Distinct Migration Modes Research Articles

Protrusion force and cell-cell adhesion play a critical role in collective tumor cluster migration.

While collective migration is shown to enhance invasive and metastatic potential in cancer, the mechanisms driving this behavior and regulating tumor migration plasticity remain poorly understood. This study provides a mechanistic framework explaining the emergence of different modes of collective migration under hypoxia-induced secretome. We focus on the interplay between cellular protrusion force and cell-cell adhesion using collectively migrating three-dimensional microtumors as models with well-defined microenvironment. Large microtumors show directional migration due to intrinsic hypoxia, while small microtumors exhibit radial migration in response to hypoxic secretome. Here, we developed the minimal multi-scale microtumor model (MSMM) to elucidate underlying mechanisms. We identified distinct migration modes within specific regions of protrusion force and cell-cell adhesion parameter space. We show that sufficient cellular protrusion force is crucial for both, radial and directional collective microtumor migration. Radial migration emerges when sufficient cellular protrusion force is generated, driving neighboring cells to move collectively in diverse directions. Within migrating tumors, strong cell-cell adhesion enhances the alignment of cell polarity, breaking the symmetric angular distribution of protrusion forces, and leading to directional microtumor migration. The integrated results from the experimental and computational models provide fundamental insights into collective migration in response to different microenvironment stimuli.

DNA nanomachines reveal an adaptive energy mode in confinement-induced amoeboid migration powered by polarized mitochondrial distribution

Energy metabolism is highly interdependent with adaptive cell migration in vivo. Mechanical confinement is a critical physical cue that induces switchable migration modes of the mesenchymal-to-amoeboid transition (MAT). However, the energy states in distinct migration modes, especially amoeboid-like stable bleb (A2) movement, remain unclear. In this report, we developed multivalent DNA framework-based nanomachines to explore strategical mitochondrial trafficking and differential ATP levels during cell migration in mechanically heterogeneous microenvironments. Through single-particle tracking and metabolomic analysis, we revealed that fast A2-moving cells driven by biomimetic confinement recruited back-end positioning of mitochondria for powering highly polarized cytoskeletal networks, preferentially adopting an energy-saving mode compared with a mesenchymal mode of cell migration. We present a versatile DNA nanotool for cellular energy exploration and highlight that adaptive energy strategies coordinately support switchable migration modes for facilitating efficient metastatic escape, offering a unique perspective for therapeutic interventions in cancer metastasis.

Precise spatial imaging of microRNAs distribution from single living cells

Accurate analysis of the spatial information of microRNAs (miRNAs) in migrating cells is challenging but also extremely significant for deciphering the molecular mechanisms involved in cancer progression and metastasis. In this work, a non-enzyme-assisted signal amplification sensing strategy was designed to achieve high-resolution imaging of miRNAs in individual migrating cells using a single-molecule fluorescence protocol. As proof of concept, miRNA let-7a was detected at the femtomolar level in vitro with accurate visualization and specificity. Next, the three-dimensional distribution of let-7a in single living cells was precisely visualized using a single-molecule imaging platform, during which the differential expression of intracellular let-7a was monitored. Significantly, the spatially resolved distribution of let-7a was further explored in cells undergoing distinct migration modes, which were induced by confined microenvironments. These results revealed that fast migration modes showed low let-7a expression. This work demonstrated an intuitive and ultrasensitive method of analyzing the spatial distribution of miRNAs in vitro, providing a new perspective for exploring regulatory mechanisms of key molecules in crowded intracellular environments in vivo.

Spatially resolved single-molecule profiling of microRNAs in migrating cells driven by microconfinement.

Cancer cells utilize a range of migration modes to navigate through a confined tissue microenvironment in vivo, while regulatory roles of key microRNAs (miRNAs) remain unclear. Precisely engineered microconfinement and the high spatial-resolution imaging strategy offer a promising avenue for deciphering the molecular mechanisms that drive cell migration. Here, enzyme-free signal-amplification nanoprobes as an effective tool are developed for three-dimensional (3D) high-resolution profiling of key miRNA molecules in single migrating cells, where distinct migration modes are precisely driven by microconfinement-engineered microchips. The constructed nanoprobes exhibit intuitive and ultrasensitive miRNA characterization in vitro by virtue of a single-molecule imaging microscope, and the differential expression and intracellular locations in different cell lines are successfully monitored. Furthermore, 3D spatial distribution of miR-141 at high resolution in flexible phenotypes of migrating cells is reconstructed in the engineered biomimetic microenvironment. The results indicate that miR-141 may be involved in the metastatic transition from a slow to a fast migration state. This work offers a new opportunity for investigating regulatory mechanisms of intracellular key biomolecules during cell migration in biomimetic microenvironments, which may advance in-depth understanding of cancer metastasis in vivo.

Discrete Modes of Migration Define Human NK Cell Development from Hematopoietic Stem Cells

Human innate lymphoid cells play a critical role in the control of infection and the shaping of the immune response. Furthermore, natural killer (NK) cells are the first reconstituting lymphocyte population after hematopoietic stem cell transplant and play a key role in shaping engraftment and moderating graft vs. leukemic effects. Relative to their adaptive immune counterparts, the molecular interactions that drive innate immune cell differentiation development are poorly understood. Human NK cells arise from bone marrow CD34+ hematopoietic stem cells (HSC) but undergo maturation in distal microenvironments, the best characterized of which is secondary lymphoid tissue. Human NK cells can also be derived in vitro from CD34+ HSC through co-culture with developmentally supportive stromal cells. Despite the requirement for direct stromal cell contact in NK cell maturation, the nature of these contacts is not known. We have previously shown that freshly isolated human NK cell developmental intermediates from in vitro culture, secondary lymphoid tissue or peripheral blood undergo spontaneous migration on the EL08.1D2 stromal cell line. We have further defined the NK cell ‘developmental synapse’, a site of productive and directional signaling in developmental intermediates arrested on stromal cells. Despite these interesting insights, the regulation of, and requirement for, NK cell migration during differentiation has not been defined.To determine the modes of migration associated with human NK cell differentiation, we performed highly resolved continuous live cell imaging of NK cells developing in vitro from CD34+ HSC over 28 days of culture. These analyses were performed in parallel with flow cytometry to monitor the progression of developmental intermediates towards a functionally mature NK cell phenotype. We found that while CD34+ HSC had constrained motility, progressive NK cell differentiation was associated with more directed migratory phenotype. NK cell maturation was associated with increased track length and velocity, and decreased frequency of time of cells spent in arrest on stromal cells. We further defined the acquisition of complex, Levy-walk behavior as has previously been described for mature T cells imaged within tissue. The consistency of these behaviors between in vitro derived cells and fresh ex vivo NK cells isolated from healthy human donors confirms that in vitro differentiation effectively recapitulates migratory phenotypes in addition to functional and cell surface phenotypes associated with NK cell maturation.Together, these data define distinct modes of migration that are acquired by human NK cells. We propose that this reflects a previously unappreciated requirement for cell migration that enables the functional maturation of human NK cells. Better defining the bidirectional signaling between developing NK cells and their microenvironment will enable a complete understanding of the signals that help shape the acquisition of key innate immune cell functions. This understanding is critical for better predicting and therapeutically enhancing lymphocyte reconstitution after transplant. DisclosuresNo relevant conflicts of interest to declare.

Integration of size separation and transport of granular particles by a posts array on a conveyor belt

We experimentally design an integrated system composed of a conveyor belt and a posts array, which can simultaneously implement the function of size separation and transportation of disk particles. Differently sized particles through the post array exhibit two types of distinct migration modes that are responsible for the size separation. Furthermore, the dividing line between the two migration modes depends on the critical radius found in experiment. The relationship between the separation efficiency and the length of the post array and the packing fraction of particles is investigated quantitatively. Finally, a simplified model is proposed to predict a critical particle radius which is verified to be in accordance with experimental measurements. This research has a potential application prospect in some fields of industry.

Mechanistic Understanding of Surface Migration Dynamics with DNA Walkers.

Dynamic DNA walkers can move cargoes on a surface through various mechanisms including enzymatic reactions and strand displacement. While they have demonstrated high processivity and speed, their motion dynamics are not well understood. Here, we utilize an enzyme-powered DNA walker as a model system and adopt a random walk model to provide new insight into migration dynamics. Four distinct migration modes (ballistic, Lévy, self-avoiding, and diffusive motions) are identified. Each mode shows unique step time and velocity distributions, which are related to mean-squared displacement (MSD) scaling. Experimental results are in excellent agreement with the theoretical predictions. With a better understanding of the dynamics, we performed a mechanistic study, elucidating the effects of cargo types and sizes, walker sequence designs, and environmental conditions. Finally, this study provides a set of design principles for tuning the behaviors of DNA walkers. The DNA walkers from this work could serve as a versatile platform for mathematical studies and open new opportunities for bioengineering.

Of Cell Shapes and Motion: The Physical Basis of Animal Cell Migration.

Motile cells have developed a variety of migration modes relying on diverse traction-force-generation mechanisms. Before the behavior of intracellular components could be easily imaged, cell movements were mostly classified by different types of cellular shape dynamics. Indeed, even though some types of cells move without any significant change in shape, most cell propulsion mechanisms rely on global or local deformations of the cell surface. In this review, focusing mostly on metazoan cells, we discuss how different types of local and global shape changes underlie distinct migration modes. We then discuss mechanical differences between force-generation mechanisms and finish by speculating on how they may have evolved.

Actomyosin contractility-dependent matrix stretch and recoil induces rapid cell migration

Cells select from a diverse repertoire of migration strategies. Recent developments in tunable biomaterials have helped identify how extracellular matrix properties influence migration, however, many settings lack the fibrous architecture characteristic of native tissues. To investigate migration in fibrous contexts, we independently varied the alignment and stiffness of synthetic 3D fiber matrices and identified two phenotypically distinct migration modes. In contrast to stiff matrices where cells migrated continuously in a traditional mesenchymal fashion, cells in deformable matrices stretched matrix fibers to store elastic energy; subsequent adhesion failure triggered sudden matrix recoil and rapid cell translocation. Across a variety of cell types, traction force measurements revealed a relationship between cell contractility and the matrix stiffness where this migration mode occurred optimally. Given the prevalence of fibrous tissues, an understanding of how matrix structure and mechanics influences migration could improve strategies to recruit repair cells to wound sites or inhibit cancer metastasis.

Correct setup of the substantia nigra requires Reelin-mediated fast, laterally-directed migration of dopaminergic neurons.

Midbrain dopaminergic (mDA) neurons migrate to form the laterally-located substantia nigra pars compacta (SN) and medially-located ventral tegmental area (VTA), but little is known about the underlying cellular and molecular processes. Here we visualize the dynamic cell morphologies of tangentially migrating SN-mDA neurons in 3D and identify two distinct migration modes. Slow migration is the default mode in SN-mDA neurons, while fast, laterally-directed migration occurs infrequently and is strongly associated with bipolar cell morphology. Tangential migration of SN-mDA neurons is altered in absence of Reelin signaling, but it is unclear whether Reelin acts directly on migrating SN-mDA neurons and how it affects their cell morphology and migratory behavior. By specifically inactivating Reelin signaling in mDA neurons we demonstrate its direct role in SN-mDA tangential migration. Reelin promotes laterally-biased movements in mDA neurons during their slow migration mode, stabilizes leading process morphology and increases the probability of fast, laterally-directed migration.

Cell migration: implications for repair and regeneration in joint disease.

Connective tissues within the synovial joints are characterized by their dense extracellular matrix and sparse cellularity. With injury or disease, however, tissues commonly experience an influx of cells owing to proliferation and migration of endogenous mesenchymal cell populations, as well as invasion of the tissue by other cell types, including immune cells. Although this process is critical for successful wound healing, aberrant immune-mediated cell infiltration can lead to pathological inflammation of the joint. Importantly, cells of mesenchymal or haematopoietic origin use distinct modes of migration and thus might respond differently to similar biological cues and microenvironments. Furthermore, cell migration in the physiological microenvironment of musculoskeletal tissues differs considerably from migration in vitro. This Review addresses the complexities of cell migration in fibrous connective tissues from three separate but interdependent perspectives: physiology (including the cellular and extracellular factors affecting 3D cell migration), pathophysiology (cell migration in the context of synovial joint autoimmune disease and injury) and tissue engineering (cell migration in engineered biomaterials). Improved understanding of the fundamental mechanisms governing interstitial cell migration might lead to interventions that stop invasion processes that culminate in deleterious outcomes and/or that expedite migration to direct endogenous cell-mediated repair and regeneration of joint tissues.

Nuclear Deformation During Neutrophil Migration at Sites of Inflammation.

Cell migration is indispensable for various biological processes including angiogenesis, wound healing, and immunity. In general, there are two different migration modes described, the mesenchymal migration mode and the amoeboid migration mode. Neutrophils rapidly migrate toward the sites of injury, infection, and inflammation using the amoeboid migration mode which is characterized by cell polarization and a high migration velocity. During site-directed trafficking of neutrophils from the blood stream into the inflamed tissue, neutrophils must first withstand shear stress while migrating on the 2-dimensional endothelial surface. Subsequently, they have to cross different physical barriers during the extravasation process including the squeezing through the compact endothelial monolayer that comprises the blood vessel, the underlining basement membrane and then the 3-dimensional meshwork of extracellular matrix (ECM) proteins in the tissue. Therefore, neutrophils have to rapidly switch between distinct migration modes such as intraluminal crawling, transmigration, and interstitial migration to pass these different confinements and mechanical barriers. The nucleus is the largest and stiffest organelle in every cell and is therefore the key cellular element involved in cellular migration through variable confinements. This review highlights the importance of nuclear deformation during neutrophil crossing of such confinements, with a focus on transendothelial migration and interstitial migration. We discuss the key molecular components involved in the nuclear shape changes that underlie neutrophil motility and squeezing through cellular and ECM barriers. Understanding the precise molecular mechanisms that orchestrate these distinct neutrophil migration modes introduces an opportunity to develop new therapeutic concepts for controlling pathological neutrophil-driven inflammation.

WASP and SCAR are evolutionarily conserved in actin-filled pseudopod-based motility

Diverse eukaryotic cells crawl through complex environments using distinct modes of migration. To understand the underlying mechanisms and their evolutionary relationships, we must define each mode and identify its phenotypic and molecular markers. In this study, we focus on a widely dispersed migration mode characterized by dynamic actin-filled pseudopods that we call "α-motility." Mining genomic data reveals a clear trend: only organisms with both WASP and SCAR/WAVE-activators of branched actin assembly-make actin-filled pseudopods. Although SCAR has been shown to drive pseudopod formation, WASP's role in this process is controversial. We hypothesize that these genes collectively represent a genetic signature of α-motility because both are used for pseudopod formation. WASP depletion from human neutrophils confirms that both proteins are involved in explosive actin polymerization, pseudopod formation, and cell migration. WASP and WAVE also colocalize to dynamic signaling structures. Moreover, retention of WASP together with SCAR correctly predicts α-motility in disease-causing chytrid fungi, which we show crawl at >30 µm/min with actin-filled pseudopods. By focusing on one migration mode in many eukaryotes, we identify a genetic marker of pseudopod formation, the morphological feature of α-motility, providing evidence for a widely distributed mode of cell crawling with a single evolutionary origin.

Live imaging reveals distinct modes of neutrophil and macrophage migration within interstitial tissues

Cell motility is required for diverse processes during immunity and inflammation. Classically, leukocyte motility is defined as an amoeboid type of migration, however some leukocytes, like macrophages, also employ a more mesenchymal mode of migration. Here, we sought to characterize the mechanisms that regulate neutrophil and macrophage migration in vivo by using real-time imaging of leukocyte motility within interstitial tissues in zebrafish larvae. Neutrophils displayed a rounded morphology and rapid protease-independent motility, lacked defined paxillin puncta, and had persistent rearward polarization of stable F-actin and the microtubule network. By contrast, macrophages displayed an elongated morphology with reduced speed and increased directional persistence and formed paxillin-containing puncta but had a less-defined polarization of the microtubule and actin networks. We also observed differential effects of protease inhibition, microtubule disruption and ROCK inhibition on the efficiency of neutrophil and macrophage motility. Taken together, our findings suggest that larval zebrafish neutrophils and macrophage display distinct modes of migration within interstitial tissues in vivo.

Leukocyte transcellular diapedesis: Rap1b is in control

The neutrophil transmigration across the blood endothelial cell barrier represents the prerequisite step of innate inflammation. It is well known that neutrophils cross the endothelial barrier by transmigrating at the endothelial cell junction (‘paracellular’). However, in vivo and in vitro evidence have clearly demonstrated occurrence of an alternate mode of migration directly through the endothelial cell body (‘transcellular’). Despite our knowledge on mechanisms of transendothelial migration, it remains unclear which factors determine distinct modes of migration. We recently found that the Ras-like Rap1b GTPase limits neutrophil transcellular migration. Rap1b restrains transcellular migration by suppressing Akt-driven invasive protrusions while leaving the paracellular route unaffected. Furthermore, Rap1b limits neutrophil tissue infiltration in mice and prevents hyper susceptibility to endotoxin shock. These findings uncover a novel role for Rap1b in neutrophil migration and inflammation. Importantly, they offer emerging evidences that paracellular and transcellular migration of neutrophils are regulated by separate mechanisms. Here, we discuss the mechanisms of neutrophil transmigration and their clinical importance for vascular integrity and innate inflammation.

T Follicular Helper Cells Have Distinct Modes of Migration and Molecular Signatures in Naive and Memory Immune Responses

B helper follicular T (Tfh) cells are critical for long-term humoral immunity. However, it remains unclear how these cells are recruited and contribute to secondary immune responses. Here we show that primary Tfh cells segregate into follicular mantle (FM) and germinal center (GC) subpopulations that display distinct gene expression signatures. Restriction of the primary Tfh cell subpopulation in the GC was mediated by downregulation of chemotactic receptor EBI2. Following collapse of the GC, memory T cells persisted in the outer follicle where they scanned CD169(+) subcapsular sinus macrophages. Reactivation and intrafollicular expansion of these follicular memory T cells in the subcapsular region was followed by their extrafollicular dissemination via the lymphatic flow. These data suggest that Tfh cells integrate their antigen-experience history to focus T cell help within the GC during primary responses but act rapidly to provide systemic T cell help after re-exposure to the antigen.

Micrometastasis in gastric cancer

Circulating tumor cells (CTCs) are a crucial contributor and indicator for cancer metastasis. The presence of CTCs and other two forms of occult disease including circulating tumor microembolus (CTMs) as well as disseminated tumor cells (DTCs) as a sign for micrometastasis has been associated with adverse survival of both localized and metastatic cancers. However, their assignments in micrometastasis are still not well understood. This mini-review is aimed to provide an overview of the biological features and the clinical impact of CTCs, DTCs and CTM in gastric cancer (GC), and to summarize the technical limitations of detection methods in addition to the significance of distinct migration modes in epithelial–mesenchymal transition (EMT) and gastric micrometastasis. Although there are controversies over current CTC identification strategies, both single and collective migration modes as indispensable parts of micrometastasis are influential participants in GC progression, diagnosis and prognosis. CTCs, CTM and DTCs are not separated forms of the occult disease; however available techniques fail to detect all subsets of them. Therefore, micrometastatic detection should be combined with conventional pathological examinations in order to make more accurate predictions of tumor outcomes. Novel markers further defining the features of CTC subsets are warranted.

Abstract C42: MLKs regulate the activities of the small GTPases, Rho and Rac, to drive both single cell and collective cell migration in breast cancer invasion

Abstract Most mortality of breast cancer patients is caused by metastasis to vital organs. One of the key steps of breast cancer metastasis is invasion through the basement membrane, which is controlled by several signaling cascades including small GTPases, Rho and Rac. Mixed lineage kinases (MLKs) are a family of MAP3Ks that activate multiple MAPK pathways, including the c-Jun N-terminal kinase (JNK) pathway. Our lab has determined that MLK3-JNK signaling is critical for migration, invasion and metastasis of triple negative breast cancer cells. Herein, the effect of a small molecule pan-MLK inhibitor on tumor cell migration in 2D wound healing and in 3D dissemination in collagen type I, the most abundant collagen in the body, was determined. Cancer cells can utilize distinct modes of migration. MDA-MB-231 and 4T1 cells were chosen as models for the study of single and collective cell migration, respectively. In addition, the effect of MLK3 on Rho-GTP and Rac-GTP levels was determined. Data from this study demonstrate that an MLK inhibitor is sufficient to block migration of multiple breast cancer cell lines in wound healing assays. Furthermore, this inhibitor dramatically reduces the dissemination of cancer cell lines that utilize single, as well as, collective cell migration. Silencing of MLK3 results in enhanced Rho activity and decreased Rac activity, as well as defective focal adhesion turnover. Inhibition of the activity of MLKs or the downstream MAPK, JNK, also increases Rho activity, which is accompanied by an increase in the number and/or size of focal adhesions and an increase in actin stress fibers. Consistent with the idea that MLK inhibition reduces migration through relieving Rho suppression, addition of a Rho/ROCK inhibitor, Y-27632, partially rescues the migratory defect of MLK inhibition by reducing peripheral focal adhesions and actin stress fibers. Taken together, these data suggest that MLK3 could play a role in controlling the balance between Rac and Rho activity required for cancer cell migration and invasion. Thus, MLK inhibitors could be therapeutically useful in the context of breast cancer metastasis. Citation Format: Chotirat Rattanasinchai, Sean Misek, Jian Chen, Kathleen A. Gallo. MLKs regulate the activities of the small GTPases, Rho and Rac, to drive both single cell and collective cell migration in breast cancer invasion. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C42.

Rac and Rho GTPases in cancer cell motility control

Rho GTPases represent a family of small GTP-binding proteins involved in cell cytoskeleton organization, migration, transcription, and proliferation. A common theme of these processes is a dynamic reorganization of actin cytoskeleton which has now emerged as a major switch control mainly carried out by Rho and Rac GTPase subfamilies, playing an acknowledged role in adaptation of cell motility to the microenvironment. Cells exhibit three distinct modes of migration when invading the 3 D environment. Collective motility leads to movement of cohorts of cells which maintain the adherens junctions and move by photolytic degradation of matrix barriers. Single cell mesenchymal-type movement is characterized by an elongated cellular shape and again requires extracellular proteolysis and integrin engagement. In addition it depends on Rac1-mediated cell polarization and lamellipodia formation. Conversely, in amoeboid movement cells have a rounded morphology, the movement is independent from proteases but requires high Rho GTPase to drive elevated levels of actomyosin contractility. These two modes of cell movement are interconvertible and several moving cells, including tumor cells, show an high degree of plasticity in motility styles shifting ad hoc between mesenchymal or amoeboid movements. This review will focus on the role of Rac and Rho small GTPases in cell motility and in the complex relationship driving the reciprocal control between Rac and Rho granting for the opportunistic motile behaviour of aggressive cancer cells. In addition we analyse the role of these GTPases in cancer progression and metastatic dissemination.

The molecular mechanisms of transition between mesenchymal and amoeboid invasiveness in tumor cells

Tumor cells exhibit at least two distinct modes of migration when invading the 3D environment. A single tumor cell’s invasive strategy follows either mesenchymal or amoeboid patterns. Certain cell types can use both modes of invasiveness and undergo transitions between them. This work outlines the signaling pathways involved in mesenchymal and amoeboid types of tumor cell motility and summarizes the molecular mechanisms that are involved in transitions between them. The focus is on the signaling of the Rho family of small GTPases that regulate the cytoskeleton-dependent processes taking place during the cell migration. The multiple interactions among the Rho family of proteins, their regulators and effectors are thought to be the key determinants of the particular type of invasiveness. Mesenchymal and amoeboid invasive strategies display different adhesive and proteolytical interactions with the surrounding matrix and the alterations influencing these interactions can also lead to the transitions.