Chemotactic Cell Migration Research Articles

TGF-β1 and FOXM1 siRNA co-loaded nanoparticles by disulfide crosslinked PEG-PDMAEMA for the treatment of triple-negative breast cancer and its bone metastases in vitro

Introduction Triple-negative breast cancer (TNBC) is characterized by higher malignancy and mortality and is prone to distant metastasis, among which bone is the most common site. It’s urgent to explore new strategies for the treatment of TNBC and its bone metastases. Methods A tumor environment responsive vector, poly-(dimethylaminoethyl methacrylate)-SS-poly(ethylene glycol)-SS-poly-(dimethylaminoethyl methacrylate) (PDMAEMA-SS-PEG-SS-PDMAEMA), was constructed to co-delivery transforming growth factor-β1 (TGF-β1) siRNA and forkhead box M1 (FOXM1) siRNA in MDA-MB-231 cells. The preparation, characterization, in vitro release, stability, and transfection efficiency of nanoparticles were measured. Cell viability, migration, and invasion of MDA-MB-231 cells were determined. Cell chemotactic migration and cell heterogeneity adhesion of MDA-MB-231 cells to the human osteoblast-like cell line MG-63 were determined. Results PDMAEMA-SS-PEG-SS-PDMAEMA self-assembled with siRNA at N/P of 15:1 into nanoparticles with a particle size of 122 nm. In vitro release exhibited redox and pH sensitivity, and the nanoparticles protected siRNA from degradation by RNase and serum protein, remaining stable at 4 °C with similar transfection efficiency with lipo2000. Nanoparticles co-loaded with TGF-β1 siRNA and FOXM1 siRNA inhibited the cell viability, migration and invasion of MDA-MB-231 cells, as well as chemotactic migration and heterogeneous adhesion of MDA-MB-231 cells to MG-63 cells, showing a synergetic effect. After gene silencing on TGF-β1 and FOXM1, the epithelial to mesenchymal transition (EMT) related molecules vimentin mRNA expression decreased while E-cadherin increased. Conclusions PDMAEMA-SS-PEG-SS-PDMAEMA was suitable for TGF-β1 siRNA and FOXM1 siRNA delivery, exhibiting a synergetic inhibition effect on TNBC and its bone metastases, which might be related to its synergetic inhibition on EMT.

Hepatocyte growth factor facilitates the repair of spinal cord injuries by driving the chemotactic migration of mesenchymal stem cells through the β-catenin/TCF4/Nedd9 signaling pathway.

Transplanted mesenchymal stem cells (MSCs) can significantly aid in repairing spinal cord injuries (SCIs) by migrating to and settling at the injury site. However, this process is typically inefficient, as only a small fraction of MSCs successfully reach the target lesion area. During SCI, the increased expression and secretion of hepatocyte growth factor (HGF) act as a chemoattractant that guides MSC migration. Nonetheless, the precise mechanisms by which HGF influences MSC migration are not fully understood. This study focused on unraveling the molecular pathways that drive MSC migration toward the SCI site in response to HGF. It was found that HGF can activate β-catenin signaling in MSCs by either phosphorylating LRP6, suppressing GSK3β phosphorylation through the AKT and ERK1/2 pathways, or enhancing the expression and nuclear translocation of TCF4. This activation leads to elevated Nedd9 expression, which promotes focal adhesion formation and F-actin polymerization, facilitating chemotactic migration. Transplanting MSCs during peak HGF expression in injured tissues substantially improves nerve regeneration, reduces scarring, and enhances hind limb mobility. Additionally, prolonging HGF release can further boost MSC migration and engraftment, thereby amplifying regenerative outcomes. However, inhibiting HGF/Met or interfering with β-catenin or Nedd9 signaling significantly impairs MSC engraftment, obstructing tissue repair and functional recovery. Together, these findings provide a theoretical basis and practical strategy for MSC transplantation therapy in SCI, highlighting the specific molecular mechanisms by which HGF regulates β-catenin signaling in MSCs, ultimately triggering their chemotactic migration.

Tumor-derived Hsp70-CD14 interaction enhances the antitumor potential of cytotoxic T cells by activating tumor-associated macrophages to express CC chemokines and CD40 costimulatory molecules

Heat shock proteins are a widely distributed group of proteins. It is constitutively expressed in almost all organisms and shows little variation throughout evolution. Previously, HSPs, particularly Hsp70, were recognized as molecular chaperones that aid in the proper three-dimensional folding of newly synthesized polypeptides in cells. Recently, researchers have focused on the potential induction of immune cells, including macrophages, antigen-specific CD8+ cytotoxic T cells, and PBMCs. It induces the expression of CC chemokines such as MIP-1α and RANTES, which are responsible for the chemotactic movement and migration of immune cells at the site of infection to neutralize foreign particles in vivo and in vitro in several cell lines but their effect on tumor-associated macrophages is still not known. These cytokines are also known to influence the movement of several immune cells, including CD8+ cytotoxic T cells, toward inflammatory sites. Therefore, the effect of tumor-derived autologous Hsp70 on the expression of MIP-lα and RANTES in tumor-associated macrophages (TAMs) was investigated. Our results indicated that Hsp70 treatment-induced MIP-lα and RANTES expression was significantly greater in TAMs than in NMOs. According to the literature, the CC chemokine shares the same receptor, CCR5, as HIV does for their action, and therefore could provide better completion to the virus for ligand binding. Furthermore, Hsp70-preactivated TAMs induced increased IL-2 and IFN-γ expression in T cells during coculture for 48 h and upregulated the antitumor immune response of the host. Therefore, the outcome of our study could be useful for developing a better approach to restricting the growth and progression of tumors.

Abstract LB166: Discovery of CT3021, a novel potent adenosine A2a/A2b/A1 receptor triple antagonist

Abstract In a CD73-positive tumor microenvironment (TME), ATP released from apoptotic tumor cells converts to adenosine, generating an adenosine-rich immunosuppressive environment. Adenosine activates G-protein coupled receptors expressed on immune cells to achieve immune suppression, with a mechanism of action involving 1) dampening antitumor effector cells (T eff and NK cells) via adenosine engagement on A2a receptor, (2) reducing dendritic cell antigen presentation and promoting MDSC differentiation by activating A2b and A2a receptors, and (3) inducing chemotactic migration of immature plasmacytoid dendritic cells to infiltrate tumor tissue via A1 receptor. Furthermore, A1 receptor expression in certain tumors mediates an adenosine-driven tumor cell proliferation, likely through A1 receptor Gi coupling to the Hippo-YAP signal transduction pathway. Thus, pharmacologic inhibition of A2a, A2b, and A1 receptors by a triple antagonist in the context of adenosine-rich TME may represent an attractive strategy to achieve a better outcome in treating solid tumors. We discovered and characterized a novel adenosine receptor antagonist CT3021. CT3021 exhibits high binding affinity to A2a, A2b, and A1, with a Ki value of 0.37 nM, 1.76 nM, and 1.26 nM, respectively, and is selective against the A3 receptor and other 84 pharmacologically and/or toxicologically relevant targets. Schild analysis demonstrated that CT3021 is a competitive antagonist with a calculated KB value of 0.18 nM on A2a receptor in a whole cell cAMP assay in transfected HEK293 cells. In functional antagonist assays, CT3021 maintains its high potency full antagonism under an adenosine-rich TME-like condition (10-100 µM adenosine) both in transfected HEK293 cells and in isolated human T lymphocytes. CT3021 has a high oral bioavailability, a limited CNS exposure, and a favorable ADME profile. CT3021 is well tolerated in rats after repeat dosing. Further toxicology and efficacy studies are underway to explore the potential of CT3021 as a best-in-class adenosine receptor antagonist immunotherapy for the treatment of CD73 positive and A1 receptor positive solid tumors. Citation Format: Sandong Han, Nam Hee Kim, Hongmei He, Zhi Liang Chu. Discovery of CT3021, a novel potent adenosine A2a/A2b/A1 receptor triple antagonist [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB166.

RSK/GSK3-mediated phosphorylation of FilGAP regulates chemotactic cancer invasion.

Cell migration plays a crucial role in various biological processes, such as gastrulation, immune response, and cancer metastasis. In response to chemoattractant-like growth factors, cells form protrusions and migrate toward the source of the signal. Rho family small GTPase Rac is a key regulator of cell migration by stimulating actin polymerization to generate lamellipodia, flat membrane protrusions at the leading edge of migrating cells. FilGAP (ARHGAP24), a Rac-specific GTPase-activating protein (GAP), suppresses lamellipodia formation, and controls tumor cell migration. In this study, we found that FilGAP is phosphorylated downstream of epidermal growth factor (EGF) signaling. Upon EGF stimulation, FilGAP is phosphorylated at Ser625 by p90 ribosomal S6 kinase (RSK) and then at Ser621 by glycogen synthase kinase 3 (GSK3). Phosphorylation of FilGAP induces its dissociation from actin filaments. We identified a novel actin-localization domain of FilGAP that is essential for stabilizing cell adhesion. Additionally, we found that phosphorylation of FilGAP inhibits its lamellipodia suppression activity. Finally, we showed the expression of nonphosphorylatable FilGAP mutant, but not wild-type FilGAP, reduced cell migration speed and persistence toward the EGF gradient. Taken together, our results suggest that phosphorylation of FilGAP downstream of EGF-signaling plays a critical role in regulating chemotactic tumor cell migration by controlling cell-matrix adhesion and protrusion formation.

Inhibition of Rho kinase (ROCK) impairs cytoskeletal contractility in human Müller glial cells without effects on cell viability, migration, and extracellular matrix production

The epiretinal membrane is a fibrocontractile tissue that forms on the inner surface of the retina, causing visual impairment ranging from mild to severe, and even retinal detachment. Müller glial cells actively participate in the formation of this membrane. Current research is constantly seeking for new therapeutic approaches that aim to prevent or treat cellular dysfunctions involved in the progression of this common fibrosis condition. The Rho GTPases signaling pathway regulates several processes associated with the epiretinal membrane, such as cell proliferation, migration, and contraction. Rho kinase (ROCK), an effector of the RhoA GTPase, is an interesting potential therapeutic target. This study aimed to evaluate the effects of a ROCK inhibitor (Y27632) on human Müller cells viability, growth, cytoskeletal organization, expression of extracellular matrix components, myofibroblast differentiation, migration, and contractility. Müller cells of the MIO-M1 lineage were cultured and treated for different periods with the inhibitor. Viability was evaluated by MTT assay and trypan blue exclusion method, and growth was evaluated by growth curve and BrdU incorporation assay. The actin cytoskeleton was stained with fluorescent phalloidin, intermediate filaments and microtubules were analyzed with immunofluorescence for vimentin and α-tubulin. Gene and protein expression of collagens I and V, laminin and fibronectin were evaluated by rt-PCR and immunofluorescence. Chemotactic and spontaneous cell migration were studied by transwell assay and time-lapse observation of live cells, respectively. Cell contractility was assessed by collagen gel contraction assay. The results showed that ROCK inhibition by Y27632 did not affect cell viability, but decreased cell growth and proliferation after 72 h. There was a change in cell morphology and organization of F-actin, with a reduction in the cell body, disappearance of stress fibers and formation of long, branched cell extensions. Microtubules and vimentin filaments were also affected, possibly because of F-actin alterations. The inhibitor also reduced gene expression and immunoreactivity of smooth muscle α-actin, a marker of myofibroblasts. The expression of extracellular matrix components was not affected by the inhibitor. Chemotactic cell migration showed no significant changes, while cell contractility was substantially reduced. No spontaneous migration of MIO-M1 cells was observed. In conclusion, pharmacological inhibition of ROCK in Müller cells could be a potentially promising approach to treat epiretinal membranes by preventing cell proliferation, contractility and transdifferentiation, without affecting cell viability.

TGFß1 Stimulates Lymphatic Endothelial Cells to Produce IL7 and IL15, Which Act as Chemotactic Factors for Breast Cancer Cells with Mesenchymal Properties

The lymphatic system is a major gateway for tumor cell dissemination but the mechanisms of how tumor cells gain access to lymphatic vessels are not completely understood. Breast cancer cells undergoing epithelial-mesenchymal transition (EMT) gain invasive and migratory properties. Overexpression of the cytokine transforming growth factor β1 (TGFβ1), a potent inducer of EMT, is frequently detected in the tumor microenvironment and correlates with invasion and lymph metastasis. Recently, we reported that TGFβ1 stimulated breast cancer cells with mesenchymal properties to migrate in a targeted fashion towards the lymphatic system via CCR7/CCL21-mediated chemotaxis, similar to dendritic cells during inflammation. Here, we aimed to identify additional chemotactic factors and corresponding receptors that could be involved in guiding breast cancer cells through the lymphatic system. Through a combination of RNA sequencing analysis, database screening and invasion assays we identified IL7/IL7R and IL15/IL15R as pairs of chemokines and receptors with potential roles in promoting chemotactic migration of breast cancer cells with mesenchymal properties towards the lymphatics. The results demonstrate the capacity of TGFβ1 to orchestrate crosstalk between tumor cells and lymphatic endothelial cells and warrant further studies to explore the roles of IL7 and IL15 in promoting lymph metastasis of breast cancer.

MCP-1/CCR2 axis is involved in the regulation of γδT cells in lupus nephritis.

γδT cells are important innate immune cells that are involved in the occurrence and development of autoimmune diseases such as systemic lupus erythematosus (SLE). Lupus nephritis (LN) is a serious complication of SLE, characterized by the accumulation of immune cells (including γδT cells) in the target organs to participate in the disease process. Therefore, clarifying how γδT cells chemotactically migrate to target organs may be a key to developing therapeutic methods against LN. Enzyme-linked immunosorbent assay (ELISA) was used to detect serum levels of chemokines in LN patients and healthy controls. Real-time polymerase chain reaction (RT-PCR) and flow cytometry were used to measure the expression of chemokine receptors on the surface of γδT cells. The chemotactic migration ability of γδT cells was detected by Transwell assay. Signalling pathway activation of γδT cells was detected by Automated Capillary Electrophoresis Immunoassay and flow cytometry. The serum levels of chemokines, including monocyte chemoattractant protein-1 (MCP-1) in LN patients, were significantly increased. CCR2, the receptor of MCP-1, was also highly expressed on the surface of peripheral γδT cells in LN patients. In addition, the exogenous addition of MCP-1 can enhance chemotactic migration of γδT cells in LN patients. MCP-1 could activate STAT3 signalling in LN patients' peripheral γδT cells. γδT cells might participate in the pathogenesis of LN through MCP-1/CCR2 axis. This finding provides new opportunities for developing treatment methods against LN by targeting MCP-1/CCR2 axis.

Study on the mechanism of CXCR3 expression in paracancer and tumor tissues of colon cancer patients.

e15506 Background: Colorectal cancer (CRC) is one of the most common types of malignant tumors, and its incidence is increasing year by year.Chemokines are small cytokines or signaling proteins secreted by cells. They have the ability to induce directional chemotactic migration of nearby responding cells and can mediate intracellular signal transduction by binding to 18 G protein-coupled receptor families.C-X-C motif chemokine receptor 3 (CXCR3) is activated by three Interferon-γ inducible ligands CXCL9, CXCL10 and CXCL11 and encodes 368 amino acids. Its molecular weight was about 41KDa and the cDNA was 1104bp in length.In recent years, a large number of studies have confirmed that the expression of CXCR3 and its ligands in the body is closely related to tumor immunity, tumorigenesis and metastasis.Therefore, the aim of this study is to investigate the expression of CXCR3 in tumor and adjacent intestinal tissues of patients with colorectal cancer and the effects of related gene detection on tumor proliferation, migration, invasion and recurrence in patients with colorectal cancer. Methods: Flow cytometry was used to detect 40 colon cancer patients who had not received neoadjuvant therapy in the Department of General Surgery of the Affiliated Cancer Hospital of Zhengzhou University. The expression of CXCR3, PD-1, CD4+, CD45+ and CD8+T cells in tumor and para-tumor specimens (taken > 2cm from the edge of the cancer) was detected, and the expression characteristics of CXCR3 in colon cancer patients and its correlation with general clinical data were analyzed. The correlation between KRAS, MSS, MSI-H gene mutations and colon cancer was analyzed under the condition of different lymphocyte expression. Results: 1.CXCR3+CD8+T cells were significantly different, and CXCR3+CD4+T cells were significantly different. These results suggest that CXCR3 expression with different lymphocytes may play an important role in tumor cell growth.2.The difference of CD45+T cells was statistically significant. This suggests that the expression of CD45+T cells may also affect tumor growth.3.CXCR3+CD4+T cells may play a role in promoting tumor metastasis. Conclusions: The expression of CXCR3+CD8+T cells, CXCR3+CD4+T cells and CD45+T lymphocytes in tumor tissues of colon cancer patients is correlated with the expression of adjacent tissues. The expression of PD-1+CD8+ lymphocytes in tumor tissues is less than that in adjacent tissues, indicating that they are related to tumor metastasis and proliferation. CXCR3 may play an important role in lymph node metastasis, and PD-1 expression may also be related to patient gender. Therefore, the expression of CXCR3 and T lymphocytes in tumor tissues and adjacent tissues of patients is helpful for the diagnosis and treatment of colon cancer, and has important value in clinical application.

YAP inhibitor verteporfin suppresses tumor angiogenesis and overcomes chemoresistance in esophageal squamous cell carcinoma.

Targeting angiogenesis is an attractive strategy for the effective treatment of cancer. This study aimed to investigate the anti-cancer activities of YAP inhibitor verteporfin (VP) in esophageal squamous cell carcinoma (ESCC) cells through its inhibitory effect on tumor angiogenesis. Cell proliferation, apoptosis, migration and invasion abilities were estimated by MTT, colony formation, DAPI staining, wound healing and transwell assays, respectively. Human umbilical vein endothelial cell (HUVEC) tube formation assay and chick embryo chorioallantoic membrane (CAM) model were used to observe angiogenesis in vitro and in vivo. The interactions between ESCC cells and HUVECs were assessed by cell chemotactic migration and adhesion assays. The expression levels of angiogenesis-related molecules were detected by Western blot. We found that VP was potential to inhibit ESCC cell proliferation, migration, invasion and induce apoptosis in the dose-dependent fashion. VP also significantly suppressed proliferation, migration, and tube formation of HUVECs and promoted apoptosis of HUVECs, and reduced angiogenesis in CAM. Moreover, VP inhibited ESCC cell-induced angiogenesis in vitro by decreasing HUVEC chemotactic migration, adhesion and tube formation, and also reduced ESCC cell-induced neovascularization of the CAM in vivo. In addition, VP suppressed the expression of pro-angiogenic molecules such as VEGFA, MMP-2 and β-catenin in ESCC cells. Furtherly, VP increased the chemosensitivity of ESCC-resistant cells to paclitaxel (PTX). The combination of VP and PTX attenuated the resistant cell-mediated angiogenesis in vitro and in vivo. These results reveal for the first time that VP potently inhibits malignant progression and overcomes chemoresistance of ESCC cells via inhibition of tumor angiogenesis. It provides insight into a new strategy for the treatment of ESCC that VP could be a potential drug candidate for targeting tumor angiogenesis.

Phthalides Isolated from the Endolichenic Arthrinium sp. EL000127 Exhibits Antiangiogenic Activity.

Endolichenic fungi (ELF) produce specialized metabolites that have various medicinal properties. Inhibition of tumor angiogenesis efficaciously suppresses many types of cancer. This study aimed to discover novel antiangiogenic agents from specialized metabolite extracts of ELF strains isolated from Korean lichens. The EtOAc extracts of 51 ELF strains were subjected to a screening pipeline consisting of cell viability, scratch wound healing, and Transwell migration assays. The EtOAc extract of Arthrinium sp. EL000127 showed the most potent inhibitory activity against the chemotactic migration of human umbilical vein endothelial cells (HUVEC). Targeted isolation on the major LC-MS peaks exhibited a previously known phthalide, 3-O-methylcyclopolic acid (1), and two unknown analogues of 1, 3-O-phenylethylcyclopolic acid (2) and 3-O-p-hydroxyphenylethylcyclopolic acid (3). The structures were characterized by MS and NMR analyses. All the isolates were acquired and applied to bioassays as racemates due to spontaneous racemization. Among the isolates, compound 3 effectively inhibits HUVEC motility by suppressing mRNA expressions of genes regulating epithelial cell survival and motility, which suggested that compound 3 is a potent antiangiogenic agent suitable for further exploration as a potential novel therapeutic against cancers.

Mathematical Modeling of Spherical Shell-Type Pattern of Tumor Invasion

Cancer cell migration, as the principal element of tumor invasion, involves different cellular mechanisms. Various modes of cell migration including single and collective motions contribute to the invasion patterns. The competition between adhesive cell–cell and cell–matrix forces is a key factor that determines such patterns. In this paper, we study a distinct shell-type mode of tumor invasion observed in brain and breast tumors. In this mode, cells at the outer layer of the tumor collectively move away from the core and form a shell-type shape. Both the core and the shell sustain a sharp interface between cells and the surrounding matrix. To model the preserved interface, we adopted a Cahn–Hilliard-type free energy relation with the contribution of the interfacial stress. This nonconvex form of free energy allows for cells to remain together and preserve the tumor core via adhesive cell–cell forces while separating the core from the surrounding matrix across a continuous sharp interface. In addition, the motion of the shell was modeled using the chemotactic migration of cells in response to the gradient of nutrients. The associated fluxes of cells were implemented in a general form of balance law. A non-Michaelis–Menten kinetics model was adopted for the proliferation rate of cells. The flux of nutrients was also modeled using a simple diffusion equation. The comparison between the model predictions and experimental observations indicates the ability of the model to manifest the salient features of the invasion pattern.

Isolation of ciliary ectosomes and analysis of amidated peptide-mediated chemotaxis in Chlamydomonas.

Ciliary ectosomes are vesicles that bud from the ciliary membrane. Isolation and analysis of these structures can shed light on their bioactive cargoes and identify proteins and biomolecules involved in intercellular communication and various physiological processes. Most published methods to isolate ciliary ectosomes are based on their size (100nm to 1μm) to separate cilia-derived vesicles from isolated cilia and/or intact cells. However, it is often difficult to determine the origin of extracellular vesicles and to distinguish ciliary ectosomes from ectosomes budded from the plasma membrane or from exosomes that derive from multivesicular bodies. Here, we describe procedures to isolate and purify ciliary ectosomes from the unicellular green alga, Chlamydomonas reinhardtii, through differential and iodixanol density gradient ultracentrifugation; in this organism, the ciliary membrane is the only membrane directly exposed to the environment and thus ectosomes are of known origin. Ciliary ectosomes contain enzymes and α-amidated peptide products required to mediate peptidergic-signaling cascades; one identified amidated peptide acts as a chemotactic modulator for C. reinhardtii gametes. Classical methods used to assess chemotaxis do not provide quantitative measurements of the chemotactic gradient or the real-time effects on the migration of fast moving cells. Consequently, we developed a chemotaxis assay protocol using microfluidic channel slides that provides quantitative and qualitative measurements of the chemotactic gradient and cell migration. Here, we describe how to establish a stable gradient of a bioactive substance in microfluidic channel slides and perform quantitative assays to assess chemotaxis of both individual cells and populations of C. reinhardtii.

Transcriptome sequencing analysis of the role of β-catenin in F-actin reorganization in embryonic palatal mesenchymal cells.

Palatogenesis is a highly regulated and coordinated developmental process that is coordinated by multiple transcription factors and signaling pathways. Our previous studies identified that defective palatal shelf reorientation due to aberrant mesenchymal β-catenin signaling is associated with Filamentous actin (F-actin) dysregulation. Herein, the underlying mechanism of mesenchymal β-catenin in regulating F-actin cytoskeleton reorganization is further investigated. Firstly, β-catenin silenced and overexpressed mouse embryonic palatal mesenchymal (MEPM) cells were established by adenovirus-mediated transduction. Subsequently, we compared transcriptomes of negative control (NC) group, β-catenin knockdown (KD) group, or β-catenin overexpression group respectively using RNA-sequencing (RNA-seq), and differentially expressed genes (DEGs) screened were further identified by quantitative real-time polymerase chain reaction (qRT-PCR). Finally, in vivo experiments further verified the expression change of critical molecules. We discovered 184 and 522 DEGs in the knockdown and overexpression groups compared to the NC group, respectively (adjusted P<0.05; |fold change| >2.0). Among these, 106 DEGs were altered in both groups. Gene Ontology (GO) enrichment analysis relating to biological functions identified cytokine-cytokine receptor interaction, and positive modulation of cellular migration. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment assessment indicated that these DEGs were chiefly linked by the regulation of signaling receptor activity and chemokine signaling pathways. Quantitative real-time polymerase chain reaction (qRT-PCR) results showed that the similar expression trend of serum amyloid A3 (Saa3) and CXC-chemokine ligand 5 (Cxcl5) possibly involved in cytoskeletal rearrangement with RNA-seq data. Experiments in vivo displayed that no significant expression change of Saa3 and Cxcl5 was observed in β-catenin knockout and overexpressed mouse models. Our study provides an expression landscape of DEGs in β-catenin silenced and overexpressed MEPM cells, which emphasizes the important role of processes such as chemotactic factor and cell migration. Our data gain deeper insight into genes associated with F-actin reorganization that is regulated by β-catenin either directly or by another route, which will contribute to further investigation of the exact mechanism of mesenchymal β-catenin/F-actin in palatal shelf reorientation.

CircRAD54L2 promotes triple-negative breast cancer progression by regulating the miR-888 family/PDK1 axis

BackgroundThe long-term prognosis of breast cancer with metastasis remains extremely poor. Genetic alterations in tumor cells result in cellular heterogeneity, promoting cancer cells invasion and colonization in some organs during the metastatic process. CircRNAs are very promising as critical biological markers and precise diagnoses in identifying disease mechanisms and developing new methods for effective treatment. However, the role of aberrant expression of circRNAs in breast cancer progression remains largely unknown. MethodsRNase R treatment and quantitative RT-PCR (qRT-PCR) were performed for circRNA detection. Transwell chamber assays were used to examine the chemotactic migration and invasion of breast cancer cells. ResultsThis study identified and characterized the circRAD54L2 originating from exon 1, 2, 3, and 4 of the RAD54L2 gene. Importantly, we found that circRAD54L2, rather than RAD54L2 linear mRNA, was significantly upregulated in breast cancer cell lines. Furthermore, we found that inhibiting circRAD54L2 expression markedly reduced the invasion, metastasis, and proliferation of breast cancer cells via sponging of the miR-888 family, which downregulated the expression of pyruvate dehydrogenase kinase 1 (PDK1). ConclusionOur results showed that circRAD54L2 could regulate PDK1 expression by sponging the miR-888 family competing for the ceRNA mechanism, indicating that circRAD54L2 may act as an essential upstream regulator and providing further mechanistic evidence to support the notion that circRAD54L2/miR-888s/PDK1 is a promising therapeutic target in the treatment of breast cancer.

Recent Advances in CXCL12/CXCR4 Antagonists and Nano-Based Drug Delivery Systems for Cancer Therapy.

Chemokines can induce chemotactic cell migration by interacting with G protein-coupled receptors to play a significant regulatory role in the development of cancer. CXC chemokine-12 (CXCL12) can specifically bind to CXC chemokine receptor 4 (CXCR4) and is closely associated with the progression of cancer via multiple signaling pathways. Over recent years, many CXCR4 antagonists have been tested in clinical trials; however, Plerixafor (AMD3100) is the only drug that has been approved for marketing thus far. In this review, we first summarize the mechanisms that mediate the physiological effects of the CXCL12/CXCR4 axis. Then, we describe the use of CXCL12/CXCR4 antagonists. Finally, we discuss the use of nano-based drug delivery systems that exert action on the CXCL12/CXCR4 biological axis.

Development of a human antibody that exhibits antagonistic activity toward CC chemokine receptor 7.

BackgroundCC chemokine receptor 7 (CCR7) is a member of G-protein-coupled receptor family and mediates chemotactic migration of immune cells and different cancer cells induced via chemokine (C–C motif) ligand 19 (CCL19) or chemokine (C–C motif) ligand 21 (CCL21). Hence, the identification of blockade antibodies against CCR7 could lead to the development of therapeutics targeting metastatic cancer.MethodsCCR7 was purified and stabilized in its active conformation, and antibodies specific to purified CCR7 were screened from the synthetic M13 phage library displaying humanized scFvs. The in vitro characterization of selected scFvs identified two scFvs that exhibited CCL19-competitive binding to CCR7. IgG4’s harboring selected scFv sequences were characterized for binding activity in CCR7+ cells, inhibitory activity toward CCR7-dependent cAMP attenuation, and the CCL19 or CCL21-dependent migration of CCR7+ cells.ResultsAntibodies specifically binding to purified CCR7 and CCR7+ cells were isolated and characterized. Two antibodies, IgG4(6RG11) and IgG4(72C7), showed ligand-dependent competitive binding to CCR7 with KD values of 40 nM and 50 nM, respectively. Particularly, IgG4(6RG11) showed antagonistic activity against CCR7, whereas both antibodies significantly blocked the ligand-induced migration and invasion activity of CCR7+ cancer cells.ConclusionsTwo antibody clones were successfully identified from a synthetic scFv-displaying phage library using purified recombinant CCR7 as an antigen. Antibodies specifically bound to the surface of CCR7+ cells and blocked CCR7+ cell migration. Particularly, 6RG11 showed antagonist activity against CCR7-dependent cAMP attenuation.

Plasminogen and plasmin can bind to human T cells and generate truncated CCL21 that increases dendritic cell chemotactic responses

Plasmin is a broad-spectrum protease and therefore needs to be tightly regulated. Active plasmin is formed from plasminogen, which is found in high concentrations in the blood and is converted by the plasminogen activators. In the circulation, high levels of α2-antiplasmin rapidly and efficiently inhibit plasmin activity. Certain myeloid immune cells have been shown to bind plasmin and plasminogen on their cell surface via proteins that bind to the plasmin(ogen) kringle domains. Our earlier work showed that T cells can activate plasmin but that they do not themselves express plasminogen. Here, we demonstrate that T cells express several known plasminogen receptors and that they bind plasminogen on their cell surface. We show T cell–bound plasminogen was converted to plasmin by plasminogen activators upon T cell activation. To examine functional consequences of plasmin generation by activated T cells, we investigated its effect on the chemokine, C-C motif chemokine ligand 21 (CCL21). Video microscopy and Western blotting confirmed that plasmin bound by human T cells cleaves CCL21 and increases the chemotactic response of monocyte-derived dendritic cells toward higher CCL21 concentrations along the concentration gradient by increasing their directional migration and track straightness. These results demonstrate how migrating T cells and potentially other activated immune cells may co-opt a powerful proteolytic system from the plasma toward immune processes in the peripheral tissues, where α2-antiplasmin is more likely to be absent. We propose that plasminogen bound to migrating immune cells may strongly modulate chemokine responses in peripheral tissues.

Extracellular Vesicles from Steatotic Hepatocytes Provoke Pro-Fibrotic Responses in Cultured Stellate Cells.

Hepatic steatosis and chronic hepatocyte damage ultimately lead to liver fibrosis. Key pathophysiological steps are the activation and transdifferentiation of hepatic stellate cells. We assessed the interplay between hepatocytes and hepatic stellate cells under normal and steatotic conditions. We hypothesized that hepatocyte-derived extracellular vesicles (EVs) modify the phenotype of stellate cells. By high speed centrifugation, EVs were isolated from conditioned media of the hepatocellular carcinoma cell line HepG2 under baseline conditions (C-EVs) or after induction of steatosis by linoleic and oleic acids for 24 h (FA-EVs). Migration of the human stellate cell line TWNT4 and of primary human stellate cells towards the respective EVs and sera of MAFLD patients were investigated using Boyden chambers. Phenotype alterations after incubation with EVs were determined by qRT-PCR, Western blotting and immunofluorescence staining. HepG2 cells released more EVs after treatment with fatty acids. Chemotactic migration of TWNT4 and primary hepatic stellate cells was increased, specifically towards FA-EVs. Prolonged incubation of TWNT4 cells with FA-EVs induced expression of proliferation markers and a myofibroblast-like phenotype. Though the expression of the collagen type 1 1 gene did not change after FA-EV treatment, expression of the myofibroblast markers, e.g., -smooth-muscle-cell actin and TIMP1, was significantly increased. We conclude that EVs from steatotic hepatocytes can influence the behavior, phenotypes and expression levels of remodeling markers of stellate cells and guides their directed migration. These findings imply EVs as operational, intercellular communicators in the pathophysiology of steatosis-associated liver fibrosis and might represent a novel diagnostic parameter and therapeutic target.

Ginger Constituent 6-Shogaol Inhibits Inflammation- and Angiogenesis-Related Cell Functions in Primary Human Endothelial Cells.

Rhizomes from Zingiber officinale Roscoe are traditionally used for the treatment of a plethora of pathophysiological conditions such as diarrhea, nausea, or rheumatoid arthritis. While 6-gingerol is the pungent principle in fresh ginger, in dried rhizomes, 6-gingerol is dehydrated to 6-shogaol. 6-Shogaol has been demonstrated to exhibit anticancer, antioxidative, and anti-inflammatory actions more effectively than 6-gingerol due to the presence of an electrophilic Michael acceptor moiety. In vitro, 6-shogaol exhibits anti-inflammatory actions in a variety of cell types, including leukocytes. Our study focused on the effects of 6-shogaol on activated endothelial cells. We found that 6-shogaol significantly reduced the adhesion of leukocytes onto lipopolysaccharide (LPS)-activated human umbilical vein endothelial cells (HUVECs), resulting in a significantly reduced transmigration of THP-1 cells through an endothelial cell monolayer. Analyzing the mediators of endothelial cell–leukocyte interactions, we found that 30 µM of 6-shogaol blocked the LPS-triggered mRNA and protein expression of cell adhesion molecules. In concert with this, our study demonstrates that the LPS-induced nuclear factor κB (NFκB) promoter activity was significantly reduced upon treatment with 6-shogaol. Interestingly, the nuclear translocation of p65 was slightly decreased, and protein levels of the LPS receptor Toll-like receptor 4 remained unimpaired. Analyzing the impact of 6-shogaol on angiogenesis-related cell functions in vitro, we found that 6-shogaol attenuated the proliferation as well as the directed and undirected migration of HUVECs. Of note, 6-shogaol also strongly reduced the chemotactic migration of endothelial cells in the direction of a serum gradient. Moreover, 30 µM of 6-shogaol blocked the formation of vascular endothelial growth factor (VEGF)-induced endothelial sprouts from HUVEC spheroids and from murine aortic rings. Importantly, this study shows for the first time that 6-shogaol exhibits a vascular-disruptive impact on angiogenic sprouts from murine aortae. Our study demonstrates that the main bioactive ingredient in dried ginger, 6-shogaol, exhibits beneficial characteristics as an inhibitor of inflammation- and angiogenesis-related processes in vascular endothelial cells.