Cyr61 promotes CD204 expression and migration of macrophages via MEK/Erk pathway in the microenvironment of human esophageal squamous cell carcinoma
ABSTRACT: The authors previously suggested that CD204 was a useful marker for tumor-associated macrophages (TAMs) of esophageal squamous cell carcinoma (ESCC). In this study, they discovered that within ESCC microenvironment not only cancer cells but also TAMs expressed cysteine-rich, angiogenic inducer, 61 (Cyr61), induced by conditioned media of ESCC cells. Levels of Cyr61 expression were associated with CD204+ macrophage infiltration in ESCC tissue. Cyr61 promoted CD204 expression and migration of macrophages via MEK/Erk pathway in vitro. To identify the molecules specifically upregulated in TAM-like THP-1 cells, we performed a cDNA microarray analysis using RNAs from THP-1 stimulated with 200 nM TPA or with 200 nM TPA followed by 50% TE-8CM. THP-1 human acute monocytic leukemia cell line was treated with 200 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 2 days to induce macrophage-like differentiation, then exposed to 50%Conditioned medium of TE-8 ESCC cell line (TE-8CM) for 2 days. Differentially expressed genes between MΦ-like and TAM-like THP-1 cells were analysed by cDNA microarray. TE-8CM was prepared by plating 5 × 106 tumor cells in 10 mL complete medium in 100-mm dishes for 24 hr, thereafter changing the medium to complete DMEM supplemented with 10% human AB serum instead of fetal bovine serum. After 2–3 days, the supernatants were harvested, centrifuged and stored in aliquots at −80°C.
Project description:The plasticity of cancer stem cells (CSCs)/tumor-initiating cells (T-ICs) indicates that multiple CSC/T-IC subpopulations exist within a tumor and multiple oncogenic pathways collaborate to maintain the CSC/T-IC state. Here, we aimed to enrich for T-ICs from clinical ESCC tissues. A chemoresistant human esophageal squamous cell carcinoma (ESCC) patient-derived xenograft model was employed to identify miRNA(s) that contribute to ESCC aggressiveness. We used microarrays to demenstrate the microRNA expression underlying different pretreated conditions. NOG mice bearing subcutaneous tumor xenografts derived from clinical ESCC cells were intraperitoneally treated with CDDP or PBS twice weekly for three weeks. Tumor cells were then isolated and re-inoculated subcutaneously into NOG mice for the next round of treatment. In the 4th round of treatment, the volume of tumors in both CDDP- and PBS-treated groups were approximately the same, suggesting that the cells in CDDP-treated tumors were becoming resistant to CDDP. microRNA expression was measured by RNA extraction from cisplatin-treated tumors (EC-CR) or PBS-treated tumors(EC-UT) after 4 round of treatment,two microarrays were performed for each sample.
Project description:Tumor-associated macrophages (TAMs) are known to be involved in progression, angiogenesis and motility of various cancers. We have previously reported the association between increased number of infiltrating TAMs with tumor progression and poor prognosis in esophageal squamous cell carcinomas (ESCCs). To study roles of TAMs in ESCC, we first exposed peripheral blood monocytes (PBMo) derived macrophages from healthy volunteers to conditioned media of TE series human ESCC cell line (TECM) and confirmed the induction of expression of M2 macrophage marker, CD204, and protumorigenic factors, interleukin (IL)-10, VEGFA and MMPs. Next, we compared gene expression profile between PBMo-derived macrophages and PBMo-derived macrophages stimulated with TECM by cDNA microarray. Based on the result, we focused on growth differentiation factor 15 (GDF15) among highly expressed genes including IL-6, IL-8 and CXCL1. Immunohistochemical study on 70 cases of surgically resected ESCCs revealed that GDF15 was detected not only in macrophages but also in cancer cells. High expression of GDF15 in ESCCs was significantly correlated with more malignant phenotypes including lymph and blood vessel invasion, lymph node metastasis as well as clinical stages. Patients with high expression of GDF15 showed poor disease-free survival (p = 0.011) and overall survival (p = 0.041). Furthermore, we found that recombinant human GDF15 promote cell proliferation and phosphorylation of both Akt and Erk1/2 in ESCC cell lines in vitro. These results indicate that GDF15 is secreted by both TAMs and cancer cells in tumor microenvironment and is associated with aberrant growth and poor prognosis of human ESCC. We exposed PBMo-derived macrophages from healthy volunteers to TECM and observed their acquisition of TAM-like characters in this study. We further investigated specific cancer-associated gene expression profile in TECM induced TAM-like macrophages by cDNA microarray analysis.
Project description:We showed that co-culture with TAMs triggered Bmi1 expression in gastrointestinal cancer cell lines. miRNAs have been found to target various oncogenes and tumor suppressors. We therefore hypothesized that the regulation of Bmi1 expression in gastrointestinal cancer cells may be mediated by miRNAs using miRNA microarray analysis. THP-1 cells were seeded in the transwell inserts (3540, Corning) for 6-well plates (1 × 106 cells/well). For preparation of M1-polarized THP-1 macrophages, 320 nM phorbol myristate acetate (PMA) was added to THP-1 cells for 6 h, followed by PMA plus 20 ng/ml interferon (IFN)-γ and 100 ng/ml lipopolysaccharide for the following 18 h. For preparation of M2-polarized THP-1 macrophages, 320 nM PMA was added to THP-1 cells for 6 h, followed by PMA plus 20 ng/ml interleukin (IL)-4/IL-13 for the following 18 h. After three washes to remove cytokines, M1- or M2-polarized THP-1 macrophages were co-cultured in upper inserts with AGS cells in 6-well plates (1 × 105 cells/well) without direct contact, in each medium without 10% FBS as described above. After 24 h of co-culture, the upper inserts containing macrophages were discarded. AGS cells were collected and analyzed to identify downregulated microRNA in a gastric cancer cell line co-cultured with M1- or M2-polarized macrophage.
Project description:One of the greatest obstacles to eliminating TB is the lack of predictive and diagnostic biomarkers. Exosomes are a promising alternative source of biomarkers for TB since they can carry mycobacterial antigens. We hypothesize that exosomes from Mtb-infected macrophages exhibit a characteristic selection of human protein that can be evaluated as TB biomarkers. We looked for the localization—exosomal membrane or lumen—of the differentially abundant proteins. Exosomes were obtained from THP-1-derived macrophages (Mtb infected and uninfected controls). The exosome population was validated by nanoparticle tracking analysis and western blot. The protein composition of exosomes was evaluated by tandem mass spectrometry. Differences in protein composition and abundances between exosomes from infected and control cells were evaluated by t-test the proteomics findings were confirmed by western blot. Using a biotinylation strategy we verified the protein localization. Forty-seven proteins were significantly more abundant in exosomes from Mtb-infected cells, 66% were predicted to be membrane associated. The biotinylation pattern confirmed the membrane-association of some of these proteins.
Project description:Tumor-immune cell interactions shape the immune cell phenotype, with microRNAs (miRs) being crucial components of this crosstalk. How they are transferred, and how they affect their target landscape, especially in tumor-associated macrophages (TAMs), is largely unknown. Here we aimed to define miRome of TAMs and identify novel miRs those are deferentially expression in TAMs and decipher their functional relevance in disease settings.
Project description:Neutrophil gelatinase associated lipocalin (NGAL), also known as oncogene 24p3, uterocalin, siderocalin or lipocalin 2 (Lcn2), is a member of the lipocalin family. Elevated NGAL involves various functions in malignant cells, even sometimes the conclusions were controversial. NGAL inhibits apoptosis in thyroid cancer and decrease invasion and angiogenesis in pancreatic cancer, but it increase proliferation and metastasis in breast and colon cancer. Nonetheless, the molecular functions and mechanisms of NGAL in ESCC are unknown. To address these issues and to identify genes and biological pathways associated with NGAL functions and mechanism, NGAL was overexpressed by pcDNA3.0 plasmid in ESCC cell line EC109 with an empty plasmid as a control, these two cells were applied for mRNA expression profile analyses to find the differentially expressed genes. Plasmids of pcDNA3.0-NGAL and pcDNA3.0 were transfected into ESCC cell line EC109, respectively. Stable transfected cell clones were selected by G418. The overexpression of NGAL protein was confirmed by western blot. Total RNAs from NGAL overexpressed cells and control cells were extracted for Agilent whole genome oligo microarray. A dye flip was performed for duplicate.
Project description:To identify candidate genes involved in enhanced tumorigenicity and metastasis of CD90+ esophageal tumor-initiating cells. The esophageal squamous carcinoma cell (ESCC) cell line KYSE-140 was sorted into CD90+ and CD90- populations by flow cytometry. Total RNA was analyzed on Affymetrix Human Genome U133 Plus GeneChip 2.0 arrays.
Project description:Lipid laden macrophages (LLM) can often be found in the airway and may indicate aspiration secondary to gastro-oesophageal reflux (GOR). GOR is often associated chronic inflammatory airways diseases. We therefore sought to determine whether lipid droplets from undigested or partially digested food had an effect on macrophage gene expression leading to bronchial inflammation. To test this, we generated an in vitro model using differentiated THP-1 cells which were treated with a high fat liquid feed. Gene expression, using the Agilent G4851C (v3) array, in phorbol myristate acetate (PMA) differentiated THP-1 cells was compared to undifferentiated cells and lipid treated differentiated THP-1 cells.
Project description:This SuperSeries is composed of the following subset Series: GSE32141: Expression analysis LPS stimulated THP-1 cells in four paired samples GSE32324: ChIP-seq analysis LPS stimulated THP-1 cells Refer to individual Series
Project description:Transcriptional changes during early infection of macrophage-like THP-1 cell line with pathogenic bacterium Mycobacterium tuberculosis. RNAseq samples were taken at 0h (THP-1 cells growing in the RPMI medium), and after 4h, 24h and 48h post infection. Bacterial enrichment was performed to increase the amount of bacterial mRNA in the samples. Non-enriched samples were used to map THP-1 cells transcripts; enriched samples were used to map M. tuberculosis transcripts the corresponding genomes.