Random Cell Motility Research Articles

Characteristics of the Speed- Acceleration Phase Diagram of Migrating Cells

Abstract The behavior of cell movement is one of the most interesting biological problems in biology, physics and medicine. In this study, we conducted experiments to investigate the characteristics of random cell motion during migration. By observing cell motion trajectories under a microscope, we employed nonlinear dynamics method to construct a speed- acceleration phase diagram. Our analysis revealed the presence of a fixed point in this phase diagram, which suggests that migrating cells possess a stable state. Cells that deviate from this stable state display a tendency to return to it, following the streamline trends of an attractor structure in the phase diagram. We derived a set of characteristic values describing cell motion, encompassing inherent speed, inherent acceleration, characteristic time for speed change, and characteristic time for acceleration change. We developed a differential equation model based on experimental data and conducted numerical calculations. The computational results align with the findings obtained from experimental results. Our research suggests that the asymmetrical characteristics observed in cell motion near an inherent speed primarily arise from the characteristics of inherent acceleration of cells.

In-vivo Evaluation of Nephroprotective Activity of Naringenin against ADPKD and MDCK-derived Cysts

Aim: Determining the in-vivo assessment of Naringenin’s nephroprotective efficacy against Autosomal dominant polycystic kidney disease (ADPKD) and Madin-Darby canine kidney (MDCK) derived cysts. Method: Our research on the molecular mechanism of naringenin, a flavonoid present in plants and berries that has been shown to limit cell growth and protect against cancer in in-vitro and animal models, was conducted using dictyostelium, a simple, controllable biomedical model. Cultured MDCK cells were used to generate differentiated tubule cells, and these findings were extrapolated to a human kidney model employing these cells. Results: While naringenin inhibited growth in dictyostelium, it had no effect on development. In a random-gene-knockout screen, a TRPP2 (polycystin-2) knockout mutant was discovered to be resistant to naringenin’s effects on growth and random-cell movement. Changes in the divalent transient receptor cause polycystic kidney disease type 2 potential cation channel TRPP2. We found that the growth of cysts and MDCK cells might be inhibited by naringenin. Partial resistance to naringenin was achieved in this model by lowering TRPP2 levels via siRNA, as evidenced by the presence of larger cysts following treatment with 3 and 10 M naringenin compared to controls. Naringenin had no effect on chloride secretion. Conclusion: Naringenin’s influence on cell proliferation is mediated by TRPP2 in both dictyostelium and mammalian kidney cells, despite their vast evolutionary distance from one another (polycystin-2). Naringenin will be the subject of more research as a possible new therapeutic treatment for ADPKD

Haptotaxis–Chemotaxis Tumor Invasion with Weighted Mass-Conservative Quantity

We consider a haptotaxis–chemotaxis tumor invasion model, which is derived by mixing two models. One model is proposed by Chaplain and Anderson in 2002 in which the haptotaxis effect is considered. The other has a conservative quantity and is proposed by Fujie, Yokota, and the author in 2014 in which the chemotaxis effect is considered. In addition, the coefficient of random motility of tumor cells is degenerate and depends on chemoattractant substance, extracellular matrix, and matrix degrading enzymes, which are unknown in the model. This situation gives quasi-variational structures to not only time-dependent subdifferentials of convex functions but also inner products on a real Hilbert space $$(H^1(\Omega ))^*$$ . In order to overcome the mathematical difficulty, which comes from such quasi-variational structures and mass-conservative quantity, we apply the general theory of quasi-variational evolution inclusions with conservative properties on real Hilbert spaces which is established by the author in 2023 and show the existence of strong solutions to the tumor invasion model under consideration.

APC-driven actin nucleation powers collective cell dynamics in colorectal cancer cells

Cell remodeling relies on dynamic rearrangements of cell contacts powered by the actin cytoskeleton. The tumor suppressor adenomatous polyposis coli (APC) nucleate actin filaments (F-actin) and localizes at cell junctions. Whether APC-driven actin nucleation acts in cell junction remodeling remains unknown. By combining bioimaging and genetic tools with artificial intelligence algorithms applied to colorectal cancer cell, we found that the APC-dependent actin pool contributes to sustaining levels of F-actin, as well as E-cadherin and occludin protein levels at cell junctions. Moreover, this activity preserved cell junction length and angle, as well as vertex motion and integrity. Loss of this F-actin pool led to larger cells with slow and random cell movement within a sheet. Our findings suggest that APC-driven actin nucleation promotes cell junction integrity and dynamics to facilitate collective cell remodeling and motility. This offers a new perspective to explore the relevance of APC-driven cytoskeletal function in gut morphogenesis.

3D-Engineered Scaffolds to Study Microtubes and Localization of Epidermal Growth Factor Receptor in Patient-Derived Glioma Cells.

A major obstacle in glioma research is the lack of in vitro models that can retain cellular features of glioma cells in vivo. To overcome this limitation, a 3D-engineered scaffold, fabricated by two-photon polymerization, is developed as a cell culture model system to study patient-derived glioma cells. Scanning electron microscopy, (live cell) confocal microscopy, and immunohistochemistry are employed to assess the 3D model with respect to scaffold colonization, cellular morphology, and epidermal growth factor receptor localization. Both glioma patient-derived cells and established cell lines successfully colonize the scaffolds. Compared to conventional 2D cell cultures, the 3D-engineered scaffolds more closely resemble in vivo glioma cellular features and allow better monitoring of individual cells, cellular protrusions, and intracellular trafficking. Furthermore, less random cell motility and increased stability of cellular networks is observed for cells cultured on the scaffolds. The 3D-engineered glioma scaffolds therefore represent a promising tool for studying brain cancer mechanobiology as well as for drug screening studies.

Dictyostelium discoideum cells retain nutrients when the cells are about to outgrow their food source.

Dictyostelium discoideum is a unicellular eukaryote that eats bacteria, and eventually outgrows the bacteria. D. discoideum cells accumulate extracellular polyphosphate (polyP), and the polyP concentration increases as the local cell density increases. At high cell densities, the correspondingly high extracellular polyP concentrations allow cells to sense that they are about to outgrow their food supply and starve, causing the D. discoideum cells to inhibit their proliferation. In this report, we show that high extracellular polyP inhibits exocytosis of undigested or partially digested nutrients. PolyP decreases plasma membrane recycling and apparent cell membrane fluidity, and this requires the G protein-coupled polyP receptor GrlD, the polyphosphate kinase Ppk1 and the inositol hexakisphosphate kinase I6kA. PolyP alters protein contents in detergent-insoluble crude cytoskeletons, but does not significantly affect random cell motility, cell speed or F-actin levels. Together, these data suggest that D. discoideum cells use polyP as a signal to sense their local cell density and reduce cell membrane fluidity and membrane recycling, perhaps as a mechanism to retain ingested food when the cells are about to starve. This article has an associated First Person interview with the first author of the paper.

Abstract A040: Intra-epithelia cell dynamics shape evolutionary dynamics and selection of therapy resistant clones in lung cancer

Abstract Lung cancer is a leading cause of cancer-related deaths. Despite enhanced characterization of genomic complexity and intra-tumor heterogeneity that underpin therapy failure, the mechanistic and predictive factors shaping and steering evolutionary dynamics remain less clear. In this study, we combined experimental, computational, and tumor molecular data analysis approach to investigate factors and rules governing evolutionary dynamics in lung cancer. A lineage tracing study in a BL/6 KRASG12D TP53mutant lung adenocarcinoma cell line model uncovers unexpected variation in the long-term fate of clones with no selective advantage or disadvantage, with clones arising near the edge of tumor colonies being favored to expand. An agent-based computational model of cell proliferation and cell-cell physical interaction, incorporating the experimentally driven assumption of compression-induced non-proliferation, recapitulated the growth patterns of tumor colonies and unequal expansion of clones. By further implementing cell random motility, the model predicts that enhanced motility destabilizes cell-cell cohesion and causes spatial clone intermixing with altered size distributions. Corroborating the observations in silico, experimental manipulations that increase the migratory capabilities of cancer cells relieve density-driven growth arrest and lead to clone intermixing in vitro. Furthermore, lung cancer cell lines with more migratory properties exhibit less propensity for suppression of proliferation. To relate findings in our models to tumor data and to infer the degree of clone intermixing in patients, we implemented a mutational process in the computational model and extracted features of variant allele frequency that enable to distinguish model runs with higher or lower degree of clone intermixing. Inference in lung adenocarcinomas in the TRACERx (TRAcking Cancer Evolution through therapy (Rx)) Lung study using these features, cross-referenced with RNA sequencing data in the same tumors, reveals notable correlation between mechanisms driving cell migration in our experimental models and inter-subclone mixing in clinical samples. Finally, modelling the competition between sensitive and mutagenesis-derived resistant clones under a virtual cytotoxic therapy predicts an accelerated selection of therapy resistant clones under conditions with enhanced cell motility. In summary, our integrated study demonstrates that intra-epithelia cell dynamics dictates the fates of clones both under neutral evolution and under selective pressures imposed by therapy. Citation Format: Xiao Fu, Ajay Bhargava, Sasha Bailey, Dhruva Biswas, Carlos Martinez Ruiz, Sunil Kumar, Paul French, Nicholas McGranahan, Charles Swanton, Paul A. Bates, Erik Sahai. Intra-epithelia cell dynamics shape evolutionary dynamics and selection of therapy resistant clones in lung cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A040.

Individual based Modelling of Biofilm Colonisation

Bacterial biofilms on implants or host tissue can cause unwanted infections. One of the main factors for the pathogenic biofilm growth is the initial adhesion of the bacteria. In this project a framework for systematic studies of the bacterial adhesion is provided. Therefore, an Individual based Model (IbM) is set up in the IbM solver NUFEB, an extension of the molecular dynamics code LAMMPS, which integrates Newton's equations of motion for the cells that interact via various forces. Experimentally measured adhesion forces are implemented for the cell-wall and cell-cell interaction. Furthermore, the random movement of planktonic cells is included into the IbM software. A sensitivity analysis of important parameters and an exemplary simulation of the attachment are performed. Thus, the attachment of the bacterial cells and the initial growth of a biofilm can be qualitatively modelled.

Cortical waves mediate the cellular response to electric fields.

Electrotaxis, the directional migration of cells in a constant electric field, is important in regeneration, development, and wound healing. Electrotaxis has a slower response and a smaller dynamic range than guidance by other cues, suggesting that the mechanism of electrotaxis shares both similarities and differences with chemical-gradient-sensing pathways. We examine a mechanism centered on the excitable system consisting of cortical waves of biochemical signals coupled to cytoskeletal reorganization, which has been implicated in random cell motility. We use electro-fused giant Dictyostelium discoideum cells to decouple waves from cell motion and employ nanotopographic surfaces to limit wave dimensions and lifetimes. We demonstrate that wave propagation in these cells is guided by electric fields. The wave area and lifetime gradually increase in the first 10 min after an electric field is turned on, leading to more abundant and wider protrusions in the cell region nearest the cathode. The wave directions display 'U-turn' behavior upon field reversal, and this switch occurs more quickly on nanotopography. Our results suggest that electric fields guide cells by controlling waves of signal transduction and cytoskeletal activity, which underlie cellular protrusions. Whereas surface receptor occupancy triggers both rapid activation and slower polarization of signaling pathways, electric fields appear to act primarily on polarization, explaining why cells respond to electric fields more slowly than to other guidance cues.

Rectified random cell motility as a mechanism for embryo elongation.

The body of vertebrate embryos forms by posterior elongation from a terminal growth zone called the tail bud. The tail bud is a source of highly motile cells that eventually constitute the presomitic mesoderm (PSM), a tissue that plays an important role in elongation movements. PSM cells establish an anterior-posterior cell motility gradient that parallels a gradient associated with the degradation of a specific cellular signal (FGF) known to be implicated in cell motility. Here, we combine the electroporation of fluorescent reporters in the PSM with time-lapse imaging in the chicken embryo to quantify cell diffusive movements along the motility gradient. We show that a simple microscopic model for random cell motility induced by FGF activity along with geometric confinement leads to rectified tissue elongation consistent with our observations. A continuum analog of the microscopic model leads to a macroscopic mechano-chemical model for tissue extension that couples FGF activity-induced cell motility and tissue rheology, and is consistent with the experimentally observed speed and extent of elongation. Together, our experimental observations and theoretical models explain how the continuous addition of cells at the tail bud combined with lateral confinement can be converted into oriented movement and drive body elongation.

A chemorepellent inhibits local Ras activation to inhibit pseudopod formation to bias cell movement away from the chemorepellent.

The ability of cells to sense chemical gradients is essential during development, morphogenesis, and immune responses. Although much is known about chemoattraction, chemorepulsion remains poorly understood. Proliferating Dictyostelium cells secrete a chemorepellent protein called AprA. AprA prevents pseudopod formation at the region of the cell closest to the source of AprA, causing the random movement of cells to be biased away from the AprA. Activation of Ras proteins in a localized sector of a cell cortex helps to induce pseudopod formation, and Ras proteins are needed for AprA chemorepulsion. Here we show that AprA locally inhibits Ras cortical activation through the G protein–coupled receptor GrlH, the G protein subunits Gβ and Gα8, Ras protein RasG, protein kinase B, the p21-activated kinase PakD, and the extracellular signal–regulated kinase Erk1. Diffusion calculations and experiments indicate that in a colony of cells, high extracellular concentrations of AprA in the center can globally inhibit Ras activation, while a gradient of AprA that naturally forms at the edge of the colony allows cells to activate Ras at sectors of the cell other than the sector of the cell closest to the center of the colony, effectively inducing both repulsion from the colony and cell differentiation. Together, these results suggest that a pathway that inhibits local Ras activation can mediate chemorepulsion.

Mechanism underlying dynamic scaling properties observed in the contour of spreading epithelial monolayer.

We found evidence of dynamic scaling in the spreading of Madin-Darby canine kidney (MDCK) cell monolayer, which can be characterized by the Hurst exponent α=0.86 and the growth exponent β=0.73, and theoretically and experimentally clarified the mechanism that governs the contour shape dynamics. Dynamic scaling refers to the roughness of the surface scales, both spatially and temporally. During the spreading of the monolayer, it is known that so-called leader cells generate the driving force and lead the other cells. Our time-lapse observations of cell behavior showed that these leader cells appeared at the early stage of the spreading and formed the monolayer protrusion. Informed by these observations, we developed a simple mathematical model that included differences in cell motility, cell-cell adhesion, and random cell movement. The model reproduced the quantitative characteristics obtained from the experiment, such as the spreading speed, the distribution of the increment, and the dynamic scaling law. Analysis of the model equation shows that the model can reproduce different scaling laws from (α=0.5,β=0.25) to (α=0.9,β=0.75), where the exponents α and β are determined by two dimensionless quantities determined by the microscopic cell behavior. From the analytical result, parameter estimation from the experimental results was achieved. The monolayer on the collagen-coated dishes showed a different scaling law, α=0.74,β=0.68, suggesting that cell motility increased ninefold. This result was consistent with the assay of the single-cell motility. Our study demonstrated that the dynamics of the contour of the monolayer were explained by the simple model, and we propose a mechanism that exhibits the dynamic scaling property.

Tracking Single Cells Motility on Different Substrates

Motility is a key property of a cell, required for several physiological processes, including embryonic development, axon guidance, tissue regeneration, gastrulation, immune response, and cancer metastasis. Therefore, the ability to examine cell motility, especially at a single cell level, is important for understanding various biological processes. Several different assays are currently available to examine cell motility. However, studying cell motility at a single cell level can be costly and/or challenging. Here, we describe a method of tracking random cell motility on different substrates such as glass, tissue-culture polystyrene, and type I collagen hydrogels, which can be modified to generate different collagen network microstructures. In this study we tracked MDA-MB-231 breast cancer cells using The CytoSMARTTM System (Lonza Group, Basel, Switzerland) for live cell imaging and assessed the average cell migration speed using ImageJ and wrMTrck plugin. Our cost-effective and easy-to-use method allows studying cell motility at a single cell level on different substrates with varying degrees of stiffness and varied compositions. This procedure can be successfully performed in a highly accessible manner with a simple setup.

The Roles of Signaling in Cytoskeletal Changes, Random Movement, Direction-Sensing and Polarization of Eukaryotic Cells.

Movement of cells and tissues is essential at various stages during the lifetime of an organism, including morphogenesis in early development, in the immune response to pathogens, and during wound-healing and tissue regeneration. Individual cells are able to move in a variety of microenvironments (MEs) (A glossary of the acronyms used herein is given at the end) by suitably adapting both their shape and how they transmit force to the ME, but how cells translate environmental signals into the forces that shape them and enable them to move is poorly understood. While many of the networks involved in signal detection, transduction and movement have been characterized, how intracellular signals control re-building of the cyctoskeleton to enable movement is not understood. In this review we discuss recent advances in our understanding of signal transduction networks related to direction-sensing and movement, and some of the problems that remain to be solved.

Automated cell cluster analysis provides insight into multi-cell-type interactions between immune cells and their targets

Understanding interactions between immune cells and their targets is an important step on the path to fully characterizing the immune system, and in doing so, learning how it combats disease. Many studies of these interactions have a narrow focus, often looking only at a binary result of whether or not a specific treatment was successful or only focusing on the interactions between two individual cells. Therefore, in an effort to more comprehensively study multicellular interactions among immune cells and their targets, we used in vitro longitudinal time-lapse imaging and developed an automated cell cluster analysis tool, or macro, to investigate the formation of cell clusters. In particular, we investigated the behavior of cancer-specific CD8+ and CD4+ T cells on how they interact around their targets: cancer cells and antigen-presenting cells. The macro that we established allowed us to examine these large-scale clustering behaviors taking place between those four cell types. Thus, we were able to distinguish directed immune cell clustering from random cell movement. Furthermore, this macro can be generalized to be applicable to systems consisting of any number of differently labeled species and can be used to track clustering behaviors and compare them to randomized simulations.

Time-lapse Imaging of Mouse Macrophage Chemotaxis

Chemotaxis is receptor-mediated guidance of cells along a chemical gradient, whereas chemokinesis is the stimulation of random cell motility by a chemical. Chemokinesis and chemotaxis are fundamental for the mobilization and deployment of immune cells. For example, chemokines (chemotactic cytokines) can rapidly recruit circulating neutrophils and monocytes to extravascular sites of inflammation. Chemoattractant receptors belong to the large family of G protein-coupled receptors. How chemoattractant (i.e., ligand) gradients direct cell migration via G protein-coupled receptor signaling is not yet fully understood. In the field of immunology, neutrophils are popular model cells for studying chemotaxis in vitro. Here we describe a real-time two-dimensional (2D) chemotaxis assay tailored for mouse resident macrophages, which have traditionally been more difficult to study. Macrophages move at a slow pace of ~1 µm/min on a 2D surface and are less well suited for point-source migration assays (e.g., migration towards the tip of a micropipette filled with chemoattractant) than neutrophils or Dictyostelium discoideum, which move an order of magnitude faster. Widely used Transwell assays are useful for studying the chemotactic activity of different substances, but do not provide information on cell morphology, velocity, or chemotactic navigation. Here we describe a time-lapse microscopy-based macrophage chemotaxis assay that allows quantification of cell velocity and chemotactic efficiency and provides a platform to delineate the transducers, signal pathways, and effectors of chemotaxis.

Time-lapse Imaging of Mouse Macrophage Chemotaxis.

Chemotaxis is receptor-mediated guidance of cells along a chemical gradient, whereas chemokinesis is the stimulation of random cell motility by a chemical. Chemokinesis and chemotaxis are fundamental for the mobilization and deployment of immune cells. For example, chemokines (chemotactic cytokines) can rapidly recruit circulating neutrophils and monocytes to extravascular sites of inflammation. Chemoattractant receptors belong to the large family of G protein-coupled receptors. How chemoattractant (i.e., ligand) gradients direct cell migration via G protein-coupled receptor signaling is not yet fully understood. In the field of immunology, neutrophils are popular model cells for studying chemotaxis in vitro. Here we describe a real-time two-dimensional (2D) chemotaxis assay tailored for mouse resident macrophages, which have traditionally been more difficult to study. Macrophages move at a slow pace of ~1 µm/min on a 2D surface and are less well suited for point-source migration assays (e.g., migration towards the tip of a micropipette filled with chemoattractant) than neutrophils or Dictyostelium discoideum, which move an order of magnitude faster. Widely used Transwell assays are useful for studying the chemotactic activity of different substances, but do not provide information on cell morphology, velocity, or chemotactic navigation. Here we describe a time-lapse microscopy-based macrophage chemotaxis assay that allows quantification of cell velocity and chemotactic efficiency and provides a platform to delineate the transducers, signal pathways, and effectors of chemotaxis.

Random Cell Motion Enhances the Capacity of Cell-Cell Communication

Cells communicate with each other via the transmission and reception of signal molecules. In this paper, we test our hypothesis that random cell motion can enhance cell-cell communication. We first conducted cell-tracking experiments to obtain motility parameters of endothelial cells, and then used the molecular communication framework to obtain the achievable rates at which cells can communicate. Our results show that random cell motion can benefit cell-cell communication when signal molecules that cells use are an order of magnitude less-diffusive than well-known vascular endothelial growth factors.

Quantitative Proteomic Analysis of Small and Large Extracellular Vesicles (EVs) Reveals Enrichment of Adhesion Proteins in Small EVs.

Extracellular vesicles (EVs) are important mediators of cell-cell communication due to their cargo content of proteins, lipids, and RNAs. We previously reported that small EVs (SEVs) called exosomes promote directed and random cell motility, invasion, and serum-independent growth. In contrast, larger EVs (LEVs) were not active in those assays, but might have unique functional properties. In order to identify protein cargos that may contribute to different functions of SEVs and LEVs, we used isobaric tags for relative and absolute quantitation (iTRAQ)-liquid chromatography (LC) tandem mass spectrometry (MS) on EVs isolated from a colon cancer cell line. Bioinformatics analyses revealed that SEVs are enriched in proteins associated with cell-cell junctions, cell-matrix adhesion, exosome biogenesis machinery, and various signaling pathways. In contrast, LEVs are enriched in proteins associated with ribosome and RNA biogenesis, processing, and metabolism. Western blot analysis of EVs purified from two different cancer cell types confirmed the enrichment of cell-matrix and cell-cell adhesion proteins in SEVs. Consistent with those data, we found that cells exhibit enhanced adhesion to surfaces coated with SEVs compared to an equal protein concentration of LEVs. These data suggest that a major function of SEVs is to promote cellular adhesion.

Activin A, a Potential Key Factor of the Malignant Bone Marrow Niche, Enhances B-Cell Precursor-Acute Lymphoblastic Leukemic Cell Migratory and Invasive Properties

The bone marrow (BM) represents a peculiar microenvironment characterized by a high concentration of growth factors and cytokines necessary for hematopoiesis, that make it a sanctuary for leukemic cell homing, survival and proliferation. B-cell precursor-Acute Lymphoblastic Leukemia (BCP-ALL) reprogram the BM stroma to create a leukemia-supporting and chemoprotective niche. Strategies to modulate the microenvironment could offer new approaches for anti-leukemia therapies. We identified ActivinA, a TGF-β family member, with a well-described promoting role in several solid malignancies, as a new potentially targetable leukemia-favoring factor.ActivinA resulted overexpressed in the BM plasma of 108 BCP-ALL pediatric patients compared to 44 Healthy Donors (HDs), as evaluated by ELISA assay. Upon in vitro culture, primary BCP-ALL cells significantly increased ActivinA secretion by mesenchymal stromal cells (MSCs) derived from HD BM. Interestingly this effect was achieved both by cell-to-cell contact-mediated mechanisms (direct contact) and by soluble factor release (transwell-mediated co-culture). Interestingly, MSCs isolated from the BM of leukemic patients showed an intrinsic ability to secrete higher amounts of ActivinA (mean=983.6±362.9 pg/mL), compared to their normal counterpart (mean=218.5±31.99 pg/mL). In addition, we demonstrated that the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α are increased in the leukemic BM and that they were able to synergize with leukemic blasts in inducing ActivinA release by MSCs (>100 fold increase compared to basal production).Both type I and type II Activin receptors were found to be expressed by leukemic cells as demonstrated by flow cytometry analysis of ALK4, ACVR2A and ACVR2B receptors and western blot analysis of ALK2. Of note, ActivinA exposure could increase the mRNA expression levels of type I receptors ALK2 and ALK4, thus suggesting a possible self-reinforcing mechanism.Gene expression analysis of ActivinA-treated BCP-ALL cells showed that this protein was able to significantly influence motility-associated molecular pathways. Accordingly, time lapse microscopy analyses revealed that ActivinA significantly increased random motility of leukemic cells (p<0.0001). In addition, ActivinA was able to almost double the migration of BCP-ALL primary cells in response to CXCL12, as demonstrated by in vitro chemotaxis assays. The specificity of the observed effect was demonstrated by using the ALK4 specific inhibitor SB431542.CXCL12 reduction is one of the typical microenvironmental alterations occurring in the leukemic BM. In line with literature data, we showed 6-fold decrese of CXCL12 levels in the BM of BCP-ALL patients compared to HDs (p<0.0001). Dose-responses chemotaxis experiments revealed that ActivinA was able to sensitize leukemic cells to suboptimal CXCL12 concentrations. On the other site, ActivinA exerted an opposite effect on CD34+ cells isolated both from HD cord blood or BM. In detail, in HD-CD34+ cells ActivinA severely impaired CXCL12-induced migration (p<0.0001). This opposite effect could be explained by ActivinA ability to increase free cytosolic calcium only in leukemic cells, both basally and after addition of CXCL12 (flow cytometry analysis of Fluo-4 NW stained cells). Of note, calcium levels of HD-CD34+ cells resulted unaffected or even decreased (in 2 out of 3 experiments performed) by ActivinA treatment. Since calcium is critically involved in boosting cytoskeleton dynamics, we analysed, by flow cytometry, actin polymerization in phalloidin stained BCP-ALL cells. Interestingly, ActivinA treatment of BCP-ALL cells significantly increased the rate of conversion of globular into filamentous actin, which is a prerequisite of site-directed migration, as soon as CXCL12 was added. Moreover, ActivinA resulted a leukemia-promoting factor: protein treatment significantly increased the in vitro migration of BCP-ALL cells through Matrigel-coated transwells in response to CXCL12, thus stimulating leukemic cell invasiveness.Overall, ActivinA resulted a key factor conferring a migratory advantage to leukemic cells over healthy hematopoiesis within the leukemic niche. Indeed, our in vitro findings provide the biological rationale for designing novel therapeutical approaches targeting the leukemia-stroma interplay. DisclosuresLocatelli:bluebird bio: Consultancy; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria.