Cell Migration Speed Research Articles

Effect of micropillar density on morphology and migration of low and high metastatic potential breast cancer cells

Study of cell migration in cancer is crucial to the comprehension of the processes and factors that govern tumor spread. Cancer cells migrate invading tissues, causing alterations in cell adhesion, cytoskeleton, and signaling pathways. Little is known about the physical attributes of cancer cells that change when interacting with microenvironments. In this work, the local topography of the ECM has been mimicked through micropillar array substrates. MDA-MB-231 and MCF-7 breast cancer cells, exhibiting high and low metastatic potential, respectively, were analyzed. Differences in morphology and migration of the cells were investigated by examining the cell spreading area, circularity, aspect ratio, migration speed, and migration path. This work encountered that none of the studied cell lines have preferential orientation migrating on uniform patterns. In contrast, cell migration on graded patterns shows preferential orientation along the longitudinal direction from sparser to denser zones which is significantly influenced by substrate stiffness and indicates that both cell lines can sense the spacing gradient and respond to this topographical cue. The migration speed of the breast cancer cell lines significantly decreases from the sparse to medium to dense zones, registering higher values for the MDA-MB-231.

Co-Targeting of DTYMK and PARP1 as a Potential Therapeutic Approach in Uveal Melanoma.

Uveal melanoma (UM) is the most common primary intraocular tumor in adults, with no standardized treatment for advanced disease. Based on preliminary bioinformatical analyses DTYMK and PARP1 were selected as potential therapeutic targets. High levels of both proteins were detected in uveal melanoma cells and correlated with increased tumor growth and poor prognosis. In vitro tests on MP41 (BAP1 positive) and MP46 (BAP1 negative) cancer cell lines using inhibitors pamiparib (PARP1) and Ymu1 (DTYMK) demonstrated significant cytotoxic effects. Combined treatment had synergistic effects in MP41 and additive in MP46 cell lines, reducing cell proliferation and inhibiting the mTOR signaling pathway. Furthermore, the applied inhibitors in combination decreased cell motility and migration speed, especially for BAP1-negative cell lines. Our hypothesis of the double hit into tumoral DNA metabolism as a possible therapeutic option in uveal melanoma was confirmed since combined targeting of DTYMK and PARP1 affected all tested cytophysiological parameters with the highest efficiency. Our in vitro findings provide insights into novel therapeutic avenues for managing uveal melanoma, warranting further exploration in preclinical and clinical settings.

Characterizing Cellular Physiological States with Three-Dimensional Shape Descriptors for Cell Membranes.

The shape of a cell as defined by its membrane can be closely associated with its physiological state. For example, the irregular shapes of cancerous cells and elongated shapes of neuron cells often reflect specific functions, such as cell motility and cell communication. However, it remains unclear whether and which cell shape descriptors can characterize different cellular physiological states. In this study, 12 geometric shape descriptors for a three-dimensional (3D) object were collected from the previous literature and tested with a public dataset of ~400,000 independent 3D cell regions segmented based on fluorescent labeling of the cell membranes in Caenorhabditis elegans embryos. It is revealed that those shape descriptors can faithfully characterize cellular physiological states, including (1) cell division (cytokinesis), along with an abrupt increase in the elongation ratio; (2) a negative correlation of cell migration speed with cell sphericity; (3) cell lineage specification with symmetrically patterned cell shape changes; and (4) cell fate specification with differential gene expression and differential cell shapes. The descriptors established may be used to identify and predict the diverse physiological states in numerous cells, which could be used for not only studying developmental morphogenesis but also diagnosing human disease (e.g., the rapid detection of abnormal cells).

Electrowriting patterns and electric field harness directional cell migration for skin wound healing

Directional cell migration is a crucial step in wound healing, influenced by electrical and topographic stimulations. However, the underlying mechanism and the combined effects of these two factors on cell migration remain unclear. This study explores cell migration under various combinations of guided straight line (SL) spacing, conductivity, and the relative direction of electric field (EF) and SL. Electrowriting is employed to fabricate conductive (multiwalled carbon nanotube/polycaprolactone (PCL)) and nonconductive (PCL) SL, with narrow (50 μm) and wide (400 μm) spacing that controls the topographic stimulation strength. Results show that various combinations of electrical and topographic stimulation yield significantly distinct effects on cell migration direction and speed; cells migrate fastest with the most directivity in the case of conductive, narrow-spacing SL parallel to EF. A physical model based on intercellular interactions is developed to capture the underlying mechanism of cell migration under SL and EF stimulations, in agreement with experimental observations. In vivo skin wound healing assay further confirmed that the combination of EF (1 V cm−1) and parallelly aligned conductive fibers accelerated the wound healing process. This study presents a promising approach to direct cell migration and enhance wound healing by optimizing synergistic electrical and topographic stimulations.

Transcriptional and functional profiling identifies inflammation and endothelial-to-mesenchymal transition as potential drivers for phenotypic heterogeneity within a cohort of endothelial colony forming cells

BackgroundEndothelial colony-forming cells (ECFCs) derived from patients can be used to investigate pathogenic mechanisms of vascular diseases like von Willebrand disease. Considerable phenotypic heterogeneity has been observed between ECFC clones derived from healthy donors. This heterogeneity needs to be well understood in order to use ECFCs as endothelial models for disease. ObjectivesTherefore, we aimed to determine phenotypic and gene expression differences between control ECFCs. MethodsA total of 34 ECFC clones derived from 16 healthy controls were analyzed. The transcriptome of a selection of ECFC clones (n = 15) was analyzed by bulk RNA sequencing and gene set enrichment analysis. Gene expression was measured in all ECFC clones by quantitative polymerase chain reaction. Phenotypic profiling was performed and migration speed of the ECFCs was measured using confocal microscopy, followed by automated quantification of cell morphometrics and migration speed. ResultsThrough hierarchical clustering of RNA expression profiles, we could distinguish 2 major clusters within the ECFC cohort. Major differences were associated with proliferation and migration in cluster 1 and inflammation and endothelial-to-mesenchymal transition in cluster 2. Phenotypic profiling showed significantly more and smaller ECFCs in cluster 1, which contained more and longer Weibel–Palade bodies. Migration speed in cluster 1 was also significantly higher. ConclusionWe observed a range of different RNA expression patterns between ECFC clones, mostly associated with inflammation and clear differences in Weibel–Palade body count and structure. We developed a quantitative polymerase chain reaction panel that can be used for the characterization of ECFC clones, which is essential for the correct analysis of pathogenic mechanisms in vascular disorders.

Abstract 6767: Multivariate analysis of ex vivo kidney 3D immune-microtumors for functional precision medicine

Abstract Immuno-oncology (IO) represents a significant paradigm shift in cancer treatment, focused on leveraging and mobilizing a patient’s own immune response and anti-tumor capabilities. Despite accounting for the majority of available treatment options, there are currently no diagnostic tests capable of predicting combination immunotherapy response in kidney cancer. We have developed a fully human 3D IO model for clinical drug efficacy prediction through multivariate analysis of the tumor-immune microenvironment (TiME) combining functional assays, artificial intelligence and multi-omics. These immune-microtumors recapitulate a patient’s unique pathophysiology and allow 3D spatio-temporal analyses of functional metrics at both bulk and single-cell resolutions. Renal cell carcinoma (RCC) resections and matched blood were collected and processed for single cell isolation of tumor and PBMCs (N=20, ISRCTN10001405). Flow cytometry (FC) and immunofluorescence (IF) were used to characterize TiME cell subpopulations (cancer, immune, stromal etc.) in pellets and 3D cultures, and to deconvolute bulk RNAseq data. Target (tumor) and effector cells (PBMCs and CD8+) were stained with fluorescent probes, activated using ɑCD3 and encapsulated in TiME-mimicking 3D matrices. 3D immune-microtumors were treated with FDA-approved regimens including immunotherapies (ipilimumab, nivolumab, pembrolizumab) and receptor-tyrosine kinase inhibitors (TKIs)(cabozantinib, lenvatinib, axitinib) as monotherapies or combination therapies. Differential drug efficacies were functionally quantified over 4 days using 3D confocal microscopy and computer vision pipelines to extract cell behavior metrics such as immune cell infiltration, immune/tumor cell migration speed, and tumor killing/viability. We devised a specialized RCC hydrogel formulation with superior viability compared to MatrigelTM alone (N=7). FC/IF/RNAseq were used to quantify relative cell subpopulations after isolation and show protein/transcriptional signature retention after 3D culture (N=7). The platform was conducive to the testing of various drugs with varying MoAs including immunotherapies, TKIs, targeted therapies and cell therapies. Treatment with pembrolizumab led to 26% higher PBMC infiltration, 14% higher CD8+ infiltration and 15% increased tumor cell death compared to control. When in combination, pembrolizumab reduced cell viability by 10% and 19% with lenvatinib and axitinib respectively (N=1). Population analyses revealed intra-patient response heterogeneity (N=7). In conclusion, we showed MoA-agnostic, patient-dependent responses to FDA-approved clinical treatment regimens via molecular and functional spatio-temporal analyses. Clinical outcome correlation is currently underway in our advanced RCC study PEAR-TREE2 as well as in breast NCT05435352 and brain NCT06038760 tumors. Citation Format: Eleonora Peerani, Keqian Nan, Demi A. Wiskerke, Thomas D. Richardson, Polina Maximchick, Jay Kearney, George R. de Fraine, Kerrie L. Loughrey, Jonathan Ient, Jessica A. Hudson, Andreas Kaffa, Aston M. Crawley, Nourdine K. Bah, Edgar G. Lopez Molina, Elli Tham, Francesco Iori, Matthew H. Williams, Maxine G. Tran, Duleek N. Ranatunga. Multivariate analysis of ex vivo kidney 3D immune-microtumors for functional precision medicine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6767.

Abstract 4240: A comprehensive in vitro tumor model: One-stop simultaneous isolation and culture of tumoroids, CAFs and TILs for advancing immuno-oncology research

Abstract The tumor microenvironment (TME), composed of a diverse array of cells, jointly participates in tumor formation and has attracted attention as a direct or indirect therapeutic target. Among the tumor-forming cells, cancer cells, cancer-associated fibroblast (CAF), and tumor-infiltrating lymphocytes (TILs) comprise major proportion and players in tumor biology. Therefore, an in vitro tumor model composed of at least above three major cellular players better mimics a patient’s tumor. Here, we developed a patient-derived tumor model containing tumoroids, CAFs and TILs isolated from the patient’s tumor tissue by using one-stop isolation culture system. Our one-stop culture system utilizes a Transwell-based method that concurrently segregates cancer cells, CAFs, and TILs based on distinct characteristics such as cell size, migration speed, and growth. Lung tumoroids, fibroblasts, and TILs segregated in Matrigel-coated upper Transwell chamber, Transwell pores and in the lower chamber, respectively. Lung tumoroids maintain the patient’s lung cancer tissue characteristics and genetic features during extended in vitro cultivation. Fibroblasts extracted from the Transwell pores exhibit significant similarity to CAFs found in patient tissue. TILs in the lower part of the Transwell chamber closely resemble those found in the original cancer tissue analyzed by FACS. We have also refined the functionality of TILs through a two-week expansion process. Patients’ tumor composed of 0.15 to 95.3 % of EpCAM positive tumor cells, 0.14 to 19.4 % of CD90 positive fibroblasts and 3.98 to 91.2 % of immune cells in FACS analysis. By reassembling three main cellular components of the tumor in a proper proportion, we developed a co-culture platform for a patient-derived tumor model reproducing patient-specific TME conditions. The co-culture platform showed a potential not only for an experimental model but also to use a predictive test of personalized immunotherapy responses for lung cancer patients. Citation Format: YOO RI KO, Sung Min Kim, Hye Seon Park, Se Jin Jang. A comprehensive in vitro tumor model: One-stop simultaneous isolation and culture of tumoroids, CAFs and TILs for advancing immuno-oncology research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4240.

Inhibition of chromatin condensation disrupts planar cell migration

ABSTRACT Cell migration involves the actin cytoskeleton, and recently recognized nuclear involvement. In this study, we explore the impact of chromatin remodeling on cell migration using NIH 3T3 cells and a scratch wound assay subjected to pharmacological interventions. We inhibit histone deacetylases (HDACs) with Trichostatin A (TSA) and methyltransferase EZH2 with GSK126 to modulate chromatin compaction. Our results indicate that chromatin modifications impair wound closure efficiency, reduce individual cell migration speed, and disrupt migration persistence. Live-cell imaging reveals dynamic intranuclear chromatin remodeling and nuclear shape parameters during migration, influenced by both small- and large-scale chromatin remodeling. The altered nuclear shape is associated with disrupted cell and nuclear mechanics, suggesting a crucial interplay between chromatin remodeling, nuclear mechanics and migration. These findings shed light on the intricate connection between intranuclear chromatin dynamics, nuclear mechanics, and cell migration, providing a basis for further investigations into the molecular mechanisms governing these processes.

CD73 mitigates ZEB1 expression in papillary thyroid carcinoma

BackgroundZEB1, a core transcription factor involved in epithelial-mesenchymal transition (EMT), is associated with aggressive cancer cell behavior, treatment resistance, and poor prognosis across various tumor types. Similarly, the expression and activity of CD73, an ectonucleotidase implicated in adenosine generation, is an important marker of tumor malignancy. Growing evidence suggests that EMT and the adenosinergic pathway are intricately linked and play a pivotal role in cancer development. Therefore, this study focuses on exploring the correlations between CD73 and ZEB1, considering their impact on tumor progression.MethodsWe employed CRISPR/Cas9 technology to silence CD73 expression in cell lines derived from papillary thyroid carcinoma. These same cells underwent lentiviral transduction of a reporter of ZEB1 non-coding RNA regulation. We conducted studies on cell migration using scratch assays and analyses of cellular speed and polarity. Additionally, we examined ZEB1 reporter expression through flow cytometry and immunocytochemistry, complemented by Western blot analysis for protein quantification. For further insights, we applied gene signatures representing different EMT states in an RNA-seq expression analysis of papillary thyroid carcinoma samples from The Cancer Genome Atlas.ResultsSilencing CD73 expression led to a reduction in ZEB1 non-coding RNA regulation reporter expression in a papillary thyroid carcinoma-derived cell line. Additionally, it also mitigated ZEB1 protein expression. Moreover, the expression of CD73 and ZEB1 was correlated with alterations in cell morphology characteristics crucial for cell migration, promoting an increase in cell polarity index and cell migration speed. RNA-seq analysis revealed higher expression of NT5E (CD73) in samples with BRAF mutations, accompanied by a prevalence of partial-EMT/hybrid state signature expression.ConclusionsCollectively, our findings suggest an association between CD73 expression and/or activity and the post-transcriptional regulation of ZEB1 by non-coding RNA, indicating a reduction in its absence. Further investigations are warranted to elucidate the relationship between CD73 and ZEB1, with the potential for targeting them as therapeutic alternatives for cancer treatment in the near future.

RSK/GSK3-mediated phosphorylation of FilGAP regulates chemotactic cancer invasion.

Cell migration plays a crucial role in various biological processes, such as gastrulation, immune response, and cancer metastasis. In response to chemoattractant-like growth factors, cells form protrusions and migrate toward the source of the signal. Rho family small GTPase Rac is a key regulator of cell migration by stimulating actin polymerization to generate lamellipodia, flat membrane protrusions at the leading edge of migrating cells. FilGAP (ARHGAP24), a Rac-specific GTPase-activating protein (GAP), suppresses lamellipodia formation, and controls tumor cell migration. In this study, we found that FilGAP is phosphorylated downstream of epidermal growth factor (EGF) signaling. Upon EGF stimulation, FilGAP is phosphorylated at Ser625 by p90 ribosomal S6 kinase (RSK) and then at Ser621 by glycogen synthase kinase 3 (GSK3). Phosphorylation of FilGAP induces its dissociation from actin filaments. We identified a novel actin-localization domain of FilGAP that is essential for stabilizing cell adhesion. Additionally, we found that phosphorylation of FilGAP inhibits its lamellipodia suppression activity. Finally, we showed the expression of nonphosphorylatable FilGAP mutant, but not wild-type FilGAP, reduced cell migration speed and persistence toward the EGF gradient. Taken together, our results suggest that phosphorylation of FilGAP downstream of EGF-signaling plays a critical role in regulating chemotactic tumor cell migration by controlling cell-matrix adhesion and protrusion formation.

Confinement primes cells for faster migration by polarizing active mitochondria.

Mechanical cues in the tumor microenvironment interplay with internal cellular processes to control cancer cell migration. Microscale pores present in tumor tissue confer varying degrees of confinement on migrating cells, increasing matrix contact and inducing cytoskeletal rearrangement. Previously, we observed that increased collagen matrix contact significantly increased cell migration speed and cell-induced strains within the matrix. However, the effects of this confinement on future cell migration are not fully understood. Here, we use a collagen microtrack platform to determine the effect of confinement on priming MDA-MB-231 cancer cells for fast migration. We show that migration through a confined track results in increased speed and accumulation of migratory machinery, including actin and active mitochondria, in the front of migrating breast cancer cells. By designing microtracks that allow cells to first navigate a region of high confinement, then a region of low confinement, we assessed whether migration in high confinement changes future migratory behavior. Indeed, cells maintain their speed attained in high confinement even after exiting to a region of low confinement, indicating that cells maintain memory of previous matrix cues to fuel fast migration. Active mitochondria maintain their location at the front of the cell even after cells leave high confinement. Furthermore, knocking out vinculin to disrupt focal adhesions disrupts active mitochondrial localization and disrupts the fast migration seen upon release from confinement. Together, these data suggest that active mitochondrial localization in confinement may facilitate fast migration post-confinement. By better understanding how confinement contributes to future cancer cell migration, we can identify potential therapeutic targets to inhibit breast cancer metastasis.

Upregulation of apoptotic genes and downregulation of target genes of Sonic Hedgehog signaling pathway in DAOY medulloblastoma cell line treated with arsenic trioxide

Sonic hedgehog (SHH) medulloblastoma etiology is associated with the SHH molecular pathway activation at different levels. We investigated the effect of arsenic trioxide as a downstream-level inhibitor of the SHH signaling pathway on morphology, cytotoxicity, migration, and SHH-related and apoptotic gene expression of DAOY cells. Cells were treated at various arsenic trioxide (ATO)concentrations (1, 2, 3, 5, and 10 μM) for different times (24 and 48 hr). Following treatments, the morphology of the cells was investigated at ×20 and ×40 magnification by an inverted microscope. Then, cytotoxicity was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and trypan blue assays. Cell migration was analyzed through the wound-healing assay. Furthermore, the expression of SHH-related (GLI1, GLI2, SMO, and MYCN) and apoptotic genes (BAX, BCL2, and TP53) was assessed by real-time quantitative polymerase chain reaction (qPCR). Finally, GLI1, SMO, and MYCN markers were analyzed through immunocytochemistry. Data were analyzed by SPSS (version 16) and P≤0.05 was considered significant. Morphological changes were seen at 3 and 2 μM in 24 and 48 hr of treatment, respectively. The MTT assay showed a dose-dependent cytotoxicity indicating an IC50 value of 3.39±0.35 and 2.05±0.64 μM in 24 and 48hr treatment, respectively. In addition, the trypan blue assay showed higher IC50 values of 4.29±0.25 and 3.92±0.22 μM in 24 and 48 hr treatment, respectively. The wound-healing assay indicated a dose-dependent reduction of cell migration speed showing a 50% reduction at 2.89±0.26 μM. Significant downregulation of GLI1 and GLI2, as well as the upregulation of BAX, BAX/BCL2 ratio, and TP53 were evident. Significant increases in GLI1 and MYCN markers were also evident in immunocytochemistry. ATO, as a downstream effective inhibitor of the SHH pathway, substantially leads to cell death, cell migration inhibition, apoptosis upregulation, and downregulation of SHH target genes in DAOY medulloblastoma. Since ATO is a toxic chemotherapeutic agent, it must be used at low concentrations (2 μM) in order not to damage healthy cells.

A multiscale theory for spreading and migration of adhesion-reinforced mesenchymal cells.

We present a chemomechanical whole-cell theory for the spreading and migration dynamics of mesenchymal cells that can actively reinforce their adhesion to an underlying viscoelastic substrate as a function of its stiffness. Our multiscale model couples the adhesion reinforcement effect at the subcellular scale with the nonlinear mechanics of the nucleus-cytoskeletal network complex at the cellular scale to explain the concurrent monotonic area-stiffness and non-monotonic speed-stiffness relationships observed in experiments: we consider that large cell spreading on stiff substrates flattens the nucleus, increasing the viscous drag force on it. The resulting force balance dictates a reduction in the migration speed on stiff substrates. We also reproduce the experimental influence of the substrate viscosity on the cell spreading area and migration speed by elucidating how the viscosity may either maintain adhesion reinforcement or prevent it depending on the substrate stiffness. Additionally, our model captures the experimental directed migration behaviour of the adhesion-reinforced cells along a stiffness gradient, known as durotaxis, as well as up or down a viscosity gradient (viscotaxis or anti-viscotaxis), the cell moving towards an optimal viscosity in either case. Overall, our theory explains the intertwined mechanics of the cell spreading, migration speed and direction in the presence of the molecular adhesion reinforcement mechanism.

Aligned electrospun fibers of different diameters for improving cell migration capacity

Electrospun fibers have gained significant attention as scaffolds in skin tissue engineering due to their biomimetic properties, which resemble the fibrous extracellular matrix. The morphological characteristics of electrospun fibers play a crucial role in determining cell behavior. However, the effects of electrospun fibers’ arrangement and diameters on human skin fibroblasts (HSFs) remain elusive. Here, we revealed the impact of electrospun fiber diameters (700 nm, 2000 nm, and 3000 nm) on HSFs' proliferation, migration, and functional expression. The results demonstrated that all fibers exhibited good cytocompatibility. HSFs cultured on nanofibers (700 nm diameter) displayed a more dispersed and elongated morphology. Conversely, fibers with a diameter of 3000 nm exhibited a reduced specific surface area and lower adsorption of adhesion proteins, resulting in enhanced cell migration speed and effective migration rate. Meanwhile, the expression levels of migration-related genes and proteins were upregulated at 48 h for the 3000 nm fibers. This study demonstrated the unique role of fiber diameters in controlling the physiological functions of cells, especially decision-making and navigating migration in complex microenvironments of aligned electrospun fibers, and highlights the utility of these bioactive substitutes in skin tissue engineering applications.

Switch of cell migration modes orchestrated by changes of three-dimensional lamellipodium structure and intracellular diffusion

Cell migration plays important roles in many biological processes, but how migrating cells orchestrate intracellular molecules and subcellular structures to regulate their speed and direction is still not clear. Here, by characterizing the intracellular diffusion and the three-dimensional lamellipodium structures of fish keratocyte cells, we observe a strong positive correlation between the intracellular diffusion and cell migration speed and, more importantly, discover a switching of cell migration modes with reversible intracellular diffusion variation and lamellipodium structure deformation. Distinct from the normal fast mode, cells migrating in the newly-found slow mode have a deformed lamellipodium with swollen-up front and thinned-down rear, reduced intracellular diffusion and compartmentalized macromolecule distribution in the lamellipodium. Furthermore, in turning cells, both lamellipodium structure and intracellular diffusion dynamics are also changed, with left-right symmetry breaking. We propose a mechanism involving the front-localized actin polymerization and increased molecular crowding in the lamellipodium to explain how cells spatiotemporally coordinate the intracellular diffusion dynamics and the lamellipodium structure in regulating their migrations.

Evaluation of the effects of glucose and oxygen on the vascular endothelial cell migration.

Glucose is essential as the main energy source for living organisms. However, excessive elevation of blood sugar levels can lead to diabetes and serious complications such as arteriosclerosis. Even though blood sugar levels as well as hypoxia associated with hyperglycemia are known to be closely related to diabetes complications, the responses of vascular endothelial cells to glucose and oxygen have not been fully investigated. In this study, using a microfluidic device that can control the oxygen concentration, we observed the behavior of vascular endothelial cell monolayers while simultaneously controlling glucose and oxygen levels. Results showed that the cell migration speed was increased by high-glucose exposure in an oxygen-rich environment, but was decreased in a hypoxic environment regardless of glucose condition. The expression of vascular endothelial-cadherin at the cell periphery, which plays a role in cell-cell adhesion, was increased by hypoxic exposure, but was largely independent of glucose condition. This suggested that cell-cell adhesion is less involved in the increase in migration caused by high glucose. Furthermore, stabilization and nuclear translocation of hypoxia-inducible factor-1α, which is involved in cellular hypoxia sensing, increased 5 h after exposure to high glucose, but decreased 3 days after the exposure. This indicated that intracellular hypoxia was generated by increased oxygen consumption in mitochondria just after the high-glucose exposure, but it was moderated within 3 days.

Matrix obstructions cause multiscale disruption in collective epithelial migration by suppressing leader cell function.

During disease and development, physical changes in extracellular matrix cause jamming, unjamming, and scattering in epithelial migration. However, whether disruptions in matrix topology alter collective cell migration speed and cell-cell coordination remains unclear. We microfabricated substrates with stumps of defined geometry, density, and orientation, which create obstructions for migrating epithelial cells. Here, we show that cells lose their speed and directionality when moving through densely spaced obstructions. Although leader cells are stiffer than follower cells on flat substrates, dense obstructions cause overall cell softening. Through a lattice-based model, we identify cellular protrusions, cell-cell adhesions, and leader-follower communication as key mechanisms for obstruction-sensitive collective cell migration. Our modeling predictions and experimental validations show that cells' obstruction sensitivity requires an optimal balance of cell-cell adhesions and protrusions. Both MDCK (more cohesive) and α-catenin-depleted MCF10A cells were less obstruction sensitive than wild-type MCF10A cells. Together, microscale softening, mesoscale disorder, and macroscale multicellular communication enable epithelial cell populations to sense topological obstructions encountered in challenging environments. Thus, obstruction-sensitivity could define "mechanotype" of cells that collectively migrate yet maintain intercellular communication.

A cell membrane model that reproduces cortical flow-driven cell migration and collective movement.

Many fundamental biological processes are dependent on cellular migration. Although the mechanical mechanisms of single-cell migration are relatively well understood, those underlying migration of multiple cells adhered to each other in a cluster, referred to as cluster migration, are poorly understood. A key reason for this knowledge gap is that many forces-including contraction forces from actomyosin networks, hydrostatic pressure from the cytosol, frictional forces from the substrate, and forces from adjacent cells-contribute to cell cluster movement, making it challenging to model, and ultimately elucidate, the final result of these forces. This paper describes a two-dimensional cell membrane model that represents cells on a substrate with polygons and expresses various mechanical forces on the cell surface, keeping these forces balanced at all times by neglecting cell inertia. The model is discrete but equivalent to a continuous model if appropriate replacement rules for cell surface segments are chosen. When cells are given a polarity, expressed by a direction-dependent surface tension reflecting the location dependence of contraction and adhesion on a cell boundary, the cell surface begins to flow from front to rear as a result of force balance. This flow produces unidirectional cell movement, not only for a single cell but also for multiple cells in a cluster, with migration speeds that coincide with analytical results from a continuous model. Further, if the direction of cell polarity is tilted with respect to the cluster center, surface flow induces cell cluster rotation. The reason why this model moves while keeping force balance on cell surface (i.e., under no net forces from outside) is because of the implicit inflow and outflow of cell surface components through the inside of the cell. An analytical formula connecting cell migration speed and turnover rate of cell surface components is presented.

Effect of extracellular matrix fiber cross-linkage on cancer cell motility and surrounding matrix deformation

Cancer incidence is increasing annually, and the invasion of cancer into the stroma significantly affects cancer metastasis. The stroma primarily comprises an abundant extracellular matrix (ECM) that interacts closely with cancer cells. Cancer cells use the ECM as a scaffold to migrate from a tumor via mechanical actions such as pushing and pulling the fibers. The purpose of this study is to clarify the effects of elastic modulus differences on cell migration behavior based on the same ECM fiber structure. We observe temporal changes in the morphology of cancer cells and the surrounding ECM to elucidate the relationship between changes in the mechanical properties of the ECM and the invasive behavior of cancer cells. We analyze the shape and migration distance of cancer cells and the displacement field of the ECM by varying the fiber elastic modulus but fixing the ECM density. Increasing the elastic modulus results in a protruding cell shape, which indicates the maximum displacement of the ECM around the cell. Additionally, differences in cell migration speed and dispersion based on the elastic modulus are observed. The behavior of cells with increasing elasticity is classified via cluster analysis. Owing to the chemical cross-linking of the fibers, some cells cannot deform the surrounding tissue. This is attributable to the gel state of the ECM and microscopic fluctuations in the fiber density around the cells. We successfully assessed the effect of changes in the ECM modulus on cell mortality and morphology to reveal the mechanism of cancer invasion.

PEGylated Paclitaxel Nanomedicine Meets 3D Confinement: Cytotoxicity and Cell Behaviors

Investigating the effect of nanomedicines on cancer cell behavior in three-dimensional (3D) platforms is beneficial for evaluating and developing novel antitumor nanomedicines in vitro. While the cytotoxicity of nanomedicines on cancer cells has been widely studied on two-dimensional flat surfaces, there is little work using 3D confinement to assess their effects. This study aims to address this gap by applying PEGylated paclitaxel nanoparticles (PEG-PTX NPs) for the first time to treat nasopharyngeal carcinoma (NPC43) cells in 3D confinement consisting of microwells with different sizes and a glass cover. The cytotoxicity of the small molecule drug paclitaxel (PTX) and PEG-PTX NPs was studied in microwells with sizes of 50 × 50, 100 × 100, and 150 × 150 μm2 both with and without a concealed top cover. The impact of microwell confinement with varying sizes and concealment on the cytotoxicity of PTX and PEG-PTX NPs was analyzed by assessing NPC43 cell viability, migration speed, and cell morphology following treatment. Overall, microwell isolation was found to suppress drug cytotoxicity, and differences were observed in the time-dependent effects of PTX and PEG-PTX NPs on NPC43 cells in isolated and concealed microenvironments. These results not only demonstrate the effect of 3D confinement on nanomedicine cytotoxicity and cell behaviors but also provide a novel method to screen anticancer drugs and evaluate cell behaviors in vitro.