Migratory Cells Research Articles

Elongation of Long-Chain Fatty Acids Is Crucial for Hematopoietic Stem Cell Engraftment and Leukemia Propagation

Lipid metabolism plays a crucial role in regulating physiological stem cells and cancer cells. Variations in length or degree of desaturation of fatty acid (FA) chains greatly contribute to lipid diversity, but whether it significantly regulates hematopoietic cell biology remains unknown. The enzyme ELOVL6 converts C16 saturated and monounsaturated FAs to C18 species. Elovl6 gene disruption (Elo6KO) in mice decreases the proportions of stearate (C18:0) and oleate (C18:1n-9) and increases those of palmitate (C16:0) and palmitoleate (C16:1n-7). Here, we report that ELOVL6 is essential for hematopoietic stem cells (HSC) engraftment after bone marrow (BM) transplantation and acute myeloid leukemia (AML) development in a mouse model. To determine whether hematopoietic cells of Elo6KO mice exhibit changes in FAs composition, we analyzed FAs in BM cell lysates. We found that the ratio of stearate to palmitate (C18:0/C16:0 ratio) was significantly decreased in Elo6KO cells compared to WT cells. To assess the effects of Elovl6 loss on the mouse HSC, we performed competitive repopulation assays. Using control or Elo6KO donor BM cells mixed in a 1:1 ratio with competitor BM cells, the chimerism of peripheral blood (PB) was about 50% in mice transplanted with control cells but remained at < 0.2% in those with Elo6KO cells at 12 weeks after transplantation. Even when transplanted Elo6KO donor cells with the competitor in a 10:1 ratio, the chimerism of PB was similarly low. Next, we investigated the roles of Elovl6 in leukemogenesis. We infected lineage-negative and c-Kit+ cells from WT or Elo6KO BM with MLL-AF9(MA9) retrovirus and transplanted lethally irradiated mice with either WT or Elo6KO MA9 cells. All mice receiving WT MA9 cells developed AML; however, AML propagation in Elo6KO MA9 cell-transplanted recipients was blocked. Elo6KO MA9 cell-transplanted mice survived until 6 months after transplantation. To assess the mechanisms underlying these phenotypes, we performed RNA-seq and gene enrichment analysis (GSEA) using CD34-, lineage-negative, Sca-1+ and c-Kit+ (CD34-LSK) cells and MA9 cells from Elo6KO or control mice. Chemotaxis, CXCR4 pathways, and their related target gene sets were enriched in Elo6KO cells, relative to control in both CD34-LSK cells and MA9 cells. We tested the chemotaxis of Elo6KO CD34-LSK and MA9 cells toward CXCL12 in a migration assay. A greater fraction of WT CD34-LSK and MA9 cells than that of these Elo6KO cells migrated to the CXCL12-containing chamber. We then examined the pharmacologic effects of inhibiting the PI3K pathway on chemotaxis. In a migration assay, the presence of PI3K inhibitor, Copanlisib, decreased the migration rate in WT MA9 cells, but not significantly in Elo6KO MA9 cells, which was already compromised. By contrast, AKT inhibitor, MK2206, did not alter WT or Elo6KO MA9 cell migration. In GSEA, Rac1-related pathways were enriched in Elo6KO cells. We thus investigated whether Rac1 activation was blocked by Elovl6 loss. The levels of the GTP-bound Rac1 were markedly increased in WT MA9 cells following CXCL12 stimulation compared to control, while those were not significantly different with/wo CXCL12 stimulation in Elo6KO MA9 cells. Given impaired chemotaxis and CXCR4-PI3K-Rac1 signaling in Elo6KO cells, we then investigated whether these cells exhibited altered actin remodeling in response to CXCL12. As expected, CXCL12 treatment induced lamellipodia formation in WT CD34-LSK cells, but not in Elo6KO CD34-LSK cells. Thus, ELOVL6 may regulate a CXCL12-CXCR4 pathway, resulting in PI3K-Rac1-mediated cytoskeletal remodeling and chemotaxis. Finally, to determine whether exogenously expressed Elovl6 could rescue Elo6KO MA9 cell phenotypes, we generated Elo6KO MA9 cells expressing Flag-tagged ELOVL6 (Elo6KO MA9Elo6) and mock controls (Elo6KO MA9mock). To evaluate the rescue of AML propagation, we transplanted lethally irradiated syngeneic mice with either Elo6KO MA9mock or Elo6KO MA9Elo6 cells. Among Elo6KO MA9mock transplanted mice, few recipients developed AML, while 10 out of 14 recipients had died due to AML after Elo6KO MA9Elo6 cell transplant. In summary, this study demonstrates novel and unexpected outcomes in normal HSCs and AML driver-transformed cells following lipid changes induced by Elovl6 deficiency. Altering lipid diversity could be a potential strategy to treat AML.

Development of the B-Cell Progenitor Niche Is Dependent on Cells Derived from the Migratory Neural Crest

INTRODUCTION: The earliest stages of B-cell production occur in the bone marrow (BM), where hematopoietic stem cells (HSCs) differentiate into common lymphoid and myeloid progenitors. Common lymphoid progenitors then undergo progressive differentiation as B cell progenitors (BCPs) prior to exiting the BM and completing their maturation in the spleen and lymph nodes. BCP development is regulated by lymphotrophic cytokines produced by BM mesenchymal stromal cells (MSCs) and osteoprogenitors (OPs). While the factors that regulate the formation and activity of the BCP niche in the BM are unknown, other laboratories have determined that signals from the peripheral nervous system (PNS) direct the formation and activity of the HSC BM niche. In this study, we hypothesized that the PNS also coordinates the formation and activity of the BCP BM niche. METHODS: To test this hypothesis, we targeted Schwann cells (SCs), which are required for peripheral nerve function, via a well-described mouse model of global peripheral nerve dysfunction (MP0-Cre+/-; Lamc1fl/fl, hereafter referred to as mutant). MP0 is expressed in the migratory neural crest from which SCs are derived, and the Lamc1 gene encodes an extracellular matrix protein (laminin-gamma-1) required for perinatal SC morphogenesis and differentiation. In mutant mice, SC development arrests at the 'immature' stage. Mutant SCs do not form myelinating or nonmyelinating SCs, which in mice takes place during the initial three weeks of postnatal development. Mutant mice die between 4-5 weeks of age; therefore 3-3.5 week old mice were used for all studies detailed below. RESULTS: In comparison to controls, mutant mice were lymphopenic. There were no differences in number of red cells, neutrophils or platelets between control and mutant mice. In the bone marrow, Pre-B-cell but not myeloid colony formation was significantly reduced in mutant mice, as were the number of both Pre and Pro B cells. Insulin Like Growth Factor Type 1 (IGF-1), an osteoprogenitor-derived lymphopoietic cytokine required for BCP maturation, was reduced by 60% in mutant bone marrow supernatant. IGF-1 transcript was also reduced in osteoprogenitor/osteoblast cells (CD45-Tert119-CD31-Sca1-CD51+) from mutant BM. Control and mutant femurs were examined by µCT, which identified significant impairment in mutant cortical and trabecular bone structure and density. A fluorescent reporter transgene (ROSAmT/eGFP) was employed to facilitate isolation and characterization of those BM niche cells genetically targeted by MP0-Cre. Using flow cytometry, we determined that less than four percent of osteoprogenitor/osteoblast cells expressed eGFP and were thus derived from the migratory neural crest. None of the MSCs (Tert119-CD31-Sca1+CD51+) expressed eGFP. The relative contribution of the migratory neural crest to each niche cell fraction did not vary between control and mutant BM. Lamc1 transcript from mutant eGFP+ cells was nearly undetectable and significantly less than from control eGFP+cells. Transcripts typically enriched in juvenile and nonmyelinating SCs (Erbb3, Sox10, GFAP, and s-100b) were enriched in eGFP+ but not eGFP- BM niche cells from both control (MP0-Cre+/-; Lamc1fl/+; ROSAmT/eGFP) and mutant (MP0-Cre+/-; Lamc1fl/fl) mice. There was no quantitative difference in IGF1 transcription between control and mutant eGFP+ cells. CONCLUSIONS: From these data, we conclude that postnatal maturation of the migratory neural crest is required for establishment of the BCP niche. While the migratory neural crest cells found within the bone marrow are enriched with SC-transcripts, they also contribute a small fraction of BM stromal cells and potentially some neurons (sympathetic and parasympathetic). Since mutant mice are paretic, it is also possible that weight bearing-induced mechanical forces contribute to osteogenic BCP niche formation. Our work to date does not differentiate between these possibilities, but does provide significant preliminary evidence to support our hypothesis that 'neuronal' development contributes to formation and activity of the BCP niche within the BM.

Key Subsets of Immune Cells in Bone Marrow Myeloid Compartment Indicate Degree of Tumor Infiltration and Avenues for Successful Immunotherapy in Multiple Myeloma

With the onset of single-cell omics, the complete gene expression landscape of immune cells can be leveraged to differentiate distinct subsets with inherently diverse functions. By analyzing the sample-specific composition of these distinct cellular groups, we can decipher the hallmarks of healthy and fully tumor-infiltrated bone marrow microenvironments as well as compositional modulations during effective immunotherapy. Unique to our approach, our analyses leverage over 500,000 existing RNA-Seq experiments packaged in the ARCHS4 repository to strengthen the biological signal and quality of our tertiary analyses by informing us in an unbiased way about lesser known immune-typologies. We applied this technique to our well-established immunocompetent mouse model of multiple myeloma (MM) which we rendered sensitive to IMiDs by introducing a human CRBN transgene: Vk*MYChCRBN. In multiple single cell experiments measuring effectiveness of Iberdomide in combination with Dexamethasone and an additional experiment comparing aged and untreated Vk*MYC mice with multiple myeloma (MM), key compositional patterns emerge in the murine bone marrow niche. Two distinct polarities of C1q expressing macrophages are present within murine bone marrow and spleen-one expressing Fcgr4 and present in untreated, tumor-bearing samples and a separate population elevated in Apoe, Vcam1, and MHC-II markers that can be found in healthy and tumor-bearing mice. Among splenocytes, we observed that the proportion of C1q macrophages correlates strongly with tumor burden and that BM and spleen samples in the process of tumor debulking via effective immunotherapy dramatically reduce relative frequencies of C1q macrophages. We hypothesize that the C1q macrophages without MHC-II features represent a tumor-associated macrophage (TAM) phenotype that is MM dependent and promotes tumor progression within the BM niche.1 Additional analyses of murine splenocytes have revealed that effective immunotherapy via an IAP antagonist induces the presence of immature or migratory dendritic cells (high expressers of Ccl5 and likely key immune recruiters) and that tumor-bearing splenocytes typically maintain large segments of immature neutrophils (high expressers of Ngp and Camp) without other neutrophils in more advanced stages of development2-while both immature and mature neutrophils are found in healthy controls. When comparing the BM composition of an aged, tumor-bearing mouse with that of a young, healthy mouse, key differences emerge. While multiple subsets of progenitors in murine BM niche can be identified via single cell, a unique population of cells over-expressing Cxcl12 that resemble Lepr+ mesenchymal stem cells from existing experiments (GSM3676085) are nearly absent in the young mouse, highly represented in the aged mouse, and may be a critical in creating a BM niche that supports tumor expansion.3 Curiously, a sizeable population of basophils is evident in the young, non-tumor bearing mouse but not found in the adult, tumor-bearing mouse. In addition, both types of C1q macrophages have expanded frequencies in the aged mouse. In total, single-cell analyses provide a snapshot of the compositional landscape of immune cell types thus illustrating hallmarks of tumor-infiltrated, healthy, and therapy modulated BM niches, providing potential new therapeutic targets.

Non-muscle myosin II and the plasticity of 3D cell migration.

Confined cells migrating through 3D environments are also constrained by the laws of physics, meaning for every action there must be an equal and opposite reaction for cells to achieve motion. Fascinatingly, there are several distinct molecular mechanisms that cells can use to move, and this is reflected in the diverse ways non-muscle myosin II (NMII) can generate the mechanical forces necessary to sustain 3D cell migration. This review summarizes the unique modes of 3D migration, as well as how NMII activity is regulated and localized within each of these different modes. In addition, we highlight tropomyosins and septins as two protein families that likely have more secrets to reveal about how NMII activity is governed during 3D cell migration. Together, this information suggests that investigating the mechanisms controlling NMII activity will be helpful in understanding how a single cell transitions between distinct modes of 3D migration in response to the physical environment.

Novel Application of 3D Scaffolds of Poly(E-Caprolactone)/Graphene as Osteoinductive Properties in Bone Defect.

Scaffolds provided a surface on which cells could attach, proliferate, and differentiate. Nowadays, bone tissue engineering offers hope for treating bone cancer. Poly(e-caprolactone) (PCL)/graphene have capability as an osteogenic and regenerative therapy. It could be used to produce bone tissue engineering scaffolds. The purpose of this study was to investigate the ability of PCL/graphene to enhance the osteoinductive mechanism. The PCL/graphene scaffold was developed utilizing a particulate-leaching process and cultured with osteoblast-like cells MG63 at 0.5, 1.5, and 2.5 wt% of graphene. We evaluated the porosity, pore size, migratory cells, and cell attachment of the scaffold. Data was expressed as the mean ± standard error of the mean and statistical analyses were performed using one-way analysis of variance and Tukey's post hoc at a level of p-value < 0.05. Porosity of scaffold with various percentage of graphene was nonsignificant (p > 0.05). There were differences in the acceleration of cell migration following wound closure between groups at 24 hours (p < 0.01) and 48 hours (p < 0.00). Adding the graphene on the scaffolds enhanced migration of osteoblast cells culture and possibility to attach. Graphene on 2.5 wt% exhibited good characteristics over other concentrations. This finding suggests that PCL/graphene composites may have potential applications in bone tissue engineering.

The critical issue linking lipids and inflammation: Clinical utility of stopping oxidative stress.

The formation of an atheroma begins when lipoproteins become trapped in the intima. Entrapped lipoproteins become oxidized and activate the innate immune system. This immunity represents the primary association between lipids and inflammation. When the trapping continues, the link between lipids and inflammation becomes chronic and detrimental, resulting in atherosclerosis. When entrapment ceases, the association between lipids and inflammation is temporary and healthy, and the atherogenic process halts. Therefore, the link between lipids and inflammation depends upon lipoprotein retention in the intima. The entrapment is due to electrostatic forces uniting apolipoprotein B to polysaccharide chains on intimal proteoglycans. The genetic transformation of contractile smooth muscle cells in the media into migratory secretory smooth muscle cells produces the intimal proteoglycans. The protein, platelet-derived growth factor produced by activated platelets, is the primary stimulus for this genetic change. Oxidative stress is the main stimulus to activate platelets. Therefore, minimizing oxidative stress would significantly reduce the retention of lipoproteins. Less entrapment decreases the association between lipids and inflammation. More importantly, it would halt atherogenesis. This review will analyze oxidative stress as the critical link between lipids, inflammation, and the pathogenesis of atherosclerosis. Through this perspective, we will discuss stopping oxidative stress to disrupt a harmful association between lipids and inflammation. Numerous therapeutic options will be discussed to mitigate oxidative stress. This paper will add a new meaning to the Morse code distress signal SOS-stopping oxidative stress.

Inhibition of invasion and metastasis in choriocarcinoma by migration and invasion inhibitory protein

IntroductionChoriocarcinoma is a highly invasive gynaecologic malignancy. Molecular mechanism of metastasis in choriocarcinoma is poorly understood. Migration and invasion inhibitory protein (MIIP) regulates cell migration and invasion. Therefore, we aimed to elucidate the function of MIIP in choriocarcinoma. MethodsChoriocarcinoma cell lines, JAR and JEG-3, were transfected with lentivirus carrying the MIIP-interfering RNA (to downregulate MIIP expression) or left untransfected (negative control). Cell migration and invasion were studied using transwell migration assays and scratch assays. In vivo tumour burden was studied using tumour xenograft models in specific-pathogen-free nude mice and live imaging. We elucidated possible molecular signalling pathways using western blotting. ResultsIn transwell migration and scratch assays MIIP-downregulated JAR and JEG-3 cells migrated and invaded faster compared to their respective negative control cells. Migration and invasion by the MIIP-upregulated SWAN cells was slower than that by negative control SWAN cells. Live imaging revealed that bioluminescence values were higher in MIIP-downregulated tumours than in the negative control tumours. Mice with MIIP-downregulated tumours had higher serum human chorionic gonadotropin (HCG) levels than those with negative control tumours. The MIIP expression was negatively correlated with that of histone deacetylase (HDAC6) and positively correlated with that of acetylated α-tubulin. DiscussionThus, MIIP—by inhibiting cellular motility in choriocarcinoma—acts as a tumour suppressor gene. This highlights a potential therapeutic target for refractory choriocarcinoma. Additionally, HDAC6 and acetylated α-tubulin may be involved in the regulatory effects of MIIP on the biobehaviour of choriocarcinoma cells.

Single‐cell/nanoparticle trajectories reveal two‐tier Lévy‐like interactions across bacterial swarms

AbstractSwarming bacteria can organize themselves into different local communities, ranging from active cell rafts to reticular biofilms. How millions of motile and immotile cells are dynamically linked together and still function as a whole is a critical interdisciplinary issue. Besides biochemical and molecular characterizations, concerns on the microscopic communication mechanisms underlying the complicated microsystem have been largely simplified to biophysics or physical interactions between individual isolated bacteria. Herein, by single‐cell tracking of fluorescent bacteria in the motile cell layer and single‐particle tracking of gold nanoparticles (AuNPs) in the upper fluid layer in different regions across a swarming colony of Bacillus subtilis, we studied collective multibacteria interactions, using the upper fluid as the medium, in detail. While the dynamic properties differ spatially, both cell migrations and AuNP transports in the swarming edge, the intermediate and the biofilm‐like center regions of the colony can be described with the Lévy‐walk‐like superdiffusion model. Moreover, the speed and motion range of the AuNPs are always much smaller than the bacteria, indicating that the intercellular fluid flow alone cannot explain long‐range cell‐to‐cell signaling. Referring to previous literature, we propose the presence of a decentralized two‐tier “active‐mixing” network in the bacterial community for efficient information exchange.

A Scribble/Cdep/Rac pathway controls follower-cell crawling and cluster cohesion during collective border-cell migration.

Collective cell movements drive normal development and metastasis. Drosophila border cells move as a cluster of 6-10 cells, where the role of the Rac GTPase in migration was first established. In border cells, as in most migratory cells, Rac stimulates leading-edge protrusion. Upstream Rac regulators in leaders have been identified; however, the regulation and function of Rac in follower border cells is unknown. Here, we show that all border cells require Rac, which promotes follower-cell motility and is important for cluster compactness and movement. We identify a Rac guanine nucleotide exchange factor, Cdep, which also regulates follower-cell movement and cluster cohesion. Scribble, Discs large, and Lethal giant larvae localize Cdep basolaterally and share phenotypes with Cdep. Relocalization of Cdep::GFP partially rescues Scribble knockdown, suggesting that Cdep is a major downstream effector of basolateral proteins. Thus, a Scrib/Cdep/Rac pathway promotes cell crawling and coordinated, collective migration invivo.

An Efficient Imaging Method for Medium-Earth-Orbit Multichannel SAR-GMTI Systems

Medium-Earth-orbit (MEO) synthetic aperture radar (SAR) has the advantages of short revisit time and wide coverage, and thus is a potential tool for implementing ground moving target indication (GMTI) tasks. In the paper, aiming at MEO SAR’s problems of low signal-to-noise ratio and limited computation resource, an efficient imaging method is proposed for MEO multichannel SAR-GMTI systems with relatively low resolution. The proposed imaging method is designed with the consideration of both static scenes and ground moving targets, and it can simultaneously correct the range cell migrations of static scenes and multiple moving targets of no Doppler ambiguity. It needs only four Fourier transforms and twice phase multiplications, and thus is computationally efficient. Moreover, moving targets’ signal characteristics, including the azimuth and range displacements and along-track interferometric phase, in the SAR image obtained by the proposed imaging method are figured out. Experimental results validate the proposed imaging method and the theoretical analyses.

DOCK2 and phosphoinositide-3 kinase δ mediate two complementary signaling pathways for CXCR5-dependent B cell migration.

Naive B cells use the chemokine receptor CXCR5 to enter B cell follicles, where they scan CXCL13-expressing ICAM-1+ VCAM-1+ follicular dendritic cells (FDCs) for the presence of antigen. CXCL13-CXCR5-mediated motility is mainly driven by the Rac guanine exchange factor DOCK2, which contains a binding domain for phosphoinositide-3,4,5-triphosphate (PIP3) and other phospholipids. While p110δ, the catalytic subunit of the class IA phosphoinositide-3-kinase (PI3K) δ, contributes to CXCR5-mediated B cell migration, the precise interdependency of DOCK2, p110δ, or other PI3K family members during this process remains incompletely understood. Here, we combined in vitro chemotaxis assays and in vivo imaging to examine the contribution of these two factors during murine naïve B cell migration to CXCL13. Our data confirm that p110δ is the main catalytic subunit mediating PI3K-dependent migration downstream CXCR5, whereas it does not contribute to chemotaxis triggered by CXCR4 or CCR7, two other chemokine receptors expressed on naïve B cells. The contribution of p110δ activity to CXCR5-driven migration was complementary to that of DOCK2, and pharmacological or genetic interference with both pathways completely abrogated B cell chemotaxis to CXCL13. Intravital microscopy of control and gene-deficient B cells migrating on FDCs confirmed that lack of DOCK2 caused a profound migration defect, whereas p110δ contributed to cell speed and directionality. B cells lacking active p110δ also displayed defective adhesion to ICAM-1; yet, their migration impairment was maintained on ICAM-1-deficient FDCs. In sum, our data uncover two complementary signaling pathways mediated by DOCK2 and p110δ, which enable CXCR5-driven naïve B cell examination of FDCs.

Chitosan-based nerve guidance conduit with microchannels and nanofibers promotes schwann cells migration and neurite growth

Peripheral nerve injury (PNI) is the leading cause of permanent dysfunction in movement and sensation. Despite the rapid development of tissue engineering in peripheral nerve regeneration, autograft remains the gold standard for treating PNI. Synthesized nerve guidance conduits (NGCs) were reported as a potential alternative treatment that could replace autograft. However, most current NGCs are hollow tubular structured, or NGCs with macro or microstructures, but not both. These simple structures could not meet the need for neurite and Schwann cell guidance and accelerate peripheral nerve regeneration. In the current study, we combine unidirectional freezing with electrospinning to produce a unique NGC with longitudinal microchannels and parallel nanofibers. The in vitro study showed the importance of having both features in promoting Schwann cell growth, migration, and PC-12 cells neurite elongation. The novel NGCs could provide desirable physical support and guidance for peripheral nerve regeneration. From the current study, we found both the micro feature and the nano feature are helpful in terms of helping cell migrating through the NGCs, and the combination of both features will have a syngeneic effect.

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

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

Annealing High Aspect Ratio Microgels into Macroporous 3D Scaffolds Allows for Higher Porosities and Effective Cell Migration.

Growing millimeter-scaled functional tissue remains a major challenge in the field of tissue engineering. Therefore, microporous annealed particles (MAPs) are emerging as promising porous biomaterials that are formed by assembly of microgel building blocks. To further vary the pore size and increase overall MAP porosity of mechanically stable scaffolds, rod-shaped microgels with high aspect ratios up to 20 are chemically interlinked into highly porous scaffolds. Polyethylene glycol based microgels (width 10µm, lengths up to 200µm) are produced via in-mold polymerization and covalently interlinked into stable 3D scaffolds via epoxy-amine chemistry. For the first time, MAP porosities can be enhanced by increasing the microgel aspect ratio (mean pore sizes ranging from 39 to 82µm, porosities from 65 to 90%). These porosities are significantly higher compared to constructs made from spherical or lower aspect ratio rod-shaped microgels. Rapid filling of the pores by either murine or primary human fibroblasts is ensured as cells migrate and grow extensively into these scaffolds. Overall, this study demonstrates that highly porous, stable macroporous hydrogels can be achieved with a very low partial volume of synthetic, high aspect ratio microgels, leading to large empty volumes available for cell ingrowth and cell-cell interactions.

Cell-cell interactions and fluctuations in the direction of motility promote directed migration of osteoblasts in direct current electrotaxis.

Under both physiological (development, regeneration) and pathological conditions (cancer metastasis), cells migrate while sensing environmental cues in the form of mechanical, chemical or electrical stimuli. In the case of bone tissue, osteoblast migration is essential in bone regeneration. Although it is known that osteoblasts respond to exogenous electric fields, the underlying mechanism of electrotactic collective movement of human osteoblasts is unclear. Here, we present a computational model that describes the osteoblast cell migration in a direct current electric field as the motion of a collection of active self-propelled particles and takes into account fluctuations in the direction of single-cell migration, finite-range cell-cell interactions, and the interaction of a cell with the external electric field. By comparing this model with in vitro experiments in which human primary osteoblasts are exposed to a direct current electric field of different field strengths, we show that cell-cell interactions and fluctuations in the migration direction promote anode-directed collective migration of osteoblasts.

Combination of the natural product mensacarcin with vemurafenib (Zelboraf) combats BRAF mutant and chemo-resistant melanoma in vitro by affecting cell metabolism and cellular migration

Melanoma is the most prevalent and aggressive form of skin cancer, often with a low survival rate due to rapid metastasis and a high occurrence of multi-drug resistance (MDR). In this study, we examine the combination treatment of the recently discovered cytotoxic bacterial metabolite mensacarcin and FDA approved therapeutic vemurafenib (Zelboraf). Vemurafenib is the standard regiment for BRAF V600E mutation positive patients with unresectable or metastatic malignant melanoma. On its own, menscarcin induces mitochondrial dysfunction in selected BRAF-mutant melanoma cell lines. In combination treatment, mensacarcin enhances vemurafenib's cytotoxicity via metabolic impairment. Specifically, it lowers cellular glucose uptake, decreases the oxygen consumption rate, reduces the abundance of glycolytic enzymes, and halts wound healing. Mensacarcin alone retains strong cytotoxic effects in three vemurafenib resistant cell lines SK-Mel-28PLXr, A375PLXr, and A2058PLXr with an average IC50 of 1 μM, inducing apoptosis via the caspase-3/7 pathway. We also find that mensacarcin lowers metastatic migration in a wound healing assay and it alters the morphology of migrating cells on a nanofiber scaffold. Using extracellular matrix (ECM) mimicking fibers, mensacarcin treated cells exhibit a decrease in single cell maximum coil width and an increase in the protrusion length limiting cellular coiling behavior which may limit metastatic migration. In summary, the bacterial metabolite mensacarcin shows great promise as a melanoma drug lead in vitro: in combination with vemurafenib, the cytotoxic activity is enhanced, and alone, mensacarcin retains activity in BRAF inhibitor chemo-resistant melanoma cell lines, and features a unique, migratory limited single cell phenotype.

Emerging concepts on the mechanical interplay between migrating cells and microenvironment in vivo.

During embryogenesis, tissues develop into elaborate collectives through a myriad of active mechanisms, with cell migration being one of the most common. As cells migrate, they squeeze through crowded microenvironments to reach the positions where they ultimately execute their function. Much of our knowledge of cell migration has been based on cells’ ability to navigate in vitro and how cells respond to the mechanical properties of the extracellular matrix (ECM). These simplified and largely passive surroundings contrast with the complexity of the tissue environments in vivo, where different cells and ECM make up the milieu cells migrate in. Due to this complexity, comparatively little is known about how the physical interactions between migrating cells and their tissue environment instruct cell movement in vivo. Work in different model organisms has been instrumental in addressing this question. Here, we explore various examples of cell migration in vivo and describe how the physical interplay between migrating cells and the neighboring microenvironment controls cell behavior. Understanding this mechanical cooperation in vivo will provide key insights into organ development, regeneration, and disease.

Topical Application of Adenosine A2-Type Receptor Agonists Prevents Contact Hypersensitivity Reactions in Mice by Affecting Skin Dendritic Cells

Adenosine (Ado) produced by skin and skin migratory CD73+ dendritic cells is critically involved in tolerance tohaptens. We therefore investigated the use of Ado receptor agonists for the treatment of contact hypersensitivity reactions. A2A- 4-[2-[[6-Amino-9-(N-ethyl-β-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino] ethyl]benzenepropanoic acid hydrochloride (CGS) and A2B- 2-[[6-Amino-3,5-dicyano-4-[4-[cyclopropylmethoxy]phenyl]-2-pyridinyl]thio]-acetamide (BAY) specific Ado receptor agonists were epicutaneously applied to the skin before sensitization and challenge with DNFB. Both agonists reduced ear swelling compared with solvent controls. This was accompanied by fewer activated T cells in the skin after the challenge and by higher numbers of T cells expressing anergic markers such as LAG-3, CD137, PD-1, CD272, and TIM-3 in the lymph nodes of CGS-treated groups. In ear tissue, Ado receptor agonist treatment reduced the production of proinflammatory cytokines and chemokines as well as the infiltration by neutrophils after sensitization. Moreover, reduced numbers of skin migratory dendritic cells producing less IL-12 and exhibiting lower expression of CD86 were recorded in lymph nodes after sensitization. In cocultures of skin migratory dendritic cells from CGS-treated mice with T cells, reduced proliferation of T cells and decreased secretion of proinflammatory cytokines compared with that of solvent controls were apparent. In conclusion, topical application of Ado receptor agonists to the skin prevents sensitization of T cells against haptens by reducing the migration and activation of skin migratory dendritic cells.

Fat2 polarizes the WAVE complex in trans to align cell protrusions for collective migration.

For a group of cells to migrate together, each cell must couple the polarity of its migratory machinery with that of the other cells in the cohort. Although collective cell migrations are common in animal development, little is known about how protrusions are coherently polarized among groups of migrating epithelial cells. We address this problem in the collective migration of the follicular epithelial cells in Drosophila melanogaster. In this epithelium, the cadherin Fat2 localizes to the trailing edge of each cell and promotes the formation of F-actin-rich protrusions at the leading edge of the cell behind. We show that Fat2 performs this function by acting in trans to concentrate the activity of the WASP family verprolin homolog regulatory complex (WAVE complex) at one long-lived region along each cell's leading edge. Without Fat2, the WAVE complex distribution expands around the cell perimeter and fluctuates over time, and protrusive activity is reduced and unpolarized. We further show that Fat2's influence is very local, with sub-micron-scale puncta of Fat2 enriching the WAVE complex in corresponding puncta just across the leading-trailing cell-cell interface. These findings demonstrate that a trans interaction between Fat2 and the WAVE complex creates stable regions of protrusive activity in each cell and aligns the cells' protrusions across the epithelium for directionally persistent collective migration.

The insufficiency of Dlx5 for ventral patterning in post-migratory neural crest cells reveals a loss of plasticity in early jaw-forming tissue

The articulated jaws of vertebrates arise from the first pharyngeal arch, the most rostral of several transient ventral structures in pharyngeal stage embryos. Migratory cranial neural crest cells from the caudal midbrain and rostral hindbrain populate the first arch as ectomesenchyme and supply the progenitors of skeletal and soft tissues that form the upper (maxillary) and lower (mandibular) jaws. Dlx genes encode key transcriptional regulators that profoundly influence jaw development through their actions in first pharyngeal arch patterning. The broadly conserved nested expression of Dlx paralogues in vertebrate embryos points to a retained ancestral role in patterning first arch tissue. Loss-of-function experiments consistently highlight the necessity of Dlx gene function for jaw morphogenesis. Specifically, the combined effects of Dlx5 and Dlx6 are required to specify ventral/mandibular fate and forced expression of Dlx5 in migrating neural crest cells results in the ectopic upregulation of ventral markers in the maxillary arch. Here, we ask whether Dlx5 is also sufficient to respecify post-migratory ectomesenchyme in the maxillary branch as mandibular. Unexpectedly, we show that Dlx5 is not sufficient to activate mandibular marker genes in the maxillary branch of PA1, highlighting a loss of plasticity in post-migratory first arch ectomesenchyme.