Cell Migration Behavior Research Articles

Application of Image Analysis to Establish Potential Relationship between Intracellular Interaction and Cell Migration Behavior

In order to predict the movement and behavior of important cellular components computational language-based image processing approach caters a lot more than the traditional approaches. In this study, a relation of PI3K protein activity during cell migration has been evaluated using MATLAB based custom coding as an image analysis tool. Cells were imaged under fluorescence microscope to detect the PI3-Kinase activity in tandem with the morphological changes. Several cell movement metrics were assessed, indicating protein activity localization. Different parametric analysis confirms that cell migration is essentially controlled by the peripheral activity rather than the overall cell activity. A quantitative relationship has been proposed as a function of high intensity region area, intensity, and angle of movement, which fit 85% of total cellular responses. Customized analysis at intracellular cellular level has the potential to reveal some fine detail that eventually could contribute to the research on the critical diseases like cancer. DUJASE Vol. 7(1) 72-81, 2022 (January)

Identification of mesenchymal stem cells based on the passaging effect using machine learning approaches.

For diseases that lack efficient therapeutic approaches, stem cell therapy is an emerging alternative treatment, wherein cell quality assessment is an essential step before cell transplantation into patients. Cell quality is generally evaluated using approaches, such as immunohistochemistry and flow cytometry, which involve complex laboratory routines. In this milieu, several studies have demonstrated the feasibility of applying image processing and analysis to assist cell quality assessment more efficiently. We developed an image-based analytical tool using cell tracking, cytometric analyses, and gating with time-lapse microscopy to investigate the migratory behavior of human mesenchymal stem cells (hMSCs) cultured on chitosan membranes. Using this tool, systematic studies were carried out to inspect variations in cell quality by extracting two morphological and seven migratory features of cells of interest. We found that chitosan membrane-induced multi-cellular spheroids move at a higher speed and in a more consistent direction than single cells. The hMSCs showed a tendency to aggregate with strong 3-D spheroid formability and increased mobility during maturation; however, both these tendencies decreased with degeneration. Furthermore, we automated the cell quality assessment procedure by utilizing machine learning. We selected 570 hMSCs, 390 adipose-derived adult stem cells (hADSCs) and 180 human placenta-derived multipotent cells (hPDMCs), with complete movement trajectories from 25 videos, at three different passages. The differentiation accuracy between hADSCs and hPDMCs in the mature passage was 96.3%, which was higher than that in the early (82.6%) and late (80.5%) passages, whereas the accuracy of passage identification was 62.8% for hADSCs and 70.6% for hPDMCs. The limited accuracy of passage identification may be owing to the insufficient number of morphological features. This study shows the potential of applying machine learning in monitoring the quality of stem cells during in vitro culture.

Modeling cellular adhesion.

Cellular adhesion strength, described by the fraction of cells that detach from a surface for a range of applied shear stresses, is predictive of cancer cell migration in 2D and 3D environments and the overall metastatic potential of these cells. Simply stated, cell adhesion strength depends on the balance between cell-surface bond formation and breakage rates. This has been previously modeled as such using the concentration of adhesion proteins such as integrins, the cell-surface interaction area, and a shear force dependent bond breakage rate between the surface ligands and adhesion proteins. However, we have shown that changes in intracellular myosin motor activity, actin stress fibers and force responsive focal adhesion complex proteins can also influence cell adhesion strength and alter cell migration behavior. Using a computational model, we explore how various intracellular factors beyond the concentration of adhesion proteins, govern cell adhesion strengths as measured by shear flow analysis. Overall, our model focuses on 5 key cytoskeletal aspects and their impact on cellular adhesion because of their apparent relevance from previous studies. These include: average number of stress fibers per cell (dictated by f-actin and myosin concentrations), available integrins per stress fiber (dictated by integrin concentration and actin branching), likelihood of bound paxillin, active myosin motors (dictated by myosin light chain phosphorylation), and finally actin polymerization and depolymerization dynamics (governed by time between depolymerization and repolymerization events). The model is further used as a predictive tool in order to determine the particular intracellular factors differentiating cancer strains. The goal of this research is to unravel biomechanical and consequently biochemical differences between cytoskeletal elements of highly metastatic and non-metastatic cancer cells.

An adaptive and versatile method to quantitate and characterize collective cell migration behaviors on complex surfaces.

Collective cell migration is critical for proper embryonic development, wound healing, and cancer cell invasion. However, much of our knowledge of cell migration has been performed using flat surfaces that lack topographical features and do not recapitulate the complex fibrous architecture of the extracellular matrix (ECM). The recent availability of synthetic fibrous networks designed to mimic in vivo ECM has been key to identify the topological features that dictate cell migration patterns as well as to determine the underlying mechanisms that regulate topography-sensing. Recent studies have underscored the prevalence of collective cell migration during cancer invasion, and these observations present a compelling need to understand the mechanisms controlling contact guidance within migratory, multicellular groups. Therefore, we designed an integrated migration analysis platform combining tunable electrospun fibers that recapitulate aspects of the biophysical properties of the ECM, and computational approaches to investigate collective cell migration. To quantitatively assess migration as a function of matrix topography, we developed an automated MATLAB code that quantifies cell migration dynamics, including speed, directionality, and the number of detached cells. This platform enables live cell imaging while providing enough cells for biochemical, proteomic, and genomic analyses, making our system highly adaptable to multiple experimental investigations.

Effects of synchrotron-based X-rays and gold nanoparticles on normal and cancer cell morphology and migration.

It has been shown lately that gold nanoparticles (AuNPs) and ionizing radiation (IR) have inhibitory effects on cancer cell migration while having promoting effects on normal cells' motility. Also, IR increases cancer cell adhesion with no significant effects on normal cells. In this study, synchrotron-based microbeam radiation therapy, as a novel pre-clinical radiotherapy protocol, is employed to investigate the effects of AuNPs on cell migration. Experiments were conducted utilizing synchrotron X-rays to investigate cancer and normal cell morphology and migration behaviour when they are exposed to synchrotron broad beams (SBB) and synchrotron microbeams (SMB). This in vitro study was conducted in two phases. In phase I two cancer cell lines - human prostate (DU145) and human lung (A549) - were exposed to various doses of SBB and SMB. Based on the phase I results, in phase II two normal cell lines were studied: human epidermal melanocytes (HEM) and human primary colon epithelial (CCD841), along with their respective cancerous counterparts, human primary melanoma (MM418-C1) and human colorectal adenocarcinoma (SW48). The results show that radiation-induced damage in cells' morphology becomes visible with SBB at doses greater than 50 Gy, and incorporating AuNPs increases this effect. Interestly, under the same conditions, no visible morphological changes were observed in the normal cell lines post-irradiation (HEM and CCD841). This can be attributed to the differences in cell metabolic and reactive oxygen species levels between normal and cancer cells. The outcome of this study highlights future applications of synchrotron-based radiotherapy, where it is possible to deliver extremely high doses to cancer tissues whilst preserving surrounding normal tissues from radiation-induced damage.

HNF4G stimulates the development of pancreatic cancer by promoting IGF2BP2 transcription.

Pancreatic cancer is a devastating and lethal malignancy. Our study investigated the effective mechanism of HNF4G on pancreatic cancer cell functions through the IGF2BP2 transcription. HNF4G and IGF2BP2 expressions in pancreatic cancer were examined. The relationship between HNF4G expression and pancreatic cancer patients' clinicopathological characteristics was evaluated. After interfering with HNF4G expression in pancreatic cancer cells, the cell proliferative, migratory, and invasive capabilities were evaluated. Also, the expression of proliferation-related gene PCNA and migration and invasion-related gene MMP2 was determined. The binding relation between HNF4G and HNF4G promoter was forecasted and testified. A tumorigenesis assay in nude mice was performed to detect the HNF4G interference's effect on the subcutaneous tumorigenic capacity of pancreatic cancer cells. HNF4G and IGF2BP2 expressions were up-regulated in pancreatic cancer. Specifically, interfering with HNF4G inhibited PANC-1 cell proliferative, invasive and migratory behaviors, and decreased PCNA and MMP2 expression. Mechanistically, HNF4G as a transcription factor could specifically bind to IGF2BP2 and promote its expression. Rescue assay findings showed that IGF2BP2 overexpression could reverse the inhibiting effect of HNF4G interference on pancreatic cancer cells. For the in vivo finding, interfering HNF4G expression retarded the subcutaneous tumorigenic ability of pancreatic cancer cells. We summarize that HNF4G as a transcription factor regulates IGF2BP2 expression to promote pancreatic cancer cell proliferation and migration capacities.

Effects of nanopillars and surface coating on dynamic traction force

The extracellular matrix serves as structural support for cells and provides biophysical and biochemical cues for cell migration. Topography, material, and surface energy can regulate cell migration behaviors. Here, the responses of MC3T3-E1 cells, including migration speed, morphology, and spreading on various platform surfaces, were investigated. Polydimethylsiloxane (PDMS) micropost sensing platforms with nanopillars, silicon oxide, and titanium oxide on top of the microposts were fabricated, and the dynamic cell traction force during migration was monitored. The relationships between various platform surfaces, migration behaviors, and cell traction forces were studied. Compared with the flat PDMS surface, cells on silicon oxide and titanium oxide surfaces showed reduced mobility and less elongation. On the other hand, cells on the nanopillar surface showed more elongation and a higher migration speed than cells on silicon oxide and titanium oxide surfaces. MC3T3-E1 cells on microposts with nanopillars exerted a larger traction force than those on flat PDMS microposts and had more filopodia and long protrusions. Understanding the relationships between platform surface condition, migration behavior, and cell traction force can potentially lead to better control of cell migration in biomaterials capable of promoting tissue repair and regeneration.

Cells function as a ternary logic gate to decide migration direction under integrated chemical and fluidic cues.

Cells sense various environmental cues and subsequently process intracellular signals to decide their migration direction in many physiological and pathological processes. Although several signaling molecules and networks have been identified in these directed migrations, it still remains ambiguous to predict the migration direction under multiple and integrated cues, specifically chemical and fluidic cues. Here, we investigated the cellular signal processing machinery by reverse-engineering directed cell migration under integrated chemical and fluidic cues. We imposed controlled chemical and fluidic cues to cells using a microfluidic platform and analyzed the extracellular coupling of the cues with respect to the cellular detection limit. Then, the cell's migratory behavior was reverse-engineered to build a cellular signal processing system as a logic gate, which is based on a "selection" gate. This framework is further discussed with a minimal intracellular signaling network of a shared pathway model. The proposed framework of the ternary logic gate suggests a systematic view to understand how cells decode multiple cues and make decisions about the migration direction.

Effect of width of microchannel device on behavior of collective cell migration

Effect of width of microchannel device on behavior of collective cell migration

Superficial groove structure in the size of focal adhesion can clarify cell-type-specific differences in force-dependent substrate mechanosensing

Mechanosensing-based cell orientation and migration in microenvironments, such as microgrooved surfaces, are essential in biological tissue growth and repair. These responses might be cell type-dependent as they are deeply involved in cellular functions. Despite their orientation and migration responses are depended on cell-substrate adhesion, which is deeply involved with focal adhesions (FAs) of cells, we have limited information on the cell behavior on FA-sized microgrooved surfaces. Here, we systematically investigated the cell orientation and migration behavior of primary porcine aortic smooth muscle cells (PASMs), embryonic rat aortic smooth muscle cells with fibroblast-like phenotype (A7r5), and human cervical cancer cells (HeLa) on a substrate comprised of superficial grooves with three widths (1 μm, 5 μm, and 10 μm) and 150-nm depth, which are the same order of magnitude of the three-dimensional size of FAs. PASM and A7r5 cells including thick bundles of actin fibers with elongated FAs showed pronounced cell polarization and directional migration on the 1 and 5 μm wide grooves. PASMs were more sensitive to the superficial grooves than A7r5 cells. HeLa cells adhering to the grooves had non-oriented actin fibers with smaller FAs, and they did not show specific orientation nor directional migration in any groove type. Atomic force microscopy elucidated that the mechanical tension of the actin fibers in live cells had a significant positive correlation with the length/width ratio of FAs, reflecting the cell alignment and directional migration on the grooves. The increase or decrease in the mechanical tension of the actin fibers improved or diminished the mechanosensing for the grooves, respectively. The results strongly suggested that the differences in cell type-specific alignment and migration become much more pronounced on the microgrooves which are similar in three-dimensional size of FA, and that is useful for clarifying slight differences in force-dependent cellular mechanosensing.

Rat cytomegalovirus efficiently replicates in dendritic cells and induces changes in their transcriptional profile.

Dendritic cells (DC) play a crucial role in generating and maintaining antiviral immunity. While DC are implicated in the antiviral defense by inducing T cell responses, they can also become infected by Cytomegalovirus (CMV). CMV is not only highly species-specific but also specialized in evading immune protection, and this specialization is in part due to characteristic genes encoded by a given virus. Here, we investigated whether rat CMV can infect XCR1+ DC and if infection of DC alters expression of cell surface markers and migration behavior. We demonstrate that wild-type RCMV and a mutant virus lacking the γ-chemokine ligand xcl1 (Δvxcl1 RCMV) infect splenic rat DC ex vivo and identify viral assembly compartments. Replication-competent RCMV reduced XCR1 and MHCII surface expression. Further, gene expression of infected DC was analyzed by bulk RNA-sequencing (RNA-Seq). RCMV infection reverted a state of DC activation that was induced by DC cultivation. On the functional level, we observed impaired chemotactic activity of infected XCR1+ DC compared to mock-treated cells. We therefore speculate that as a result of RCMV infection, DC exhibit diminished XCR1 expression and are thereby blocked from the lymphocyte crosstalk.

Analysis of Vascular Morphogenesis in Zebrafish.

Analysis of cardiovascular development in zebrafish embryos has become a major driver of vascular research in recent years. Imaging-based analyses have allowed the discovery or verification of morphologically distinct processes and mechanisms of, e.g., endothelial cell migration, angiogenic sprouting, tip or stalk cell behavior, and vessel anastomosis. In this chapter, we describe the techniques and tools used for confocal imaging of zebrafish endothelial development in combination with general experimental approaches for molecular dissection of involved signaling pathways.

E-cadherin-dependent coordinated epithelial rotation on a two-dimensional discoidal pattern.

In vivo, cells collectively migrate in a variety of developmental and pathological contexts. Coordinated epithelial rotation represents a unique type of collective cell migrations, which has been modeled in vitro under spatially confined conditions. Although it is known that the coordinated rotation depends on intercellular interactions, the contribution of E-cadherin, a major cell-cell adhesion molecule, has not been directly addressed on two-dimensional (2D) confined substrates. Here, using well-controlled fibronectin-coated surfaces, we tracked and compared the migratory behaviors of MDCK cells expressing or lacking E-cadherin. We observed that wild-type MDCK II cells exhibited persistent and coordinated rotations on discoidal patterns, while E-cadherin knockout cells migrated in a less coordinated manner without large-scale rotation. Our comparison of the collective dynamics between these two cell types revealed a series of changes in migratory behavior caused by the loss of E-cadherin, including a decreased global migration speed, less regularity in quantified coordination, and increased average density of topological defects. Taken together, these data demonstrate that spontaneous initiation of collective epithelial rotations depends on E-cadherin under 2D discoidal confinements.

Self-assembly of mesoscale collagen architectures and applications in 3D cell migration

3D in vitro tumor models have recently been investigated as they can recapitulate key features in the tumor microenvironment. Reconstruction of a biomimetic scaffold is critical in these models. However, most current methods focus on modulating local properties, e.g. micro- and nano-scaled topographies, without capturing the global millimeter or intermediate mesoscale features. Here we introduced a method for modulating the collagen I-based extracellular matrix structure by disruption of fibrillogenesis and the gelation process through mechanical agitation. With this method, we generated collagen scaffolds that are thickened and wavy at a larger scale while featuring global softness. Thickened collagen patches were interconnected with loose collagen networks, highly resembling collagen architecture in the tumor stroma. This thickened collagen network promoted tumor cell dissemination. In addition, this novel modified scaffold triggered differences in morphology and migratory behaviors of tumor cells. Altogether, our method for altered collagen architecture paves new ways for studying in detail cell behavior in physiologically relevant biological processes. Statement of significanceTumor progression usually involves chronic tissue damage and repair processes. Hallmarks of tumors are highly overlapped with those of wound healing. To mimic the tumor milieu, collagen-based scaffolds are widely used. These scaffolds focus on modulating microscale topographies and mechanics, lacking global architecture similarity compared with in vivo architecture. Here we introduced one type of thick collagen bundles that mimics ECM architecture in human skin scars. These thickened collagen bundles are long and wavy while featuring global softness. This collagen architecture imposes fewer steric restraints and promotes tumor cell dissemination. Our findings demonstrate a distinct picture of cell behaviors and intercellular interactions, highlighting the importance of collagen architecture and spatial heterogeneity of the tumor microenvironment.

Sirt1 Regulates Corneal Epithelial Migration by Deacetylating Cortactin.

Silent information regulator 1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD+) dependent deacetylase, which plays an essential role in cellular metabolism, autophagy, and chromatin accessibility. Our study aimed to determine its role in controlling corneal epithelial wound healing (CEWH). Corneal epithelial (CE)-specific Sirt1 deletion mice were created using the Cre-lox system. CE debridement was used to create a CEWH model. Corneal epithelial cells (CECs) were collected with an Algerbrush. Western blot analysis and RT-qPCR were performed to determine protein and mRNA expression levels. SiRNA transfection technology knocked down SIRT1 and cortactin expression levels in human corneal epithelial cells. Scratch wound assay, MTS assay, and TUNEL assay determined cell migratory, proliferative, and apoptotic behavior, respectively. Co-immunoprecipitation probed for SIRT1 and cortactin interaction. Immunofluorescence staining evaluated the location and expression levels of SIRT1, cortactin, acetylated-cortactin, and F-actin. During CEWH, increases in SIRT1 mRNA and protein expression levels accompanied the downregulation of acetylated lysine in non-histone proteins. The loss of SIRT1 function reduced cell migration and, in turn, delayed CEWH. SIRT1 bound to and deacetylated cortactin in vitro and in vivo. Loss of either SIRT1 or cortactin suppressed wound edge lamellipodia formation, which is consistent with migration retardation. During CEWH, SIRT1 upregulation and its modification of cortactin boost CEC migration by increasing the development of lamellipodia at the wound edge. Therefore SIRT1 may serve as a potential target to enhance CEWH.

Δ133p53 coordinates ECM-driven morphogenesis and gene expression in three-dimensional mammary epithelial acini.

Growing evidence indicates that p53 (encoded by TP53) has a crucial role in normal tissue development. The role of the canonical p53 (p53α) and its 12 isoforms in development and homeostasis of healthy tissue remains poorly understood. Here, we demonstrate that the Δ133p53 isoforms, the three short isoforms of p53, respond specifically to laminin-111 and play an important regulatory role in formation of mammary organoids in concert with p53α. We demonstrate that down-modulation of Δ133p53 isoforms leads to changes in gene expression of the extracellular matrix molecules fibronectin (FN), EDA+-FN, laminin α5 and laminin α3 in human breast epithelial cells. These changes resulted in increased actin stress fibers and enhanced migratory behavior of cells in two-dimensional culture. We found that α5β1-integrin coupled with the extracellularly deposited EDA+-FN activates the Akt signaling pathway in three-dimensional (3D) culture when Δ133p53 is dysregulated. Cells that do not express detectable Δ133p53 isoforms or express low levels of these isoforms failed to form polarized structures in 3D. These results uncover that Δ133p53 isoforms coordinate expression and deposition of organ-specific ECM molecules that are critical for maintenance of tissue architecture and function.

Electromagnetic interactions in regulations of cell behaviors and morphogenesis.

Emerging evidence indicates that the cellular electromagnetic field regulates the fundamental physics of cell biology. The electromagnetic oscillations and synchronization of biomolecules triggered by the internal and external pulses serve as the physical basis of the cellular electromagnetic field. Recent studies have indicated that centrosomes, a small organelle in eukaryotic cells that organize spindle microtubules during mitosis, also function as a nano-electronic generator in cells. Additionally, cellular electromagnetic fields are defined by cell types and correlated to the epigenetic status of the cell. These interactions between tissue-specific electromagnetic fields and chromatin fibers of progenitor cells regulate cell differentiation and organ sizes. The same mechanism is implicated in the regulation of tissue homeostasis and morphological adaptation in evolution. Intercellular electromagnetic interactions also regulate the migratory behaviors of cells and the morphogenesis programs of neural circuits. The process is closely linked with centrosome function and intercellular communication of the electromagnetic fields of microtubule filaments. Clearly, more and more evidence has shown the importance of cellular electromagnetic fields in regulatory processes. Furthermore, a detailed understanding of the physical nature of the inter- and intracellular electromagnetic interactions will better our understanding of fundamental biological questions and a wide range of biological processes.

Quantitative Analysis of Collective Migration by Single-Cell Tracking Aimed at Understanding Cancer Metastasis.

Metastasis is a major complication of cancer treatments. Studies of the migratory behavior of cells are needed to investigate and control metastasis. Metastasis is based on the epithelial–mesenchymal transition, in which epithelial cells acquire mesenchymal properties and the ability to leave the population to invade other regions of the body. In collective migration, highly migratory “leader” cells are found at the front of the cell population, as well as cells that “follow” these leader cells. However, the interactions between these cells are not well understood. We examined the migration properties of leader–follower cells during collective migration at the single-cell level. Different mixed ratios of “leader” and “follower” cell populations were compared. Collective migration was quantitatively analyzed from two perspectives: cell migration within the colony and migration of the entire colony. Analysis of the effect of the cell mixing ratio on migration behavior showed that a small number of highly migratory cells enhanced some of the migratory properties of other cells. The results provide useful insights into the cellular interactions in collective cell migration of cancer cell invasion.

Understanding the Cell's Response to Chemical Signals: Utilisation of Microfluidic Technology in Studies of Cellular and Dictyostelium discoideum Chemotaxis.

Cellular chemotaxis has been the subject of a variety of studies due to its relevance in physiological processes, disease pathogenesis, and systems biology, among others. The migration of cells towards a chemical source remains a closely studied topic, with the Boyden chamber being one of the earlier techniques that has successfully studied cell chemotaxis. Despite its success, diffusion chambers such as these presented a number of problems, such as the quantification of many aspects of cell behaviour, the reproducibility of procedures, and measurement accuracy. The advent of microfluidic technology prompted more advanced studies of cell chemotaxis, usually involving the social amoeba Dictyostelium discoideum (D. discoideum) as a model organism because of its tendency to aggregate towards chemotactic agents and its similarities to higher eukaryotes. Microfluidic technology has made it possible for studies to look at chemotactic properties that would have been difficult to observe using classic diffusion chambers. Its flexibility and its ability to generate consistent concentration gradients remain some of its defining aspects, which will surely lead to an even better understanding of cell migratory behaviour and therefore many of its related biological processes. This paper first dives into a brief introduction of D. discoideum as a social organism and classical chemotaxis studies. It then moves to discuss early microfluidic devices, before diving into more recent and advanced microfluidic devices and their use with D. discoideum. The paper then closes with brief opinions about research progress in the field and where it will possibly lead in the future.

Platelet-Derived Extracellular Vesicles Stimulate Migration through Partial Remodelling of the Ca2+ Handling Machinery in MDA-MB-231 Breast Cancer Cells.

Background: Platelets can support cancer progression via the release of microparticles and microvesicles that enhance the migratory behaviour of recipient cancer cells. We recently showed that platelet-derived extracellular vesicles (PEVs) stimulate migration and invasiveness in highly metastatic MDA-MB-231 cells by stimulating the phosphorylation of p38 MAPK and the myosin light chain 2 (MLC2). Herein, we assessed whether the pro-migratory effect of PEVs involves the remodelling of the Ca2+ handling machinery, which drives MDA-MB-231 cell motility. Methods: PEVs were isolated from human blood platelets, and Fura-2/AM Ca2+ imaging, RT-qPCR, and immunoblotting were exploited to assess their effect on intracellular Ca2+ dynamics and Ca2+-dependent migratory processes in MDA-MB-231 cells. Results: Pretreating MDA-MB-231 cells with PEVs for 24 h caused an increase in Ca2+ release from the endoplasmic reticulum (ER) due to the up-regulation of SERCA2B and InsP3R1/InsP3R2 mRNAs and proteins. The consequent enhancement of ER Ca2+ depletion led to a significant increase in store-operated Ca2+ entry. The larger Ca2+ mobilization from the ER was required to potentiate serum-induced migration by recruiting p38 MAPK and MLC2. Conclusions: PEVs stimulate migration in the highly metastatic MDA-MB-231 breast cancer cell line by inducing a partial remodelling of the Ca2+ handling machinery.