Chemerin-conditioned medium increases cell adhesion, proliferation and migration.

<p><i>A</i>. Increased adhesion of MSCs (1x10⁵) after treatment with chemerin (Ch) for 30 min. <i>B</i>. Conditioned medium (CM) from MSCs treated with chemerin increased adhesion of normal gastric myofibroblasts and addition of the ChemR23 antagonist CCX832 only slightly reduced the response. <i>C</i>. CM from MSCs treated with chemerin stimulated migration of myofibroblasts in Boyden chambers and addition of the ChemR23 antagonist CCX832 only slightly reduced the response. <i>D</i>. CM from MSCs treated with chemerin stimulated proliferation of myofibroblasts and addition of the ChemR23 antagonist CCX832 only slightly reduced the response. Means ± SE, n = 3; horizontal arrows, p<0.05.</p

Western blot validation of proteomic studies of the MSC secretome.

<p>MSCs were treated with chemerin or IGF-II and media or cell extracts probed by western blot for proteins identified by SILAC. Six proteins that exhibited increased secreted in proteomic studies were also increased in western blots response to IGF (MMP-2, TGFβig-h3, MIF, IGFBP-7, decorin and lumican) and all except one (IGFBP-7) were also stimulated by chemerin. SPARC was not identified as exhibiting stimulated exocytosis in proteomic studies and neither did it respond to IGF-II or chemerin in western blot studies. Cellular content of SPARC and GAPDH was not influenced by IGF-II or chemerin.</p

Enriched molecular functions, protein classes and biological processes in the MSC secretome.

<p><i>A</i>, The main molecular functions shown by PANTHER for 62 proteins in the MSC secretome identified as “secreted” and as exhibiting increased abundance after IGF-II treatment. <i>B</i>. Enriched protein classes, <i>C</i>. Enriched biological processes. P, probability.</p

Localisation of proteins in secretory vesicles.

<p><i>A</i>. localisation of TGFβig-h3 (green) and MMP2, MIF or SPACR (red) in MSCs revealed by immunocytochemistry. In each case there are images for the red channel, green channel, a marge of these two images, and finally a merged image at higher magnification. There are clear examples of TGFβig-h3 and either MIF or MMP-2 in similar vesicular populations (yellow or orange), while TGFβig-h3 and SPARC are localised to distinct vesicular structures (red or green vesicular structures). Nuclei stained blue with DAPI. Scale bars 5 or 10 μm. <i>B</i>. TEM identifies dense core secretory vesicles in MSCs. representative photomicrograph taken at a magnification of 60,000x show dense core secretory vesicles (closed arrows) in MSCs the Golgi apparatus is clearly visible (open arrows). Scale bar 500nm.</p

Secretion of TGFβig-h3 is stimulated by chemerin and IGF.

<p><i>A</i>. Western blot analysis of TGFβig-h3 in media from MSC cells shows stimulation by chemerin and IGF-II and inhibition by CCX832 and AG1042, respectively. <i>B</i>. Stimulated secretion of TGFβig-h3 is maintained after cycloheximide treatment. TGFβig-h3 abundance in cell extracts was unchanged (middle panel); GAPDH was used as a loading control for the cell extracts (bottom panel). <i>C</i>. The calcium ionophore, ionomycin (1μM) stimulated TGFβig-h3 secretion comparable to IGF-I (50ng.ml^-1) and IGF-II (100ng.ml^-1). <i>D</i>. In calcium-free medium stimulated secretion in response to IGF-II is inhibited.</p

Mapping Proteolytic Processing in the Secretome of Gastric Cancer-Associated Myofibroblasts Reveals Activation of MMP-1, MMP-2, and MMP‑3

Cancer progression involves changes in extracellular proteolysis, but the contribution of stromal cell secretomes to the cancer degradome remains uncertain. We have now defined the secretome of a specific stromal cell type, the myofibroblast, in gastric cancer and its modification by proteolysis. SILAC labeling and COFRADIC isolation of methionine containing peptides allowed us to quantify differences in gastric cancer-derived myofibroblasts compared with myofibroblasts from adjacent tissue, revealing increased abundance of several proteases in cancer myofibroblasts including matrix metalloproteinases (MMP)-1 and -3. Moreover, N-terminal COFRADIC analysis identified cancer-restricted proteolytic cleavages, including liberation of the active forms of MMP-1, -2, and -3 from their inactive precursors. In vivo imaging confirmed increased MMP activity when gastric cancer cells were xenografted in mice together with gastric cancer myofibroblasts. Western blot and enzyme activity assays confirmed increased MMP-1, -2, and -3 activity in cancer myofibroblasts, and cancer cell migration assays indicated stimulation by MMP-1, -2, and -3 in cancer-associated myofibroblast media. Thus, cancer-derived myofibroblasts differ from their normal counterparts by increased production and activation of MMP-1, -2, and -3, and this may contribute to the remodelling of the cancer cell microenvironment

Mapping Proteolytic Processing in the Secretome of Gastric Cancer-Associated Myofibroblasts Reveals Activation of MMP-1, MMP-2, and MMP‑3

Cancer progression involves changes in extracellular proteolysis, but the contribution of stromal cell secretomes to the cancer degradome remains uncertain. We have now defined the secretome of a specific stromal cell type, the myofibroblast, in gastric cancer and its modification by proteolysis. SILAC labeling and COFRADIC isolation of methionine containing peptides allowed us to quantify differences in gastric cancer-derived myofibroblasts compared with myofibroblasts from adjacent tissue, revealing increased abundance of several proteases in cancer myofibroblasts including matrix metalloproteinases (MMP)-1 and -3. Moreover, N-terminal COFRADIC analysis identified cancer-restricted proteolytic cleavages, including liberation of the active forms of MMP-1, -2, and -3 from their inactive precursors. In vivo imaging confirmed increased MMP activity when gastric cancer cells were xenografted in mice together with gastric cancer myofibroblasts. Western blot and enzyme activity assays confirmed increased MMP-1, -2, and -3 activity in cancer myofibroblasts, and cancer cell migration assays indicated stimulation by MMP-1, -2, and -3 in cancer-associated myofibroblast media. Thus, cancer-derived myofibroblasts differ from their normal counterparts by increased production and activation of MMP-1, -2, and -3, and this may contribute to the remodelling of the cancer cell microenvironment

Chemerin stimulates transendothelial migration of MSCs and requires MMP-2.

<p><i>A</i>, Representative fields from MSC transendothelial migration experiments showing migration of PKH67-labelled MSCs (left). CCX832 (1 µM) inhibited chemerin- (center) and CAM-CM stimulated MSC transendothelial migration but CCX826 (1 µM) had no effect (right). <i>B</i>, Chemerin, and IGF-II used as a positive control, promptly (30 min) stimulated proMMP2 abundance in media as detected by Western blot but had no effect on cellular proMMP2 abundance (left); chemerin significantly increased MMP-2 enzyme activity in MSC media detected by the selective substrate MCA-Pro-Leu-Ala-Nva-Dpa-Ala-Arg-NH₂ (right). <i>C</i>, Human recombinant MMP-2 (80 ng/ml) stimulated transendothelial migration and there was dose-dependent inhibition by an MMP-2 selective inhibitor (MMP-2 inhibitor I) (left). The MMP-2 inhibitor (60 µM) significantly inhibited chemerin-stimulated MSC transendothelial migration (centre). Horizontal arrows, p<0.05, t- test (n = 3).</p

Increased MSC homing to xenografts seeded with CAMs and inhibition of homing by the chemR23 receptor antagonist, CCX832.

<p><i>A</i>, Visualisation of PKH67-labelled MSCs in representative fields from xenografts established with OE21 cancer cells alone or co-injected with CAMs followed by treatment with vehicle (top) or CCX832 (bottom) and iv injection of PKH67-labelled MSCs. <i>B</i>, In xenografts with OE21 cancer cells and CAMs there was increased MSC homing expressed as labelled cells per unit area of xenograft compared with xenografts of OE21 cancer cell alone; treatment with CCX832 inhibited homing (OE21/vehicle, n = 3; OE21/CCX832, n = 4; OE21 and CAMs/vehicle, n = 6; OE21 and CAMs/CCX832, n = 6). Horizontal arrows, p<0.05, ANOVA.</p

ChemR23 mediates chemerin stimulation of MSC migration via PKC and MAP kinases.

<p><i>A</i>, Representative images from MSCs stained for vimentin (positive control) and chemR23 revealing knock-down (KD) after ChemR23 siRNA treatment (left). Knockdown of ChemR23, but not GPR1, inhibited MSC migration in response to chemerin (100 ng/ml)(center) and CAM-CM (right). <i>B</i>, Concentration-dependent inhibition of MSC migration in response to chemerin by the ChemR23 antagonist CCX832 (left) but not the control compound CCX826 (1 µM) (center). MSC migration in response to CAM-CM was inhibited similarly by chemerin neutralising antibody, and CCX832, but not CCX826 (1 µM)(right). <i>C</i>, Representative Western blot shows increased phosphorylation of p42/44, p38 and JNK-II kinases in MSCs treated with chemerin (100 ng/ml)(left). In Boyden chamber assays, chemerin-stimulated MSC migration was inhibited by the JNK-II inhibitor, SP600125 (50 µM), the p42/44 inhibitor, UO126 (10 µM), p38 inhibitor SB202190 (3 µM), and the PKC inhibitor Ro320432 (2 µM) but not by PIK3 inhibitor LY294002 (50 µM) (right). Horizontal arrows, p<0.05, ANOVA (n = 3 in each case).</p