Fibronectin-coated Plates Research Articles

Establishment of porcine embryonic stem cells in simplified serum free media and feeder free expansion

BackgroundThe establishment of stable porcine embryonic stem cells (pESCs) can contribute to basic and biomedical research, including comparative developmental biology, as well as assessing the safety of stem cell-based therapies. Despite these advantages, most pESCs obtained from in vitro blastocysts require complex media and feeder layers, making routine use, genetic modification, and differentiation into specific cell types difficult. We aimed to establish pESCs with a single cell-passage ability, high proliferative potency, and stable in long-term culture from in vitro-derived blastocysts using a simplified serum-free medium.MethodsWe evaluated the establishment efficiency of pESCs from in vitro blastocysts using various basal media (DMEM/F10 (1:1), DMEM/F12, and a-MEM) and factors (FGF2, IWR-1, CHIR99021, and WH-4-023). The pluripotency and self-renewal capacity of the established pESCs were analyzed under feeder or feeder-free conditions. Ultimately, we developed a simplified culture medium (FIW) composed of FGF2, IWR-1, and WH-4-023 under serum-free conditions.ResultsThe pESC-FIW lines were capable of single-cell passaging with short cell doubling times and expressed the pluripotency markers POU5F1, SOX2, and NANOG, as well as cell surface markers SSEA1, SSEA4, and TRA-1-60. pESC-FIW showed a stable proliferation rate and normal karyotype, even after 50 passages. Transcriptome analysis revealed that pESC-FIW were similar to reported pESC maintained in complex media and showed gastrulating epiblast cell characteristics. pESC-FIW were maintained for multiple passages under feeder-free conditions on fibronectin-coated plates using mTeSR™, a commercial medium used for feeder-free culture, exhibiting characteristics similar to those observed under feeder conditions.ConclusionsThese results indicated that inhibition of WNT and SRC was sufficient to establish pESCs capable of single-cell passaging and feeder-free expansion under serum-free conditions. The easy maintenance of pESCs facilitates their application in gene editing technology for agriculture and biomedicine, as well as lineage commitment studies.

Current Strategies of Modeling Human Trophoblast Using Human Pluripotent Stem Cells in vitro.

We previously established a trophoblast differentiation protocol from primed human pluripotent stem cells (PSC). To induce this lineage, we use a combination of Bone Morphogenetic Protein-4 (BMP4) and the WNT inhibitor IWP2. This protocol has enabled us to obtain a pure population of trophectoderm (TE)-like cells that could subsequently be terminally differentiated into syncytiotrophoblasts (STB) and extravillous trophoblasts (EVT). However, the resulting TE-like cells could only be terminally differentiated to a variable mixture of STB and EVT, with a bias toward the STB lineage. Recently, methods have been developed for derivation and culture of self-renewing human trophoblast stem cells (TSC) from human embryos and early gestation placental tissues. These primary TSCs were further able to differentiate into either STB or EVT with high efficiency using the lineage specific differentiation protocols. Based partly on these protocols, we have developed methods for establishing self-renewing TSC-like cells from PSC, and for efficient lineage-specific terminal differentiation. Here, we describe in detail the protocols to derive and maintain PSC-TSC, from both embryonic stem cells (ESC) and patient-derived induced pluripotent stem cells (iPSC), and their subsequent terminal differentiation to STB and EVT. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Trophoblast Differentiation into TE-like Cells Basic Protocol 2: Conversion of PSC-Derived TE-like Cells to TSC Basic Protocol 3: Passaging PSC-Derived TSC in iCTB Complete Medium Basic Protocol 4: STB Differentiation from PSC-derived TSC Basic Protocol 5: EVT Differentiation from PSC-derived TSC Support Protocol 1: Geltrex-coated tissue culture plate preparation Support Protocol 2: Collagen IV-coated tissue culture plate preparation Support Protocol 3: Fibronectin-coated tissue culture plate preparation.

Decellularized Lung Extracellular Matrix Scaffold Promotes Human Embryonic Stem Cell Differentiation towards Alveolar Progenitors.

Efficient production of functional and mature alveolar epithelial is a major challenge for developing any cell replacement therapy for lung degenerative diseases. The extracellular matrix (ECM) pro-vides a dynamic environment and mediates cellular responses during development and maintenance of tissue functions. The decellularized ECM (dECM) which retains its native-like structure and bio-chemical composition can provide the induction of embryonic stem cell (ESC) differentiation toward the tissue-specific lineages during in vitro culture. Therefore, the aim of this study was to evaluate the effect of sheep lung dECM-derived scaffold on differentiation and further maturation of ESC-derived lung progenitor cells. This study was an experimental study. In the first step, a sheep lung was decellularized to achieve dECM scaffolds and hydrogels. Afterwards, the obtained dECM scaffold was evaluated for collagen and glycosaminoglycan contents, DNA quantification, and its ultrastructure. Next, the three experimental groups: i. Sheep lung dECM-derived scaffold, ii. Sheep lung dECM-derived hydrogel, and iii. Fibronectin-coated plates were compared in their abilities to induce further differentiation of human embryonic stem cells (hESCs)-derived definitive endoderm (DE) into lung progenitor cells. The comparison was evaluated by immuno-staining and real-time polymerase chain reaction (PCR) assessments. We found that the dECM-derived scaffold preserved its composition and native porous structures while lacking nuclei and intact cells. All experimental groups displayed lung progenitor cell differen-tiation as revealed by the RNA and protein expression of NKX2.1, P63 and CK5. DE cells differenti-ated on dECM-derived scaffold and dECMderived hydrogel showed significant upregulation of SOX9 gene expression, a marker of the distal airway epithelium. DE cells differentiated on the dECM-derived scaffold compared to the two other groups, showed enhanced expression of SFTPC (type 2 alveolar epithelial [AT2] cell marker), FOXJ1 (ciliated cell marker), and MUC5A (secretory cell marker) genes. Overall, our results suggest that dECM-derived scaffold improves the differentiation of DE cells towards lung alveolar progenitor cells in comparison with dECM-derived hydrogel and fibronectin-coated plates.

SPAD-1, a serine proteinase associated disintegrin from Russell's viper venom disrupts adhesion of MCF7 human breast cancer cells

Serine Proteinase Associated Disintegrin-1 (SPAD-1) is a low molecular mass (26 kDa) positively charged protein purified from Russell's viper venom (RVV) possessing cytotoxic activity on MCF7, human breast cancer cells. Primary sequence analysis of the protein confirms that it is a novel Snake Venom Serine Proteinase (SVSP) and a member of the trypsin family. SPAD-1 contains a conserved triad of Histidine (H), Aspartic acid(D) and Serine(S) residues at its active site for proteinase activity and also an adjacent histidine-glycine-aspartic acid (HGD) disintegrin-like motif. The serine proteinase and disintegrin parts are functionally active and independent. SPAD-1 showed proteolytic digestion of fibrinogen and fibronectin, but laminin digestion was below the detectable limit. Proteolytically inactivated SPAD-1 inhibited collagen and ADP-induced platelet aggregation. This study proposes considering Serine Proteinase Associated Disintegrin (SPAD) as a new group of snake venom proteins. Members of this group contain a serine proteinase catalytic triad and a disintegrin-like motif. SPAD-1 caused visible morphological changes in MCF7 cells, including a reduction of the cell-to-cell attachments, rounding of cell shape and death, in vitro. SPAD-1 also showed a dose-dependent significant decrease in the invasive potency of breast cancer cells. Confocal microscopic analysis revealed the breakage of nuclei of the SPAD-1-treated cells. SPAD-1 also increased cell detachment from the poly L-lysine-coated, laminin-coated and fibronectin-coated culture plate matrices, confirming the disintegrin activity. This study concludes that SPAD-1 may be a good candidate for anti-tumour drug design in the future.

Effects of titanium dioxide microparticles and nanoparticles on cytoskeletal organization, cell adhesion, migration, and proliferation in human gingival fibroblasts in the presence of lipopolysaccharide.

Titanium wear particles may participate in the etiology of peri-implantitis. However, the influence of titanium wear particles on biological behavior of human gingival fibroblasts (HGFs) in the presence of LPS is still not clear. The present study demonstrated the effects of titanium dioxide micro- and nanoparticles (TiO2 MPs and NPs) on HGF cell viability, cytoskeletal organization, adhesion, migration, and proliferation in vitro, and LPS was used to mimic the in vivo condition. Primary HGFs were treated with TiO2 MPs (primaryparticle size <5μm, 0.1mg/ml) and NPs (primary particle size <100nm, 0.1mg/ml) with or without 1μg/ml LPS. The effects of TiO2 MPs and NPs on HGFs cell viability was measured by CCK-8 assay. The proliferation of HGF was detected by Ki67 nuclear staining. The confocal laser scanning microscope (CLSM) was used to detect the internalization of TiO2 MPs and NPs in HGFs as well as the arrangement of F-actin, vinculin, and vimentin organization. Wound healing assay and transwell assay were performed to measure the migration of HGFs induced by TiO2 MPs and NPs. Cell adhesion was measured using fibronectin-coated plates. The relative mRNA and protein expression of adhesion relative protein such as focal adhesion kinase (FAK), fibronectin (FN), and type I collagen (COL1) were measured using quantitative RT-PCR and western blot analysis. One-way analysis of variance (ANOVA) and Student's t-test were used to analyze the statistical significance, and p<.05 was considered statistically significant. TiO2 NPs significantly inhibited HGF cell viability, proliferation, and migration compared with TiO2 MPs group and control group. Compared with control group (2.64±0.09), the mean absorbance of the cells in 1mg/ml TiO2 MPs group and 0.25mg/ml TiO2 NPs group were significantly decreased to 1.93±0.33 (p<.05) and 2.22±0.18 (p<.01), respectively. The cytoskeleton disruption was found in TiO2 NPs group. The mRNA and protein expression were significantly downregulated by TiO2 NPs. Furthermore, both TiO2 NPs and MPs induced more adverse effects on HGFs in the presence of LPS. Our results indicate that TiO2 NPs but not TiO2 MPs significantly disrupt the cytoskeletal organization and inhibited cell adhesion, migration, and proliferation of HGFs. However, in the presence of LPS, TiO2 MPs, and TiO2 NPs enhance these negative effects in HGFs. Titanium wear particles are probably involved in the initiation and progression of peri-implant diseases.

Maternal physiology and blastocyst morphology are correlated with an inherent difference in peri-implantation human embryo development

To determine what patient and embryo characteristics are correlated with the developmental potential of the peri-implantation embryo. Retrospective study. Research laboratory. Six hundred fifty-one cryopreserved human blastocysts donated for research with informed patient consent. Not applicable. Blastocyst attachment to fibronectin-coated plates, trophectoderm outgrowth area, epiblast cell number, total cell number, human chorionic gonadotropin secretion. Patients' body mass index, age, follicle-stimulating hormone: luteinizing hormone ratio on menstrual cycle day 3, antral follicle count on menstrual cycle day 3, antimüllerian hormone level on menstrual cycle day 3, and blastocyst morphological grade were correlated with peri-implantation development outcomes. After controlling for good-quality morphological grades, blastocysts from patients of advanced maternal age developed fewer epiblast cells than blastocysts from younger patients. Extended embryo culture during the peri-implantation period mirrors several disparities in fertility treatment outcome that we see clinically, including those from patients with advanced maternal age, high body mass index, and low ovarian reserve and from embryos with lower-quality morphological grades. This model system may be useful by providing an alternative or more sensitive endpoint assessment in studying patient, clinical, or laboratory factors that may influence preimplantation embryo developmental potential.

Dermal fibroblasts promote cancer cell proliferation and exhibit fibronectin overexpression in early mycosis fungoides

BackgroundMycosis fungoides (MF) is caused by proliferation of malignant T-cells in the skin and may progress to involve blood, lymph nodes, and viscera. While the skin microenvironment is essential for the initiation and progression of MF in early stages, little is known about the impact of skin stroma on the growth and survival of malignant lymphocytes. ObjectiveWe investigated the effect of dermal fibroblasts and their product, fibronectin, on the survival and proliferation of malignant MF cells. MethodsFibroblasts and malignant MF CD4 T-cells were isolated from skin of patients with early-stage MF. Fibroblast-lymphocyte co-culture experiments and lymphocyte cultures on fibronectin-coated plates were established utilizing the cells derived from lesional skin, blood, and MF cell lines. The survival and proliferation rates of lymphocytes were assessed via Annexin V and carboxyfluorescein succinimidyl ester assays respectively. Additionally, integrin and fibronectin expressions in MF skin were assessed via immunofluorescence. ResultsWe found that dermal fibroblasts increased the proliferation rates of MF cells, but not normal skin or blood CD4 T-cells. However, fibroblasts did not rescue MF cells from apoptosis in co-cultures. In MF skin, we found an overexpression of a fibronectin isoform not normally found in healthy skin. MF cells expressed fibronectin-binding integrins and adhered to fibronectin but did not exhibit adhesion-mediated survival via fibronectin-integrin interactions. ConclusionOverall, our results suggest a direct role for fibroblasts, independent of fibronectin-mediated adhesion, in promoting MF cell proliferation. These findings have implications in understanding and targeting the malignant skin stromal microenvironment in cutaneous lymphomas.

Fibronectin Adherent Cell Populations Derived From Avascular and Vascular Regions of the Meniscus Have Enhanced Clonogenicity and Differentiation Potential Under Physioxia.

The meniscus is composed of an avascular inner region and vascular outer region. The vascular region has been shown to contain a progenitor population with multilineage differentiation capacity. Strategies facilitating the isolation and propagation of these progenitors can be used to develop cell-based meniscal therapies. Differential adhesion to fibronectin has been used to isolate progenitor populations from cartilage, while low oxygen or physioxia (2% oxygen) enhances the meniscal phenotype. This study aimed to isolate progenitor populations from the avascular and vascular meniscus using differential fibronectin adherence and examine their clonogenicity and differentiation potential under hyperoxia (20% oxygen) and physioxia (2% oxygen). Human vascular and avascular meniscus cells were seeded onto fibronectin-coated dishes for a short period and monitored for colony formation under either hyperoxia or physioxia. Non-fibronectin adherent meniscus cells were also expanded under both oxygen tension. Individual fibronectin adherent colonies were isolated and further expanded, until approximately ten population doublings (passage 3), whereby they underwent chondrogenic, osteogenic, and adipogenic differentiation. Physioxia enhances clonogenicity of vascular and avascular meniscus cells on plastic or fibronectin-coated plates. Combined differential fibronectin adhesion and physioxia isolated a progenitor population from both meniscus regions with trilineage differentiation potential compared to equivalent hyperoxia progenitors. Physioxia isolated progenitors had a significantly enhanced meniscus matrix content without the presence of collagen X. These results demonstrate that combined physioxia and fibronectin adherence can isolate and propagate a meniscus progenitor population that can potentially be used to treat meniscal tears or defects.

Purple sweet potato extract and vitamin C increase the proliferation of endothelial progenitor cells from stable coronary artery disease patients

Introduction: Endothelial progenitor cell (EPC) numbers are reduced in stable coronary artery disease (CAD), partly due to oxidative stress. This study aimed to evaluate the effect of different antioxidants, purple sweet potato (PSP) extract and vitamin C, on EPC proliferation in stable CAD patients.Methods: Peripheral blood mononuclear cells were isolated and cultivated on fibronectin-coated plates with the colony-forming unit (CFU)-Hill medium for three days. Non-adherent cells were divided into control, PSP extract (1, 5, 25 μg/ml), and vitamin C (10, 50, 250 μg/ml) groups; then cultured for two days. EPC proliferation was assessed with MTT Cell Proliferation Assay Kit. EPCs were identified by detecting the expression of CD34. Resulted CFU-Hill colonies were counted under an inverted light microscope.Results: EPC proliferation was increased in low, moderate, and high doses of PSP extract and vitamin C compared to control (all groups vs. control, p&lt;0.001). The high dose of PSP extract increased EPC proliferation non significantly compared to the moderate dose (p=0.289). Vitamin C increased EPC proliferation better than PSP extract in moderate and high dose groups (p=0.042 and p&lt;0.01, respectively). Meanwhile, the low dose of both treatments increased the EPC proliferation equally (p=0.353). CFU numbers, representing EPC differentiation capability, were highest in the groups given PSP extract compared to control and vitamin C groups.Conclusions: PSP extract and vitamin C increased EPC proliferation dose-dependently. Vitamin C induced EPC proliferation better than PSP extract. Furthermore, PSP extract was presumably a better EPC differentiation inducer.

The molecular functions of Biodentine and mineral trioxide aggregate in lipopolysaccharide-induced inflamed dental pulp cells.

To explore the proliferation, adhesion and differentiation response and the underlying mechanisms that occur in lipopolysaccharide (LPS)-induced inflamed dental pulp cells (DPCs) in contact with Biodentine and mineral trioxide aggregate (MTA). The DPCs were isolated from three healthy donors and named DPC-H1 to DPC-H3. The DPCs were pre-cultured with 2 or 5μgmL-1 LPS for 24h to induce inflammation. The expression of inflammation marker miR-146a was detected by q-PCR. The normal and LPS-induced DPCs were further treated with 0.14mgmL-1 Biodentine or 0.13mgmL-1 MTA for 24h. MTT assay and adhesion assay were used to analyse the changes of cell phenotypes. DSPP, AKT and ERK expressions were detected by Western blotting. The data were analysed by Mann-Whitney test or two-way anova. Differences were considered statistically significant when P<0.05. In LPS-induced DPCs, Biodentine and MTA treatment neither induced nor aggravated LPS-induced inflammation, but their presence did increase the expression of the odontogenic differentiation marker DSPP. Under 2 or 5μgmL-1 LPS-induced inflammation, Biodentine and MTA promoted the proliferation of DPC cells, and significantly in DPC-H2 (P<0.0001 for both reagents). With the treatment of 2μgmL-1 LPS, the cell adhesion of DPCs on the fibronectin-coated culture plates was increased significantly by Biodentine (P=0.0413) and MTA (P<0.0001). Biodentine and MTA regulated cell adhesion on the fibronectin-coated culture plates (P<0.0001 for both reagents) and proliferation (P<0.0001 for both reagents) via the AKT pathway. However, the AKT pathway was not involved in the expression of DSPP induced by Biodentine and MTA. Biodentine and MTA enhanced the proliferation, adhesion and differentiation of LPS-induced DPCs. The proliferation and adhesion process induced by Biodentine and MTA was via the AKT pathway. However, the cellular differentiation process might not use the same pathway, and this needs to be explored in future studies.

Cucurbitacin B Diminishes Metastatic Behavior of Cholangiocarcinoma Cells by Suppressing Focal Adhesion Kinase.

Cholangiocarcinoma (CCA) is a malignant tumor with aggressive metastatic property resulted from dysregulation of metastasis-regulated signaling pathways. The aim of this study was to investigate the effect of cucurbitacin B on metastatic behavior of CCA cells through modulation of focal adhesion kinase (FAK) protein. KKU-452 cells were treated with a specific FAK inhibitor, FAK inhibitor-14, or cucurbitacin B at various concentrations for 24 h. Cell viability was assessed by sulforhodamine B assay. The migratory and invasive abilities of the cells were investigated using wound healing and transwell invasion assays, respectively. The fibronectin-coated plate was used for adhesion assay. The effects of the test compounds on FAK activation and the expression of metastasis-associated proteins were determined by Western blot analysis. The amount of MMP-9 was evaluated using a commercial ELISA Kit. FAK inhibitor-14 and cucurbitacin B at concentrations which minimally affected KKU-452 cell viability could suppress FAK activation, evidently by decreased level of phospho-FAK protein after exposure to the compound. At these conditions, cucurbitacin B suppressed metastatic behavior including migration, invasion and adhesion abilities of CCA cells similar to FAK inhibitor-14. Further molecular studies demonstrated that FAK inhibitor-14 and cucurbitacin B downregulated the expression of metastasis-associated proteins including MMP-9, ICAM-1 and VEGF. Consequently, exposure to cucurbitacin B inhibited the production of MMP-9 enzyme in CCA cells similar to FAK inhibitor-14 treatment. FAK participated in regulation of metastatic behavior of KKU-452 CCA cells. Cucurbitacin B suppressed FAK activation in the cells which was associated with inhibition of metastasis essential steps and their related metastatic proteins. The compound may be developed as a novel therapeutic agent for CCA metastasis therapy.<br />.

Inhibition of \u03b1v\u03b23 integrin impairs adhesion and uptake of tumor-derived small extracellular vesicles

BackgroundExtracellular vesicles (EVs) are lipid-bound particles that are naturally released from cells and mediate cell-cell communication. Integrin adhesion receptors are enriched in small EVs (SEVs) and SEV-carried integrins have been shown to promote cancer cell migration and to mediate organ-specific metastasis; however, how integrins mediate these effects is not entirely clear and could represent a combination of EV binding to extracellular matrix and cells.MethodsTo probe integrin role in EVs binding and uptake, we employed a disintegrin inhibitor (DisBa-01) of integrin binding with specificity for αvβ3 integrin. EVs were purified from MDA-MB-231 cells conditioned media by serial centrifugation method. Isolated EVs were characterized by different techniques and further employed in adhesion, uptake and co-culture experiments.ResultsWe find that SEVs secreted from MDA-MB-231 breast cancer cells carry αvβ3 integrin and bind directly to fibronectin-coated plates, which is inhibited by DisBa-01. SEV coating on tissue culture plates also induces adhesion of MDA-MB-231 cells, which is inhibited by DisBa-01 treatment. Analysis of EV uptake and interchange between cells reveals that the amount of CD63-positive EVs delivered from malignant MDA-MB-231 breast cells to non-malignant MCF10A breast epithelial cells is reduced by DisBa-01 treatment. Inhibition of αvβ3 integrin decreases CD63 expression in cancer cells suggesting an effect on SEV content.ConclusionIn summary, our findings demonstrate for the first time a key role of αvβ3 integrin in cell-cell communication through SEVs.6Ak9CS6CfK5Ku3JS4e63coVideo Graphical abstract

RAP1-RAC1 Signaling Has an Important Role in Adhesion and Migration in HNSCC

Cell-cell adhesion is a key mechanism to control tissue integrity and migration. In head and neck squamous cell carcinoma (HNSCC), cell migration facilitates distant metastases and is correlated with poor prognosis. RAP1, a ras-like protein, has an important role in the progression of HNSCC. RAC1 is an integrin-linked, ras-like protein that promotes cell migration. Here we show that loss of cell-cell adhesion is correlated with inactivation of RAP1 confirmed by 2 different biochemical approaches. RAP1 activation is required for cell-matrix adhesion confirmed by adhesion to fibronectin-coated plates with cells that have biochemically activated RAP1. This effect is reversed when RAP1 is inactivated. In addition, RAP1GTP-mediated adhesion is only facilitated through α5β1 integrin complex and is not a function of either α5 or β1 integrin alone. Moreover, the inside-out signaling of RAP1 activation is coordinated with RAC1 activation. These findings show that RAP1 has a prominent role in cell-matrix adhesion via extracellular matrix molecule fibronectin-induced α5β1 integrin and supports a critical role for the RAP1/RAC1 signaling axis in HNSCC cell migration.

High Dose Allicin with Vitamin C Improves EPCs Migration from the Patient with Coronary Artery Disease

Endothelial Progenitor Cells (EPCs) have an important role in endothelial dysfunction repairment through neovasculogenesis and cardiac myocytes regeneration. However, EPCs migration is greatly reduced in the patient with Coronary Artery Disease (CAD). Allicin and Vitamin C are hypothesized to improve EPCs migration due to its antioxidant properties. Objective: To investigate the effect of Allicin and its combination with Vitamin C in EPCs migration of CAD patients. Material and Method: Mononuclear cells were isolated from CAD patients and cultured on fibronectin-coated plates with colony-forming unit Hill medium. The cells were divided into untreated (control), Allicin treatment (dose 100 mcg/ml, 200 mcg/ml, 400 mcg/ ml), and each dose of Allicin combined with 250 mcg/mL of Vitamin C. EPCs migration was assessed with Transwell Migration Assay Kit and evaluated by using statistical tests. Results: This research shows that EPC migration was significantly higher in the treatment. Allicin at all dose (dose 100 mcg/ml, 200 mcg/ml, 400 mcg/ml) and its combination with 250 mcg/mL of vitamin C compared to untreated group (p<0.05). Allicin increase EPCs migration in a dosedependent manner. However, the only combination of 400 mcg/ml Allicin with 250 mcg/mL of vitamin C which has significantly higher EPCs migration compared to Allicin treatment alone. Conclusion: Allicin improves EPCs migration in a dose-dependent manner. Improvement of the migration only observed on the Allicin dose 400 mcg/ml with Vitamin C.

LMW-PTP targeting potentiates the effects of drugs used in chronic lymphocytic leukemia therapy

BackgroundLow molecular weight protein tyrosine phosphatase (LMW-PTP) is overexpressed in different cancer types and its expression is related to more aggressive disease, reduced survival rate and drug resistance. Morin is a natural polyphenol which negatively modulates, among others, the activity of LMW-PTP, leading to the potentiation of the effects of different antitumoral drugs, representing a potential beneficial treatment against cancer.MethodsLMW-PTP levels were measured by immunoblot analysis both in CLL cells from patients and in chronic lymphocytic leukemia (CLL)-derived Mec-1 cells. Cell viability was assessed in Mec-1 cells treated with morin alone or in combination with either fludarabine or ibrutinib or following siRNA-mediated LMW-PTP knockdown. Furthermore, the expression levels of VLA-4 and CXCR4 were assessed by both qRT-PCR and flow cytometry and both adhesion to fibronectin-coated plates and migration toward CXCL12 were analyzed in Mec-1 cells treated with morin alone or in combination with fludarabine or ibrutinib.ResultsWe observed that LMW-PTP is highly expressed in Mec-1 cells as well as in leukemic B lymphocytes purified from CLL patients compared to normal B lymphocytes. Morin treatment strongly decreased LMW-PTP expression levels in Mec-1 cells and potentiated the anticancer properties of both fludarabine and ibrutinib by increasing their apoptotic effects on leukemic cells. Moreover, morin negatively regulates adhesion and CXCL12-dependent migration of Mec-1 cells by affecting VLA-4 integrin expression and CXCR4 receptor recycling.ConclusionsMorin treatment in CLL-derived Mec-1 cell line synergizes with conventional anticancer drugs currently used in CLL therapy by affecting leukemic cell viability and trafficking.

Reawakening of dormant estrogen-dependent human breast cancer cells by bone marrow stroma secretory senescence

BackgroundDormant estrogen receptor positive (ER+) breast cancer micrometastases in the bone marrow survive adjuvant chemotherapy and recur stochastically for more than 20 years. We hypothesized that inflammatory cytokines produced by stromal injury can re-awaken dormant breast cancer cells.MethodsWe used an established in vitro dormancy model of Michigan Cancer Foundation-7 (MCF-7) breast cancer cells incubated at clonogenic density on fibronectin-coated plates to determine the effects of inflammatory cytokines on reactivation of dormant ER+ breast cancer cells. We measured induction of a mesenchymal phenotype, motility and the capacity to re-enter dormancy. We induced secretory senescence in murine stromal monolayers by oxidation, hypoxia and estrogen deprivation with hydrogen peroxide (H2O2), carbonyl-cyanide m-chlorophenylhydrazzone (CCCP) and Fulvestrant (ICI 182780), respectively, and determined the effects on growth of co-cultivated breast cancer cells.ResultsExogenous recombinant human (rh) interleukin (IL)-6, IL-8 or transforming growth factor β1 (TGFβ1) induced regrowth of dormant MCF-7 cells on fibronectin-coated plates. Dormant cells had decreased expression of E-cadherin and estrogen receptor α (ERα) and increased expression of N-cadherin and SNAI2 (SLUG). Cytokine or TGFβ1 treatment of dormant clones induced formation of growing clones, a mesenchymal appearance, increased motility and an impaired capacity to re-enter dormancy. Stromal injury induced secretion of IL-6, IL-8, upregulated tumor necrosis factor alpha (TNFα), activated TGFβ and stimulated the growth of co-cultivated MCF-7 cells. MCF-7 cells induced secretion of IL-6 and IL-8 by stroma in co-culture.ConclusionsDormant ER+ breast cancer cells have activated epithelial mesenchymal transition (EMT) gene expression programs and downregulated ERα but maintain a dormant epithelial phenotype. Stromal inflammation reactivates these cells, induces growth and a mesenchymal phenotype. Reactivated, growing cells have an impaired ability to re-enter dormancy. In turn, breast cancer cells co-cultured with stroma induce secretion of IL-6 and IL-8 by the stroma, creating a positive feedback loop.

Abstract LB-168: Inhibitory activities of Sesamum indicum L. leaf extract against the growth, invasion, migration, and adhesion of human colon cancer cells

Abstract Colorectal cancer is one of the most common cancers in both incidence and mortality among men and women worldwide. Sesame (Sesamum indicum L.) leaves (SL) are used as vegetables and traditional medicines in Asian and African countries. In the current study, we aimed to investigate the effects of ethanol extract of sesame leaves (SLE) against key characteristics of cancer cells, including unrestricted growth, resisted apoptosis, and activated metastasis, and to evaluate whether thermal treatment of SL alter the inhibitory effects. Two human colon cancer cell lines were used in this study, HT29 colon adenocarcinoma cells and HCT116 colorectal carcinoma cells. In 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, treatment with SLE at the concentrations of 50-500 µg/mL for 24-72h resulted in dose-dependent and time-dependent growth inhibition in HT29 and HCT116 cells. In cell cycle analyses, SLE caused significantly increased cell population at sub-G1 (in HT29 and HCT116) and G2/M (in HCT116) phases, suggesting that the observed growth inhibition is associated with induction of apoptosis and cell cycle arrest. SLE at non-cytotoxic concentrations was effective in inhibiting critical events during metastasis in HT29 and HCT116, such as invasion (determined by matrigel-coated transwell assay), migration (determined by wound-healing assay), and adhesion (determined using fibronectin-coated plates). Thermal treatment of SL (at 150°C for 15 min) resulted in significantly enhanced inhibitory activities of SLE against the invasion, migration and adhesion in HCT116 cells, but did not change the growth-inhibitory and apoptosis-inducing activities. These results suggest that SLE possesses in vitro anti-cancer activities against colon cancers and that thermal treatment of SL enhances the anti-metastatic activities of SLE. Further studies are needed to verify whether similar effects are reproduced in vivo and to identify active ingredients of SLE responsible for the inhibitory activities. Citation Format: Seoyun Kim, Jihyeung Ju. Inhibitory activities of Sesamum indicum L. leaf extract against the growth, invasion, migration, and adhesion of human colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-168.

Abstract 54: Tumor dormancy in breast cancer cells: Identification of junctional adhesion molecule-A as a novel regulator

Abstract In tumor dormancy, a sub-population of tumor cells becomes quiescent but can re-enter the cell cycle upon certain environmental cues. The re-awakening of dormant populations has been associated with tumor cell proliferation and poor patient outcomes. Tumor dormancy can be modelled in vitro by exposing cancer cells on fibronectin-coated plates to bFGF-2, a mammary differentiation factor abundant in the bone marrow stroma, which causes partial re-differentiation, cell spreading and re-expression of integrin-α5β1. We have uncovered a novel potential role for the adhesion protein Junctional Adhesion Molecule-A (JAM-A) in tumor dormancy. JAM-A overexpression has previously been linked to increased risk of metastasis in breast cancer patients, and its expression regulates the angiogenic functions of bFGF-2. Furthermore, since loss of JAM-A downregulates the α5β1 downstream effector FAK, we hypothesized that JAM-A is required for maintenance of tumor dormancy. To investigate this, JAM-A was transiently silenced in MCF-7 breast cancer cells grown on fibronectin-coated plates and exposed to FGF-2. JAM-silenced cells exhibited expressional downregulations in integrin-α5β1 and FAK proteins, and failed to exhibit the cortical actin redistribution and morphological spreading which characterize dormant cells. in parallel, pharmacologically-induced loss of JAM-A (using an anti-tumor antibiotic) prevented tumor dormancy and was cytotoxic to dormant cells. We next investigated a potential relationship between JAM-A expression and pro-inflammatory cytokines linked with the re-awakening of dormant cells in post-menopausal women. Exposure of MCF-7 cells to IL-1β, TNF-α, IL-6 and IL-8 increased the protein expression of matrix metalloproteinase ADAM17, which in turn caused JAM-A cleavage and loss from the cell membrane. Analysis of bone metastasis sections from post-menopausal breast cancer patients revealed significant heterogeneity in JAM-A expression, with some regions strongly positive for JAM-A while other regions were negative. Nonetheless, 20/24 metastatic sections expressed either moderate or high JAM-A levels, suggesting that even if cells transiently lose JAM-A to reawaken from tumor dormancy, they may later acquire high JAM-A expression during disease progression. The fact that JAM-high dormant cells had high Aldefluor activity, a feature of stemness, might also support an important role for JAM-A in tumor evolution or progression. To conclude, our data support a model whereby JAM-A is important for the maintenance of dormancy in breast cancer cells in a simulated bone marrow microenvironment, and represents a novel therapeutic target worthy of investigation in breast cancer. This work was financially supported by Science Foundation Ireland (grant 13/IA/1994 to AMH). Citation Format: Sri HariKrishna Vellanki, Rodrigo G. Cruz, Katherine M. Sheehan, Elaine W. Kay, Ann M. Hopkins. Tumor dormancy in breast cancer cells: Identification of junctional adhesion molecule-A as a novel regulator [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 54.

Mitogen activated protein kinases (MAPK) and protein phosphatases are involved in Aspergillus fumigatus adhesion and biofilm formation

The main characteristic of biofilm formation is extracellular matrix (ECM) production. The cells within the biofilm are surrounded by ECM which provides structural integrity and protection. During an infection, this protection is mainly against cells of the immune system and antifungal drugs. A. fumigatus forms biofilms during static growth on a solid substratum and in chronic aspergillosis infections. It is important to understand how, and which, A. fumigatus signal transduction pathways are important for the adhesion and biofilm formation in a host during infection. Here we investigated the role of MAP kinases and protein phosphatases in biofilm formation. The loss of the MAP kinases MpkA, MpkC and SakA had an impact on the cell surface and the ECM during biofilm formation and reduced the adherence of A. fumigatus to polystyrene and fibronectin-coated plates. The phosphatase null mutants ΔsitA and ΔptcB, involved in regulation of MpkA and SakA phosphorylation, influenced cell wall carbohydrate exposure. Moreover, we characterized the A. fumigatus protein phosphatase PphA. The ΔpphA strain was more sensitive to cell wall-damaging agents, had increased β-(1,3)-glucan and reduced chitin, decreased conidia phagocytosis by Dictyostelium discoideum and reduced adhesion and biofilm formation. Finally, ΔpphA strain was avirulent in a murine model of invasive pulmonary aspergillosis and increased the released of tumor necrosis factor alpha (TNF-α) from bone marrow derived macrophages (BMDMs). These results show that MAP kinases and phosphatases play an important role in signaling pathways that regulate the composition of the cell wall, extracellular matrix production as well as adhesion and biofilm formation in A. fumigatus.

Endoplasmic reticulum–resident protein 57 (ERp57) oxidatively inactivates human transglutaminase 2

Transglutaminase 2 (TG2) is a ubiquitously expressed, intracellular as well as extracellular protein with multiple modes of post-translational regulation, including an allosteric disulfide bond between Cys-370-Cys-371 that renders the enzyme inactive in the extracellular matrix. Although recent studies have established that extracellular TG2 is switched "on" by the redox cofactor protein thioredoxin-1 (TRX), it is unclear how TG2 is switched "off." Here, we demonstrate that TG2 oxidation by small-molecule biological oxidants, including glutathione, cystine, and hydrogen peroxide, is unlikely to be the inactivation mechanism. Instead, endoplasmic reticulum (ER)-resident protein 57 (ERp57), a protein in the ER that promotes folding of nascent proteins and is also present in the extracellular environment, has the cellular and biochemical characteristics for inactivating TG2. We found that ERp57 colocalizes with extracellular TG2 in cultured human umbilical vein endothelial cells (HUVECs). ERp57 oxidized TG2 with a rate constant that was 400-2000-fold higher than those of the aforementioned small molecule oxidants. Moreover, its specificity for TG2 was also markedly higher than those of other secreted redox proteins, including protein disulfide isomerase (PDI), ERp72, TRX, and quiescin sulfhydryl oxidase 1 (QSOX1). Lastly, siRNA-mediated ERp57 knockdown in HUVECs increased TG2-catalyzed transamidation in the extracellular environment. We conclude that, to the best of our knowledge, the disulfide bond switch in human TG2 represents the first example of a post-translational redox regulatory mechanism that is reversibly and allosterically modulated by two distinct proteins (ERp57 and TRX).