Macrophage Infiltration Induces Gastric Cancer Invasiveness by Activating the β-Catenin Pathway - Fig 4

<p>(A) Migration activity of THP-1 monocyte co-cultured with different gastric cancer cell lines (AGS, N87, MKN-45 and TSGH). THP-1 monocyte will be recruited by different gastric cancer cell lines and the recruiting ability is dependent on the degree of malignancy. (B) Invasion ability of GC cells treated by macrophage CM. Four different types of GC cells (AGS, MKN45, N87 and TSGH) were seeded in a Boyden chamber and co-cultured with or without macrophage cells or macrophage CM for 24 hours. Invasion abilities of each cell line were measured <i>in vitro</i> for 24 hours. All data represent the arithmetic mean ± SEM. * p < 0.05, ** p < 0.01. (C) Effect of macrophage CM co-cultured with cells. N87 and AGS cells were co-cultured with macrophage CM respectively for 24 hours and cell viability was determined by MTT assay.</p

Immunohistochemistry analysis.

<p>Immunostaining against CD68 was performed on tissue slides from gastric cancer patients. CD68+ macrophage staining was sparse in the normal gastric mucosa (A). CD68+ cells were detected in both the stroma and tumor nest (B & C). The density of CD68+ macrophages in pathological tumor staging (pT) 1 (D) and pT4 (E) tumor lesions.</p

Effect of macrophage CM on β-catenin pathway.

<p>(A) β-catenin accumulates in nucleus after treatment with macrophage CM for 30 minutes in N87 cells. (B) Immunofiuorescent images were observed with a confocal microscope. β-catenin positive cells were analyzed by using an anti-β-catenin antibody, which is recognized by secondary rabbit antibody conjugated with FITC, depicted by green fluorescence; Nuclear staining was detected by counterstaining cells with 4', 6-Diamidino-2-phenylindole (DAPI), represented as blue fluorescence. Co-culturing with macrophage CM, immunofiuorescence staining showed β-catenin-FITC complexes translocated into nucleus. (C) Invasion ability of GC cells treated by macrophage CM.</p

Immunohistochemistry analysis.

<p>β-catenin was highly expressed in tumors. (A) In most cases, β-catenin expression was primarily located in the cytoplasm. However, (B) strong nuclear staining of β-catenin in GC cells was positively associated with CD68+ macrophages in the tumor lesions.</p

Kaplan-Meier overall survival curves for gastric cancer patients as TAM density (Low: TAM<671, blue line; High: TAM>671, brown line; <i>p</i> = 0.0073).

<p>Kaplan-Meier overall survival curves for gastric cancer patients as TAM density (Low: TAM<671, blue line; High: TAM>671, brown line; <i>p</i> = 0.0073).</p

Macrophage Infiltration Induces Gastric Cancer Invasiveness by Activating the β-Catenin Pathway - Fig 6

<p>(A) Effect of macrophage CM on mRNA and protein expression of β-catenin down-stream genes. N87 cells were cultured in the presence or absence of macrophage CM. For RNA and protein level, cells were collected after 6 hours and 24 hour treatment respectively. (B) N87 cells with or without knock-down of β-catenin utilizing shRNA-2 were co-treated in the presence or absence of macrophage CM. N87 cells were collected after 24 hours treatment and analyzed with western blot. (C) Effect of MMP7 neutralized antibody on GC cells invasion. N87 cells in the presence of macrophage CM for 24 hours were pre-treated with various doses MMP7 neutralized antibody (0, 0.625, 1.25 and 2.5μg/ml) for 1 hour Isotype IgG was used as a blocking control. (D) Effect of CD44 neutralized antibody on GC cell invasion. N87 cells in the presence of macrophage CM for 24 hours were pre-treated with various doses CD44 neutralized antibody (0, 1.25, 2.5, 5 and 10 μg/ml) for 1 hour. Isotype IgG was used as a blocking control. All data represent the arithmetic mean ± SEM. * <i>p</i> < 0.05.</p

Associations of <i>VEGF-C</i> Genetic Polymorphisms with Urothelial Cell Carcinoma Susceptibility Differ between Smokers and Non-Smokers in Taiwan

<div><p>Background</p><p>Vascular endothelial growth factor (VEGF)-C is associated with lymphangiogenesis, pelvic regional lymph node metastasis, and an antiapoptotic phenotype in urothelial cell carcinoma (UCC). Knowledge of potential roles of <i>VEGF-C</i> genetic polymorphisms in susceptibility to UCC is lacking. This study was designed to examine associations between <i>VEGF-C</i> gene variants and UCC susceptibility and evaluate whether they are modified by smoking.</p><p>Methodology/Principal Findings</p><p>Five single-nucleotide polymorphisms (SNPs) of <i>VEGF-C</i> were analyzed by a TaqMan-based real-time polymerase chain reaction (PCR) in 233 patients with UCC and 520 cancer-free controls. A multivariate logistic regression was applied to model associations between genetic polymorphisms and UCC susceptibility, and to determine if the effect was modified by smoking. We found that after adjusting for other covariates, individuals within the entire population and the 476 non-smokers carrying at least one A allele at <i>VEGF-C</i> rs1485766 respectively had 1.742- and 1.834-fold risks of developing UCC than did wild-type (CC) carriers. Among the 277 smokers, we found that <i>VEGF-C</i> rs7664413 T (CT+TT) and rs2046463 G (AG+GG) allelic carriers were more prevalent in UCC patients than in non-cancer participants. Moreover, UCC patients with the smoking habit who had at least one T allele of <i>VEGF-C</i> rs7664413 were at higher risk of developing larger tumor sizes (<i>p</i> = 0.021), compared to those patients with CC homozygotes.</p><p>Conclusions</p><p>Our results suggest that the involvement of <i>VEGF-C</i> genotypes in UCC risk differs among smokers compared to non-smokers among Taiwanese. The genetic polymorphism of <i>VEGF-C</i> rs7664413 might be a predictive factor for the tumor size of UCC patients who have a smoking habit.</p></div

Adjusted odds ratios (ORs, AORs) and 95% confidence intervals (CIs) of clinical statuses associated with genotypic frequencies of MTNR1A rs13140012 in oral cancer among 478 betel quid chewers.

<p>AORs with their 95% CIs were estimated by multiple logistic regression models, after controlling for gender, age, and alcohol and tobacco consumption.</p><p>> T2: multiple tumors more than 2 cm.</p><p>Cell differentiated grade: grade I: well differentiated; grade II: moderately differentiated; grade III: poorly differentiated.</p><p>* <i>p</i> < 0.05, statistically significant.</p><p>Adjusted odds ratios (ORs, AORs) and 95% confidence intervals (CIs) of clinical statuses associated with genotypic frequencies of MTNR1A rs13140012 in oral cancer among 478 betel quid chewers.</p

Additional file 1: Table S1. of The Gαh-PLCδ1 signaling axis drives metastatic progression in triple-negative breast cancer

The paired primers used in the study. Table S2. Cox univariate analysis of disease-free survival for the protein of cytosol Gαh [Gαh (C)], extracellular Gαh [Gαh (E)] and PLC-δ1 and the stage of pathologic T and N. Table S3. Cox multivariate analysis of disease-free survival for the protein of Gαh (C) and Gαh (E) and the stage of pathologic T and N. Table S4. Cox multivariate analysis of disease-free survival for the protein of Gαh (C) and PLC-δ1 and the stage of pathologic T and N. Figure S1. Clinical relevance of Gαh and PLC-δ1 in breast cancer patients. (DOCX 223 kb

Distributions of demographical characteristics in 560 controls and 618 patients with oral cancer.

<p>* <i>p</i> < 0.05, statistically significant.</p><p>Distributions of demographical characteristics in 560 controls and 618 patients with oral cancer.</p