Cell Surface Adhesion Research Articles

Polydopamine modified polycaprolactone powder for fabrication bone scaffold owing intrinsic bioactivity

Polycaprolactone (PCL) has been extensively investigated in the field of bone tissue engineering due to its good biocompatibility, biodegradability and processability. Nevertheless, the lack of bioactivity results in poor osseointegration between artificial implant and host bone tissue, which severely restricts its application for bone regeneration. In this study, PCL powder was first surface functionalized with polydopamine (PDA) coating through oxidative self-polymerization of monomer dopamine, and the PDA modified PCL powder was used for the fabrication of bone scaffold with intrinsic bioactivity via selective laser sintering (SLS). The results suggested that PDA was modified on the PCL powder surface successfully and still remained after laser sintering. The 3D printed scaffold achieved a transition from bioinert to bioactive as evaluated by the formation of apatite on its surface, which was the result of the interaction between the abundant active catechol groups in the introduced PDA and calcium ion from simulated body fluid. Besides, the scaffold provided more hydrophilic surfaces for cell adhesion and growth than the pure PCL scaffold due to the presence of amino and hydroxyl functional group.

Bi-allelic variants in SPATA5L1 lead to intellectual disability, spastic-dystonic cerebral palsy, epilepsy, and hearing loss

Spermatogenesis-associated 5 like 1 (SPATA5L1) represents an orphan gene encoding a protein of unknown function. We report 28 bi-allelic variants in SPATA5L1 associated with sensorineural hearing loss in 47 individuals from 28 (26 unrelated) families. In addition, 25/47 affected individuals (53%) presented with microcephaly, developmental delay/intellectual disability, cerebral palsy, and/or epilepsy. Modeling indicated damaging effect of variants on the protein, largely via destabilizing effects on protein domains. Brain imaging revealed diminished cerebral volume, thin corpus callosum, and periventricular leukomalacia, and quantitative volumetry demonstrated significantly diminished white matter volumes in several individuals. Immunofluorescent imaging in rat hippocampal neurons revealed localization of Spata5l1 in neuronal and glial cell nuclei and more prominent expression in neurons. In the rodent inner ear, Spata5l1 is expressed in the neurosensory hair cells and inner ear supporting cells. Transcriptomic analysis performed with fibroblasts from affected individuals was able to distinguish affected from controls by principal components. Analysis of differentially expressed genes and networks suggested a role for SPATA5L1 in cell surface adhesion receptor function, intracellular focal adhesions, and DNA replication and mitosis. Collectively, our results indicate that bi-allelic SPATA5L1 variants lead to a human disease characterized by sensorineural hearing loss (SNHL) with or without a nonprogressive mixed neurodevelopmental phenotype.

Fluid Flow Dependency in Immunoselective Cell Capture via Liquid Biopsy.

Liquid biopsy targets rare cells that overexpress disease-specific membrane markers and capture these cells via immunoaffinity. The diagnosis efficiency of liquid biopsy can be impaired by the presence of healthy adherent cells also expressing the same biomarkers. Here, we investigated the effect of settling times and rinsing flow rates on the efficiency of EpCAM-based immunocapture using both simulation and experiments with three different cell types. Cell-surface adhesion forces and shear rates were calculated to define the range of rinsing flow rates to test experimentally. Healthy adherent cells did not adhere to blocked immunofunctionalized surfaces within the timeframe of the experiment; however, healthy EpCAM positive cells did bind to the surface to some extent. The greatest difference in capture efficiency was obtained using a high rinsing flow rate of 25 mL/min following 40 min static incubation, indicating that optimizing rinsing flow rates could be a viable option to capture, more specifically, cancer cells overexpressing EpCAM.

Cell spheroids containing bioactive molecule-immobilized porous particles with a leaf-stacked structure

Despite the widespread use of cell spheroids in tissue engineering for the regeneration of large tissues and organs and for high-throughput screening in pharmacology and toxicology, the clinical challenges include cellular heterogeneity, low structural stability, and uncontrolled cell differentiation. Using human bone marrow-derived mesenchymal stem cells (hBMSCs), we developed cell spheroids containing bioactive molecule (bone morphogenetic protein-2, BMP-2)-immobilized polycaprolactone (PCL) particles with a leaf-stacked structure (LSS). The LSS particles were fabricated via simple heating–cooling method, and the BMP-2 was continuously released from LSS particles for 19 days. Based on in vitro and in vivo observations of the cell spheroids, we found that (i) the porous LSS particles prevent cellular heterogeneity via sufficient diffusion of oxygen/nutrients, (ii) the cell adhesive surface on LSS particles improved the structural stability, (iii) the BMP-2 released from LSS particles induced effective osteogenic differentiation of stem cells, and (iv) the BMP-2-immobilized LSS particles induced new bone formation. Therefore, the cell spheroid containing bioactive molecule-immobilized LSS particles represent a potential strategy to overcome the inherent limitations of conventional cell spheroids. We further suggest that the cell spheroid containing bioactive molecule-immobilized LSS particles is an elegant platform for the regeneration of various tissues and organs as well as high-throughput screening in pharmacology and toxicology studies.

Phenotype and pathological significance of MCAM+ (CD146+) T cell subset in psoriatic arthritis

BackgroundCD146 (MCAM-melanoma cell adhesion molecule) is a cell surface adhesion molecule for Laminin 411. T cells expressing MCAM are mainly responsible for IL-17 production. IL-17 secreting T helper cells (Th17 cells) are critical for the pathogenesis of psoriatic arthritis (PsA). Here we hypothesized enrichment of CD146+IL-17+ memory T cells in PsA synovium and studied the association of CD146 expression and CD4+IL-17+ activated memory (CD11a+CD45RO+) T cells in synovial fluid and blood of PSA, rheumatoid arthritis (RA, a positive control) and osteoarthritis (OA) patients.MethodsHi-D FACS studies were done to identify IL-17 in CD4+CD146+CD45RO+ and CD8+CD146+CD45RO+ T cells.ResultsWe observed that effector CD146+(MCAM+) T cells are enriched at the synovial inflammation site in PsA.ConclusionAs CD146+ T cells are a key resource for IL-17 it is likely that the enrichment of these MCAM+ pathologic cells are critical for the disease process of PsA.

Biological Evaluation of Alkyl Triphenylphosphonium Ostruthin Derivatives as Potential Anti-Inflammatory Agents Targeting the Nuclear Factor κB Signaling Pathway in Human Lung Adenocarcinoma A549 Cells

Ostruthin (6-geranyl-7-hydroxycoumarin) is one of the constituents isolated from Paramignya trimera and has been classified as a simple coumarin. We recently reported the synthesis of alkyl triphenylphosphonium (TPP) derivatives from ostruthin and evaluated their anticancer activities. In the present study, we demonstrated that alkyl TPP ostruthin derivatives inhibited the up-regulation of cell-surface intercellular adhesion molecule-1 (ICAM-1) in human lung adenocarcinoma A549 cells stimulated with tumor necrosis factor-α (TNF-α) without affecting cell viability, while ostruthin itself exerted cytotoxicity against A549 cells. The heptyl TPP ostruthin derivative (termed OS8) attenuated the up-regulation of ICAM-1 mRNA expression at concentrations higher than 40 µM in TNF-α-stimulated A549 cells. OS8 inhibited TNF-α-induced nuclear factor κB (NF-κB)-responsive luciferase reporter activity at concentrations higher than 40 µM, but did not affect the translocation of the NF-κB subunit RelA in response to the TNF-α stimulation at concentrations up to 100 µM. A chromatin immunoprecipitation assay showed that OS8 at 100 µM prevented the binding of RelA to the ICAM-1 promoter. We also showed that OS8 at 100 µM inhibited the TNF-α-induced phosphorylation of RelA at Ser 536. Moreover, the TNF-α-induced phosphorylation of an inhibitor of NF-κB α and extracellular signal-regulated kinase was reduced by OS8. These results indicate that OS8 has potential as an anti-inflammatory agent that targets the NF-κB signaling pathway.

Retrospective study for classifying hypertensive disorders of pregnancy by time of onset

Preeclampsia is characterised by systemic endothelial cell dysfunction thought to be triggered by toxic/dangerous factors from the placenta, including placental extracellular vesicles (EVs). Why placental EVs become toxic is unknown. We previously reported that preeclamptic sera produced toxic/dangerous placental macrovesicles but whether small EVs are also toxic/dangerous in preeclampsia is unknown.First trimester placental explants were treated with 10% preeclamptic or control sera (n = 10) for 24 h. Micro- and nano-vesicles were harvested by sequential centrifugation. Micro- or nano-vesicles were also exposed to monolayers of endothelial cells in the presence or absence of nifedipine (50 μg/ml) or labetalol (0.5 μg/ml) which are well-known anti-hypertensives in clinical practices.The number and size of micro- and nano-vesicles were counted. Endothelial cell-surface intercellular adhesion molecule 1 (ICAM-1) and high mobility group box 1 (HMGB1) levels in micro- or nano-vesicles were measured by immunoassays.Neither the amount nor size of both micro- and nano-vesicles was different after treating placental explants with preeclamptic or control sera. The levels of HMGB1 were significantly increased in both micro- and nano-vesicles from preeclamptic sera treated placental explants (p < 0.03). Exposing endothelial cells to micro- or nano-vesicles from preeclamptic sera-treated placental explants induced endothelial activation, but it was reversed by co-incubation with nifedipine (p = 0.004) or labetalol (p = 0.002).Our data demonstrate that preeclamptic sera produce toxic/dangerous micro- and nano-placental EVs which activated endothelial cells. This effect was reversed by antihypertensives. The increased levels of HMGB1 in EVs may contribute to endothelial cell activation.

Neuropilin-1 is required for endothelial cell adhesion to soluble vascular endothelial growth factor receptor 1.

Neuropilin‐1 (NRP‐1) is a semaphorin receptor involved in neuron guidance, and a co‐receptor for selected isoforms of the vascular endothelial growth factor (VEGF) family. NRP‐1 binding to several VEGF‐A isoforms promotes growth factor interaction with VEGF receptor (VEGFR)‐2, increasing receptor phosphorylation. Additionally, NRP‐1 directly interacts with VEGFR‐1, but this interaction competes with NRP‐1 binding to VEGF‐A165 and does not enhance VEGFR‐1 activation. In this work, we investigated in detail the role of NRP‐1 interaction with the soluble isoform of VEGFR‐1 (sVEGFR‐1) in angiogenesis. sVEGFR‐1 acts both as a decoy receptor for VEGFs and as an extracellular matrix protein directly binding to α5β1 integrin on endothelial cells. By combining cell adhesion assays and surface plasmon resonance experiments on purified proteins, we found that sVEGFR‐1/NRP‐1 interaction is required both for α5β1 integrin binding to sVEGFR‐1 and for endothelial cell adhesion to a sVEGFR‐1‐containing matrix. We also found that a previously reported anti‐angiogenic peptide (Flt2‐11), which maps in the second VEGFR‐1 Ig‐like domain, specifically binds NRP‐1 and inhibits NRP‐1/sVEGFR‐1 interaction, a process that likely contributes to its anti‐angiogenic activity. In view of potential translational applications, we developed a five‐residue‐long peptide, derived from Flt2‐11, which has the same ability as the parent Flt2‐11 peptide to inhibit cell adhesion to, and migration towards, sVEGFR‐1. Therefore, the Flt2‐5 peptide represents a potential anti‐angiogenic compound per se, as well as an attractive lead for the development of novel angiogenesis inhibitors acting with a different mechanism with respect to currently used therapeutics, which interfere with VEGF‐A165 binding.

Integrated Bioinformatic Analysis of the Expression and Prognosis of Caveolae-Related Genes in Human Breast Cancer.

Caveolae-related genes, including CAVs that encodes caveolins and CAVINs that encodes caveolae-associated proteins cavins, have been identified for playing significant roles in a variety of biological processes including cholesterol transport and signal transduction, but evidences related to tumorigenesis and cancer progression are not abundant to correlate with clinical characteristics and prognosis of patients with cancer. In this study, we investigated the expression of these genes at transcriptional and translational levels in patients with breast cancer using Oncomine, Gene Expression Profiling Interactive Analysis (GEPIA), cBioPortal databases, and immunohistochemistry of the patients in our hospital. Prognosis of patients with breast cancer based on the expressions of CAVs and CAVINs was summarized using Kaplan-Meier Plotter with their correlation to different subtyping. The relevant molecular pathways of these genes were further analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database and Gene Set Enrichment Analysis (GSEA). Results elucidated that expression levels of CAV1, CAV2, CAVIN1, CAVIN2, and CAVIN3 were significantly lower in breast cancer tissues than in normal samples, while the expression level of CAVIN2 was correlated with advanced tumor stage. Furthermore, investigations on survival of patients with breast cancer indicated outstanding associations between prognosis and CAVIN2 levels, especially for the patients with estrogen receptor positive (ER+) breast cancer. In conclusion, our investigation indicated CAVIN2 is a potential therapeutic target for patients with ER+ breast cancer, which may relate to functions of cancer cell surface receptors and adhesion molecules.

Effect of trypsin concentration on living SMCC-7721 cells studied by atomic force microscopy.

Trypsin is playing an important role in the processes of cancer proliferation, invasion and metastasis which require the precise information of morphology and mechanical properties on the nano-scale for the related research. In this work, living human hepatoma (SMCC-7721) cells were treated with different concentrations of trypsin solution. The morphology and mechanical properties of the cells were measured via atomic force microscope (AFM). Statistical analyses of measurement data indicated that with the increase of trypsin concentration, the average cell height and the surface roughness were both increased, but the cell viability, the cell surface adhesion and the elasticity modulus were decreased significantly. The force required to puncture the cells was also gradually reduced. It indicates that trypsin not only hydrolyses the proteins between the cell and the substrate but also the membrane proteins. The results offer valuable clues for the cancerous process study, pathological analysis and trypsin inhibitor drug development. And this work provides an effective way for overcoming the cell membrane in drug injection for cell-targeted therapy.

Chondroitin Sulfate/Dermatan Sulfate-Protein Interactions and Their Biological Functions in Human Diseases: Implications and Analytical Tools.

Chondroitin sulfate (CS) and dermatan sulfate (DS) are linear anionic polysaccharides that are widely present on the cell surface and in the cell matrix and connective tissue. CS and DS chains are usually attached to core proteins and are present in the form of proteoglycans (PGs). They not only are important structural substances but also bind to a variety of cytokines, growth factors, cell surface receptors, adhesion molecules, enzymes and fibrillary glycoproteins to execute series of important biological functions. CS and DS exhibit variable sulfation patterns and different sequence arrangements, and their molecular weights also vary within a large range, increasing the structural complexity and diversity of CS/DS. The structure-function relationship of CS/DS PGs directly and indirectly involves them in a variety of physiological and pathological processes. Accumulating evidence suggests that CS/DS serves as an important cofactor for many cell behaviors. Understanding the molecular basis of these interactions helps to elucidate the occurrence and development of various diseases and the development of new therapeutic approaches. The present article reviews the physiological and pathological processes in which CS and DS participate through their interactions with different proteins. Moreover, classic and emerging glycosaminoglycan (GAG)-protein interaction analysis tools and their applications in CS/DS-protein characterization are also discussed.

Identification of miRNA-target gene regulatory networks in liver fibrosis based on bioinformatics analysis.

BackgroundLiver cirrhosis is one of the leading causes of death worldwide. MicroRNAs (miRNAs) can regulate liver fibrosis, but the underlying mechanisms are not fully understood, and the interactions between miRNAs and mRNAs are not clearly elucidated.MethodsmiRNA and mRNA expression arrays of cirrhotic samples and control samples were acquired from the Gene Expression Omnibus database. miRNA-mRNA integrated analysis, functional enrichment analysis and protein-protein interaction (PPI) network construction were performed to identify differentially expressed miRNAs (DEMs) and mRNAs (DEGs), miRNA-mRNA interaction networks, enriched pathways and hub genes. Finally, the results were validated with in vitro cell models.ResultsBy bioinformatics analysis, we identified 13 DEMs between cirrhotic samples and control samples. Among these DEMs, six upregulated (hsa-miR-146b-5p, hsa-miR-150-5p, hsa-miR-224-3p, hsa-miR-3135b, hsa-miR-3195, and hsa-miR-4725-3p) and seven downregulated (hsa-miR-1234-3p, hsa-miR-30b-3p, hsa-miR-3162-3p, hsa-miR-548aj-3p, hsa-miR-548x-3p, hsa-miR-548z, and hsa-miR-890) miRNAs were further validated in activated LX2 cells. miRNA-mRNA interaction networks revealed a total of 361 miRNA-mRNA pairs between 13 miRNAs and 245 corresponding target genes. Moreover, PPI network analysis revealed the top 20 hub genes, including COL1A1, FBN1 and TIMP3, which were involved in extracellular matrix (ECM) organization; CCL5, CXCL9, CXCL12, LCK and CD24, which participated in the immune response; and CDH1, PECAM1, SELL and CAV1, which regulated cell adhesion. Functional enrichment analysis of all DEGs as well as hub genes showed similar results, as ECM-associated pathways, cell surface interaction and adhesion, and immune response were significantly enriched in both analyses.ConclusionsWe identified 13 differentially expressed miRNAs as potential biomarkers of liver cirrhosis. Moreover, we identified 361 regulatory pairs of miRNA-mRNA and 20 hub genes in liver cirrhosis, most of which were involved in collagen and ECM components, immune response, and cell adhesion. These results would provide novel mechanistic insights into the pathogenesis of liver cirrhosis and identify candidate targets for its treatment.

Diploid genomic architecture of Nitzschia inconspicua, an elite biomass production diatom

A near-complete diploid nuclear genome and accompanying circular mitochondrial and chloroplast genomes have been assembled from the elite commercial diatom species Nitzschia inconspicua. The 50 Mbp haploid size of the nuclear genome is nearly double that of model diatom Phaeodactylum tricornutum, but 30% smaller than closer relative Fragilariopsis cylindrus. Diploid assembly, which was facilitated by low levels of allelic heterozygosity (2.7%), included 14 candidate chromosome pairs composed of long, syntenic contigs, covering 93% of the total assembly. Telomeric ends were capped with an unusual 12-mer, G-rich, degenerate repeat sequence. Predicted proteins were highly enriched in strain-specific marker domains associated with cell-surface adhesion, biofilm formation, and raphe system gliding motility. Expanded species-specific families of carbonic anhydrases suggest potential enhancement of carbon concentration efficiency, and duplicated glycolysis and fatty acid synthesis pathways across cytosolic and organellar compartments may enhance peak metabolic output, contributing to competitive success over other organisms in mixed cultures. The N. inconspicua genome delivers a robust new reference for future functional and transcriptomic studies to illuminate the physiology of benthic pennate diatoms and harness their unique adaptations to support commercial algae biomass and bioproduct production.

Bio-clogging mitigation in vertical subsurface flow constructed wetlands using rhamnolipids-citric acid compound

Sustainable operation of constructed wetlands (CWs) is challenging due to bio-clogging caused by the excessive aggregation of extracellular polymeric substances (EPS) in substrate interspaces. In this study, rhamnolipids (RLs) and citric acid (CA) were used as solubilizers in situ with the influent to mitigate bio-clogging in CWs for the first time. Four lab-scale CWs with vertical subsurface flow were set to validate the efficiency of CA-RLs. After solubilization treatment with CA-RLs, the effective porosity of CWs restored to about 83.0% of the initial. RLs could solubilize and disperse blockage, and CA further promote the effects by destroying the stable structure of EPS. The surface contact angle became larger after CA-RLs treatment, as did the absolute zeta potential of EPS, attributed to the solubilization of hydrophobic EPS. CA-RLs inhibited the surface adhesion of cells and dissociated EPS into small particles, reduced the aggregation ability of EPS. The EEM-PARAFAC analysis showed solubilization treatment increased the tryptophan-like or protein-like components in effluent. Furthermore, the rehabilitative porosity after CA-RLs solubilization treatment provided suitable conditions for the growth of denitrifiers (especially Rhodopseudomonas and Rubrivivax), conducive to recover the performance of CWs. After bio-clogging mitigation, the removal rates of NH4+-N (25–30%) and COD (50–55%) in CWs gradually recovered to 55–70% and 70–75%, respectively. These findings provided a better understanding of the bio-clogging characteristics, which were helpful to mitigate bio-clogging in CWs.

Identifying Main Genes and Pathways by Using Gene Expression Profiling in Primary Immunodeficiency HOIL-1/RBCK1 Disorder Patients

HOIL-1/RBCK1 deficiency is a new autosomal receiving disorder with dysfunctional cellular responses to pro-inflammatory cytokines, leading to auto-inflammation, pyogenic bacterial disease, and muscle amylopectinosis growth. Our study with integrated bioinformatics studies of the feature genes and the correlative gene functions, investigated the molecular mechanisms of RBCK1 deficiency. GSE31064 dataset expression profile was downloaded from the Omnibus Gene Expression database. Between RBCK1, MYDK88, NEMO deficient fibroblast, and healthy fibroblast specimens, differentially expressed genes (DEGs) were defined. Gene ontology (GO) gene role enrichment analysis and the Kyoto Encyclopedia of Gene and Genome (KEGG) pathway analysis were performed using the Annotation, Visualization and Integrated Discovery Database (DAVID). The protein-protein interaction (PPI) of these DEGs was visualized using Cytoscape. GO analysis revealed that the “Skeletal system development, Extracellular matrix organization, Positive regulation of cell migration, Negative regulation of canonical Wnt signaling pathway, Cell adhesion, Angiogenesis and Negative regulation of BMP signaling pathway, Serine-type carboxypeptidase activity, Polysaccharide binding, Calcium ion binding, frizzled binding, Neuropilin binding, and cell adhesion molecule binding, extracellular exosome, extracellular space, extracellular region, lysosomal lumen, endoplasmic reticulum lumen, cell surface and focal adhesion to BP, MF, and CC, respectively. The study of the KEGG pathway showed that the complement and coagulation cascade, interactions of the ECM receptor, PI3K-Akt signaling pathway, PPAR signaling pathway, TGF-beta signaling pathway, cancer pathway, viral carcinogenesis and focal adhesion pathway were closely correlated with the incidence of RBCK1 deficiency. Importantly, it has been predicted that TK1, AURKB, CDCA2, UBE2C, KIFC1, CEP55, CDCA3, GINS2, MCM6 and CDC45 are significantly associated with RCBK1 deficiency. Our study offers a record of damaged genes and pathways in RCBK1, which will boost the understanding of RBCK1 deficiency pathogenesis and other inherent immunodeficiency diseases. This research has the potential and can possibly use in the clinic for diagnosis and targeted therapy of HOIL-1/RBCK1 disorder and other inherent immunodeficiencies.

Appearance of peanut agglutinin in the blood circulation after peanut ingestion promotes endothelial secretion of metastasis-promoting cytokines.

Peanut agglutinin (PNA) is a carbohydrate-binding protein in peanuts that accounts for ~0.15% peanut weight. PNA is highly resistant to cooking and digestion and is rapidly detectable in the blood after peanut consumption. Our previous studies have shown that circulating PNA mimics the actions of endogenous galactoside-binding protein galectin-3 by interaction with tumour cell-associated MUC1 and promotes circulating tumour cell metastatic spreading. The present study shows that circulating PNA interacts with micro- as well as macro-vascular endothelial cells and induces endothelial secretion of cytokines MCP-1 (CCL2) and IL-6 in vitro and in vivo. The increased secretion of these cytokines autocrinely/paracrinely enhances the expression of endothelial cell surface adhesion molecules including integrins, VCAM and selectin, leading to increased tumour cell-endothelial adhesion and endothelial tubule formation. Binding of PNA to endothelial surface MCAM (CD146), via N-linked glycans, and subsequent activation of PI3K-AKT-PREAS40 signalling is here shown responsible for PNA-induced secretion of MCP-1 and IL-6 by vascular endothelium. Thus, in addition to its influence on promoting tumour cell spreading by interaction with tumour cell-associated MUC1, circulating PNA might also influence metastasis by enhancing the secretion of metastasis-promoting MCP-1 and IL-6 from the vascular endothelium.

Abstract 1858: SBT6290, a systemically administered Nectin4-directed TLR8 ImmunoTAC™ product candidate, is designed for tumor-localized activation of myeloid cells

Abstract SBT6290 is a novel product candidate comprised of a selective TLR8 agonist conjugated to a Nectin4-specific monoclonal antibody, designed for systemic delivery and tumor-localized activation of myeloid cells. Nectin4 is a cell surface adhesion molecule that is overexpressed in multiple solid tumor types including bladder, triple negative breast, squamous head and neck, and non-small cell lung cancers, with limited expression in normal tissues. Nectin4-expressing solid tumors display substantial myeloid cell infiltrate. Activation of these myeloid cells in the tumor microenvironment has emerged as a promising approach in overcoming resistance mechanisms to current cancer immunotherapies. TLR8 is highly expressed in myeloid cell types prevalent in human tumors, including conventional dendritic cells (DCs) and macrophages. TLR8 agonism in human myeloid cells activates a broad spectrum of anti-tumor immune mechanisms, including proinflammatory cytokine production, repolarization of suppressive myeloid cells, and the priming of CTL responses. Here, we show that SBT6290 activates human myeloid cells by SBT6290 in a Nectin4-dependent manner and that a SBT6290 mouse surrogate confers single agent anti-tumor activity in preclinical studies. In vitro studies with human immune cells demonstrate that SBT6290 induces multiple anti-tumor immune mechanisms including proinflammatory cytokine and chemokine production, inflammasome activation, direct activation of DCs and indirect activation of T and NK cell cytolytic activity. This activity is Nectin4-specific and requires SBT6290 engagement of Fcγ receptors on the surface of myeloid cells to facilitate uptake of the conjugate into myeloid cells. Notably, SBT6290 is &amp;gt;100-fold more active than free, unconjugated TLR8 agonist and demonstrates activity on tumor cells with Nectin4 overexpression that correlates with levels found in primary tumor samples. Nectin4 was recently described to be a ligand for T cell immunoreceptor with Ig and ITIM domains (TIGIT), an inhibitory receptor considered to be a promising new target for cancer immunotherapy (J Immunother Cancer. 2020; 8(1): e000266). The SBT6290 binding domain blocks the interaction between TIGIT and Nectin4, potentially contributing to the T and NK cell activation induced by SBT6290. Systemic administration of a SBT6290 surrogate in mice bearing Nectin4-expressing tumors results in intra-tumoral myeloid and T cell activation and increased overall survival. The preclinical data described here demonstrate the potential for SBT6290 to drive anti-tumor responses and support continued preclinical development of SBT6290 for Nectin4-expressing tumors. Citation Format: Michael R. Comeau, Heather Metz, Brenda Stevens, Damion Winship, Jamie Brevik, Marcus Rhodehamel, Monica Childs, Elsa Hay, Jenny Chang, Li-Qun Fan, Hengyu Xu, Jonathan Grey, Jeffrey Adamo, Ben Setter, Ray Carillo, Sean W. Smith, Phil Tan, Robert Dubose, Yvette Latchman, Peter Baum, Valerie Odegard. SBT6290, a systemically administered Nectin4-directed TLR8 ImmunoTAC™ product candidate, is designed for tumor-localized activation of myeloid cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1858.

Defining the Transcriptional Code That Specifies Sinusoidal Endothelial Cells in the HSPC Niche

Hematopoietic stem and progenitor cell (HSPC) niches are specialized microenvironments that support the maintenance and self-renewal of HSPCs. In the fetal liver, bone marrow and spleen, vascular endothelial cells are thought to function as core components of the HSPC niche. In the bone marrow, sinusoidal endothelial cells are known to secrete soluble factors and express cell surface adhesion molecules that promote the recruitment, maintenance and function of HSPCs. Using tomo-seq (RNA tomography) - a technique that involves extracting and barcoding the RNA from sequentially collected cryosections and then performing RNA-seq - we identified ~300 genes enriched in the zebrafish caudal hematopoietic tissue (CHT; the hematopoietic equivalent of the mammalian fetal liver). We also used FACS with tissue-specific fluorescent reporter transgenes to isolate different cell populations from whole embryos and then performed RNA-seq. This included endothelial cells, macrophages, neutrophils and erythrocytes. By overlaying these datasets with the tomo-seq results, we determined the cell types in which many of the CHT-enriched genes were expressed. Unexpectedly, we found a set of ~20 genes that appeared to be enriched specifically in endothelial cells within the CHT. Whole embryo in situ hybridization revealed that these genes had similar spatial and temporal expression patterns, being specifically enriched in sinusoidal endothelial cells in the CHT but absent in other endothelial cells throughout the body. We cloned the upstream promoter regions for several of these genes, fused them to a GFP reporter and then injected them into embryos. Of note, we found that a 5.3 kb sequence upstream of selectin-e (sele ; a gene known to be expressed by bone marrow endothelial cells where it promotes HSPC homing) drove GFP expression specifically in sinusoidal endothelial cells in the CHT. By crossing this sele:GFP transgene to a pan-endothelial marker (kdrl:mCherry), we were able to use FACS to isolate CHT endothelial cells, as well as endothelial cells from tissues outside the CHT. We performed ATAC-seq - a transposon-based method used to detect regions of open chromatin - on these two cell populations and identified 6,710 peaks (regions of open chromatin) that were unique to sele:GFP+; kdrl:mCherry+ CHT niche endothelial cells. Several of these peaks were associated with CHT endothelial genes we had initially identified by tomo-seq, including mrc1a, gpr182, stab1, stab2 and exoc3l2a . Informatic analysis of the sequences under the 6,710 CHT endothelial peaks identified Ets, Sox, Nuclear Hormone and Gata sites as the most enriched transcription factor binding motifs compared to peaks from the non-CHT endothelial cells. These results suggested a transcription factor code involving members of these families might specify sinusoidal endothelial cells in the HSPC niche. Upon closer examination of the sele locus itself, we observed a narrow ATAC-seq peak within the 5.3 kb upstream sequence that was uniquely open in the sele:GFP+; kdrl:mCherry+ CHT niche endothelial cells. Promoter truncation experiments identified a short 158 bp sequence that perfectly aligned with this unique ATAC-seq peak and which was sufficient to drive GFP expression in CHT endothelial cells. This 158 bp sequence contained three Ets, three Sox and one RORA (nuclear hormone receptor) binding sites. Synthetic variants were made in which the different transcription factor binding sites within the 158 bp sequence were disrupted. Injection of these variants driving GFP demonstrated that the Sox and RORA sites were required for expression in CHT endothelial cells. Disruption of the Ets sites, in contrast, led to dramatically enhanced GFP expression, suggesting these sites could be repressive. Together these studies demonstrate that the transcriptional regulation of niche endothelial cells include transcription factors that bind to Ets, Sox, and RORA motifs. The results of this work has important implications for culturing HSPCs in vitro with endothelial cells or for modulating the HSPC niche in the context of human disease or transplantation. DisclosuresZon:Fate, Inc.: Consultancy, Equity Ownership; Marauder, Inc.: Consultancy, Equity Ownership; Scholar Rock, Inc: Consultancy, Equity Ownership; Stemgent: Consultancy.

Staphylococcus epidermidis Has Growth Phase Dependent Affinity for Fibrinogen and Resulting Fibrin Clot Elasticity.

Bacterial infection and thrombosis are highly correlated, especially in patients with indwelling medical devices. Coagulase-negative staphylococci, typified by Staphylococcus epidermidis, are a common cause of medical device infections owing to their biofilm forming capacity which provides protection from antibiotics and host immune response. Attention has been drawn to the interaction between S. epidermidis and host proteins, specifically fibrinogen. However, little is known regarding the impact of the transition from planktonic to biofilm forming phenotype on this interaction. Here we investigate the growth phase dependence of bacteria-fibrinogen interaction and the resulting effect on fibrin clot formation, structure, and mechanics. Flow cytometry demonstrated growth phase dependent affinity for fibrinogen. To mimic intravascular device seeding, we quantified the adhesion of S. epidermidis to a fibrinogen coated surface under continuous flow conditions in vitro. The bacterial deposition rate onto fibrinogen was significantly greater for stationary (5,360 ± 1,776 cells/cm2s) versus exponential phase (2,212 ± 264, cells/cm2 s). Furthermore, the expression of sdrG–a cell surface adhesion protein with specificity for fibrinogen–was upregulated ∼twofold in the stationary versus the exponential phase. Rheometry and confocal microscopy demonstrated that stationary phase S. epidermidis slows clot formation and generates a more heterogeneous fibrin network structure with greater elasticity (G′ = 5.7 ± 1.0 Pa) compared to sterile fibrinogen (G′ = l.5 ± 0.2 Pa), while exponential phase cells had little effect. This work contributes to the current understanding of the growth phase dependent regulation of bacterial virulence factors and the correlation between bacterial infection and thrombosis.

Nuclear Syndecan-1 Regulates Epithelial-Mesenchymal Plasticity in Tumor Cells.

Simple SummaryThe major cause of death in cancer is that tumor cells metastasize (i.e., spread to vital organs such as the lungs and liver). Our knowledge of how metastasis occurs is incomplete and consequently, there is a lack of therapeutic strategies that target metastasis. Recent data show that inflammatory signals may influence tumor cells to undergo an identity switch, whereby they develop metastatic properties. Understanding how this identity switch is regulated is important to develop novel therapeutics. Recently, syndecan-1, a protein found on the surface of various cells, was shown to regulate the identity switch. In this study, we report that re-localization of syndecan-1 from the cell surface to the nucleus contributes to its capacity to regulate the identity switch in tumor cells. These results identify nuclear syndecan-1 as a regulator of the identity switch and open up to further studies to elucidate the mechanisms involved.Tumor cells undergoing epithelial-mesenchymal transition (EMT) lose cell surface adhesion molecules and gain invasive and metastatic properties. EMT is a plastic process and tumor cells may shift between different epithelial-mesenchymal states during metastasis. However, how this is regulated is not fully understood. Syndecan-1 (SDC1) is the major cell surface proteoglycan in epithelial cells and has been shown to regulate carcinoma progression and EMT. Recently, it was discovered that SDC1 translocates into the cell nucleus in certain tumor cells. Nuclear SDC1 inhibits cell proliferation, but whether nuclear SDC1 contributes to the regulation of EMT is not clear. Here, we report that loss of nuclear SDC1 is associated with cellular elongation and an E-cadherin-to-N-cadherin switch during TGF-β1-induced EMT in human A549 lung adenocarcinoma cells. Further studies showed that nuclear translocation of SDC1 contributed to the repression of mesenchymal and invasive properties of human B6FS fibrosarcoma cells. The results demonstrate that nuclear translocation contributes to the capacity of SDC1 to regulate epithelial-mesenchymal plasticity in human tumor cells and opens up to mechanistic studies to elucidate the mechanisms involved.