Fig 3

M2 macrophage-secreted CHI3L1 mediates cancer cell metastasis. a Screening for different proteins secreted by M1 and M2 macrophages. The secreted proteins were separated using SDS-PAGE and were stained with Coomassie Blue, after which they were identified by mass spectrometry. The arrow indicates the protein identified using mass spectrometry. b The expression of CHI3L1 in M2 macrophages derived from the peripheral blood monocytes of healthy humans. The peripheral blood monocytes were induced to differentiate into M1 and M2 macrophages. Then monocyte-derived M1 and M2 macrophages or their culture supernatants were subjected to western blotting analysis using anti-CHI3L1 IgG. c Effect of recombinant CHI3L1 protein on gastric and breast cancer cell migration in Boyden chamber assays. Gastric cancer cells (MKN-45 and AGS), breast cancer cells (MDA-MB-231, MDA-MB-435, and MDA-MB-468), or melanoma cells (A375) were cultured with the recombinant CHI3L1 protein at different concentrations. 12 h later, the cancer cells were examined by phase-contrast microscopy. Scalebar, 100 μm. d Roles of recombinant CHI3L1 protein in gastric and breast cancer cell adhesion. Gastric cancer cells (MKN-45 and AGS) or breast cancer cells (MDA-MB-231, MDA-MB-435, and MDA-MB-468) were suspended in serum-free medium and were allowed to adhere to a culture plate coated with fibronectin in the presence of recombinant CHI3L1 protein. After 30 min of incubation, the cells were analyzed using the adhesion assay. Scalebar, 100 μm. e Effects of recombinant CHI3L1 protein on gastric and breast cancer cell invasion. The invasive capacities of gastric cancer cells (MKN-45 and AGS) or breast cancer cells (MDA-MB-231, MDA-MB-435, and MDA-MB-468) in the presence of the CHI3L1 protein were assessed. The cells were examined after 24 h of treatment. Scalebar, 100 μm. f The effects of anti-CHI3L1 neutralization on gastric or breast cancer cell migration in Boyden chamber assays. Gastric cancer cells (MKN-45 and AGS) or breast cancer cells (MDA-MB-231, MDA-MB-435, and MDA-MB-468) were co-cultured with the peripheral blood monocytes-derived M2 macrophages of healthy humans in the presence or absence of anti-CHI3L1 antibody; IgG was used as the control. 12 h later, the cancer cells were examined by phase-contrast microscopy. Scalebar, 100 μm. g The influence of neutralizing CHI3L1 protein on gastric or breast cancer cell adhesion. The adhesion capacity of cancer cells in the presence of the peripheral blood monocytes-derived M2 macrophages and anti-CHI3L1 was evaluated. Scalebar, 100 μm. h Evaluation of the role of anti-CHI3L1 neutralization in gastric or breast cancer cell invasion. Cancer cells and isolated M2 macrophages were co-cultured and were then subjected to the invasion assay in the presence or absence of the anti-CHI3L1 antibody. Scalebar, 100 μm. i CHI3L1 gene expression silencing in isolated M2 macrophages from the peripheral blood monocytes of healthy humans. Isolated M2 macrophages were transfected with CHI3L1-siRNA or CHI3L1-siRNA-scrambled. At 24 h after transfection, the CHI3L1 protein level was determined using western blotting. β-actin served as a loading control. j Effects of CHI3L1 silencing in isolated M2 macrophages on gastric and breast cancer cell migration. The CHI3L1-silenced isolated M2 macrophages were co-cultured with cancer cells and subjected to cell migration assays. Scalebar, 100 μm. k The impact of CHI3L1 silencing on gastric and breast cancer cell adhesion. The CHI3L1 gene expression was knocked down in the isolated M2 macrophages. 24 h later, the M2 macrophages were co-cultured with cancer cells, and the adhesion capacities of cancer cells were examined. Scalebar, 100 μm. l The influence of CHI3L1 silencing on gastric and breast cancer cell invasion. Cancer cells were co-cultured with the CHI3L1-silenced isolated M2 macrophages and then cell invasion assays were conducted. Scalebar, 100 μm. m Immunohistochemical staining of CHI3L1 protein (up) and immunofluorescent double staining of CD206 (green) and CHI3L1 (red) (down) in solid tumors and pericarcinous tissues from patients with gastric cancer. In immunohistochemical staining, the arrows indicate cancer cells, and the triangles indicate CHI3L1 protein. Scalebar, 20 μm. In immunofluorescent double staining, the arrows show double-positive macrophages. Scalebar, 10 μm. In all the panels, the significant differences are indicated by asterisks (*p < 0.05, **p < 0.01)