Ameboid Migration Research Articles

YAP silencing by RB1 mutation is essential for small-cell lung cancer metastasis

Small cell lung cancer (SCLC) is highly lethal due to its prevalent metastasis. Most SCLCs have inactivating mutations in TP53 and RB1. We find that loss of YAP expression is key for SCLC cells to acquire rapid ameboid migration and high metastatic potential. YAP functions through its target genes CCN1/CCN2 to inhibit SCLC ameboid migration. RB1 mutation contributes to YAP transcriptional silencing via E2F7, which recruits the RCOR co-repressor complex to YAP promoter. We discover that benzamide family HDAC inhibitors stimulate YAP expression by inhibiting the RCOR-HDAC complex, thereby suppressing SCLC metastasis and improving survival in a mouse model. Our study unveils the molecular and cellular basis underlying SCLC’s high metastatic potential, the previously unrecognized role of YAP in suppressing ameboid migration and tumor metastasis, and the mechanism of YAP transcription regulation involving E2F7, RCOR, and Sin3 HDAC. This study reveals a therapeutic potential of benzamides for SCLC treatment.

Ameboid cell migration through regular arrays of micropillars under confinement

Migrating cells often encounter a wide variety of topographic features—including the presence of obstacles—when navigating through crowded biological environments. Unraveling the impact of topography and crowding on the dynamics of cells is key to better understand many essential physiological processes such as the immune response. We study the impact of geometrical cues on ameboid migration of HL-60 cells differentiated into neutrophils. A microfluidic device is designed to track the cells in confining geometries between two parallel plates with distance h, in which identical micropillars are arranged in regular pillar forests with pillar spacing e. We observe that the cells are temporarily captured near pillars, with a mean contact time that is independent of h and e. By decreasing the vertical confinement h, we find that the cell velocity is not affected, while the persistence reduces; thus, cells are able to preserve their velocity when highly squeezed but lose the ability to control their direction of motion. At a given h, we show that by decreasing the pillar spacing e in the weak lateral confinement regime, the mean escape time of cells from effective local traps between neighboring pillars grows. This effect, together with the increase of cell-pillar contact frequency, leads to the reduction of diffusion constant D. By disentangling the contributions of these two effects on D in numerical simulations, we verify that the impact of cell-pillar contacts on cell diffusivity is more pronounced at smaller pillar spacing.

Effects of vimentin on the migration, search efficiency, and mechanical resilience of dendritic cells

Dendritic cells use amoeboid migration to pass through narrow passages in the extracellular matrix and confined tissue in search for pathogens and to reach the lymph nodes and alert the immune system. Amoeboid migration is a migration mode that, instead of relying on cell adhesion, is based on mechanical resilience and friction. To better understand the role of intermediate filaments in ameboid migration, we studied the effects of vimentin on the migration of dendritic cells. We show that the lymph node homing of vimentin-deficient cells is reduced in our in vivo experiments in mice. Lack of vimentin also reduces the cell stiffness, the number of migrating cells, and the migration speed in vitro in both 1D and 2D confined environments. Moreover, we find that lack of vimentin weakens the correlation between directional persistence and migration speed. Thus, vimentin-expressing dendritic cells move faster in straighter lines. Our numerical simulations of persistent random search in confined geometries verify that the reduced migration speed and the weaker correlation between the speed and direction of motion result in longer search times to find regularly located targets. Together, these observations show that vimentin enhances the ameboid migration of dendritic cells, which is relevant for the efficiency of their random search for pathogens.

Perivascular infiltration reflects subclinical lymph node metastasis in invasive lobular carcinoma.

Invasive lobular carcinoma (ILC) is characterized by discohesive cells due to irreversible loss of E-cadherin expression and multiple satellites, where individual cell migration is evident without disturbance of the stroma. Neoplastic cells sometimes infiltrate the surrounding vessel in satellites. Here, we aimed to clarify the specific role of perivascular infiltration (PVI) and ameboid migration, characterized by nondisturbance of the background stromal structure, in ILCs. A total of 139 cases with ILC and 122 cases with invasive breast carcinoma of no special type (IBC-NST) were evaluated retrospectively. PVI was significantly more common in ILC than in IBC-NST (50% [70 of 139 cases] vs. 9% [11 of 122 cases], p < 0.001). ILC cases with PVI showed a larger pathological tumour size than clinical tumour size (p < 0.01), a higher frequency of pathological node status pN2-pN3 when limited to clinically node-negative cases (p < 0.01) and lower circularity of tumour morphology on imaging (p < 0.01) than ILC cases without PVI. In the pathological evaluation, the intensity and occupancy of tumour cells expressing phospho-myosin light chain 2, which is a hallmark of ameboid migration, were significantly higher in ILC cases with PVI than in those without PVI at the tumour margins (p < 0.05). ILC with PVI is associated with irregular, poorly defined tumour margins and lymph node metastasis without adenopathy, which is difficult to assess using imaging. PVI may be caused by ameboid migration, as shown by the positive expression of phospho-myosin light chain 2. The presence of PVI may be a predictor for clinically node-negative pN2-pN3 in ILC patients.

Computational models of migration modes improve our understanding of metastasis.

Tumor cells migrate through changing microenvironments of diseased and healthy tissue, making their migration particularly challenging to describe. To better understand this process, computational models have been developed for both the ameboid and mesenchymal modes of cell migration. Here, we review various approaches that have been used to account for the physical environment's effect on cell migration in computational models, with a focus on their application to understanding cancer metastasis and the related phenomenon of durotaxis. We then discuss how mesenchymal migration models typically simulate complex cell–extracellular matrix (ECM) interactions, while ameboid migration models use a cell-focused approach that largely ignores ECM when not acting as a physical barrier. This approach greatly simplifies or ignores the mechanosensing ability of ameboid migrating cells and should be reevaluated in future models. We conclude by describing future model elements that have not been included to date but would enhance model accuracy.

Abstract 485: Epidermal growth factor-like repeats of Tenascin C promote melanoma cell invasion, possibly by enabling mesenchymal to ameboid migration transition

Abstract In malignant melanoma, the most dangerous skin cancer, Tenascin-C (TNC) is overexpressed, especially at the invasive fronts of invasive melanomas. We found that the migration and invasiveness of a set of melanoma cell lines correlates with levels of TNC expression suggesting, surprisingly, that the melanoma cells themselves provide much of this matrix component. Adding TNC to matrices enhanced individual cell migration in cells that did not express much TNC, whereas it did not enhance the rapid motility of the cells expressing high levels. As TNC is a multidomain protein, the signaling pathways that promote this motogenic invasiveness are not obvious. Herein, we examined the potential role of the EGF-like repeats of TNC that have been shown to preferentially activate motogenic-signaling cascades. To analyze the effects of EGFL signaling on melanoma migration and invasion, we overexpressed TNC fragment containing the assembly and EGFL domains of TNC (TNCEGFL). Interestingly, this led to impaired cell migration in in vitro wound healing assays and live cell tracking experiments. This was likely due to a shift in the adhesion-contractility balance during cell migration. TNCEGFL expressing cells presented a spindle-shaped morphology and formed cords in culture. Expression of TNCEGFL delayed melanoma cell attachment and spreading, as determined in cell adhesion and inverted centrifugation assays. This anti-adhesive phenotype was concomitant with increased MLC2 phosphorylation. Inhibition of ROCK activity, which drives transcellular contractility, restored adhesion of TNCEGFL expressing cells and improved migration in 2D. In distinction to the findings in 2D, TNCEGFL expressing cells had higher invasion potential in Matrigel invasion assays, with cells expressing high TNC having rounded “ameboid” morphology, underlying the importance of morphological shifts, and possibly ROCK signaling, in 3D movement through barrier matrices during invasion and dissemination. These results imply that TNCEGFL repeats are involved in melanoma invasion both by directly influencing cell migration and indirectly changing the tumor microenvironment composition, possibly allowing transition from mesenchymal to ameboid migrational mode. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 485. doi:1538-7445.AM2012-485

What makes cells move: requirements and obstacles for spontaneous cell motility

Movement of individual cells and of cellular cohorts, chains or sheets requires physical forces that are established through interactions of cells with their environment. In vivo, migration occurs extensively during embryonic development and in adults during wound healing and tumorigenesis. In order to identify the molecular events involved in cell movement, in vitro systems have been developed. These have contributed to the definition of a number of molecular pathways put into play in the course of migratory behaviours, such as mesenchymal and amoeboid movement. More recently, our knowledge of migratory modes has been enriched by analyses of cells exploring and moving through three-dimensional (3D) matrices. While the cells' morphologies differ in 2D and 3D environments, the basic mechanisms that put a cellular body into motion are remarkably similar. Thus, in both 2D and 3D, the polarity of the migrating cell is initially defined by a specific subcellular localization of signalling molecules and components of molecular machines required for motion. While the polarization can be initiated either in response to extracellular signalling or be a chance occurrence, it is reinforced and sustained by positive feedback loops of signalling molecules. Second, adhesion to a substratum is necessary to generate forces that will propel the cell engaged in either mesenchymal or ameboid migration. For collective cell movement, intercellular coordination constitutes an additional requirement: a cell cohort remains stationary if individual cells pull in opposite directions. Finally, the availability of space to move into is a general requirement to set cells into motion. Lack of free space is probably the main obstacle for migration of most healthy cells in an adult multicellular organism. Thus, the requirements for cell movement are both intrinsic to the cell, involving coordinated signalling and interactions with molecular machines, and extrinsic, imposed by the physicochemical nature of the environment. In particular, the geometry and stiffness of the support act on a range of signalling pathways that induce specific cell migratory responses. These issues are discussed in the present review in the context of published work and our own data on collective migration of hepatocyte cohorts.

Cytohesin-1 controls the activation of RhoA and modulates integrin-dependent adhesion and migration of dendritic cells

AbstractAdhesion and motility of mammalian leukocytes are essential requirements for innate and adaptive immune defense mechanisms. We show here that the guanine nucleotide exchange factor cytohesin-1, which had previously been demonstrated to be an important component of beta-2 integrin activation in lymphocytes, regulates the activation of the small GTPase RhoA in primary dendritic cells (DCs). Cytohesin-1 and RhoA are both required for the induction of chemokine-dependent conformational changes of the integrin beta-2 subunit of DCs during adhesion under physiological flow conditions. Furthermore, use of RNAi in murine bone marrow DCs (BM-DCs) revealed that interference with cytohesin-1 signaling impairs migration of wild-type dendritic cells in complex 3D environments and in vivo. This phenotype was not observed in the complete absence of integrins. We thus demonstrate an essential role of cytohesin-1/RhoA during ameboid migration in the presence of integrins and further suggest that DCs without integrins switch to a different migration mode.

P311-induced myofibroblasts exhibit ameboid-like migration through RalA activation

We previously showed that P311, an intracellular protein involved in cell migration, is found in human wound myofibroblast precursors (proto-myofibroblasts) and myofibroblasts. Furthermore, by binding to the TGF-β1 latency associated protein (LAP), P311 induced NIH 3T3 cells to transform into non-fibrogenic myofibroblasts characterized by lack of TGF-β1 production. Here we demonstrate that P311-induced myofibroblasts migrate in an ameboid rather than a mesenchymal pattern. Ameboid migration is characterized by lack of focal adhesions and stress fibers, absence of integrins and MMPs clustering/activation and changes in small GTPases activity, all leading to increased cell motility. P311-induced ameboid migration depended on activation of the GTPase RalA and was reverted to mesenchymal-type migration by RalA RNA interference. Ameboid migration was conserved in cells plated on fibrin, the initial wound matrix, but was switched back to mesenchymal-type migration by collagen I, the main ECM component in late stages of wound healing. TGF-β1, the major stimulus of collagen production during wound repair, also reversed the ameboid phenotype to mesenchymal. Our studies therefore suggest that, by inducing RalA activity, P311 promotes a motile proto-myofibroblast and myofibroblast phenotype specifically adapted to rapidly populate the initial wound matrix.

The biology of cell locomotion within three-dimensional extracellular matrix.

Cell migration in three-dimensional (3-D) extracellular matrix (ECM) is not a uniform event but rather comprises a modular spectrum of interdependent biophysical and biochemical cell functions. Haptokinetic cell migration across two-dimensional (2-D) surfaces consists of at least three processes: (i) the protrusion of the leading edge for adhesive cell-substratum interactions is followed by (ii) contraction of the cell body and (iii) detachment of the trailing edge. In cells of flattened morphology migrating slowly across 2-D substrate, contact-dependent clustering of adhesion receptors including integrins results in focal contact and stress fiber formation. While haptokinetic migration is predominantly a function of adhesion and deadhesion events lacking spatial barriers towards the advancing cell body, the biophysics of the tissues require a set of cellular strategies to overcome matrix resistance. Matrix barriers force the cells to adapt their morphology and change shape and/or enzymatically degrade ECM components, either by contact-dependent proteolysis or by protease secretion. In 3-D ECM, in contrast to 2-D substrate, the cell shape is mostly bipolar and the cytoskeletal organization is less stringent, frequently lacking discrete focal contacts and stress fibers. Morphologically large spindle-shaped cells (i.e., fibroblasts, endothelial cells, and many tumor cells) of high integrin expression and strong cytoskeletal contractility utilize integrin-dependent migration strategies that are coupled to the capacity to reorganize ECM. In contrast, a more dynamic ameboid migration type employed by smaller cells expressing low levels of integrins (i.e., T lymphocytes, dendritic cells, some tumor cells) is characterized by largely integrin-independent interaction strategies and flexible morphological adaptation to preformed fiber strands, without structurally changing matrix architecture. In tumor invasion and angiogenesis, migration mechanisms further comprise the migration of entire cell clusters or strands maintaining stringent cell-cell adhesion and communication while migrating. Lastly, cellular interactions, enzyme and cytokine secretion, and tissue remodeling provided by reactive stroma cells (i.e. fibroblasts and macrophages) contribute to cell migration. In conclusion, depending on the cellular composition and tissue context of migration, diverse cellular and molecular migration strategies can be developed by different cell types.

Studies on the cytodynamics of human endometrial regeneration: III. In vitro short-term incubation historadioautography

The proliferation kinetics of endometrial regeneration were assessed by tracing nucleoprotein precursor, radiothymidine. Increased isotope uptake occurred by cycle day 3 and was confined to the gland cells of denuded stratum basale (basal layer) and persistent uterine lining, bordering peripheral areas of denudation. This increase resulted in the formation of flattened surface epithelial cells, which initially were devoid of significant deoxyribonucleic acid synthesis. Such alterations are consistent with changes in cells involved in ameboid migration. Maturation of migrating cells was accompanied by a significant rise in labeling indices and replication at cycle days 3 and 4. By cycle day 5, endometrial repair was complete. The findings indicate that, following physiologic loss of stratum functionale, endometrial healing involves both migration and replication of surface cells, which originate from the gland stumps of the residual basal layer and the persistent surface epithelium adjacent to the periphery of denuded endometrium.

Studies on the cytodynamics of experimental endometrial regeneration in the rabbit: I. Historadioautography and ultrastructure

The kinetic and fine structural characteristics and site of origin of the resurfacing uterine lining, as well as the influence of ovarian hormonal stimuli on the reparative processes of experimentally injured endometrium in the rabbit, were studied by means of in vivo historadioautography and electron microscopy. The first-generation, resurfacing spindle-shaped cells lacked mitosis and expanded by ameboid migration. This was followed by a rapid increase in nuclear deoxyribonucleic acid synthesis, which presumably provided for accelerated mitosis-mediated surface epithelial proliferation and maturation. By Day 3 after injury, endometrial regeneration was completed with a retum of labeling index to base-line levels. The newly formed surface lining was derived exclusively from the persistent surface epithelium bordering the denuded regions. The isotope uptake and morphologic alterations of the regenerating endometrium in castrated rabbits which were similar to those of intact rabbits suggest that endometrial repair is not mediated by the hormones of the reproductive system.

A study of neurogenesis in the forebrain of opossum pouch young.

Morphological changes of growing axons and dendrites are traced in rapid Golgi preparations of the olfactory bulb and the striatum and the parietal, cingulate, and hippocampal cortex. In age-graded series of opossum pouch young changes in the form and position of the differentiating neurons are related to the genesis of the neuronal architecture of each region. In the olfactory bulb there is a specific sequence of changes, starting with the formation of the olfactory glomeruli and the apical dendrites of the mitral cells and ending with the formation of the granule cell processes and the lateral dendrites of the mitral cells. In the ganglionic eminence, the external processes of neuroblasts form axons as they pass from the matrix zone into the presumptive striatal neuropil. Subsequently the perikarya move through the external processes from the matrix zone to the origins of the axons, the external processes disappear, and the dendrites of the striatal neurons start to form. The region of neuronal differentiation is defined by the intersection of the primitive processes of the neuroblasts with the growth cones of specific afferent axons. In the cerebral cortex the neuroblasts resemble primitive epithelial cells (spongioblasts). Their perikarya are in the matrix zone; their primitive processes extend between the internal and external limiting membranes. When afferent growth cones appear in the mantle zone of the parietal area, the perikarya of the pyramidal neuroblasts move to the external limiting layer through their primitive processes. Next these processes disappear; then axons and dendrites differentiate. The order of these events differs in other cortical areas. There is no sign of free, ameboid migration of the neuroblasts. The classical problems of neurogenesis are redefined in the light of these findings.

Effects of in Vitro X-Irradiation on the Ameboid Migration of Human Leukocytes

Human venous blood was x irradiated in vitro, and the ameboid migration of polymorphonuclear leukocytes was measured by the capillary tube technique. Capillary tubes filled with heparinized blood were centrifuged, resulting in a basal layer of red cells, an intermediate buffy coat of leukocytes, and an uppermost layer of plasma. After irradiation, the tubes were incubated vertically, and the distance the leukocytes migrated into the plasma was measured by means of an ocular micrometer. The migration of leukocytes in blood x irradiated with doses ranging from 25 to 150,000 rads was not significantly altered. Irradiation with 200 krad, however, caused a marked inhibition of leukocyte migration. When nonirradiated cells were combined with plasma irradiated without cells, the same degree of inhibition occurred as when cells and plasma were irradiated together. This latter finding indicates that the observed inhibition resulted not from an effect of irradiation on the leukocytes directly, but indirectly through a change in the plasma. (auth)

The effect of certain psychopharmacological agents on the ameboid migration of human leucocytes in vitro

THE ameboid migration of human leucocytes in capillary tubes has been shown to be inhibited by relatively low concentrations of hydrocortisonel. Further, this inhibitory effect was found to be reversed by the addition of prochlorperazine 2. The sensitivity of leucocyte migration to these agents suggested that the capillary tube technique might be useful for the study of pharmacological agents at the cellular level. The present report presents some preliminary findings which indicate that certain psychopharmacological agents do have a measurable effect on the ameboid migration of leucocytes.

Physiological and radioistopic evidence for the binding of hydrocortisone by human leucocytes in vitro.

The ameboid migration of human leucocyles, as measured in capillary tubes, is inhibited by the addition of 10 μg/ml. of hydrocortisone. If the leucocytes are incubated with 10 10 μg/ml, hydrocortisone, washed, and then put into plasma to which no hydrocortisone has been added, the inhibition still occurs. If leucocytes are incubated with 10 10 μg/ml, of hydrocortisone, washed, and then put in plasma to which 10 10 μg/ml, of hydrocortisone is added, no more inhibition occurs than if the hydrocortisone were present only during the incubation or only during the migration period. A period of 5 minutes is sufficient for the incubation with hydrocortisone to cause an inhibition in leucocyte migration. Incubation of leucocytes with the hydrocortisone is effective in a non-protein salt-solution, as well as in plasma. This physiological evidence of the binding of hydrocortisone by leucocytes in vitro is substantiated by the detection of radioactivity in leucocytes after incubation with hydrocortisone- 4-C14.

The in vitro action of hydrocortisone on leucocyte migration.

Hydrocortisone inhibits the ameboid migration of human leucocytes when added in vitro. The dose-response curve for the reaction between this steroid and leucocytes can be best expressed by a logarithmic plot of the steroid concentrations. Tetrahydrocortisone and desoxycorticosterone had no effect on in vitro leucotyte migration.