Fig 2: Macrophages secrete higher levels of cytokines in interaction with C1GalT1 suppressed tumour cells than with shCon cells.Macrophage interaction with SW620shC1GT and HCT116F3shC1GT tumour cells leads to higher macrophage secretion of IL-6 and IL-10 in comparison to interaction with shCon cells. SW620 (a) or HCT116F3 (b) cells were introduced to M1 (a, b) or M2 (c, d) macrophages for 90 min and the concentrations of IL-6 (a, b) or IL-10 (c and d) in the culture medium were analysed by ELISAs. Data are expressed as mean ± SEM of three independent experiment, each in triplicate. e, f, shC1alT1 tumour cells show reduced viability than shCon cells after interaction with M1 macrophages. M1 macrophages were incubated with SW620 (e) or HCT116F3 (f) cells for 24 h before cell proliferation as well as cell viability were separately analysed. Data are expressed as mean ± SEM of thee independent experiments, each in triplicate. Percentage cell viability/proliferation of shC1GT cells in compassion to shCon cells are shown in (e) and (f). *p < 0.05, **p < 0.01, ***p < 0.001 (ANOVA, followed by Bonferroni). g More macrophage-tumour phagocytosis is seen in M1 macrophage (DiO labelling, green) interaction with shC1GT cells (DiI labelling, red) than with shCon cancer cells (DiI labelling, red). Representative images are shown (arrows point to phagocytotic events). h Number of phagocytotic events in five randomly selected FOVs were quantified and data are shown as mean ± SEM from three independent experiment, *p < 0.05 (T test).

Fig 3: C1GalT suppression reduces galectin-3 binding but increases MGL binding to human colon cancer cells.Binding of galectin-3 (Gal3) (a, c) and MGL (b, d) to SW620shCon and SW620shC1GT cells (a, b) and HCT116F3shCon and HCT116F3shC1GT (c, d) was assessed by flow cytometry using biotinylated galectin-3 or MGL followed by anti-MGL antibody and fluorescence-conjugated secondary antibody. Representative flow cytometry histograms are shown. Percentage changes of the mean fluorescent intensity (MFI) (% of shCon cells), shown in the right on each histogram, are expressed as mean ± SD from three independent experiment. *p < 0.05, **p < 0.01, ***p < 0.001 (T-test).

Fig 4: C1GalT suppression reduces tumour growth in chicken embryos and more macrophages and dendritic cells are accumulated in the tumour microenvironment of ME C1galt1-/-/Erb mice than of C1galt1f/f/Erb mice.GFP-SW620shC1GT and GFP-SW620shCon cells (1.8 × 106 cells) without or with 5 µg galectin-3 was implanted in the CAMs at Day 7 and tumour sizes were measured at Day 14 of embryonation. Representative images of the tumours grown on CAM at Day 14 are shown in (a). Tumour volume are shown in (b) (number of eggs with detectable tumour/total number of eggs at Day 14 are also shown on top of each bar). Data are expressed as mean ± SD, *p < 0.05, **p < 0.01 (T test). c, d Fewer macrophages and dendritic cells were seen in the tumour microenvironment of C1galt1f/f/Erb mice than in ME C1galt1-/-/Erb mice. Lower expression of TF and higher expression of Tn were found in the tumours of ME C1galt1-/-/Erb mice compared with C1galt1f/f/Erb mice. Higher number of macrophages (F4/80 positive, MGL-positive) and dendritic cells (CD11c positive, MGL positive) are seen in the tumours of ME C1galt1-/-/Erb mice compared with C1galt1f/f/Erb mice. Two mice in each group were randomly selected and analyzed and representative images are shown.

Fig 5: C1GalT suppression increases MGL-mediated macrophage-tumour cell interaction.Equal numbers (5 × 105 cells/ml) of Dil-labelled cancer cells and DiO-labelled macrophages were mixed in the absence or presence of anti-MGL antibody (20 µg/ml) for 40 min before analysis by flow cytometry. The top right (blue) in each of the bivariate correlation plots that contain both DiO-labelled macrophages and Dil-labelled cancer cells are defined as tumour-macrophage aggregates/interaction. Representative flow cytometry plots from two independent experiments are shown in (a) (numbers in the correlation plots show percentage cell population). Percentage changes of M1 and M2 macrophages-tumour cell aggregates of shC1alT1 cells, without or with anti-MGL antibody, in comparison to shCon cells are shown in (b) (SW620) and (c) (HCT116F3). Data are expressed as mean ± SD from three independent experiments. *p < 0.05 (T test).