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

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

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

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

Cysteine-Rich Protein 61 Plays a Proinflammatory Role in Obstructive Kidney Fibrosis

<div><p>Cysteine-rich protein 61 (Cyr61) is a secreted matrix-associated protein that regulates a broad spectrum of biological and cellular activities. This study aimed to investigate the role of Cyr61 in progressive kidney fibrosis induced by unilateral ureteral obstruction (UUO) surgery in mice. The expression of Cyr61 transcripts and proteins in the obstructed kidneys were increased from day 1 and remained high until day 10 after surgery. Immunohistochemistry indicated that Cyr61 was expressed mainly in renal tubular epithelial cells. The upregulated Cyr61 in UUO kidneys was reduced in mice treated with pan-transforming growth factor-β (TGF-β) antibody. The role of TGF-β in tubular Cyr61 upregulation after obstructive kidney injury was further supported by experiments showing that TGF-β1 stimulated Cyr61 expression in cultured tubular epithelial cells. Notably, the upregulation of Cyr61 in UUO kidneys was followed by a marked increase in monocyte chemoattractant protein 1 (<em>MCP-1</em>) transcripts and macrophage infiltration, which were attenuated in mice treated with anti-Cyr61 antibodies. This proinflammatory property of Cyr61 in inducing <em>MCP-1</em> expression was further confirmed in tubular epithelial cells cultured with Cyr61 protein. The anti-Cyr61 antibody in UUO mice also reduced the levels of collagen type 1-α1 transcripts, collagen fibril accumulation evaluated by picrosirius red staining, and the levels of α-smooth muscle actin (<em>α-SMA</em>) transcripts and proteins on day 4 after surgery; however, the antifibrotic effect was not sustained. In conclusion, the TGF-β-mediated increase in tubular Cyr61 expression involved renal inflammatory cell infiltration through MCP-1 induction during obstructive kidney injury. The Cyr61 blockade attenuated kidney fibrosis in the early phase, but the antifibrotic effect could not be sustained.</p> </div

Primer sequences used for quantitative-PCR.

<p>Primer sequences used for quantitative-PCR.</p

Cyr61 expression in cultured renal tubular epithelial cells.

<p>(<b>A</b>) Cultured rat proximal tubular epithelial cells (NRK-52E) were treated with various concentrations of recombinant TGF-β1 for 2 hours. Q-PCR showed <i>Cyr61</i> upregulation by TGF-β1 at doses of 0.25 ng/mL or greater. N = 4/group. (<b>B</b>) Recombinant Cyr61 was added to NRK-52E cells for 2 or 6 hours. Q-PCR showed increased expression of <i>MCP-1</i> transcripts by Cyr61 in a dose-dependent manner. N = 4/group. The values are the mean+SD. *P<0.05 vs. control.</p

Effect of Cyr61 blockade on renal fibrosis in UUO kidneys.

<p>The mice received an intraperitoneal injection of 10 µg/g BW of the control rabbit IgG (filled bar) or anti-Cyr61 antibody (open bar) 2 hours before the UUO surgery and then one dose per day until euthanasia. (<b>A–B</b>) Q-PCR showing renal <i>Col 1-α1</i> (A) and α-smooth muscle actin (<i>α-SMA</i>) (B) transcripts after UUO. N = 8/group. (<b>C</b>) Representative images of picrosirius red staining for interstitial fibrillar collagen (red) in the UUO kidneys (magnification 200×, scale bar = 100 µm). The bar charts are a summary of the picrosirius red-stained percentage in the field. N = 8/group. (<b>D</b>) Representative images of Western blots of renal α-SMA protein expression in the UUO kidneys. (<b>E–F</b>) Q-PCR showing no difference in renal <i>CTGF</i> (E) and <i>TGF-β1</i> (F) transcripts between the 2 groups. N = 8/group. All of the Q-PCR data are expressed as the fold differences compared to the sham operation kidneys (gray bar). The values are the mean+SD. *P<0.05 vs. sham operation; #P<0.05 vs. control IgG.</p