Boyden Chamber Transwell Assay Research Articles

Retraction Note to: Slug down-regulation by RNA interference inhibits invasion growth in human esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive carcinomas of the gastrointestinal tract. We assessed the relevance of Slug in measuring the invasive potential of ESCC cells in vitro and in vivo in immunodeficient mice. We utilized RNA interference to knockdown Slug gene expression, and effects on survival and invasive carcinoma were evaluated using a Boyden chamber transwell assay in vitro. We evaluated the effect of Slug siRNA-transfection and Slug cDNA-transfection on E-cadherin and Bcl-2 expression in ESCC cells. A pseudometastatic model of ESCC in immunodeficient mice was used to assess the effects of Slug siRNA transfection on tumor metastasis development. The EC109 cell line was transfected with Slug-siRNA to knockdown Slug expression. The TE13 cell line was transfected with Slug-cDNA to increase Slug expression. EC109 and TE13 cell lines were tested for the expression of apoptosis-related genes bcl-2 and metastasis-related gene E-cadherin identified previously as Slug targets. Bcl-2 expression was increased and E-cadherin was decreased in Slug siRNA-transfected EC109 cells. Bcl-2 expression was increased and E-cadherin was decreased in Slug cDNA-transfected TE13 cells. Invasion of Slug siRNA-transfected EC109 cells was reduced and apoptosis was increased whereas invasion was greater in Slug cDNA-transfected cells. Animals injected with Slug siRNA-transfected EC109 cells exhihited fewer seeded nodes and demonstrated more apoptosis. Slug down-regulation promotes cell apoptosis and decreases invasion capability in vitro and in vivo. Slug inhibition may represent a novel strategy for treatment of metastatic ESCC.

Abstract 1978: ING4 deficiency results in recurrent crosstalk between Cxcl10/Cxcr3 and Egfr in breast cancer cells

Abstract Cxcl10 is a chemokine that attracts immune cells by binding to the Cxcr3 receptor during a normal immune response. Cxcl10 or Cxcr3 expression has been correlated with aggressive breast cancer. Inhibitor of Growth 4 (ING4) has previously shown to repress CXCL10 expression in breast cancer cells. We investigated the effects of Cxcl10/Cxcr3 in ING4-deficient breast cancer. First, we correlated expression levels of ING4, CXCL10, and CXCR3 with patient survival outcomes using the GDS806 data set. To determine the biological effects Cxcl10/Cxcr3 signaling in ING4-deficient cells, we established ING4 knockout cell lines using CRISPR/Cas9 system in MDAmb231 and MDAmb468 breast cancer cells. We compared proliferation and migration of ING4 wild-type vs. ING4-deleted cells with or without exogenous Cxcl10 using SRB growth assays and Boyden chamber transwell assays, respectively. We used a Cxcr3 inhibitor, AMG-487, or an Egfr inhibitor, erlotinib, to determine a signaling requirement(s) for Cxcl10-induced effects. Immunofluorescence (IF) confocal microscopy was used to evaluate Cxcr3/Egfr interactions. Analysis of the GDS806 gene expression data set showed that patients with tumors containing CXCL10-high/ING4-low had significantly reduced disease-free survival, compared to low CXCL10-low/ING4-high tumors (H.R.= 5.20, 95% CI 2.06-13.09, p = 0.003), suggesting that Cxcl10 may contributed to aggressive progression of ING4 deficient tumors. ING4 deletion with or without Cxcl10 did not affect cell growth. Cxcl10 induced migration of ING4-deleted cells, but not of ING4 wild type cells, suggesting that Cxcl10-induced migration may be responsible for aggressive behavior of ING4-deficient tumors. AMG-487 or erlotinib inhibited Cxcl10 induced cell migration of ING4-deleted cells, indicating that both Cxcr3 and Egfr were required for Cxcl10-induced cell migration. IF results showed that Cxcr3 and Egfr were co-localized in the presence of Cxcl10 at the 10-minute time point in both ING4-wild type and ING4-deleted cells, whereas Cxcr3/Egfr colocalization was present at the 24-hour time point only in ING4-deleted cells. These results demonstrated that Cxcl10-mediated Cxcr3/Egfr co-localization recurred only in the absence of ING4. Cxcr3/Egfr colocalization was inhibited by AMG-487, but not by erlotinib. Taken together, these results indicated that Cxcl10 binding to Cxcr3 induced recurrent colocalization of Egfr only in ING4-deleted cells, which in turn signals downstream components to facilitate cell migration. These results further suggest Cxcl10/Cxcr3/Egfr axis as a novel therapeutic target for ING4-deficient aggressive breast cancer. Citation Format: Emily Tsutsumi, Jeremiah Stricklin, Emily Arnold, Suwon Kim. ING4 deficiency results in recurrent crosstalk between Cxcl10/Cxcr3 and Egfr in breast cancer 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 1978.

Metastatic Dissemination Mimicked in a Multicellular Transwell Assay.

Tumor progression and metastasis are multistep processes that are critically dependent on the interaction of metastasizing tumor cells with other cells of the local microenvironment. Mimicking the single steps of the metastatic cascade in vitro is therefore challenging when investigating not only tumor cell behavior alone but also cellular crosstalk between different cell populations. In particular, the crosstalk of tumor cells with pericytes and endothelial cells when accessing the bloodstream is of great importance for successful intravasation and metastatic dissemination. To examine metastatic intravasation and analyze the interaction of tumor cells with pericytes, which reside within the basement membrane and endothelial cells, aligning the vessel wall, we have designed a 3D in vitro transwell assay mimicking tumor cell intravasation into a vessel. Modifying the Boyden chamber transwell assay by seeding first an endothelial cell layer onto the transwell membrane and covering it with pericytes before adding the tumor cells allows us to investigate the role of pericytes and endothelial cells on tumor cell intravasation and at the same time to study their crosstalk at the molecular level and how this interaction influences tumor cell behavior. It further allows the manipulation of the system for proof-of-principle experimentation.

Abstract P2-01-09: ESR1 mutations drive breast cancer metastasis by context-dependent alterations in adhesive and migratory properties

Abstract Background: Estrogen receptor alpha (ERα/ESR1) is mutated in 30-40% of endocrine resistant ER+ breast cancer. These mutations, primarily located in the ligand binding domain, are associated with worse outcome in patients, and preclinical studies have shown that they cause ligand independent growth. An open question is whether these mutations contribute to actual metastatic process, or merely endocrine resistance. Methods: Using Y537S and D538G genome-edited MCF7 and T47D cells, 3D growth was assessed in ultralow attachment plates. Cell-cell adhesion was determined using calcein-labelled adhesion assay and quantitative microfluidic fluorescence microscope (qMFM). Collagen-based adhesion and spheroid invasion assays were used to test adhesive and invasive properties. Wound scratching, spheroid collective migration and Boyden chamber transwell assays were applied to monitor cell migratory phenotypes. Mutated ER cistromes were profiled using ChIP-sequencing. ESR1 mutations in clinical samples were characterized using ddPCR. Results: Visual inspection of cells grown in suspension culture revealed more compressed multicellular spheroids in ESR1 mutant cells, indicative of increased cell-cell interactions. This observation was confirmed in both static and microfluidic conditions. This effect was more pronounced in MCF7 than T47D cells, correlating with increased expression of desmosome and gap junction genes. Pharmacological blockade of gap junctions decreased cell-cell adhesion. Decreased attachment and increased invasion to collagen were discerned in all mutant cell types. Further functional analysis identified alterations in the TIMP3-MMP axis causing these phenotypes. The cell-cell adhesion phenotypes were restricted to MCF7-Y537S/D538G and T47D-Y537S, whereas T47D-D538G cells showed significantly increased migration. A GSEA screen identified Wnt signaling as uniquely induced in this context, and combination treatment using the Wnt inhibitor LGK974 and Fulvestrant led to synergistic inhibition of migration. ChIP-seq identified mutation-specific cistromes with an overall increased ligand-independent ER binding. However, it did not reveal binding sites in any candidate metastases genes, suggesting secondary epigenetic mechanisms. The motif analysis revealed the enrichment of FOXA1 motifs in mutated ER cistromes except T47D-D538G cells. However, knockdown of FOXA1 induced significantly higher inhibition of T47D-D538G migration than Fulvestrant treatment alone, indicating a FOXA1-dominated mechanism. Collectively, these data show that ESR1 mutant cells gain metastatic properties, in addition to endocrine resistance. To prove this using clinical samples, we measured ESR1 mutations in a well-defined cohort of endocrine resistant local or distant recurrence. Significant enrichment of ESR1 mutations in distant (9/55) vs local (0/27) recurrences confirms critical role of mutant ERα in metastases. Conclusion: Further analysis of context dependent changes in cell-cell adhesion and migration of ESR1 mutant cells might guide the design and development of drugs targeting ERα-mutant tumors, such as inhibitors of gap junction, FOXA1, MMP, and Wnt signaling pathways. Disclosure: The authors declare no conflict of interest. Citation Format: Li Z, Bahreini A, Levine KM, Wang P, Tasdemir N, Montanez MA, Sundd P, Wallace CT, Watkins SC, Chu D, Park BH, Hou W, Mooring MS, Zhu L, Tseng GC, Carroll JS, Atkinson JM, Lee AV, Oesterreich S. ESR1 mutations drive breast cancer metastasis by context-dependent alterations in adhesive and migratory properties [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-01-09.

Abstract A70: ESR1 mutations confer novel metastatic functions in genome-edited breast cancer models

Abstract Background: Estrogen receptor alpha (ERα), encoded by the ESR1 gene, is expressed in approximately 70% of breast cancers. Recent studies conducted by us and others have shown that somatic mutations in ESR1 gene play a key role in conferring endocrine resistance in ER+ breast cancer. These hotspot mutations mainly occur on the ligand-binding domain of ERα, leading to poor outcomes in 25-30% of patients with ER+ metastatic breast cancer in clinic. The mechanisms behind the potential enhanced metastasis of these mutations have become an urgent issue to be addressed, but they are not well understood due to a lack of ESR1 mutant models. Methods: We generated and characterized genome-edited T47D and MCF7 breast cancer cell lines with the two most common ESR1 mutations (Y537S and D538G), using CRIPSR/Cas9 and rAAV systems, respectively. Multiple clones for each mutant were sorted and the mutation frequencies were detected using digital droplet PCR (ddPCR). Levels of total or phosphorylated ER were determined by Western blot. We subsequently performed an RNA-sequencing and ChIP-sequencing to deeply differentiate the gene expression and ER-DNA binding patterns in these mutants at transcriptome and cistrome. The growth of these pooled mutant-cells was determined in both 2D and 3D ultralow attachment plates. The cell-matrix adhesions were measured based on ECM array, and 84-ECM adhesion related genes were further tested by qPCR array. IncuCyte real-time image system and Boyden chamber trans-well assays were used to monitor the cell migration and chemotaxis. Tail vein injections were performed on nude mice. H&E and immunofluorescent staining of lung and liver tissues with human specific cytokeratin 19 were utilized to evaluate in vivo metastatic capacities of the mutant cell models. Results: We first identified the robust mutation frequencies at both RNA and DNA levels in our cell models. The RNA-seq and ChIP-seq exhibits multiple ligand-independent genes and ER binding events overlapping between either cell lines or mutants, which were further conformed by qPCR and ChIP. We also found that both Y537S and D538G mutants present ligand-independent growth in 2D and 3D ultralow attachment plates. Using wound-scratching assay, we observed significant higher migration rate in D538G mutant of T47D cell lines on both Matrigel and type I collagen, indicating a cell-line and mutant-specific phenotype. We also detected lower attachment of both mutants on type I collagen in both cell lines, and our qPCR array revealed that alterations in the MMP pathways could be one of the major mechanism causing this phenotype. Finally, tail vein injection of T47D-D538G mutant cells in nude mice derived more micrometatsatic spots in the lung and liver tissues. Conclusion: In sum, our study presents the first in-depth metastatic functional analysis of the biology of ESR1 mutations in genomic knockin cell models, pointing out the enhanced migration and decreased cell-matrix adhesion as a potential novel gain-of-function of the Y537S and D538G mutant cells in vitro and in vivo. These findings suggest the potential role of enhanced metastasis of these ESR1 mutations through remodeling of transcriptional profiles, shedding lights towards the development of efficient therapies of ESR1 mutant breast cancer. Citation Format: Zheqi Li, Amir Bahreini, Peilu Wang, Kevin M. Levine, Nilgun Tasdemir, David Chu, Ben H. Park, Adrian V. Lee, Steffi Oesterreich. ESR1 mutations confer novel metastatic functions in genome-edited breast cancer models [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A70.

Chondroitin sulfate proteoglycans as novel drivers of leucocyte infiltration in multiple sclerosis

Multiple sclerosis presents with profound changes in the network of molecules involved in maintaining central nervous system architecture, the extracellular matrix. The extracellular matrix components, particularly the chondroitin sulfate proteoglycans, have functions beyond structural support including their potential interaction with, and regulation of, inflammatory molecules. To investigate the roles of chondroitin sulfate proteoglycans in multiple sclerosis, we used the experimental autoimmune encephalomyelitis model in a time course study. We found that the 4-sulfated glycosaminoglycan side chains of chondroitin sulfate proteoglycans, and the core protein of a particular family member, versican V1, were upregulated in the spinal cord of mice at peak clinical severity, correspondent with areas of inflammation. Versican V1 expression in the spinal cord rose progressively over the course of experimental autoimmune encephalomyelitis. A particular structure in the spinal cord and cerebellum that presented with intense upregulation of chondroitin sulfate proteoglycans is the leucocyte-containing perivascular cuff, an important portal of entry of immune cells into the central nervous system parenchyma. In these inflammatory perivascular cuffs, versican V1 and the glycosaminoglycan side chains of chondroitin sulfate proteoglycans were observed by immunohistochemistry within and in proximity to lymphocytes and macrophages as they migrated across the basement membrane into the central nervous system. Expression of versican V1 transcript was also documented in infiltrating CD45+ leucocytes and F4/80+ macrophages by in situ hybridization. To test the hypothesis that the chondroitin sulfate proteoglycans regulate leucocyte mobility, we used macrophages in tissue culture studies. Chondroitin sulfate proteoglycans significantly upregulated pro-inflammatory cytokines and chemokines in macrophages. Strikingly, and more potently than the toll-like receptor-4 ligand lipopolysaccharide, chondroitin sulfate proteoglycans increased the levels of several members of the matrix metalloproteinase family, which are implicated in the capacity of leucocytes to cross barriers. In support, the migratory capacity of macrophages in vitro in a Boyden chamber transwell assay was enhanced by chondroitin sulfate proteoglycans. Finally, using brain specimens from four subjects with multiple sclerosis with active lesions, we found chondroitin sulfate proteoglycans to be associated with leucocytes in inflammatory perivascular cuffs in all four patients. We conclude that the accumulation of chondroitin sulfate proteoglycans in the perivascular cuff in multiple sclerosis and experimental autoimmune encephalomyelitis boosts the activity and migration of leucocytes across the glia limitans into the central nervous system parenchyma. Thus, chondroitin sulfate proteoglycans represent a new class of molecules to overcome in order to reduce the inflammatory cascades and clinical severity of multiple sclerosis.

Abstract PD8-08: ESR1 mutations confer novel metastatic functions in genome-edited breast cancer cell models

Abstract Background: Estrogen receptor alpha (ERα), encoded by the ESR1 gene, is expressed in approximately 70% of breast cancers. Recent studies conducted by us and others have shown that somatic mutations in ESR1 gene play a key role in conferring endocrine resistance in ER+ breast cancer. These hotspot mutations mainly occur on the ligand binding domain of ERα, leading to poor outcomes in 25-30% of patients with ER+ metastatic breast cancer in clinic. The mechanisms behind the potential enhanced metastasis of these mutations have become an urgent issue to be addressed, but they are not well understood due to a lack of ESR1 mutant models. Methods: We generated and characterized genome-edited T47D and MCF7 breast cancer cell lines with the two most common ESR1 mutations (Y537S and D538G), using CRIPSR/Cas9 and rAAV systems respectively. Multiple clones for each mutant were sorted and the mutation frequencies were detected using digital droplet PCR (ddPCR). We subsequently performed an RNA-sequencing to deeply differentiate the gene expression patterns in these mutants. The growth of these pooled mutant-cells was determined in both 2D and 3D plates. The cell-matrix adhesions were measured based on ECM array, and 84-ECM adhesion related genes were further tested by qPCR array. IncuCyte real-time image system and boyden chamber transwell assays were used to monitor the cell migration and chemotaxis. Tail vein injection were performed on nude mice, and immunofluorescent staining of lung tissues with human specific cytokeratin 19 were utilized to evaluate in vivo metastatic capacities of the mutant cell models. Results: We first identified the robust mutation frequencies at both RNA and DNA levels in our cell models. The RNA-seq exhibits multiple ligand-independent genes overlapping between either cell lines or mutants, which were further conformed by qPCR. We also found that both Y537S and D538G mutants present ligand-independent growth in 2D and 3D ultra-low attachment plates. Using wound-scratching assay, we observed significant higher migration rate in D538G mutant of T47D cell lines on both matrigel and type I collagen, indicating a cell-line and mutant-specific phenotype. We also detected lower attachment of both mutants on type I collagen in both cell lines, and our qPCR array revealed that alterations in the MMP pathways could be one of the major mechanism causing this phenotype. Finally, tail vein injection of T47D mutant-cells in nude mice derived more micrometatsatic spots in the lung tissues. Conclusion: In sum, our study presents the first in-depth metastatic functional analysis of the biology of ESR1 mutations in genomic knock-in cell models, pointing out the enhanced migration and decreased cell-matrix adhesion as a potential novel gain-of-function of the Y537S and D538G mutant-cells in vitro and in vivo. These findings suggest the potential role of enhanced metastasis of these ESR1 mutations through remodeling of transcriptional profiles, shedding lights towards the development of efficient therapies of ESR1 mutant breast cancer. Citation Format: Oesterreich S, Li Z, Bahreini A, Wang P, Levine KM, Tasdemir N, Chu D, Park BH, Lee AV. ESR1 mutations confer novel metastatic functions in genome-edited breast cancer cell models [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD8-08.

HDAC1 knockdown inhibits invasion and induces apoptosis in non-small cell lung cancer cells.

Non-small cell lung cancer (NSCLC) is a common malignant tumor. Although the abnormal expression and potential clinical prognostic value of histone deacetylase 1 (HDAC1) were recently discovered in many kinds of cancer, the roles and molecular mechanisms of HDAC1 in NSCLC is still limited. The CCK-8 assay is used to evaluate the viability of NSCLC cells. Downregulation of HDAC1 by shRNA. The TUNEL assay was used to evaluate the role of HDAC1 in NSCLC apoptosis. To evaluate the role of HDAC1 in NSCLC cells migration, the Boyden chamber transwell assay and wound healing assay were used. To evaluate the cells invasion, the matrigel precoated Transwell assay was used. Enzyme-linked immunosorbent assays (ELISAs) were used to detect the level of vascular endothelial growth factor (VEGF) and IL-8 in NSCLC. To investigate the role of HDAC1 in angiogenesis, the tube formation assay was investigated. In this study, we showed that HDAC1 expression was elevated in NSCLC lines compared to that in normal liver cells LO2. Furthermore, downregulation of HDAC1 inhibited cell proliferation, prevented cell migration, decreased cell invasion, reduced tumor angiogenesis and induced cell apoptosis. In summary, HDAC1 may be regarded as a potential indicator for NSCLC patient treatment.

High mannose N-glycan binding lectin from Remusatia vivipara (RVL) limits cell growth, motility and invasiveness of human breast cancer cells

Breast cancer known for its high metastatic potential is responsible for large mortality rate amongst women; hence it is imperative to search for effective anti-metastatic molecules despite anticancer drugs. The current study describes the potential of Remusatia vivipara lectin (RVL), inducing apoptosis in breast cancer cells there by limiting motility and invasiveness. RVL binds to the cell surface glycans of MDA-MB-468 and MCF-7 cells, exhibiting strong glycan mediated cytotoxic effect, but show marginal effect on non-tumorigenic MCF-10A cells. RVL elicits increased cellular stress, apoptotic vacuoles and nuclear disintegration in both MDA-MB-468 and MCF-7 cells accompanied by depletion of G0/G1, S and G2/M phases. Lectin interaction induced production of reactive oxygen species through altering mitochondrial membrane potential progressing to apoptosis. Further, RVL strongly elicited reproductive cell death in MDA-MB-468 cells and showed strong inhibitory effect on neovascularization demonstrated in chorioallantoic membrane assay. Treatment of MDA-MB-468 cells with RVL, suppress the motility and invasive property as shown by scratch wound heal and Boyden chamber transwell assays respectively. These results provide an insight into significance of interaction of RVL with specific cell surface high mannose N-glycans resulting in curtailing the metastatic ability of cancer cells.

Celastrol negatively regulates cell invasion and migration ability of human osteosarcoma via downregulation of the PI3K/Akt/NF-κB signaling pathway in vitro.

Osteosarcoma (OS) is a primary malignant tumor of the bone, with a tendency to metastasize early. Despite the advances in treatment options, more than 30% of patients develop distant metastases, and the prognosis of these patients with metastases is extremely poor. Celastrol has been demonstrated to manifest multiple pharmacological activities, including induction of apoptosis in numerous types of cancer cell lines. Our previous studies have also suggested that Celastrol is capable of inducing apoptosis of human osteosarcoma cells via the mitochondrial-dependent pathway. The purpose of this study was to investigate the effects of Celastrol on the migration and invasion of human osteosarcoma U-2OS cells in vitro. Cell migration and invasion were investigated using wound healing and Boyden chamber Transwell assays. We observed that Celastrol suppressed cell invasion and migration in human osteosarcoma U-2OS cells. Furthermore, protein expression levels of phosphorylated phosphatidylinositol 3-kinase (PI3K), Akt, inhibitor of κB kinase α/β, inhibitor of κB α, nuclear factor-κB (NF-κB subunit p65) and matrix metalloproteinase (MMP)-2 and -9 were measured by western blot analysis. We observed that the PI3K/Akt/NF-κB signaling pathway was inhibited following Celastrol treatment. In addition, the expression levels of MMP-2 and -9 proteins were also reduced significantly following Celastrol treatment. Therefore, we confirmed that Celastrol suppressed osteosarcoma U-2OS cell metastasis via downregulation of the PI3K/Akt/NF-κB signaling pathway in vitro.

In vitro and in vivo evaluation of FGF2 and FGF9 dual-loaded poly(ester amide) fibers for therapeutic angiogenesis

Event Abstract Back to Event In vitro and in vivo evaluation of FGF2 and FGF9 dual-loaded poly(ester amide) fibers for therapeutic angiogenesis Somiraa Said1, Caroline O’neil2, Hao Yin2, Zengxuan Nong2, Mai Elfarnawany1, 2, Hon Leong3, James Lacefield1, 4, 5, Geoffrey Pickering2, 5, 6, 7 and Kibret Mequanint1, 8 1 The University of Western Ontario, Biomedical Engineering Graduate Program, Canada 2 The University of Western Ontario, Robarts Research Institute, Canada 3 The University of Western Ontario, Department of Surgery (Urology), Canada 4 The University of Western Ontario, Department of Electrical and Computer Engineering, Canada 5 The University of Western Ontario, Department of Medical Biophysics, Canada 6 The University of Western Ontario, Department of Biochemistry, Canada 7 The University of Western Ontario, Department of Medicine (Cardiology), Canada 8 The University of Western Ontario, Department of Chemical and Biochemical Engineering, Canada Introduction: In therapeutic angiogenesis, the delivery of angiogenic factors either by bolus injection or infusion results in their rapid clearance from the site of interest due to their short biological half-life[1]. The angiogenesis process is complex, therefore therapeutic angiogenesis regimens should include the administration of multiple growth factors and recapitulate temporal presentation of these growth factors[2],[3]. In this study, we are reporting controlled delivery of a different ‘cocktail’ of growth factors; an angiogenic factor − fibroblast growth factor-2 (FGF2) and an arteriogenic factor − fibroblast growth factor-9 (FGF9). We are aiming to achieve sustained and differential release of these two growth factors from biodegradable poly(ester amide) (PEA) electrospun fibers towards targeting neovascular formation and stabilization. Materials and Methods: FGF2 and FGF9 were dual loaded into PEA fibers using a mixed blend and emulsion electrospinning technique. In vitro release kinetics of FGF2/FGF9 dual-loaded PEA fibers was studied in PBS (pH 7.4) at 37 ºC for 28 days using ELISA as previously described[4]. In vitro angiogenesis assays including Matrigel tube formation assay and Boyden chamber transwell assay were used to evaluate endothelial cell (EC) tube formation, directed smooth muscle cell (SMC) migration, and EC-SMC interaction. Ex ovo chick chorioallantoic membrane (CAM) model coupled with power Doppler ultrasound imaging was employed to assess the in vivo angiogenic capacity of our delivery system. Results and Discussion: In vitro release studies showed controlled and differential release of both factors in a bioactive form. Co-released FGF2 and FGF9 from dual loaded PEA fibers enhanced EC tube formation, directed-migration of SMCs towards PDGF-BB, and EC/SMC tube stabilization. The CAM assay showed a higher percentage increase in vascular density of the full CAM surface treated with FGF2/FGF9 dual-loaded fibers with time (Figure 1). Power Doppler 3D volumes showed the formation of smaller vessels near the CAM surface in the case of dual-loaded PEA fibers and soluble growth factors treated CAMs that were not observed in the case of unloaded PEA fibers treated CAMs and the negative control, which may indicate a localized angiogenic effect (Figure 2). Conclusion: Localized angiogenesis was observed at the interface between the dual-loaded fibrous mat and the CAM surface. The CAM model had some limitations in detecting a statistically significant difference in the full CAM vasculature due to its rapid vascular growth that did not emulate the controlled and relatively slow release of FGF2 and FGF9 from the PEA fibers. Despite the aforementioned limitations, the in vitro angiogenesis assays data together with the preliminary CAM assay data suggests that FGF2/FGF9 dual loaded PEA fibers can have a potential therapeutic angiogenesis application for treatment of ischemic vascular diseases. Moving forward, a more clinically relevant model, the ischemic hind limb mouse model will be employed to better investigate the angiogenic effects of the FGF2/FGF9 dual-loaded PEA fibers. The Authors acknowledge the financial support from The Heart and Stroke Foundation of Canada, the Canadian Institutes for Health Research (FRN 11715), the Canadian Cancer Society (Grant #701080), and the Natural Sciences and Engineering Research Council of Canada.

The decrease of cyclin B2 expression inhibits invasion and metastasis of bladder cancer

ObjectiveIt has been shown that cyclin B2 is commonly overexpressed in many malignant tumors. This study aimed to investigate the potential role of cyclin B2 in bladder cancer. Method and materialFixed tissues for immunohistochemistry and fresh tissues for western blotting and quantitative real-time polymerase chain reaction assay were randomly selected from Nanfang hospital. Normal bladder urothelial cell and bladder cancer cell lines was stored in our laboratory, the bladder cancer cells were transfected to develop bladder cancer cell clones expressing decreased cyclin B2 levels, the clones were used for cell growth and metastasis experiments in vitro and in vivo. ResultsWestern blot and immunohistochemical analysis both showed that the cyclin B2 protein expression was higher in bladder urothelial carcinoma than in normal bladder mucosa, especially in invasive cancer. Real-time polymerase chain reaction showed that the cyclin B2 messenger RNA expression exhibited the same trend. Results of cell lines experiments also showed higher cyclin B2 expression in cancer cells. In vitro tests the decrease of cyclin B2 expression that had little effect on cell growth and cell cycling according to the MTT assay and the Edu assay, whereas in the Boyden chamber transwell assay, the cyclin B2 low-expressing clones significantly inhibits the cells׳ invasion and metastatic abilities. This result was consistent with the scratch-wound assay result showing that the target clone needed more time for healing the wound. The in vivo experiment in nude mice produced similar results, the lung and liver target cell metastasis nodules were smaller and less than those of the negative control by the hepatic subcapsular injection assay, and the mice of the target clone group has longer survival time in no intervention observed test. ConclusionThese results indicate that the cyclin B2 was overexpressed in bladder cancer, and the down-regulation of cyclin B2 expression in bladder cancer greatly inhibits the cell׳s invasion and metastatic abilities, and it prolonged the survival time of nude mice in vivo.

Establishment and characterization of gemcitabine-resistant human cholangiocarcinoma cell lines with multidrug resistance and enhanced invasiveness.

To establish and characterize the gemcitabine-resistant cholangiocarcinoma (CCA) cell lines, CCA KKU‑M139 and KKU‑M214 cell lines were exposed stepwisely to increasing gemcitabine (GEM). The resultant drug-resistant cell lines, KKU‑M139/GEM and KKU‑M214/GEM, retained the resistant phenotype in drug-free medium at least for 2 months. Sulforhodamine B assay demonstrated that KKU‑M139/GEM and KKU‑M214/GEM were 25.88- and 62.31-fold more resistant to gemcitabine than their parental cells. Both gemcitabine-resistant cell lines were cross-resistant to 5-fluorouracil (5-FU), doxorubicin and paclitaxel indicating their multidrug-resistant nature. Using reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR and western blot analyses, gemcitabine-resistant cells showed upregulation of RRM1 and downregulation of hENT1 and dCK. In relation to multidrug resistance, these cell lines showed upregulation of multidrug resistance protein 1 (MRP1) leading to an increase of drug efflux. Using cell adhesion and Boyden chamber transwell assays, these cell lines also showed higher cell adhesion, migration and invasion capabilities via the activations of protein kinase C (PKC), focal adhesion kinase (FAK), extracellular signal-regulated kinase-1/2 (ERK1/2) and nuclear factor-κB (NF-κB). Higher activity of matrix metalloproteinase-9 (MMP-9) and urokinase plasminogen activator (uPA) was also observed by a gelatin zymography assay and a casein-plasminogen zymography assay. Flow cytometry analysis indicated the G2/M arrest regulated by downregulation of cyclin B1 and cyclin-dependent kinase 1 (Cdk1) resulted in an extended population doubling time. Using Annexin V/propidium iodide staining, evasion of apoptosis via an intrinsic pathway was observed in both cell lines in association with upregulation of Bcl-2 and downregulation of Bax. Interestingly, Fas was additionally downregulated in KKU‑M214/GEM supporting the view of its higher GEM resistant characteristics. These findings indicate that long-term exposure of CCA cell lines to gemcitabine induce not only multidrug resistance but also enhance their invasiveness.

AMPK-dependent signaling modulates the suppression of invasion and migration by fenofibrate in CAL 27 oral cancer cells through NF-κB pathway.

Fenofibrate, a peroxisome proliferator-activated receptor alpha (PPARα) agonist and lipid-lowering agent, has been used worldwide for treatment of hyperlipidemia. The clinical trials demonstrate that fenofibrate possesses multiple pharmacological activities, including antitumor effects. However, the precise mechanisms in oral squamous cell carcinoma (OSCC) remain unclear. In this study, we investigated the anticancer effects of fenofibrate on the migration and invasion of human oral cancer CAL 27 cells. Fenofibrate inhibited the cell migration and invasion of CAL 27 cells by the wound healing and Boyden chamber transwell assays, respectively. In addition, fenofibrate reduced the protein expressions of MMP-1, MMP-2, MMP-7, and MMP-9 by Western blotting and inhibited enzyme activities of MMP-2/-9 using gelatin zymography assay. Results from immunoblotting analysis showed that the proteins of p-LKB1 (Ser428), LKB1, p-AMPKα (Thr172), p-AMPKα1/α2 (Ser425/Ser491), p-AMPKβ1 (Ser108), and AMPKγ1 were upregulated by fenofibrate; the levels of p-IKKα/β (Ser176) and p-IκBα were reduced in fenofibrate-treated cells. Also, fenofibrate suppressed the expressions of nuclear NF-κB p65 and p50 by immunoblotting and NF-κB DNA binding activity by EMSA assay. The anti-invasive effect of fenofibrate was attenuated by compound C [an adenosine 5'-monophosphate-activated protein kinase (AMPK) inhibitor] or dominant negative form of AMPK (DN-AMPKα1). Thus, fenofibrate considerably inhibited metastatic behaviors of CAL 27 cells might be mediated through blocking NF-κB signaling, resulting in the inhibition of MMPs; these effects were AMPK-dependent rather than PPARα signaling. Our findings provide a molecular rationale, whereby fenofibrate exerts anticancer effects and additional beneficial effects for the treatment of cancer patients. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 866-876, 2016.

Heparan sulfate modulates Slit3-induced endothelial cell migration.

Heparan sulfate is a long, linear polysaccharide with sulfation modifications and belongs to the glycosaminoglycan family. Our recent studies elucidated that the axon guidance molecule Slit3 is a new heparan sulfate-binding protein and a novel angiogenic factor by interacting with its cognate receptor Robo4, which is specifically expressed in endothelial cells. Here we describe using heparan sulfate-deficient mouse endothelial cells to determine the co-reception function of heparan sulfate in Slit3-induced endothelial cell migration in a Boyden chamber trans-well migration assay.

Possible contribution of aminopeptidase N(APN/CD13) to migration and invasion of human osteosarcoma cell lines.

Osteosarcoma is the most common primary malignancy of the bone. Aminopeptidase N (APN/CD13), a Zn+2-dependent ectopeptidase localized on the cell surface, is widely considered to influence the invasion mechanism. This study explores the potential involvement of APN in migration and invasion of human osteosarcoma cells in vitro using inhi-bitors and activators of APN. Cells treated with APN inhibitor bestatin displayed decreased migration and invasion in a Boyden chamber Transwell assay. Western blotting revealed reduced levels of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathway proteins, reduced phosphorylation of p38, ERK1/2 and JNK and decreased levels of NF-κB. Bestatin treatment also lowered APN, matrix metalloproteinase (MMP)-2 and -9 enzymatic activity and their mRNA expression. Reduced MMP-2 and -9 protein levels were also observed. By comparison, cells treated with cytokine interleukin-6 (IL-6), a stimulator of APN, displayed increased migration and invasion. Western blotting revealed increased levels of MAPK and PI3K pathway proteins, phosphorylated p38, ERK1/2 and JNK, and NF-κB. IL-6 treatment also increased APN and MMP-2 and -9 enzymatic activity. An increase of APN, MMP-2 and -9 mRNA levels, and MMP-2 and -9 protein levels was also observed. Together these experiments reveal potential enzymatic and signalling roles for APN in osteosarcoma and establish a starting point for an in-depth analysis of the role of APN in regulating invasiveness. A deeper knowledge about the regulatory mechanisms of APN may contribute to the development of anti-metastatic therapies.

CXCR4 antibody treatment suppresses metastatic spread to the lung of intratibial human osteosarcoma xenografts in mice

Current combined surgical and neo-adjuvant chemotherapy of primary metastatic osteosarcoma (OS) is ineffective, reflected by a 5-year survival rate of affected patients of less than 20 %. Studies in experimental OS metastasis models pointed to the CXCR4/CXCL12 homing axis as a novel target for OS metastasis-suppressive treatment. The present study investigated for the first time the CXCR4-blocking principle in a spontaneously metastasizing human 143B OS cell line-derived orthotopic xenograft mouse model. The highly metastatic 143B cells, unlike the parental non-metastatic HOS cells, express functional CXCR4 receptors at the cell surface, as revealed in this study by RT/PCR of gene transcripts, by FACS analysis with the monoclonal anti CXCR4 antibody 12G5 (mAb 12G5) and by CXCL12 time- and dose-dependent stimulation of AKT and ERK phosphorylation. A significantly (p < 0.05) higher CXCL12 dose-dependent chemotactic response of 143B compared to HOS cells in a Boyden chamber trans-well migration assay suggested a crucial role of the CXCL12/CXCR4 homing axis in 143B cell lung metastasis. Repetitive treatment of mice with 143B cell-derived intratibial tumors given intravenous bolus injections of mAb12G5 indeed inhibited significantly (p < 0.01) the number of X-gal-stainable lung micrometastases of lacZ-transduced 143B cells. Antibody treatment had also a mild inhibitory effect on primary tumor growth associated with remarkably less osteolysis, but it did not affect the number of developing lung macrometastases. In conclusion, these results demonstrate considerable potential of high-affinity CXCR4-blocking agents for OS tumor cell homing suppressive treatment in metastasizing OS complementary to current (neo)-adjuvant chemotherapy.

Dicer is down-regulated in clear cell renal cell carcinoma and in vitro Dicer knockdown enhances malignant phenotype transformation

ObjectivesAlthough emerging evidence has shown that the deregulation of micro-ribonucleic acid (RNA) biogenesis machinery is involved in various human malignancies, this role has not been investigated in clear cell renal cell carcinoma (ccRCC). This study aims to determine whether Dicer, a key enzyme responsible for biogenesis of microRNA, is deregulated in ccRCC. The biological roles of Dicer in vitro are also determined. Materials and methodsThe expression of Dicer at messenger RNA and protein levels was detected by real-time quantitative polymerase chain reaction and western blot, respectively, in human kidney tubule epithelial cell line, nonmetastatic 786-O ccRCC cell line, and metastatic ACHN ccRCC cell line, as well as in 42 cases of ccRCC surgical specimens including 14 cases with distant metastasis and their corresponding adjacent normal renal tissues. Dicer expression levels in specimens were also measured by immunohistochemical staining. Knockdown of Dicer expression in 786-O and ACHN ccRCC cell lines was achieved by transfecting short interfering RNA against Dicer. The effects of Dicer on cell proliferation, migration, and invasion were detected by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, flow cytometric analyses, and Boyden chamber Transwell assay, respectively. ResultsCompared with human kidney tubule epithelial cell line, Dicer expression levels were significantly down-regulated in 786-O and ACHN ccRCC cell lines, with the metastatic ACHN ccRCC cell line having even lower levels. Meanwhile, Dicer expression levels were significantly down-regulated in ccRCC surgical specimens compared with adjacent normal renal tissues, with the metastatic ones further reduced, and Dicer messenger RNA levels were significantly correlated with overall tumor-node-metastasis stage of ccRCC. In vitro, the knockdown of Dicer significantly promoted cell proliferation, migration, and invasion. ConclusionsReduced expression of Dicer may play a role in the tumorigenesis of ccRCC and further decline may be associated with distant metastasis of ccRCC.

Luteolin reduces the invasive potential of malignant melanoma cells by targeting β3 integrin and the epithelial-mesenchymal transition

To investigate whether luteolin, a highly prevalent flavonoid, reverses the effects of epithelial-mesenchymal transition (EMT) in vitro and in vivo and to determine the mechanisms underlying this reversal. Murine malignant melanoma B16F10 cells were exposed to 1% O(2) for 24 h. Cellular mobility and adhesion were assessed using Boyden chamber transwell assay and cell adhesion assay, respectively. EMT-related proteins, such as E-cadherin and N-cadherin, were examined using Western blotting. Female C57BL/6 mice (6 to 8 weeks old) were injected with B16F10 cells (1×10(6) cells in 0.2 mL per mouse) via the lateral tail vein. The mice were treated with luteolin (10 or 20 mg/kg, ip) daily for 23 d. On the 23rd day after tumor injection, the mice were sacrificed, and the lungs were collected, and metastatic foci in the lung surfaces were photographed. Tissue sections were analyzed with immunohistochemistry and HE staining. Hypoxia changed the morphology of B16F10 cells in vitro from the cobblestone-like to mesenchymal-like strips, which was accompanied by increased cellular adhesion and invasion. Luteolin (5-50 μmol/L) suppressed the hypoxia-induced changes in the cells in a dose-dependent manner. Hypoxia significantly decreased the expression of E-cadherin while increased the expression of N-cadherin in the cells (indicating the occurrence of EMT-like transformation), which was reversed by luteolin (5 μmol/L). In B16F10 cells, luteolin up-regulated E-cadherin at least partly via inhibiting the β3 integrin/FAK signal pathway. In experimental metastasis model mice, treatment with luteolin (10 or 20 mg/kg) reduced metastatic colonization in the lungs by 50%. Furthermore, the treatment increased the expression of E-cadherin while reduced the expression of vimentin and β3 integrin in the tumor tissues. Luteolin inhibits the hypoxia-induced EMT in malignant melanoma cells both in vitro and in vivo via the regulation of β3 integrin, suggesting that luteolin may be applied as a potential anticancer chemopreventative and chemotherapeutic agent.

Antitumor effects of the novel quinazolinone MJ-33: Inhibition of metastasis through the MAPK, AKT, NF-κB and AP-1 signaling pathways in DU145 human prostate cancer cells

Quinazolinone compounds have been shown to have antitumor activity in many human cancer cell lines. In the present study, we investigated the anti-metastatic activity of MJ-33 (2-(3-ethoxyphenyl)-6-pyrrolidinylquinazolinone), a novel quinazolinone derivate, and the signaling pathway of MJ-33 in human prostate cells. MJ-33 exhibited a growth inhibitory effect on DU145, LNCaP and PC-3 cells by MTT assay. DU145 cells showed greater sensitivity to the growth inhibition of MJ-33 than that of LNCaP and PC-3 cells. MJ-33 also had an inhibitory effect on the invasion, migration and adhesion of DU145 cells using Boyden chamber transwell assays, wound-healing and adhesion assay. In addition, MJ-33 inhibited cell metastasis through the reduction of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and urokinase-type plasminogen activator (u-PA) enzyme activities and protein levels by gelatin zymography assay and western blot analysis, respectively. MJ-33 reduced the protein levels of p-JNK, p-p38, p-ERK, p-AKT and nuclear NF-κB (p65), c-fos and c-Jun protein levels by western blotting. Using electrophoretic mobility-shift assay (EMSA), we demonstrated that MJ-33 blocked the activation of transcription factor AP-1 (activator protein-1) and NF-κB, which led to the inhibition of MMP-2 and MMP-9 expression. Collectively, our data showed that MJ-33 decreased protein levels of MAPKs (mitogen-activated protein kinases), AKT, AP-1 and NF-κB, resulting in the inhibition of matrix metalloproteinases. Downregulation of MMP-2 and MMP-9 reduces the invasion, migration and adhesion activities of DU145 cells. MJ-33 may be a promising agent against prostate cancer metastasis.