells in a more complex microenvironment.     

Role of mTOR signaling in tumor cell motility, invasion and metastasis

Tumor cell migration and invasion play fundamental roles in cancer metastasis. The mammalian target of rapamycin (mTOR), a highly conserved and ubiquitously expressed serine/threonine (Ser/Thr) kinase, is a central regulator of cell growth, proliferation, differentiation and survival. Recent studies have shown that mTOR also plays a critical role in the regulation of tumor cell motility, invasion and cancer metastasis. Current knowledge indicates that mTOR functions as two distinct complexes, mTORC1 and mTORC2. mTORC1 phosphorylates p70 S6 kinase (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1), and regulates cell growth, proliferation, survival and motility. mTORC2 phosphorylates Akt, protein kinase C α (PKCα) and the focal adhesion proteins, and controls the activities of the small GTPases (RhoA, Cdc42 and Rac1), and regulates cell survival and the actin cytoskeleton. Here we briefly review recent knowledge of mTOR complexes and the role of mTOR signaling in tumor cell migration and invasion. We also discuss recent efforts about the mechanism by which rapamycin, a specific inhibitor of mTOR, inhibits cell migration, invasion and cancer metastasis.     

Betulonic Acid,as One of the Active Components of the Celastrus orbiculatus Extract,Inhibits the Invasion and Metastasis of Gastric Cancer Cells by Mediating Cytoskeleton Rearrangement In Vitro

Gastric cancer is a type of malignant tumor that seriously threatens human life and health. Invasion and metastasis present difficulties in the treatment of gastric cancer, and the remodeling of the tumor cytoskeleton plays an important role in mediating the ability of tumor cells to achieve invasion and metastasis. Previous experimental results suggest that Celastrus orbiculatus extract can regulate cytoskeletal remodeling in gastric cancer, but the active component has not been determined. Betulonic acid, as an effective component of COE, inhibits the invasion and metastasis of gastric cancer cells by regulating cytoskeletal remodeling in vitro; its specific mechanisms have been studied here. After betulonic acid was dissolved, it was diluted to various working concentrations in RPMI-1640 medium and added to AGS, HGC-27 and GES-1 cell lines. Cell viability was assessed by CCK-8 and colony formation assays. Cytoskeleton staining was used to detect changes in cytoskeleton morphology. Functional assays including wound healing assays and transwell assays were used to detect the invasion and migration of cells. The effect of betulonic acid on cell invasion and migration was clearly and precisely observed by high-content imaging technology. Western blotting was used to detect the regulation of matrix metalloproteinase-related proteins and epithelial–mesenchymal transformation-related proteins. We found that betulonic acid inhibited the migration and invasion of gastric cancer cells. Therefore, betulonic acid inhibits the invasion and metastasis of gastric cancer cells by mediating cytoskeletal remodeling and regulating epithelial mesenchymal transformation.     

Multi-step microfluidic device for studying cancer metastasis

This paper describes a multi-step microfluidic device for studying the deformation and extravasation of primary tumor cells. Prior to extravasation, primary tumor cells undergo sequential steps of deformation through the capillaries, before adhering and transmigrating through the endothelial lining and basement membrane. To study this cascade of events, we fabricated a multi-step microfluidic device whose microgaps were coated with Matrigel to mimic the basement membrane. The microchannel was lined with human microvascular endothelial cells (HMECs) to replicate the endothelial lining. Analysis of deformation, biological and migratory capabilities of various tumor cell lines viz. HepG2, HeLa, and MDA-MB 435S were quantified using the fabricated device. After deformation, the cells' viabilities were significantly reduced and their doubling times were simultaneously increased, indicating changes in their biological capability. However, cell deformation did not significantly reduce their cell motility. Cell motility was co-assessed using the cell's migration rate and the overall population's percentage migration under various conditions (no barrier, Matrigel and Matrigel-HMEC). The device was also used to quantify the effects of Matrigel and the endothelial lining on cell migration. Our results suggest that both played an independent role in inhibiting cell extravasation, with the Matrigel significantly slowing down cell movement and the endothelial lining reducing the total number of transmigrated cells.     

Engineering Photocleavable Protein-decorated Hydrogels to Regulate Cell Adhesion and Migration

Cell adhesion and migration play essential roles in tissue development and maintenance, and abnormal cell migration is involved in life-threatening diseases, including vascular disease, tumor formation, and metastasis. The advances in hydrogel-based 3D cell culture development facilitated the investigation of cell motility behavior, including cell-cell and cell-matrix adhesion and cell migration in a microenvironment more related to in vivo situations. Establishing advanced methods for these in vitro studies is thus necessary. Photo-sensitive proteins show advantages in remote and non-invasive regulation of hydrogels' properties, and thus are of great potential in regulating 3D cultured cells' behavior. In the presented study, we engineered photocleavable protein(PhoCl)-decorated hydrogels to regulate cell adhesion and migration of MDA-MB-231. The integrin-binding motif RGD was fused to the PhoCl and was decorated on the hydrogel. After being exposed to light at 405 nm, the PhoCl was cleaved and the RGD motif was released, resulting in detachment of the binding cells. The regulatory effect of the light illumination showed a time-dependent and cell density-dependent manner. Furthermore, the elimination of RGD by patterned light exposure completely suspended the cell migration to the corresponding region, suggesting a controllable regulation of the cell migration direction.     

The Effects of Photodynamic Therapy on Human Neutrophil Migration Using Bacteriochlorin a

Abstract— Bacteriochlorin a photodynamic therapy (BCA-PDT) caused inhibition of interleukin (IL)-8-activated neutrophil migration, at concentrations that did not induce membrane damage. Random migration and migration induced by other chemoattractants were also inhibited, indicating that the effect of BCA-PDT was not at the level of chemoattractant-receptor interaction but down stream. The BCA-PDT completely blocked superoxide production of phorbol ester-stimulated neutrophils, indicating that superoxide production by neutrophils present in the tumor before and during BCA-PDT is not the cause of inactivation of tumor cells. Both type I and type II quenchers prevented inhibition by BCA-PDT but only in electroporated cells. Confocal laser scanning microscopy showed that the fluorescence of BCA was located inside the cell. These results show that the effects of BCA-PDT are intracellular and of a mixed type I/type II character and that the neutrophils present in the tumor during illumination probably do not contribute to tumor eradication by releasing reactive oxygen species.     

Surface Modification of Intact Poly(dimethylsiloxane) for Cell Culture and Cell Migration

Cell migration plays a crucial role in various biological processes including embryogenests,wound healing,immune response,and tissue development~([1]).Exploring and understanding the mechanisms and related factors underlying cell migration arc also very important for emerging areas of biotedmology which focus on cellular transplantation and the manufacture of artificial tissues,as well as for the development of new therapeutic strategies for controlling invasive tumor cells~([2]).     

Lipoic Acid-Modified Oligoethyleneimine-Mediated miR-34a Delivery to Achieve the Anti-Tumor Efficacy

MiR-34a, an important tumor suppressor, has been demonstrated to possess great potential in tumor gene therapy. To achieve the upregulation of miR-34a expression level, an oligoethyleneimine (OEI) derivative was constructed and employed as the carrier through the modification with lipoic acid (LA), namely LA-OEI. In contrast to OEI, the derivative LA-OEI exhibited superior transfection efficiency measured by confocal laser scanning microscopy and flow cytometry, owing to rapid cargo release in the disulfide bond-based reduction sensitive pattern. The anti-proliferation and anti-migration effects were tested after the miR-34a transfection to evaluate the anti-tumor response, using human cervical carcinoma cell line HeLa as a model. The delivery of LA-OEI/miR-34a nanoparticles could achieve obvious anti-proliferative effect caused by the induction of cell apoptosis and cell cycle arrest at G1 phase. In addition, it could inhibit the migration of tumor cells via the downregulation of MMP-9 and Notch-1 level. Overall, the LA-OEI-mediated miR-34a delivery was potential to be used as an effective way in the tumor gene therapy.     

Chemistry and biology of moverastins, inhibitors of cancer cell migration, produced by Aspergillus

Cancer cell migration is a required step in cancer metastasis. We screened for inhibitors of cancer cell migration of microbial origin, and obtained moverastin, a member of the cylindrol family, from Aspergillus sp. F7720. However, the results of an NMR spectroscopic analysis raised the possibility that moverastin is a mixture of two diastereomers. Separation of the C-10 epimers of synthetic moverastin and a bioassay revealed that both diastereomers (moverastins A and B) had inhibitory effects on cell migration. Furthermore, we demonstrated that moverastins A and B inhibited FTase in vitro, and they also inhibited both the membrane localization of H-Ras and the activation of the PI3K/Akt pathway in EC17 cells. Thus, moverastins inhibited the migration of tumor cells by inhibiting the farnesylation of H-Ras, and subsequent H-Ras-dependent activation of the PI3K/Akt pathway.     

Engineered Bacteriorhodopsin May Induce Lung Cancer Cell Cycle Arrest and Suppress Their Proliferation and Migration

Highly expressible bacteriorhodopsin (HEBR) is a light-triggered protein (optogenetic protein) that has seven transmembrane regions with retinal bound as their chromophore to sense light. HEBR has controllable photochemical properties and regulates activity on proton pumping. In this study, we generated HEBR protein and incubated with lung cancer cell lines (A549 and H1299) to evaluate if there was a growth-inhibitory effect with or without light illumination. The data revealed that the HEBR protein suppressed cell proliferation and induced the G₀/G₁ cell cycle arrest without light illumination. Moreover, the migration abilities of A549 and H1299 cells were reduced by ~17% and ~31% after incubation with HEBR (40 μg/mL) for 4 h. The Snail-1 gene expression level of the A549 cells was significantly downregulated by ~50% after the treatment of HEBR. In addition, HEBR significantly inhibited the gene expression of Sox-2 and Oct-4 in H1299 cells. These results suggested that the HEBR protein may inhibit cell proliferation and cell cycle progression of lung cancer cells, reduce their migration activity, and suppress some stemness-related genes. These findings also suggested the potential of HEBR protein to regulate the growth and migration of tumor cells, which may offer the possibility for an anticancer drug.     

Transformable peptide nanoparticles inhibit the migration of N-cadherin overexpressed cancer cells

About 90% cancer-related mortality results from the cancer metastasis, which generally undergoes after epithelial-mesenchymal transition (EMT) process. N-Cadherin, overexpressed on cancer cell surface during EMT, can enhance the migration of cancer cells. Herein, we design and synthesize a transformable peptide BP-KLVFF-SWTLYTPSGQSK (BFS) that can block N-cadherin for inhibiting cancer migration and metastasis. The peptide BFS consists of three modules including (1) the hydrophobic bis-pyrene (BP) unit for forming and locating nanoparticles, (2) the KLVFF peptide sequence for forming and stabilizing fibrous structures and (3) the targeting peptide sequence SWTLYTPSGQSK that can specifically bind to N-cadherin. The peptide BFS can form nanoparticles in PBS, which can transform to nanofibers when targeting and binding to N-cadherin. The nanofibers inhibit the migration of N-cadherin overexpressed MDA-MB-436 cancer cells. The peptide BFS shows 83.6% inhibiting rate in cells wound healing assay. In addition, the inhibition rate is 67.9% when the BFS applied in transwell migration assay. These results indicate that the BFS has excellent ability to inhibit migration of cancer cells. This self-assembly strategy could be potentially utilized to regulate the key protein during EMT for inhibiting the tumor metastasis.     

Lysophosphatidic acid receptor 2 and Gi/Src pathway mediate cell motility through cyclooxygenase 2 expression in CAOV-3 ovarian cancer cells

Lysophosphatidic acid (LPA) is a bioactive phospholipids and involves in various cellular events, including tumor cell migration. In the present study, we investigated LPA receptor and its transactivation to EGFR for cyclooxygenase-2 (COX-2) expression and cell migration in CAOV-3 ovarian cancer cells. LPA induced COX-2 expression in a dose-dependent manner, and pretreatment of the cells with pharmacological inhibitors of Gi (pertussis toxin), Src (PP2), EGF receptor (EGFR) (AG1478), ERK (PD98059) significantly inhibited LPA-induced COX-2 expression. Consistent to these results, transfection of the cells with selective Src siRNA attenuated COX-2 expression by LPA. LPA stimulated CAOV-3 cell migration that was abrogated by pharmacological inhibitors and antibody of EP2. Higher expression of LPA2 mRNA was observed in CAOV-3 cells, and transfection of the cells with a selective LPA2 siRNA significantly inhibited LPA-induced activation of EGFR and ERK, as well as COX-2 expression. Importantly, LPA2 siRNA also blocked LPA-induced ovarian cancer cell migration. Collectively, our results clearly show the significance of LPA2 and Gi/Src pathway for LPA-induced COX-2 expression and cell migration that could be a promising drug target for ovarian cancer cell metastasis.     

The HGF-met signaling axis: emerging themes and targets of inhibition

The Met tyrosine kinase receptor is the only known receptor for hepatocyte growth factor (HGF). Downstream Met signaling is essential for embryonic development; however, aberrant Met signaling promotes tumor progression by facilitating cell proliferation, survival, migration, invasion, and metastasis. Tumor cell invasion is considered an important step in distant metastatic foci formation. Several recent reviews have focused on the pleiotropic effects of Met signaling in both tumor cells and in the surrounding stromal cells. This review will summarize the currently described mechanisms driving Met induced tumor cell progression and invasion, the role played by cells in the tumor stroma, and therapeutic approaches to block receptor activity. In addition, this review will also highlight two new areas of development: 1) attenuation of Met signaling via multiple mechanisms of action targeting tumor cells and cells in the surrounding stroma using plant-derived polyphenols and 2) the induction by HGF of atypical lysosome trafficking, leading to increased protease secretion and tumor cell invasion. These new areas of research will help to uncover novel therapeutic targets to block the HGF/Met signaling axis to slow cancer progression.     

Total Syntheses and Biological Reassessment of Lactimidomycin,Isomigrastatin and Congener Glutarimide Antibiotics

Lactimidomycin ( 1 ) was described in the literature as an exquisitely potent cell migration inhibitor. Encouraged by this claim, we developed a concise and scalable synthesis of this bipartite glutarimide‐macrolide antibiotic, which relies on the power of ring‐closing alkyne metathesis (RCAM) for the formation of the unusually strained 12‐membered head group. Subsequent deliberate digression from the successful path to 1 also brought the sister compound isomigrastatin ( 2 ) as well as a series of non‐natural analogues of these macrolides into reach. A careful biological re‐evaluation of this compound collection showed 1 and progeny to be potently cytotoxic against a panel of cancer cell lines, even after one day of compound exposure; therefore any potentially specific effects on tumor cell migration were indistinguishable from the acute effect of cell death. No significant cell migration inhibition was observed at sub‐toxic doses. Although these findings cannot be reconciled with some reports in the literature, they are in accord with the notion that lactimidomycin is primarily a ribosome‐binder able to effectively halt protein biosynthesis at the translation stage.     

Profiling Functional and Biochemical Phenotypes of Circulating Tumor Cells Using a Two‐Dimensional Sorting Device

During cancer progression, tumors shed circulating tumor cells (CTCs) into the bloodstream. CTCs that originate from the same primary tumor can have heterogeneous phenotypes and, while some CTCs possess benign properties, others have high metastatic potential. Deconstructing the heterogeneity of CTCs is challenging and new methods are needed that can sort small numbers of cancer cells according to their phenotypic properties. Here we describe a new microfluidic approach that profiles, along two independent phenotypic axes, the behavior of heterogeneous cell subpopulations. Cancer cells are first profiled according to expression of a surface marker using a nanoparticle‐enabled approach. Along the second dimension, these subsets are further separated into subpopulations corresponding to migration profiles generated in response to a chemotactic agent. We deploy this new technique and find a strong correlation between the surface expression and migration potential of CTCs present in blood from mice with xenografted tumors. This system provides an important new means to characterize functional diversity in circulating tumor cells.