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OBJECTIVE: The dysregulation of the axon guidance pathway is common in pancreatic ductal adenocarcinoma (PDAC), yet our understanding of its biological relevance is limited. Here, we investigated the functional role of the axon guidance cue SEMA3A in supporting PDAC progression. DESIGN: We integrated bulk and single-cell transcriptomic datasets of human PDAC with in situ hybridisation analyses of patients tissues to evaluate SEMA3A expression in molecular subtypes of PDAC. Gain and loss of function experiments in PDAC cell lines and organoids were performed to dissect how SEMA3A contributes to define a biologically aggressive phenotype. RESULTS: In PDAC tissues, SEMA3A is expressed by stromal elements and selectively enriched in basal-like/squamous epithelial cells. Accordingly, expression of SEMA3A in PDAC cells is induced by both cell-intrinsic and cell-extrinsic determinants of the basal-like phenotype. In vitro, SEMA3A promotes cell migration as well as anoikis resistance. At the molecular level, these phenotypes are associated with increased focal adhesion kinase signalling through canonical SEMA3A-NRP1 axis. SEMA3A provides mouse PDAC cells with greater metastatic competence and favours intratumoural infiltration of tumour-associated macrophages and reduced density of T cells. Mechanistically, SEMA3A functions as chemoattractant for macrophages and skews their polarisation towards an M2-like phenotype. In SEMA3Ahigh tumours, depletion of macrophages results in greater intratumour infiltration by CD8+T cells and better control of the disease from antitumour treatment. CONCLUSIONS: Here, we show that SEMA3A is a stress-sensitive locus that promotes the malignant phenotype of basal-like PDAC through both cell-intrinsic and cell-extrinsic mechanisms.

BACKGROUND: Pulmonary hypertension (PH) represents an important phenotype in heart failure with preserved ejection fraction (HFpEF). However, management of PH-HFpEF is challenging because mechanisms involved in the regulation of PH-HFpEF remain unclear. METHODS: We used a mass spectrometry-based comparative plasma proteomics approach as a sensitive and comprehensive hypothesis-generating discovery technique to profile proteins in patients with PH-HFpEF and control subjects. We then validated and investigated the role of one of the identified proteins using in vitro cell cultures, in vivo animal models, and independent cohort of human samples. RESULTS: Plasma proteomics identified high protein abundance levels of B2M (β2-microglobulin) in patients with PH-HFpEF. Interestingly, both circulating and skeletal muscle levels of B2M were increased in mice with skeletal muscle SIRT3 (sirtuin-3) deficiency or high-fat diet-induced PH-HFpEF. Plasma and muscle biopsies from a validation cohort of PH-HFpEF patients were found to have increased B2M levels, which positively correlated with disease severity, especially pulmonary capillary wedge pressure and right atrial pressure at rest. Not only did the administration of exogenous B2M promote migration/proliferation in pulmonary arterial vascular endothelial cells but it also increased PCNA (proliferating cell nuclear antigen) expression and cell proliferation in pulmonary arterial vascular smooth muscle cells. Finally, B2m deletion improved glucose intolerance, reduced pulmonary vascular remodeling, lowered PH, and attenuated RV hypertrophy in mice with high-fat diet-induced PH-HFpEF. CONCLUSIONS: Patients with PH-HFpEF display higher circulating and skeletal muscle expression levels of B2M, the magnitude of which correlates with disease severity. Our findings also reveal a previously unknown pathogenic role of B2M in the regulation of pulmonary vascular proliferative remodeling and PH-HFpEF. These data suggest that circulating and skeletal muscle B2M can be promising targets for the management of PH-HFpEF.

Regenerative cell therapy to replenish the missing neurons and glia in the aganglionic segment of Hirschsprung disease represents a promising treatment option. However, the success of cell therapies for this condition are hindered by poor migration of the transplanted cells. This limitation is in part due to a markedly less permissive extracellular environment in the postnatal gut than that of the embryo. Coordinated interactions between enteric neural crest-derived cells (ENCDCs) and their local environment drive migration along the embryonic gut during development of the enteric nervous system. Modifying transplanted cells, or the postnatal extracellular environment, to better recapitulate embryonic ENCDC migration could be leveraged to improve the engraftment and coverage of stem cell transplants. We compared the transcriptomes of ENCDCs from the embryonic intestine to that of postnatal-derived neurospheres and identified 89 extracellular matrix (ECM)-associated genes that are differentially expressed. Agrin, a heparin sulfate proteoglycan with a known inhibitory effect on ENCDC migration, was highly over-expressed by postnatal-derived neurospheres. Using a function-blocking antibody and a shRNA-expressing lentivirus, we show that inhibiting agrin promotes ENCDC migration in vitro and following cell transplantation ex vivo and in vivo. This enhanced migration is associated with an increased proportion of GFAP + cells, whose migration is especially enhanced.

PURPOSE: Triple-negative breast cancer (TNBC) often metastasizes to the central nervous system (CNS) and has the highest propensity among breast cancer subtypes to develop leptomeningeal disease (LMD). LMD is a spread of cancer into leptomeningeal space that speeds up the disease progression and severely aggravates the prognosis. LMD has limited treatment options. We sought to test whether the common anti-helminthic drug mebendazole (MBZ) may be effective against murine TNBC LMD. METHODS: A small-molecule screen involving TNBC cell lines identified benzimidazoles as potential therapeutic agents for further study. In vitro migration assays were used to evaluate cell migration capacity and the effect of MBZ. For in vivo testing, CNS metastasis was introduced into BALB/c athymic nude mice through internal carotid artery injections of brain-tropic MDA-MB-231-BR or MCF7-BR cells. Tumor growth and spread was monitored by bioluminescence imaging and immunohistochemistry. MBZ was given orally at 50 and 100 mg/kg doses. MBZ bioavailability was assayed by mass spectrometry. RESULTS: Bioinformatic analysis and migration assays revealed higher migratory capacity of TNBC compared to other breast cancer subtypes. MBZ effectively slowed down migration of TNBC cell line MDA-MB-231 and its brain tropic derivative MDA-MB-231-BR. In animal studies, MBZ reduced leptomeningeal spread, and extended survival in brain metastasis model produced by MDA-MB-231-BR cells. MBZ did not have an effect in the non-migratory MCF7-BR model. CONCLUSIONS: We demonstrated that MBZ is a safe and effective oral agent in an animal model of TNBC CNS metastasis. Our findings are concordant with previous efforts involving MBZ and CNS pathology and support the drugs potential utility to slow down leptomeningeal spread.

Extracellular matrix (ECM) pathologic remodeling underlies many disorders, including muscular dystrophy. Tissue decellularization removes cellular components while leaving behind ECM components. We generated on-slide decellularized tissue slices from genetically distinct dystrophic mouse models. The ECM of dystrophin- and sarcoglycan-deficient muscles had marked thrombospondin 4 deposition, while dysferlin-deficient muscle had excess decorin. Annexins A2 and A6 were present on all dystrophic decellularized ECMs, but annexin matrix deposition was excessive in dysferlin-deficient muscular dystrophy. Muscle-directed viral expression of annexin A6 resulted in annexin A6 in the ECM. C2C12 myoblasts seeded onto decellularized matrices displayed differential myoblast mobility and fusion. Dystrophin-deficient decellularized matrices inhibited myoblast mobility, while dysferlin-deficient decellularized matrices enhanced myoblast movement and differentiation. Myoblasts treated with recombinant annexin A6 increased mobility and fusion like that seen on dysferlin-deficient decellularized matrix and demonstrated upregulation of ECM and muscle cell differentiation genes. These findings demonstrate specific fibrotic signatures elicit effects on myoblast activity.

BACKGROUND: Loss of AZGP1 expression is a biomarker associated with progression to castration resistance, development of metastasis, and poor disease-specific survival in prostate cancer. However, high expression of AZGP1 cells in prostate cancer has been reported to increase proliferation and invasion. The exact role of AZGP1 in prostate cancer progression remains elusive. METHOD: AZGP1 knockout and overexpressing prostate cancer cells were generated using a lentiviral system. The effects of AZGP1 under- or over-expression in prostate cancer cells were evaluated by in vitro cell proliferation, migration, and invasion assays. Heterozygous AZGP1± mice were obtained from European Mouse Mutant Archive (EMMA), and prostate tissues from homozygous knockout male mice were collected at 2, 6 and 10 months for histological analysis. In vivo xenografts generated from AZGP1 under- or over-expressing prostate cancer cells were used to determine the role of AZGP1 in prostate cancer tumor growth, and subsequent proteomics analysis was conducted to elucidate the mechanisms of AZGP1 action in prostate cancer progression. AZGP1 expression and microvessel density were measured in human prostate cancer samples on a tissue microarray of 215 independent patient samples. RESULT: Neither the knockout nor overexpression of AZGP1 exhibited significant effects on prostate cancer cell proliferation, clonal growth, migration, or invasion in vitro. The prostates of AZGP1-/- mice initially appeared to have grossly normal morphology; however, we observed fibrosis in the periglandular stroma and higher blood vessel density in the mouse prostate by 6 months. In PC3 and DU145 mouse xenografts, over-expression of AZGP1 did not affect tumor growth. Instead, these tumors displayed decreased microvessel density compared to xenografts derived from PC3 and DU145 control cells, suggesting that AZGP1 functions to inhibit angiogenesis in prostate cancer. Proteomics profiling further indicated that, compared to control xenografts, AZGP1 overexpressing PC3 xenografts are enriched with angiogenesis pathway proteins, including YWHAZ, EPHA2, SERPINE1, and PDCD6, MMP9, GPX1, HSPB1, COL18A1, RNH1, and ANXA1. In vitro functional studies show that AZGP1 inhibits human umbilical vein endothelial cell proliferation, migration, tubular formation and branching. Additionally, tumor microarray analysis shows that AZGP1 expression is negatively correlated with blood vessel density in human prostate cancer tissues. CONCLUSION: AZGP1 is a negative regulator of angiogenesis, such that loss of AZGP1 promotes angiogenesis in prostate cancer. AZGP1 likely exerts heterotypical effects on cells in the tumor microenvironment, such as stromal and endothelial cells. This study sheds light on the anti-angiogenic characteristics of AZGP1 in the prostate and provides a rationale to target AZGP1 to inhibit prostate cancer progression.

It is well established that neutrophils adopt malleable polymorphonuclear shapes to migrate through narrow interstitial tissue spaces1-3. However, how polymorphonuclear structures are assembled remains unknown4. Here we show that in neutrophil progenitors, halting loop extrusion-a motor-powered process that generates DNA loops by pulling in chromatin5-leads to the assembly of polymorphonuclear genomes. Specifically, we found that in mononuclear neutrophil progenitors, acute depletion of the loop-extrusion loading factor nipped-B-like protein (NIPBL) induced the assembly of horseshoe, banded, ringed and hypersegmented nuclear structures and led to a reduction in nuclear volume, mirroring what is observed during the differentiation of neutrophils. Depletion of NIPBL also induced cell-cycle arrest, activated a neutrophil-specific gene program and conditioned a loss of interactions across topologically associating domains to generate a chromatin architecture that resembled that of differentiated neutrophils. Removing NIPBL resulted in enrichment for mega-loops and interchromosomal hubs that contain genes associated with neutrophil-specific enhancer repertoires and an inflammatory gene program. On the basis of these observations, we propose that in neutrophil progenitors, loop-extrusion programs produce lineage-specific chromatin architectures that permit the packing of chromosomes into geometrically confined lobular structures. Our data also provide a blueprint for the assembly of polymorphonuclear structures, and point to the possibility of engineering de novo nuclear shapes to facilitate the migration of effector cells in densely populated tumorigenic environments.

Thrombospondin-4 (TSP-4) belongs to the extracellular matrix glycoprotein family of thrombospondins (TSPs). The multidomain, pentameric structure of TSP-4 allows its interactions with numerous extracellular matrix components, proteins and signaling molecules that enable its modulation to various physiological and pathological processes. Characterization of TSP-4 expression under development and pathogenesis of disorders has yielded important insights into mechanisms underlying the unique role of TSP-4 in mediating various processes including cell-cell, cell-extracellular matrix interactions, cell migration, proliferation, tissue remodeling, angiogenesis, and synaptogenesis. Maladaptation of these processes in response to pathological insults and stress can accelerate the development of disorders including skeletal dysplasia, osteoporosis, degenerative joint disease, cardiovascular diseases, tumor progression/metastasis and neurological disorders. Overall, the diverse functions of TSP-4 suggest that it may be a potential marker or therapeutic target for prognosis, diagnosis, and treatment of various pathological conditions upon further investigations. This review article highlights recent findings on the role of TSP-4 in both physiological and pathological conditions with a focus on what sets it apart from other TSPs.

Peyers patches (PPs) are lymphoid structures situated adjacent to the intestinal epithelium that support B cell responses that give rise to many intestinal IgA-secreting cells. Induction of isotype switching to IgA in PPs requires interactions between B cells and TGFβ-activating conventional dendritic cells type 2 (cDC2s) in the subepithelial dome (SED). However, the mechanisms promoting cDC2 positioning in the SED are unclear. Here, we found that PP cDC2s express GPR35, a receptor that promotes cell migration in response to various metabolites, including 5-hydroxyindoleacetic acid (5-HIAA). In mice lacking GPR35, fewer cDC2s were found in the SED, and frequencies of IgA+ germinal center (GC) B cells were reduced. IgA plasma cells were reduced in both the PPs and lamina propria. These phenotypes were also observed in chimeric mice that lacked GPR35 selectively in cDCs. GPR35 deficiency led to reduced coating of commensal bacteria with IgA and reduced IgA responses to cholera toxin. Mast cells were present in the SED, and mast cell-deficient mice had reduced PP cDC2s and IgA+ cells. Ablation of tryptophan hydroxylase 1 (Tph1) in mast cells to prevent their production of 5-HIAA similarly led to reduced PP cDC2s and IgA responses. Thus, mast cell-guided positioning of GPR35+ cDC2s in the PP SED supports induction of intestinal IgA responses.

The temporal lobe of the human brain contains the entorhinal cortex (EC). This region of the brain is a highly interconnected integrative hub for sensory and spatial information; it also has a key role in episodic memory formation and is the main source of cortical hippocampal inputs1-4. The human EC continues to develop during childhood5, but neurogenesis and neuronal migration to the EC are widely considered to be complete by birth. Here we show that the human temporal lobe contains many young neurons migrating into the postnatal EC and adjacent regions, with a large tangential stream persisting until the age of around one year and radial dispersal continuing until around two to three years of age. By contrast, we found no equivalent postnatal migration in rhesus macaques (Macaca mulatta). Immunostaining and single-nucleus RNA sequencing of ganglionic eminence germinal zones, the EC stream and the postnatal EC revealed that most migrating cells in the EC stream are derived from the caudal ganglionic eminence and become LAMP5+RELN+ inhibitory interneurons. These late-arriving interneurons could continue to shape the processing of sensory and spatial information well into postnatal life, when children are actively interacting with their environment. The EC is one of the first regions of the brain to be affected in Alzheimers disease, and previous work has linked cognitive decline to the loss of LAMP5+RELN+ cells6,7. Our investigation reveals that many of these cells arrive in the EC through a major postnatal migratory stream in early childhood.

While the involvement of actin polymerization in cell migration is well-established, much less is known about the role of transmembrane water flow in cell motility. Here, we investigate the role of water influx in a prototypical migrating cell, the neutrophil, which undergoes rapid, directed movement to sites of injury, and infection. Chemoattractant exposure both increases cell volume and potentiates migration, but the causal link between these processes are not known. We combine single-cell volume measurements and a genome-wide CRISPR screen to identify the regulators of chemoattractant-induced neutrophil swelling, including NHE1, AE2, PI3K-gamma, and CA2. Through NHE1 inhibition in primary human neutrophils, we show that cell swelling is both necessary and sufficient for the potentiation of migration following chemoattractant stimulation. Our data demonstrate that chemoattractant-driven cell swelling complements cytoskeletal rearrangements to enhance migration speed.

Large-scale cell flow characterizes gastrulation in animal development. In amniote gastrulation, particularly in avian gastrula, a bilateral vortex-like counter-rotating cell flow, called 'polonaise movements', appears along the midline. Here, through experimental manipulations, we addressed relationships between the polonaise movements and morphogenesis of the primitive streak, the earliest midline structure in amniotes. Suppression of the Wnt/planar cell polarity (PCP) signaling pathway maintains the polonaise movements along a deformed primitive streak. Mitotic arrest leads to diminished extension and development of the primitive streak and maintains the early phase of the polonaise movements. Ectopically induced Vg1, an axis-inducing morphogen, generates the polonaise movements, aligned to the induced midline, but disturbs the stereotypical cell flow pattern at the authentic midline. Despite the altered cell flow, induction and extension of the primitive streak are preserved along both authentic and induced midlines. Finally, we show that ectopic axis-inducing morphogen, Vg1, is capable of initiating the polonaise movements without concomitant PS extension under mitotic arrest conditions. These results are consistent with a model wherein primitive streak morphogenesis is required for the maintenance of the polonaise movements, but the polonaise movements are not necessarily responsible for primitive streak morphogenesis. Our data describe a previously undefined relationship between the large-scale cell flow and midline morphogenesis in gastrulation.

The collective migration of keratinocytes during wound healing requires both the generation and transmission of mechanical forces for individual cellular locomotion and the coordination of movement across cells. Leader cells along the wound edge transmit mechanical and biochemical cues to ensuing follower cells, ensuring their coordinated direction of migration across multiple cells. Despite the observed importance of mechanical cues in leader cell formation and in controlling coordinated directionality of cell migration, the underlying biophysical mechanisms remain elusive. The mechanically-activated ion channel PIEZO1 was recently identified to play an inhibitory role during the reepithelialization of wounds. Here, through an integrative experimental and mathematical modeling approach, we elucidate PIEZO1's contributions to collective migration. Time-lapse microscopy reveals that PIEZO1 activity inhibits leader cell formation at the wound edge. To probe the relationship between PIEZO1 activity, leader cell formation and inhibition of reepithelialization, we developed an integrative 2D continuum model of wound closure that links observations at the single cell and collective cell migration scales. Through numerical simulations and subsequent experimental validation, we found that coordinated directionality plays a key role during wound closure and is inhibited by upregulated PIEZO1 activity. We propose that PIEZO1-mediated retraction suppresses leader cell formation which inhibits coordinated directionality between cells during collective migration.

Focal adhesion kinase (FAK) relays integrin signaling from outside to inside cells and contributes to cell adhesion and motility. However, the spatiotemporal dynamics of FAK activity in single FAs is unclear due to the lack of a robust FAK reporter, which limits our understanding of these essential biological processes. Here we have engineered a genetically encoded FAK activity sensor, dubbed FAK-separation of phases-based activity reporter of kinase (SPARK), which visualizes endogenous FAK activity in living cells and vertebrates. Our work reveals temporal dynamics of FAK activity during FA turnover. Most importantly, our study unveils polarized FAK activity at the distal tip of newly formed single FAs in the leading edge of a migrating cell. By combining FAK-SPARK with DNA tension probes, we show that tensions applied to FAs precede FAK activation and that FAK activity is proportional to the strength of tension. These results suggest tension-induced polarized FAK activity in single FAs, advancing the mechanistic understanding of cell migration.

Objective To investigate the role and prognostic value of matrix metalloproteinase-7（MMP7） in the migration and immune cell infiltration of hepatocellular carcinoma. Methods MMP7_siRNA for downregulating the target gene MMP7 and pMMP7 for upregulating MMP7 were constructed and were used to transfect hepatocellular carcinoma cell line （MHCC97H）. RT-qPCR and Western Blot were used to measure the mRNA and protein expression levels of the target gene in cells. Scanning electron microscopy and Transwell assay were used to observe the changes in cell pseudopodia and migration ability, and bioinformatics methods were used to investigate the correlation of MMP7 with immune cells and immune infiltration score in TCGA and TIMER databases in patients with hepatocellular carcinoma, as well as the association between MMP7 and the prognosis of patients with hepatocellular carcinoma. The Spearman method was used for correlation analysis. Sanger Box online tool was used to assess the value of MMP7 in the overall survival curve and diseasespecific survival of hepatocellular carcinoma. The Kaplan-Meier method was used to plot survival curves, and the Log-rank test was used for comparison of prognosis between different samples. Results After MHCC97H cells were transfected with MMP7_siRNA or pMMP7, there was a significant reduction or increase in the expression of the target gene MMP7； after downregulation of MMP7, there were significant reductions in the number and length of the pseudopodia, while after MMP7 overexpression, there were significant increases in the number and length of filopodia with radial arrangement. The Transwell chamber assay showed that MMP7_siRNA2 significantly reduced the migration ability of cells （P<0.05), and there was a significant increase in migration ability after pMMP7 transfection. The expression of MMP7 was significantly correlated with B lymphocytes （r=0.37, P<0.05), CD4+ T lymphocytes （r=0.40, P<0.05), neutrophils （r=0.49, P<0.05), macrophages （r=0.49, P<0.05), and dendritic cells （r=0.47, P<0.05）. In the TCGA database, the patients with hepatocellular carcinoma were divided into MMP7 high expression group with 267 patients and MMP7 low expression group with 146 patients based on overall survival, and the results showed that the MMP7 high expression group had a significantly shorter overall survival time than the MMP7 low expression group （P<0.05）； based on the disease-specific survival time, the patients were divided into MMP7 high expression group with 257 patients and MMP7 low expression group with 145 patients, and the analysis showed that the MMP7 high expression group also had a significantly shorter disease-specific survival time than the MMP7 low expression group （P<0.05）. Conclusion MMP7 promotes the migration of hepatocellular carcinoma cells and plays a major role in immune cell infiltration, and the expression of MMP7 is also significantly associated with the prognosis of hepatocellular carcinoma. [ABSTRACT FROM AUTHOR]

The enteric nervous system (ENS) is principally derived from vagal neural crest cells that migrate caudally along the entire length of the gastrointestinal tract, giving rise to neurons and glial cells in two ganglionated plexuses. Incomplete migration of enteric neural crest-derived cells (ENCDC) leads to Hirschsprung disease, a congenital disorder characterized by the absence of enteric ganglia along variable lengths of the colorectum. Our previous work strongly supported the essential role of the avian ceca, present at the junction of the midgut and hindgut, in hindgut ENS development, since ablation of the cecal buds led to incomplete ENCDC colonization of the hindgut. In situ hybridization shows bone morphogenetic protein-4 (BMP4) is highly expressed in the cecal mesenchyme, leading us to hypothesize that cecal BMP4 is required for hindgut ENS development. To test this, we modulated BMP4 activity using embryonic intestinal organ culture techniques and retroviral infection. We show that overexpression or inhibition of BMP4 in the ceca disrupts hindgut ENS development, with GDNF playing an important regulatory role. Our results suggest that these two important signaling pathways are required for normal ENCDC migration and enteric ganglion formation in the developing hindgut ENS.

Neutrophils are the most abundant leukocyte in humans and provide a critical early line of defense as part of our innate immune system. We perform a comprehensive, genome-wide assessment of the molecular factors critical to proliferation, differentiation, and cell migration in a neutrophil-like cell line. Through the development of multiple migration screen strategies, we specifically probe directed (chemotaxis), undirected (chemokinesis), and 3D amoeboid cell migration in these fast-moving cells. We identify a role for mTORC1 signaling in cell differentiation, which influences neutrophil abundance, survival, and migratory behavior. Across our individual migration screens, we identify genes involved in adhesion-dependent and adhesion-independent cell migration, protein trafficking, and regulation of the actomyosin cytoskeleton. This genome-wide screening strategy, therefore, provides an invaluable approach to the study of neutrophils and provides a resource that will inform future studies of cell migration in these and other rapidly migrating cells.

Objective To investigate the effect of Bcl-2-associated athanogene 3 (BAG3) on the proliferation and migration abilities of non-small cell lung cancer (NSCLC) cells. Methods TCGA database was searched to perform a pan-cancer ana-lysis of BAG3 expression in different tumors. Western blotting was used to measure the relative expression level of BAG3 protein in NSCLC cell lines (H1299, A549, H460, Anip973, PC9, H358, and 95-D cells) and the human normal lung epithelial cell line Beas-2B. H1299 cells were transfected with Flag (group A) or Flag-BAG3 plasmid (group B), and A549 cells were transfected with siControl (group C) or siBAG3 (group D). Western blot was used to measure the transfection efficiency of plasmid and small interfering RNAs; CCK-8 assay and plate colony formation assay were used to observe the effect of BAG3 on the proliferation ability of NSCLC cells; cell scratch assay and Transwell assay were used to observe the effect of BAG3 on the migration ability of NSCLC cells. Results The mRNA expression level of BAG3 in six types of tumor was significantly higher than that in normal tissue (t=8.45-24.13,P

Matrix stiffening is ubiquitous in solid tumors and can direct epithelial-mesenchymal transition (EMT) and cancer cell migration. Stiffened niche can even cause poorly invasive oral squamous cell carcinoma (OSCC) cell lines to acquire a less adherent, more migratory phenotype, but mechanisms and durability of this acquired "mechanical memory" are unclear. Here, we observed that contractility and its downstream signals could underlie memory acquisition; invasive SSC25 cells overexpress myosin II (vs. noninvasive Cal27 cells) consistent with OSCC. However, prolonged exposure of Cal27 cells to a stiff niche or contractile agonists up-regulated myosin and EMT markers and enabled them to migrate as fast as SCC25 cells, which persisted even when the niche softened and indicated "memory" of their prior niche. Stiffness-mediated mesenchymal phenotype acquisition required AKT signaling and was also observed in patient samples, whereas phenotype recall on soft substrates required focal adhesion kinase (FAK) activity. Phenotype durability was further observed in transcriptomic differences between preconditioned Cal27 cells cultured without or with FAK or AKT antagonists, and such transcriptional differences corresponded to discrepant patient outcomes. These data suggest that mechanical memory, mediated by contractility via distinct kinase signaling, may be necessary for OSCC to disseminate.

Membrane tension is thought to be a long-range integrator of cell physiology. Membrane tension has been proposed to enable cell polarity during migration through front-back coordination and long-range protrusion competition. These roles necessitate effective tension transmission across the cell. However, conflicting observations have left the field divided as to whether cell membranes support or resist tension propagation. This discrepancy likely originates from the use of exogenous forces that may not accurately mimic endogenous forces. We overcome this complication by leveraging optogenetics to directly control localized actin-based protrusions or actomyosin contractions while simultaneously monitoring the propagation of membrane tension using dual-trap optical tweezers. Surprisingly, actin-driven protrusions and actomyosin contractions both elicit rapid global membrane tension propagation, whereas forces applied to cell membranes alone do not. We present a simple unifying mechanical model in which mechanical forces that engage the actin cortex drive rapid, robust membrane tension propagation through long-range membrane flows.

The objective of this study is to examine IL-11-induced mechanisms of inflammatory cell migration to the central nervous system (CNS). We report that IL-11 is produced at highest frequency by myeloid cells among the peripheral blood mononuclear cell (PBMC) subsets. Patients with relapsing-remitting multiple sclerosis (RRMS) have an increased frequency of IL-11+ monocytes, IL-11+ and IL-11R+ CD4+ lymphocytes, and IL-11R+ neutrophils in comparison to matched healthy controls. IL-11+ and granulocyte-macrophage colony-stimulating factor (GM-CSF)+ monocytes, CD4+ lymphocytes, and neutrophils accumulate in the cerebrospinal fluid (CSF). The effect of IL-11 in-vitro stimulation, examined using single-cell RNA sequencing, revealed the highest number of differentially expressed genes in classical monocytes, including up-regulated NFKB1, NLRP3, and IL1B. All CD4+ cell subsets had increased expression of S100A8/9 alarmin genes involved in NLRP3 inflammasome activation. In IL-11R+-sorted cells from the CSF, classical and intermediate monocytes significantly up-regulated the expression of multiple NLRP3 inflammasome-related genes, including complement, IL18, and migratory genes (VEGFA/B) in comparison to blood-derived cells. Therapeutic targeting of this pathway with αIL-11 mAb in mice with RR experimental autoimmune encephalomyelitis (EAE) decreased clinical scores, CNS inflammatory infiltrates, and demyelination. αIL-11 mAb treatment decreased the numbers of NFκBp65+, NLRP3+, and IL-1β+ monocytes in the CNS of mice with EAE. The results suggest that IL-11/IL-11R signaling in monocytes represents a therapeutic target in RRMS.

Increased secretion of hyaluronic acid (HA), a glycosaminoglycan abundant in the brain extracellular matrix (ECM), correlates with worse clinical outcomes for glioblastoma (GBM) patients. GBM cells aggressively invade the brain parenchyma while encountering spatiotemporal changes in their local ECM, including HA concentration. To investigate how varying HA concentrations affect GBM invasion, patient-derived GBM cells are cultured within a soft, 3D matrix in which HA concentration is precisely varied and cell migration observed. Data demonstrate that HA concentration can determine the invasive activity of patient-derived GBM cells in a biphasic and highly sensitive manner, where the absolute concentration of HA at which cell migration peaked is specific to each patient-derived line. Furthermore, evidence that this response relies on phosphorylated ezrin, which interacts with the intracellular domain of HA-engaged CD44 to effectively link the actin cytoskeleton to the local ECM is provided. Overall, this study highlights CD44-HA binding as a major mediator of GBM cell migration that acts independently of integrins and focal adhesion complexes and suggests that targeting HA-CD44-ezrin interactions represents a promising therapeutic strategy to prevent tumor cell invasion in the brain.

Background and purpose: Lung adenocarcinoma has the characteristics of difficult early detection, rapid tumor progression and low surgical resection rate. Although studies on immunotherapy alone and immunotherapy combined with chemotherapy have shown initial success in improving prognosis and overcoming drug resistance, the majority of lung adenocarcinoma patients still receive limited benefits. Therefore, there is an urgent need to identify novel biomarkers with relatively high sensitivity and specificity to improve the prognosis of lung adenocarcinoma. Cell division cycle protein 20 (CDC20) is involved in the occurrence and development of various tumors, but its biological role and mechanism in lung adenocarcinoma remain unclear. The aim of this study was to investigate the expression of CDC20 in lung adenocarcinoma and its predictive value for the prognosis of patients with lung adenocarcinoma, and to further explore the effects of CDC20 on the proliferation and invasion capabilities of lung adenocarcinoma cells. Methods: Utilizing immunohistochemistry (IHC) to detect the expression of CDC20 in lung adenocarcinoma tissues, we analyzed its correlation with poor prognosis in combination with bioinformatics and clinicopathological parameters. Kaplan-Meier survival curves were employed to illustrate the impact of CDC20 on postoperative survival rates in lung adenocarcinoma patients. COX multivariate regression analysis was conducted to identify independent prognostic factors influencing postoperative survival rates. Additionally, receiver operating characteristic (ROC) curves were applied to assess the diagnostic value of CDC20 expression in lung adenocarcinoma patients. Real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR) and Western blot were used to measure the expression levels of CDC20 in normal human lung epithelial cells (BEAS-2B) and human lung adenocarcinoma cells (A549 and H1299). In cellular experiments, CDC20 was knocked down in lung adenocarcinoma cells, which were divided into three groups: si-NC (control group), si-CDC20#1 (knockdown group 1) and si-CDC20#2 (knockdown group 2). Cell counting kit-8 (CCK-8), colony formation, transwell and wound healing assays were conducted to assess cell proliferation, migration and invasion capabilities. Functional enrichment analysis using Geng Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways was conducted to explore the biological roles of CDC20 in lung adenocarcinoma. Finally, gene set enrichment analysis (GSEA) was employed to investigate potential regulatory pathways of CDC20 in lung adenocarcinoma. This study was approved by the Ethics Committee of Hebei Chest Hospital (Number: 2022051). Results: The results of both bioinformatics analysis and IHC demonstrated a significantly high expression of CDC20 in lung adenocarcinoma tissues (P

Objective To investigate the effect of different concentrations of bufotalin (BT) on the proliferation, migration, invasion, and epithelial-mesenchymal transition of human colorectal cancer HCT116 cells. Methods CCK-8 assay was used to measure cell viability after 24 and 48 h of BT treatment at different concentrations (0, 10, 20, 40, 80, 160, and 320 nmol/L), and the half-maximal inhibitory concentration (IC50) was calculated. The plate colony formation assay was used to verify the effect of BT treatment at the concentrations of 0, 12.5, and 25.0 nmol/L for 14 days on the colony formation ability of HCT116 cells (established as groups A, B, and C). The wound healing assay and the Transwell assay were used to verify the effect of BT treatment at the concentrations of 0, 25, and 50 nmol/L for 24 h on the migration and invasion abilities of HCT116 cells (established as groups A, C, and D). Western blot was used to measure the protein expression levels of E-cadherin and N-cadherin in HCT116 cells after 24 h of treatment in groups A, C and D. Results CCK-8 assay showed that after HCT116 cells were treated by BT for 24 or 48 h, the inhibitory effect of BT on the proliferation of HCT116 cells increased significantly with the increase in the concentration of BT (F=2 106.00,3 725.00,P

Objective To investigate the effects of chronic starvation stress on the proliferation and migration of colorectal cancer cells, as well as the underlying mechanisms. Methods By using prolonged serum starvation to simulate chronic starvation stress in tumor cells, we established enduring serum-deprived models of SW480 and DLD-1 cells and observed cellular morphological change. Effects of prolonged serum starvation on SW480 and DLD-1 proliferative and migratory capabilities were assessed using CCK-8 and Transwell assays. Differential gene-expression analysis on SW480 cultured with 1% FBS or 10% FBS medium was followed by GO and KEGG pathway assessments. Migration-related protein interactions were explored using String database and Metascape software, leading to 16 genes being selected for RT-qPCR validation. Protein levels of ITGB1 and key molecules in the relevant pathways were measured. Mobility changes in SW480 were observed through Transwell assay after ITGB1 knockdown or STAT3 inhibition. Results Prolonged serum starvation significantly inhibited the proliferation of SW480 and DLD-1 cells, and DLD-1 mobility, while enhanced SW480 migration. Transcriptome analysis revealed that prolonged serum deprivation caused the upregulation of 3016 genes, among which 283 were involved in cell migration. Metascape analysis identified the correlations among potential core genes ITGB1, CD44, TNS1, STAT3, etc. Prolonged serum deprivation increased the mRNA levels of VTN, TNS1, VEGFA, STAT3, and ITGB1 while also increasing the protein levels of ITGB1 and MMP2 and the phosphorylation levels of JAK2 and STAT3. Mobility reduction in prolonged serum-starved SW480 cells was achieved through ITGB1 knockdown or a STAT3 inhibitor. Conclusion Colorectal cancer cells can endure chronic starvation stress which enhances migration capability by upregulating ITGB1 expression.

Cell polarity and movement are fundamental to many biological functions. Experimental and theoretical studies have indicated that interactions of certain proteins lead to the cell polarization which plays a key role in controlling the cell movement. We study the cell polarity and movement based on a class of biophysical models that consist of reaction-diffusion equations for different proteins and the dynamics of a moving cell boundary. Such a moving boundary is often simulated by a phase-field model. We first apply the matched asymptotic analysis to give a rigorous derivation of the sharp-interface model of the cell boundary from a phase-field model. We then develop a robust numerical approach that combines the level-set method to track the sharp boundary of a moving cell and accurate discretization techniques for solving the reaction-diffusion equations on the moving cell region. Our extensive numerical simulations predict the cell polarization under various kinds of stimuli and capture both the linear and the circular trajectories of a moving cell for a long period of time. In particular, we have identified some key parameters controlling different cell trajectories that are less accurately predicted by reduced models. Our work has linked different models and also developed tools that can be adapted for the challenging three-dimensional simulations. [ABSTRACT FROM AUTHOR]

Cell-cell adhesion plays a vital role in the development and maintenance of multicellular organisms. One of its functions is regulation of cell migration, such as occurs, e.g. during embryogenesis or in cancer. In this work, we develop a versatile multiscale approach to modelling a moving self-adhesive cell population that combines a careful microscopic description of a deterministic adhesion-driven motion component with an efficient mesoscopic representation of a stochastic velocity-jump process. This approach gives rise to mesoscopic models in the form of kinetic transport equations featuring multiple non-localities. Subsequent parabolic and hyperbolic scalings produce general classes of equations with non-local adhesion and myopic diffusion, a special case being the classical macroscopic model proposed in Armstrong et al. (J Theoret Biol 243(1): 98–113, 2006). Our simulations show how the combination of the two motion effects can unfold. Cell-cell adhesion relies on the subcellular cell adhesion molecule binding. Our approach lends itself conveniently to capturing this microscopic effect. On the macroscale, this results in an additional non-linear integral equation of a novel type that is coupled to the cell density equation. [ABSTRACT FROM AUTHOR]

Objective To investigate the effect of ankyrin-repeat domain-containing protein 22 (ANKRD22) on the proliferation, invasion, and migration of human hepatoma cells and its molecular mechanism. Methods The TCGA database was used to analyze the expression level of ANKRD22 in normal liver tissue and hepatocellular carcinoma tissue and its association with prognosis. Western Blot and qRT-PCR were used to measure the expression of ANKRD22 in human normal liver cells (L-02) and human hepatoma cells (Huh7, HepG2, MHCC-97H, SK-HEP-1, and SMMC-7721); CCK-8 assay, EdU, wound healing assay, and Transwell assay were used to observe the effect of ANKRD22 on the proliferation, invasion, and migration of hepatoma cells; Western Blot was used to investigate the association of ANKRD22 with cyclins and EMTrelated proteins; KEGG and ssGSEA analyses were performed to investigate the mechanism of action of ANKRD22 in hepatoma cells, and related experiments were conducted for validation. The independent-samples t-test was used for comparison of continuous data between two groups; a one-way analysis of variance was used for comparison between multiple groups, and the least significant difference t-test was used for further comparison between two groups. Results In the TCGA database, the expression level of ANKRD22 in hepatoma tissue was significantly higher than that in normal liver tissue (t= 5.083, P<0.05), and the patients with a high expression level of ANKRD22 had longer overall survival and disease-related survival than those with a low expression level of ANKRD22 (P<0.05). The expression level of ANKRD22 in various human hepatoma cell lines was higher than that in human normal liver cells (all P<0.05). Cell proliferation assay showed that the ANKRD22 overexpression group had significantly higher EdU positive rate and proliferation rate than the Vector group (t= 19.60 and 6.72, both P<0.001), and compared with the si-NC group, the si-ANKRD22#2 group and the si-ANKRD22#3 group had significantly lower EdU positive rate and proliferation rate (all P<0.001). Compared with the Vector group, the overexpression group had significantly higher expression levels of Cyclin E1, Cyclin D1, CDK7, and CDK4 (t=3.54, 4.95, 6.34, and 5.19, all P<0.01), and the si-ANKRD22#2 group and the si-ANKRD22#3 group had significantly lower expression levels than the si-NC group (all P<0.001). The overexpression group had a significantly lower expression level of P27 than the Vector group (t=6.12, P<0.001), and the si-ANKRD22#2 group and the si-ANKRD22#3 group had a significantly higher expression level than the si-NC group (both P<0.001). Invasion and migration experiments showed that compared with the Vector group, the ANKRD22 overexpression group had significantly higher migration rate and number of crossings through the membrane (migration group and invasion group) (t=5.01, 25.60, and 3.67, all P<0.05), and compared with the si-NC group, thesi-ANKRD22#2 group and the si-ANKRD22#3 group had significantly lower migration rate and number of crossings through the membrane (migration group and invasion group) (all P<0.01). The overexpression group had significantly higher expression levels of N-cadherin, Vimentin, and Snail than the Vector group (t=12.13, 8.85, and 13.97, all P<0.001), and the si-ANKRD22#2 group and the si-ANKRD22#3 group had significantly lower expression levels than the si-NC group (all P< 0.001); the overexpression group had a significantly lower expression level of E-cadherin than the Vector group (t=4.98, P< 0.01), and the si-ANKRD22#2 group and the si-ANKRD22#3 group had a significantly higher expression level than the si-NC group (both P<0.001). The KEGG enrichment analysis and the ssGSEA analysis showed that ANKRD22 was associated with the PI3K/AKT/mTOR signaling pathway in hepatocellular carcinoma, and the overexpression group had significantly higher expression levels of p-AKT/AKT, p-PI3K/PI3K, and p-mTOR/mTOR than the Vector group (t=12.21, 3.43, and 9.75, all P< 0.01), and the si-ANKRD22#2 group and the si-ANKRD22#3 group had significantly lower expression levels than the si-NC group (all P<0.001). Conclusion ANKRD22 is highly expressed in hepatoma cells and can promote the proliferation, invasion, and migration of hepatoma cells and the activation of the PI3K/AKT/mTOR signaling pathway. [ABSTRACT FROM AUTHOR]

Objective To explore effects of MCF-7 breast cancer cell conditioned medium on proliferation, apoptosis and migration of bone marrow mesenchymal stem cells (BMSC) and its molecular mechanism. Methods BMSC cultured under normal environment was used as the control group. BMSC cultured with MCF-7 cell conditioned medium was selected as the MCF-7 conditioned medium group. The MCF-7 conditioned medium group supplemented with 10 nmol/L GSK690693 (Akt inhibitor) was used as the Akt inhibitor group. The MCF-7 conditioned medium group added with 10 µmol/L Reparixin (CXCR1/2 inhibitor) was used as the CXCR1/2 inhibitor group. BMSC proliferation was detected by MTT assay. Annexin V-FITC/PI double labeled flow cytometry was used to detect BMSC apoptosis rate in each group. Transwell cell migration assay was used to detect the migration ability of BMSC in each group. The contents of interleukin (IL)-8 protein in two kinds of cell culture supernatant and MCF-7 cell conditioned medium were detected by enzyme-linked immunosorbent assay. The protein expression levels of protein kinase B (Akt)/phosphorylated Akt (p-Akt) and mammalian target of rapamycin (mTOR)/phosphorylated mTOR (p-mTOR) in BMSC of each group were detected by Western blot assay. Results Compared with the control group, cell proliferation level, migration number and the relative expression levels of p-Akt, p-mTOR protein of BMSC were increased in the MCF-7 conditioned medium group, and the apoptosis rate was decreased (P＜0.05). Compared with the MCF-7 conditioned medium group, cell proliferation level, migration number and the relative expression levels of p-Akt, p-mTOR protein of BMSC were decreased in the CXCR1/2 inhibitor group and the Akt inhibitor group, and the apoptosis rate was increased (P＜0.05). The contents of IL-8 protein in MCF-7 cell conditioned medium and MCF-7 culture supernatant were higher than that in the BMSC culture supernatant (P＜0.05). Conclusion MCF-7 cell conditioned medium promotes BMSC proliferation and migration and inhibits BMSC apoptosis by activating Akt-mTOR signaling pathway, in which IL-8-CXCR1/2 axis plays a key role. [ABSTRACT FROM AUTHOR]

Objective To investigate the effects of chronic starvation stress on the proliferation and migration of colorectal cancer cells, as well as the underlying mechanisms. Methods By using prolonged serum starvation to simulate chronic starvation stress in tumor cells, we established enduring serum-deprived models of SW480 and DLD-1 cells and observed cellular morphological change. Effects of prolonged serum starvation on SW480 and DLD-1 proliferative and migratory capabilities were assessed using CCK-8 and Transwell assays. Differential gene-expression analysis on SW480 cultured with 1% FBS or 10% FBS medium was followed by GO and KEGG pathway assessments. Migration-related protein interactions were explored using String database and Metascape software, leading to 16 genes being selected for RT-qPCR validation. Protein levels of ITGB1 and key molecules in the relevant pathways were measured. Mobility changes in SW480 were observed through Transwell assay after ITGB1 knockdown or STAT3 inhibition. Results Prolonged serum starvation significantly inhibited the proliferation of SW480 and DLD-1 cells, and DLD-1 mobility, while enhanced SW480 migration. Transcriptome analysis revealed that prolonged serum deprivation caused the upregulation of 3016 genes, among which 283 were involved in cell migration. Metascape analysis identified the correlations among potential core genes ITGB1, CD44, TNS1, STAT3, etc. Prolonged serum deprivation increased the mRNA levels of VTN, TNS1, VEGFA, STAT3, and ITGB1 while also increasing the protein levels of ITGB1 and MMP2 and the phosphorylation levels of JAK2 and STAT3. Mobility reduction in prolonged serum-starved SW480 cells was achieved through ITGB1 knockdown or a STAT3 inhibitor. Conclusion Colorectal cancer cells can endure chronic starvation stress which enhances migration capability by upregulating ITGB1 expression. [ABSTRACT FROM AUTHOR]

The velocity of the gliding movement of filamentous cyanobacteria on a solid surface usually has a strong temperature dependency, and the higher the temperature, the faster the speed. Former studies on this phenomenon were conducted using filamentous cyanobacteria with straight morphology. We examined the velocity of the gliding movement of Arthrospira platensis NIES-39 along its longitudinal axis to see if the same was true for this cyanobacterium with helicoid trichomes. Experimental results showed little temperature dependency in the velocity in a wide temperature range in this cyanobacterium. However, when we examined the velocity using mutants with straight trichomes, their velocity was strongly affected by temperature, like other formerly analyzed filamentous cyanobacteria. This result indicates that the helicoid morphology of A. platensis trichomes confers temperature compensation to their migration velocity, enabling them to keep a relatively constant velocity under various temperatures. Migration of wild-type trichomes is considerably suppressed compared to the straight-trichome mutants on solid media. The temperature compensation in the locomotion of this organism appears to be established as part of such a suppression. It was also found that the velocity of this cyanobacterium depended on the trichome length when they were atypically short (<250 µm); the shorter the trichomes, the slower the gliding movement tended to be. This result indicates that the coordinated action of a high number of cells constituting the trichome is required for efficient gliding movement. [ABSTRACT FROM AUTHOR]

Esophageal squamous cell carcinoma (ESCC) is a deadly disease with few prevention or treatment options. ESCC development in humans and rodents is associated with Zn deficiency (ZD), inflammation, and overexpression of oncogenic microRNAs: miR-31 and miR-21. In a ZD-promoted ESCC rat model with upregulation of these miRs, systemic antimiR-31 suppresses the miR-31-EGLN3/STK40-NF-κB-controlled inflammatory pathway and ESCC. In this model, systemic delivery of Zn-regulated antimiR-31, followed by antimiR-21, restored expression of tumor-suppressor proteins targeted by these specific miRs: STK40/EGLN3 (miR-31), PDCD4 (miR-21), suppressing inflammation, promoting apoptosis, and inhibiting ESCC development. Moreover, ESCC-bearing Zn-deficient (ZD) rats receiving Zn medication showed a 47% decrease in ESCC incidence vs. Zn-untreated controls. Zn treatment eliminated ESCCs by affecting a spectrum of biological processes that included downregulation of expression of the two miRs and miR-31-controlled inflammatory pathway, stimulation of miR-21-PDCD4 axis apoptosis, and reversal of the ESCC metabolome: with decrease in putrescine, increase in glucose, accompanied by downregulation of metabolite enzymes ODC and HK2. Thus, Zn treatment or miR-31/21 silencing are effective therapeutic strategies for ESCC in this rodent model and should be examined in the human counterpart exhibiting the same biological processes.

PURPOSE: This study aims to elucidate the electrotaxis response of alveolar epithelial cells (AECs) in direct-current electric fields (EFs), explore the impact of EFs on the cell fate of AECs, and lay the foundation for future exploitation of EFs for the treatment of acute lung injury. METHODS: AECs were extracted from rat lung tissues using magnetic-activated cell sorting. To elucidate the electrotaxis responses of AECs, different voltages of EFs (0, 50, 100, and 200 mV/mm) were applied to two types of AECs, respectively. Cell migrations were recorded and trajectories were pooled to better demonstrate cellular activities through graphs. Cell directionality was calculated as the cosine value of the angle formed by the EF vector and cell migration. To further demonstrate the impact of EFs on the pulmonary tissue, the human bronchial epithelial cells transformed with Ad12-SV40 2B (BEAS-2B cells) were obtained and experimented under the same conditions as AECs. To determine the influence on cell fate, cells underwent electric stimulation were collected to perform Western blot analysis. RESULTS: The successful separation and culturing of AECs were confirmed through immunofluorescence staining. Compared with the control, AECs in EFs demonstrated a significant directionality in a voltage-dependent way. In general, type Ⅰ alveolar epithelial cells migrated faster than type Ⅱ alveolar epithelial cells, and under EFs, these two types of cells exhibited different response threshold. For type Ⅱ alveolar epithelial cells, only EFs at 200 mV/mm resulted a significant difference to the velocity, whereas for, EFs at both 100 mV/mm and 200 mV/mm gave rise to a significant difference. Western blotting suggested that EFs led to an increased expression of a AKT and myeloid leukemia 1 and a decreased expression of Bcl-2-associated X protein and Bcl-2-like protein 11. CONCLUSION: EFs could guide and accelerate the directional migration of AECs and exert antiapoptotic effects, which indicated that EFs are important biophysical signals in the re-epithelialization of alveolar epithelium in lung injury.

By acting both upstream of and downstream from biochemical organizers of the cytoskeleton, physical forces function as central integrators of cell shape and movement. Here we use a combination of genetic, pharmacological, and optogenetic perturbations to probe the role of the conserved mechanosensitive mTOR complex 2 (mTORC2) programs in neutrophil polarity and motility. We find that the tension-based inhibition of leading-edge signals (Rac, F-actin) that underlies protrusion competition is gated by the kinase-independent role of the complex, whereas the regulation of RhoA and myosin II-based contractility at the trailing edge depend on mTORC2 kinase activity. mTORC2 is essential for spatial and temporal coordination of the front and back polarity programs for persistent migration under confinement. This mechanosensory pathway integrates multiple upstream signals, and we find that membrane stretch synergizes with biochemical co-input phosphatidylinositol (3,4,5)-trisphosphate to robustly amplify mTORC2 activation. Our results suggest that different signaling arms of mTORC2 regulate spatially and molecularly divergent cytoskeletal programs for efficient coordination of neutrophil shape and movement.

Recent research has elucidated mechanochemical pathways of single cell polarization, but much less is known about collective motility initiation in adhesive cell groups. We used galvanotactic assays of zebrafish keratocyte cell groups, pharmacological perturbations, electric field switches, particle imaging velocimetry, and cell tracking to show that large cell groups initiate motility in minutes toward the cathode. Interestingly, while PI3K-inhibited single cells are biased toward the anode, inhibiting PI3K does not affect the cathode-directed cell group migration. We observed that control groups had the fastest cathode-migrating cell at the front, while the front cells in PI3K-inhibited groups were the slowest. Both control and PI3K-inhibited groups rapidly repolarized when the electric field direction was reversed, and the group migration continued after the electric field was switched off. Inhibiting myosin disrupted the cohesiveness of keratocyte groups and abolished the collective directionality and ability to switch direction when the electric field is reversed. Our data are consistent with a model according to which cells in the group sense the electric field individually and mechanical integration of the cells results in coherent group motility.

Maintaining persistent migration in complex environments is critical for neutrophils to reach infection sites. Neutrophils avoid getting trapped, even when obstacles split their front into multiple leading edges. How they re-establish polarity to move productively while incorporating receptor inputs under such conditions remains unclear. Here, we challenge chemotaxing HL60 neutrophil-like cells with symmetric bifurcating microfluidic channels to probe cell-intrinsic processes during the resolution of competing fronts. Using supervised statistical learning, we demonstrate that cells commit to one leading edge late in the process, rather than amplifying structural asymmetries or early fluctuations. Using optogenetic tools, we show that receptor inputs only bias the decision similarly late, once mechanical stretching begins to weaken each front. Finally, a retracting edge commits to retraction, with ROCK limiting sensitivity to receptor inputs until the retraction completes. Collectively, our results suggest that cell edges locally adopt highly stable protrusion/retraction programs that are modulated by mechanical feedback.

Although cancer stem cells (CSCs) play a major role in tumorigenesis and metastasis, the role of genetic alterations in invasiveness of CSCs is still unclear. Tumor microenvironment signals, such as extracellular matrix (ECM) composition, significantly influence cell behaviors. Unfortunately, these signals are often lost in in vitro cell culture. This study determines putative CSC populations, examines genetic changes during tumorigenesis of human breast epithelial stem cells, and investigates single-cell migration properties on ECM-mimetic platforms. Whole exome sequencing data indicate that tumorigenic cells have a higher somatic mutation burden than non-tumorigenic cells, and that mutations exclusive to tumorigenic cells exhibit higher predictive deleterious scores. Tumorigenic cells exhibit distinct somatic copy number variations (CNVs) including gain of duplications in chromosomes 5 and 8. ECM-mimetic topography selectively enhances migration speed of tumorigenic cells, but not of non-tumorigenic cells, and results in a wide distribution of tumorigenic single-cell migration speeds, suggesting heterogeneity in cellular sensing of contact guidance cues. This study identifies mutations and CNVs acquired during breast tumorigenesis, which can be associated with enhanced migration of breast tumorigenic cells, and demonstrates that a nanotopographically-defined platform can be applied to recapitulate an ECM structure for investigating cellular migration in the simulated tumor microenvironment.

Marinobazzanan (1), a new bazzanane-type sesquiterpenoid, was isolated from a marine-derived fungus belonging to the genus Acremonium. The chemical structure of 1 was elucidated using NMR and mass spectroscopic data, while the relative configurations were established through the analysis of NOESY data. The absolute configurations of 1 were determined by the modified Mosher's method as well as vibrational circular dichroism (VCD) spectra calculation and it was determined as 6R, 7R, 9R, and 10R. It was found that compound 1 was not cytotoxic to human cancer cells, including A549 (lung cancer), AGS (gastric cancer), and Caco-2 (colorectal cancer) below the concentration of 25 μM. However, compound 1 was shown to significantly decrease cancer-cell migration and invasion and soft-agar colony-formation ability at concentrations ranging from 1 to 5 μM by downregulating the expression level of KITENIN and upregulating the expression level of KAI1. Compound 1 suppressed β-catenin-mediated TOPFLASH activity and its downstream targets in AGS, A549, and Caco-2 and slightly suppressed the Notch signal pathway in three cancer cells. Furthermore, 1 also reduced the number of metastatic nodules in an intraperitoneal xenograft mouse model.

Oligodendrocyte precursor cells (OPCs) undergo an extensive and coordinated migration in the developing CNS, using the pre-formed scaffold of developed blood vessels as their physical substrate for migration. While OPC association with vasculature is critical for dispersal, equally important for permitting differentiation and proper myelination of target axons is their appropriate and timely detachment, but regulation of this process remains unclear. Here we demonstrate a correlation between the developmental formation of astrocytic endfeet on vessels and the termination of OPC perivascular migration. Ex vivo and in vivo live imaging shows that astrocyte endfeet physically displace OPCs from vasculature, and genetic abrogation of endfoot formation hinders both OPC detachment from vessels and subsequent differentiation. Astrocyte-derived semaphorins 3a and 6a act to repel OPCs from blood vessels at the cessation of their perivascular migration and, in so doing, permit subsequent OPC differentiation by insulating them from a maturation inhibitory endothelial niche.

The mature mammalian cortex is composed of 6 architecturally and functionally distinct layers. Two key steps in the assembly of this layered structure are the initial establishment of the glial scaffold and the subsequent migration of postmitotic neurons to their final position. These processes involve the precise and timely regulation of adhesion and detachment of neural cells from their substrates. Although much is known about the roles of adhesive substrates during neuronal migration and the formation of the glial scaffold, less is understood about how these signals are interpreted and integrated within these neural cells. Here, we provide in vivo evidence that Cas proteins, a family of cytoplasmic adaptors, serve a functional and redundant role during cortical lamination. Cas triple conditional knock-out (Cas TcKO) mice display severe cortical phenotypes that feature cobblestone malformations. Molecular epistasis and genetic experiments suggest that Cas proteins act downstream of transmembrane Dystroglycan and β1-Integrin in a radial glial cell-autonomous manner. Overall, these data establish a new and essential role for Cas adaptor proteins during the formation of cortical circuits and reveal a signaling axis controlling cortical scaffold formation.

BackgroundIn light of the growing appreciation for the role of collective cell motility in metastasis, a deeper understanding of the underlying signaling pathways will be critical to translating these observations to the treatment of advanced cancers. Here, we examine the contribution of Wnt/planar cell polarity (Wnt/PCP), one of the non-canonical Wnt signaling pathways and defined by the involvement of the tetraspanin-like proteins Vangl1 and Vangl2, to breast tumor cell motility, collective cell invasiveness and mammary tumor metastasis.MethodsVangl1 and Vangl2 knockdown and overexpression and Wnt5a stimulation were employed to manipulate Wnt/PCP signaling in a battery of breast cancer cell lines representing all breast cancer subtypes, and in tumor organoids from MMTV-PyMT mice. Cell migration was assessed by scratch and organoid invasion assays, Vangl protein subcellular localization was assessed by confocal fluorescence microscopy, and RhoA activation was assessed in real time by fluorescence imaging with an advanced FRET biosensor. The impact of Wnt/PCP suppression on mammary tumor growth and metastasis was assessed by determining the effect of conditional Vangl2 knockout on the MMTV-NDL mouse mammary tumor model.ResultsWe observed that Vangl2 knockdown suppresses the motility of all breast cancer cell lines examined, and overexpression drives the invasiveness of collectively migrating MMTV-PyMT organoids. Vangl2-dependent RhoA activity is localized in real time to a subpopulation of motile leader cells displaying a hyper-protrusive leading edge, Vangl protein is localized to leader cell protrusions within leader cells, and actin cytoskeletal regulator RhoA is preferentially activated in the leader cells of a migrating collective. Mammary gland-specific knockout of Vangl2 results in a striking decrease in lung metastases in MMTV-NDL mice, but does not impact primary tumor growth characteristics.ConclusionsWe conclude that Vangl-dependent Wnt/PCP signaling promotes breast cancer collective cell migration independent of breast tumor subtype and facilitates distant metastasis in a genetically engineered mouse model of breast cancer. Our observations are consistent with a model whereby Vangl proteins localized at the leading edge of leader cells in a migrating collective act through RhoA to mediate the cytoskeletal rearrangements required for pro-migratory protrusion formation.

Bone metastasis resulting from advanced breast cancer causes osteolysis and increases mortality in patients. Kalkitoxin (KT), a lipopeptide toxin derived from the marine cyanobacterium Moorena producens (previously Lyngbya majuscula), has an anti-metastatic effect on cancer cells. We verified that KT suppressed cancer cell migration and invasion in vitro and in animal models in the present study. We confirmed that KT suppressed osteoclast-soup-derived MDA-MB-231 cell invasion in vitro and induced osteolysis in a mouse model, possibly enhancing/inhibiting metastasis markers. Furthermore, KT inhibits CXCL5 and CXCR2 expression, suppressing the secondary growth of breast cancer cells on the bone, brain, and lungs. The breast-cancer-induced osteolysis in the mouse model further reveals that KT plays a protective role, judging by micro-computed tomography and immunohistochemistry. We report for the first time the novel suppressive effects of KT on cancer cell migration and invasion in vitro and on MDA-MB-231-induced bone loss in vivo. These results suggest that KT may be a potential therapeutic drug for the treatment of breast cancer metastasis.

BackgroundLong non-coding RNAs (lncRNAs) have critical functions within esophageal squamous cell carcinoma (ESCC). However, the function and mechanism underlying ESCC-associated lncRNA-1 (ESCCAL-1) in ESCC tumorigenesis have not been well clarified.MethodsESCCAL-1, miR-590 and LRP6 were quantified using qRT-PCR. Cell viability, migration and invasion abilities were measured using CCK-8 assay and transwell assays. The protein pression was determined with western blot assay. The xenograft model assays were used to examine the impact of ESCCAL-1 on tumorigenic effect in vivo. Direct relationships among ESCCAL-1, miR-590 and LRP6 were confirmed using dual-luciferase reporter assays.ResultsThe present work discovered the ESCCAL-1 up-regulation within ESCC. Furthermore, ESCCAL-1 was found to interact with miR-590 and consequently restrict its expression. Functionally, knocking down ESCCAL-1 or over-expressing miR-590 hindered ESCC cell growth, invasion, and migration in vitro. Moreover, inhibition of miR-590 could reverse the effect of knockdown of ESCCAL-1 on cells. Importantly, it was confirmed that LRP6 was miR-590's downstream target and LRP6 over-expression also partly abolished the role of miR-590 overexpression in ESCC cells.ConclusionWe have uncovered a novel regulatory network comprising aberrant interaction of ESCCAL-1/miR-590/LRP6 participated in ESCC progression.

The incidence of human papillomavirus (HPV)-associated anogenital and oropharyngeal cancer in human immunodeficiency virus (HIV)-infected individuals is substantially higher than in HIV-uninfected individuals. HIV may also be a risk factor for the development of HPV-negative head and neck, liver, lung, and kidney cancer. However, the molecular mechanisms underlying HIV-1-associated increase of epithelial malignancies are not fully understood. Here, we showed that HPV-16-immortalized anal AKC-2 and cervical CaSki epithelial cells that undergo prolonged exposure to cell-free HIV-1 virions or HIV-1 viral proteins gp120 and tat respond with the epithelial-mesenchymal transition (EMT) and increased invasiveness. Similar responses were observed in HPV-16-infected SCC-47 and HPV-16-negative HSC-3 oral epithelial cancer cells that were cultured with these viral proteins. EMT induced by gp120 and tat led to detachment of poorly adherent cells from the culture substratum; these cells remained capable of reattachment, upon which they coexpressed both E-cadherin and vimentin, indicative of an intermediate stage of EMT. The reattached cells also expressed stem cell markers CD133 and CD44, which may play a critical role in cancer cell invasion and metastasis. Inhibition of transforming growth factor (TGF)-β1 and MAPK signaling and vimentin expression, and restoration of E-cadherin expression reduced HIV-induced EMT and the invasive activity of HPV-16-immortalized anal and cervical epithelial cells. Collectively, our results suggest that these approaches along with HIV viral suppression with antiretroviral therapy (ART) might be useful to limit the role of HIV-1 infection in the acceleration of HPV-associated or HPV-independent epithelial neoplasia. IMPORTANCE HPV-16-immortalized genital and oral epithelial cells and HPV-negative oral cancer cells that undergo prolonged contact with cell-free HIV-1 virions or with viral proteins gp120 and tat respond by becoming more invasive. EMT cells induced by HIV-1 in cultures of HPV-16-immortalized anal and cervical epithelial cells express the stem cell markers CD133 and CD44. These results suggest that the interaction of HIV-1 with neoplastic epithelial cells may lead to their de-differentiation into cancer stem cells that are resistant to apoptosis and anti-cancer drugs. Thus, this pathway may play a critical role in the development of invasive cancer. Inhibition of TGF-β1 and MAPK signaling and vimentin expression, and restoration of E-cadherin expression reduced HIV-induced EMT and the invasiveness of HPV-16-immortalized anal and cervical epithelial cells. Taken together, these results suggest that these approaches might be exploited to limit the role of HIV-1 infection in the acceleration of HPV-associated or HPV-independent epithelial neoplasia.

Epithelial-to-mesenchymal transition (EMT) is a dramatic change in cellular physiology during development and metastasis, which requires coordination between cell signaling, adhesion, and membrane protrusions. These processes all involve dynamic changes in the plasma membrane; yet, how membrane lipid content regulates membrane function during EMT remains incompletely understood. By screening for differential expression of lipid-modifying genes over the course of EMT in the avian neural crest, we have identified the ceramide-producing enzyme neutral sphingomyelinase 2 (nSMase2) as a critical regulator of a developmental EMT. nSMase2 expression begins at the onset of EMT, and in vivo knockdown experiments demonstrate that nSMase2 is necessary for neural crest migration. We find that nSMase2 promotes Wnt and BMP signaling and is required to activate the mesenchymal gene expression program. Mechanistically, we show that nSMase2-dependent ceramide production is necessary for and sufficient to up-regulate endocytosis and is required for Wnt co-receptor internalization. Finally, inhibition of endocytosis in the neural crest mimics the loss of migration and Wnt signaling observed following nSMase2 knockdown. Our results support a model in which nSMase2 is expressed at the onset of neural crest EMT to produce ceramide and facilitate receptor-mediated endocytosis of Wnt and BMP signaling complexes, thereby activating promigratory gene expression. These results highlight the critical role of plasma membrane lipid metabolism in regulating transcriptional changes during developmental EMT programs.

Immunoglobulin family and carbohydrate vascular addressins encoded by Madcam1 and St6gal1 control lymphocyte homing into intestinal tissues, regulating immunity and inflammation. The addressins are developmentally programmed to decorate endothelial cells lining gut post-capillary and high endothelial venules (HEV), providing a prototypical example of organ- and segment-specific endothelial specialization. We identify conserved NKX-COUP-TFII composite elements (NCCE) in regulatory regions of Madcam1 and St6gal1 that bind intestinal homeodomain protein NKX2-3 cooperatively with venous nuclear receptor COUP-TFII to activate transcription. The Madcam1 element also integrates repressive signals from arterial/capillary Notch effectors. Pan-endothelial COUP-TFII overexpression induces ectopic addressin expression in NKX2-3+ capillaries, while NKX2-3 deficiency abrogates expression by HEV. Phylogenetically conserved NCCE are enriched in genes involved in neuron migration and morphogenesis of the heart, kidney, pancreas and other organs. Our results define an NKX-COUP-TFII morphogenetic code that targets expression of mucosal vascular addressins.

Cell clusters that collectively migrate from primary tumors appear to be far more potent in forming distant metastases than single cancer cells. A better understanding of the collective cell migration phenomenon and the involvement of various cell types during this process is needed. Here, an in vitro platform based on inverted-pyramidal microwells to follow and quantify the collective migration of hundreds of tumor cell clusters at once is developed. These results indicate that mesenchymal stromal cells (MSCs) or cancer-associated fibroblasts (CAFs) in the heterotypic tumor cell clusters may facilitate metastatic dissemination by transporting low-motile cancer cells in a Rac-dependent manner and that extracellular vesicles secreted by mesenchymal cells only play a minor role in this process. Furthermore, in vivo studies show that cancer cell spheroids containing MSCs or CAFs have faster spreading rates. These findings highlight the active role of co-traveling stromal cells in the collective migration of tumor cell clusters and may help in developing better-targeted therapies.

Piezo1 is a bona fide mechanosensitive ion channel ubiquitously expressed in mammalian cells. The distribution of Piezo1 within a cell is essential for various biological processes including cytokinesis, cell migration, and wound healing. However, the underlying principles that guide the subcellular distribution of Piezo1 remain largely unexplored. Here, we demonstrate that membrane curvature serves as a key regulator of the spatial distribution of Piezo1 in the plasma membrane of living cells. Piezo1 depletes from highly curved membrane protrusions such as filopodia and enriches to nanoscale membrane invaginations. Quantification of the curvature-dependent sorting of Piezo1 directly reveals the in situ nano-geometry of the Piezo1-membrane complex. Piezo1 density on filopodia increases upon activation, independent of calcium, suggesting flattening of the channel upon opening. Consequently, the expression of Piezo1 inhibits filopodia formation, an effect that diminishes with channel activation.

The velocity of the gliding movement of filamentous cyanobacteria on a solid surface usually has a strong temperature dependency, and the higher the temperature, the faster the speed. Former studies on this phenomenon were conducted using filamentous cyanobacteria with straight morphology. We examined the velocity of the gliding movement of Arthrospira platensis NIES-39 along its longitudinal axis to see if the same was true for this cyanobacterium with helicoid trichomes. Experimental results showed little temperature dependency in the velocity in a wide temperature range in this cyanobacterium. However, when we examined the velocity using mutants with straight trichomes, their velocity was strongly affected by temperature, like other formerly analyzed filamentous cyanobacteria. This result indicates that the helicoid morphology of A. platensis trichomes confers temperature compensation to their migration velocity, enabling them to keep a relatively constant velocity under various temperatures. Migration of wild-type trichomes is considerably suppressed compared to the straight-trichome mutants on solid media. The temperature compensation in the locomotion of this organism appears to be established as part of such a suppression. It was also found that the velocity of this cyanobacterium depended on the trichome length when they were atypically short (

Objective To investigate the effect of regulator of ribosome synthesis 1 (RRS1) on the proliferation and metastasis abilities of human breast cancer cells. Methods Western Blot was used to measure the protein expression level of RRS1 in human breast cancer cell lines (MDA-MB-231, MDA-MB-468, BT549, and MCF-7) and normal human breast epithelial cells. MDA-MB-468 cells were infected with sh-RRS1 lentivirus (sh-RRS1 group) and negative-control lentivirus (Con group), and MDA-MB-468 cells without infection were established as Blank group. Lentiviral infection efficiency was observed under a fluorescence microscope for the Con group and the sh-RRS1 group, and quantitative real-time PCR and Western blot were used to measure the mRNA and protein expression levels of RRS1 in cells; CCK-8 assay was used to observe the effect of RRS1 on the viability of MDA-MB-468 cells; scratch assay, Transwell assay, and invasion assay were used to observe the effect of RRS1 on the invasion and migration abilities of MDA-MB-468 cells. Results The expression level of RRS1 in the breast cancer cell lines was significantly higher than that in normal human breast epithelial cells (F=28.71,P