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. 2023 Aug 23;14(8):547.
doi: 10.1038/s41419-023-06082-7.

C1GalT1 expression reciprocally controls tumour cell-cell and tumour-macrophage interactions mediated by galectin-3 and MGL with double impact on cancer development and progression

Yangu Wan  1 , Kareena Adair  2 , Anne Herrmann  3 , Xindi Shan  4 , Lijun Xia  4 , Carrie A Duckworth  5 , Lu-Gang Yu  6
Affiliations

C1GalT1 expression reciprocally controls tumour cell-cell and tumour-macrophage interactions mediated by galectin-3 and MGL with double impact on cancer development and progression

Yangu Wan et al. Cell Death Dis. .

Abstract

Although most cell membrane proteins are modified by glycosylation, our understanding of the role and actions of protein glycosylation is still very limited. β1,3galactosyltransferase (C1GalT1) is a key glycosyltransferase that controls the biosynthesis of the Core 1 structure of O-linked mucin type glycans and is overexpressed by many common types of epithelial cancers. This study reports that suppression of C1GalT1 expression in human colon cancer cells caused substantial changes of protein glycosylation of cell membrane proteins, many of which were ligands of the galactoside-binding galectin-3 and the macrophage galactose-type lectin (MGL). This led to significant reduction of cancer cell proliferation, adhesion, migration and the ability of tumour cells to form colonies. Crucially, C1GalT1 suppression significantly reduced galectin-3-mediated tumour cell-cell interaction and galectin-3-promoted tumour cell activities. In the meantime, C1GalT1 suppression substantially increased MGL-mediated macrophage-tumour cell interaction and macrophage-tumour cell phagocytosis and cytokine secretion. C1GalT1-expressing cancer cells implanted in chick embryos resulted in the formation of significantly bigger tumours than C1GalT1-suppressed cells and the presence of galectin-3 increased tumour growth of C1GalT1-expressing but not C1GalT1-suppressed cells. More MGL-expressing macrophages and dendritic cells were seen to be attracted to the tumour microenvironment in ME C1galt1-/-/Erb mice than in C1galt1f/f /Erb mice. These results indicate that expression of C1GalT1 by tumour cells reciprocally controls tumour cell-cell and tumour-macrophage interactions mediated by galectin-3 and MGL with double impact on cancer development and progression. C1GalT1 overexpression in epithelial cancers therefore may represent a fundamental mechanism in cancer promotion and in reduction of immune response/surveillance in cancer progression.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. shRNA C1GalT1 suppression reduces TF and increases Tn expression but has little influence on expressions of terminal sialic acid and Core 3 carbohydrate structures in human colon cancer cells.
shRNA C1GalT1 transfection led to substantial reduction of C1GalT1 in SW620 (a) and HC116F3 (c) cells assessed by C1GalT1 immunoblotting. b, d shRNA C1GalT1 suppression led to reduction of TF and increase of Tn structures of cellular proteins of SW620 (b) and HCT116F3 (d) cells when analysed by lectin blotting using TF-binding PNA and Tn-binding VVA. No change of terminal sialic acids (SNA binding) and Core 3 (GSL-II) structures occurs in control and C1GalT1 suppressed cells. e, f Analysis of cell surface expressions of TF, Tn, terminal sialic acid and Core 3 structures by lectin blot and flow cytometry and show similar results as by lectin blots of SW620 (e) and HCT116F3 (f) cells. Representative blots and flow cytometry histograms from two independent experiments are shown.
Fig. 2
Fig. 2. C1GalT suppression alters cancer cell activities.
C1GalT1 suppression reduces migration of SW620 (a, b) and HCT116F3 (f, g) cells. The distance between the two-cell migrating fronts was quantified and is shown as percentage of 0 h (b, g). C1GalT1 suppression in SW620 (c) and HCT116F3 (h) cells leads to reduction of cell proliferation and colony formation of SW620 (d) and HCT116F3 (i) cells. The bar charts show average number of colonies with >80 cells. C1GalT1 suppression reduces adhesion of SW620 (e) and HCT116F3 (j) cells to matrix proteins. Number of adherent cells from at least 10 randomly selected FOVs for SW620 and three for HCT116F3 cells were quantified. Data are expressed as mean ± SEM from three independent experiment, each in triplicate, *p < 0.05, **p < 0.01, ***p < 0.001 (ANOVA followed by Bonferroni).
Fig. 3
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
Fig. 4. C1GalT1 suppression reduces galectin-3-mediated tumour cell-cell interaction and cell adhesion to basement proteins.
a, b Equal number (5 × 105 cells/ml) of DiO- and Dil-labelled SW620shCon or SW620shC1GalT1 (a); HCT116F3shCon or HCT116F3shC1GalT1 (b) cells were mixed without or with 5ug/ml recombinant galectin-3 for 90 min before analysis by flow cytometry. Cell population occurs at the top right (blue) in the bivariate correlation plot which contains both DiO- and Dil-labled cells are defined in this study as cell-cell aggregates/interaction. Representative flow cytometry plots are shown in (a) and (b) . Percentage changes of cell aggregates of shC1GT in comparison to shCon cells are expressed as mean ± SD from four (c, SW620) and three (d, HCT116F3) independent experiments. *p < 0.05 (T-test). e, f C1GalT suppression reduces galectin-3-mediated tumour cell adhesion to matrix proteins. Percentage adhesion of shC1GT cells in comparison to shCon cells are shown in e (SW620) and f (HCT116F3). Data are expressed as mean ± SEM of three independent experiments, each in triplicate *p < 0.05 (ANOVA, followed by Bonferroni). g, h MUC1 immunoblots show reduction of MUC1 molecular size in HCT116F3shC1GT (g) and SW620sgC1GT (h) cells in comparison to control cells. i C1GalT1 suppression reduces galectin-3-mediated EGFR phosphorylation in cell response to EGF in HCT116F3 cells. Representative blots from three independent experiments are shown.
Fig. 5
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).
Fig. 6
Fig. 6. 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. 7
Fig. 7. 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.
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