Journal: Nature Communications

Article Title: MGAT1-Guided complex N-Glycans on CD73 regulate immune evasion in triple-negative breast cancer

doi: 10.1038/s41467-025-58524-9

Figure Lengend Snippet: a MGAT1 complex was purified with a tandem-affinity purification protocol followed by mass spectrometry analysis in MDA-MB468-Flag/HA-MGAT1 cells. Silver staining of the purified MGAT1 complex is illustrated. CD73 was identified as a binding partner of MGAT1, and the representative spectra are shown. b , c The biochemical interaction between MGAT1 and CD73 in MDA-MB468 cells was validated by reciprocal coimmunoprecipitation of ectopic Flag-MGAT1 ( b ) and endogenous CD73 ( c ). The samples derived from the same experiment but different gels for CD73 MGAT1, and β-ACTIN were processed in parallel. d The colocalization of immunostained MGAT1 (green) and CD73 (red) in MDA-MB468 and MDA-MB231 cells was visualized by confocal imaging. e The subcellular localization of MGAT1 was detected through colocalization of MGAT1 (green) and Golgi indicator, GM-130 (red), in MDA-MB468 and MDA-MB231 cells by confocal imaging. f The intracellular interaction between MGAT1 (green) and CD73 (red) in MDA-MB468 cells was visualized by immunofluorescence stimulated emission depletion microscopy imaging followed by 3D reconstruction by Imaris. g The intracellular interaction between MGAT1 and CD73 was validated by a proximity ligation assay (PLA-red) with anti-MGAT1 and anti-CD73 antibodies or control IgG followed by confocal imaging. h Adenosine levels were determined in WT or CD73-KD MDA-MB231/MDA-MB468 breast cancer cells with MGAT1 OE or MGAT1 KD. i Spearman’s rank correlation analysis shows that CD73 protein expression is highly positively correlated with several N-glycan biosynthesis genes, and MGAT1 is the most positively correlated one. j Representative IHC staining of MGAT1 and CD73 in human TNBC tissue sections ( n = 23). Quantification of positive staining areas using QuPath reveals a significant positive correlation between MGAT1 and CD73 expression. Data (mean ± SEM), images, and western blots are representative of at least three independent experiments. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test ( h ) or simple linear regression ( j ). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies targeting MGAT1 (Sigma-Aldrich, SAB1400165) and CD73 (Cell Signaling Technology, #13160) were applied and incubated overnight at 4 °C on a shaker.

Techniques: Purification, Affinity Purification, Mass Spectrometry, Silver Staining, Binding Assay, Derivative Assay, Imaging, Immunofluorescence, Microscopy, Proximity Ligation Assay, Control, Expressing, Glycoproteomics, Immunohistochemistry, Staining, Western Blot

Journal: Nature Communications

Article Title: MGAT1-Guided complex N-Glycans on CD73 regulate immune evasion in triple-negative breast cancer

doi: 10.1038/s41467-025-58524-9

Figure Lengend Snippet: a Schematic diagram of human MGAT1 domains and strategy to engineer a series of MGAT1 truncation and deletion mutants. b The interactions between CD73 and MGAT1 fragments were examined by co-IP experiments in HEK-293T cells. Red text indicates the smallest truncation mutant that bound CD73 (left) and the deletion mutant with reduced binding to CD73 (right). Amino acids 321–370 were identified as the region on MGAT1 that mediates the interaction with CD73. The samples derived from the same experiment but different gels for Flag V5, and β-ACTIN were processed in parallel. c Schematic diagram of human CD73 domains and strategy to engineer a series of CD73 truncation and deletion mutants. d The interactions between MGAT1 and CD73 fragments were examined by co-IP experiments in HEK-293T cells. Amino acids 79–128 on CD73 were identified as the region that mediates the interaction between MGAT1 and CD73. The samples derived from the same experiment but different gels for V5 and HA, and β-ACTIN were processed in parallel. e The top CD73-MGAT1 complex model predicted by ClusPro with an estimated docking occupancy of 10.5% and binding affinity of − 15.4 kcal/mol. The interacting domains discovered by mapping were highlighted. f , g The time evolution of the RMSDs of the MGAT1-CD73 complex in five 200-ns MD simulations of the complex formed with MGAT1-CD73 using the start points in ( e ) is shown in ( f ). The RMSD was evaluated after structurally aligning the conformers observed during MD trajectories with respect to the initial ClusPro-predicted complex. The corresponding time evolution of residue-residue interactions between MGAT1 and CD73 residues is shown in ( g ), and regions shaded in gray refer to time intervals during which the indicated residue pairs made interfacial contacts. Data and western blots are representative of at least three independent experiments. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies targeting MGAT1 (Sigma-Aldrich, SAB1400165) and CD73 (Cell Signaling Technology, #13160) were applied and incubated overnight at 4 °C on a shaker.

Techniques: Co-Immunoprecipitation Assay, Mutagenesis, Binding Assay, Derivative Assay, Residue, Western Blot

Journal: Nature Communications

Article Title: MGAT1-Guided complex N-Glycans on CD73 regulate immune evasion in triple-negative breast cancer

doi: 10.1038/s41467-025-58524-9

Figure Lengend Snippet: a The CD73 protein levels and molecular sizes in MDA-MB468 and MDA-MB231 cells with MGAT1 OE/KD were determined by immunoblotting. The samples derived from the same experiment but different gels for CD73 and MGAT1, and β-ACTIN were processed in parallel. b Immunoblotting of CD73 in MGAT1 OE/KD MDA-MB468 lysates treated with Endo H/PNGase F. c Flow cytometry analysis of membrane CD73 in MGAT1 OE/KD MDA-MB468 cells. d The membrane distribution of CD73 (green) was visualized with stimulated emission depletion microscopy imaging in MDA-MB468 cells with MGAT1 OE/KD with membrane dye (red). e , f CD73 dimerization in MGAT1 OE/KD MDA-MB468 cells was assessed by glutaraldehyde cross-linking ( e ) and semi-native gel ( f ) immunoblotting. g Structural analysis of CD73 dimerization in inactive (PDB: 4H2G) and active (PDB: 6TVX) states, highlighting N-glycosylation sites (cyan) and C-terminal dimerization interface (magenta). h The scheme of investigating CD73 dimerization with a split-GFP assay. The GFP 1-10 or GFP 11 × 7 was attached to the N-terminus of WT CD73, CD73-4NQ, or CD73Δ480-537 and co-transfected into cells to investigate their dimerization abilities. i The split-GFP signal generated from dimerized WT CD73, CD73-4NQ, or CD73Δ480-537 was visualized by confocal microscopy in HEK-293T cells. j Flow cytometry of membrane CD73 + cells in MGAT1 OE/KD MDA-MB231 and MDA-MB468 with or without VAMP3 KD. k Schematic model showing how MGAT1-mediated glycosylation of CD73 orchestrates its dimerization and further translocation to cell membranes. Failure of CD73 glycosylation impedes CD73 dimerization and membrane translocation. Created in BioRender. Zhu, Y. (2025) https://BioRender.com/i34w446 . l Spearman’s rank correlation analysis shows the MGAT1 protein expression is highly positively correlated with the THBS1 signaling pathways. m Adenosine levels in MGAT1 OE/KD MDA-MB468 and MDA-MB231 cells treated with THBS1. Data (mean ± SEM), images, western blot, and flow cytometry are representative of at least three independent experiments. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test ( j ) or two-tailed unpaired t test ( m ). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies targeting MGAT1 (Sigma-Aldrich, SAB1400165) and CD73 (Cell Signaling Technology, #13160) were applied and incubated overnight at 4 °C on a shaker.

Techniques: Western Blot, Derivative Assay, Flow Cytometry, Membrane, Microscopy, Imaging, Glycoproteomics, Split GFP Assay, Transfection, Generated, Confocal Microscopy, Translocation Assay, Expressing, Protein-Protein interactions, Two Tailed Test

Journal: Nature Communications

Article Title: MGAT1-Guided complex N-Glycans on CD73 regulate immune evasion in triple-negative breast cancer

doi: 10.1038/s41467-025-58524-9

Figure Lengend Snippet: a Schematic of an MGAT1 function-based drug screening assay converting enzymatic activity to a luminance signal. Key components include MGAT1, mannose-5-AEAD (substrate), UDP-GlcNAc (sugar donor), and a compound library. MGAT1 inhibition reduces free UDP, decreasing luminance. Created in BioRender. Zhu, Y. (2025) https://BioRender.com/t24b827 . b Normalized MGAT1 enzymatic function in high-throughput screening with an anti-cancer compound library (~ 3500 compounds). c AutoDock Vina-predicted most energy-favorable binding of TW-37 (pink) to MGAT1, with interacting residues (within 3.5 Å) shown. d Pharmacophore model 1 (PM1) based on binding of TW-37 to MGAT1. e Pharmit-predicted binding of the top 10 compounds (colored differently) screened against PM_1. f Normalized MGAT1 enzyme function upon treatment with 15 screened compounds was measured by function-based screening assay. g Flow cytometry analysis of membrane CD73 in MDA-MB468 cells treated with 15 compounds. h , i The membrane-fractionated protein levels of CD73 in MDA-MD468 cells upon treatment with screened compounds were determined by ( h ) immunostaining and ( i ) immunoblotting. j The chemical structures of two top-ranked compounds (No.8 and No.9). k Dose-dependent MGAT1 inhibition by No.2, No.8, and No.9 in the function-based assay. l – o MDA-MB468 cells were cocultured with PBMC cells and the indicated compounds. Percentages of TNFα + (l), IFNγ + (m), Ki-67 + ( n ), and Granzyme B + ( o ) in CD8 + T cells were measured using flow cytometry. p , q Time-lapse quantification of MDA-MB468 survival in PBMC coculture treated with No.2/No.8(W-GTF01)/No.9 ( p ) or No.8 in MDA-MB468-WT/CD73-KD cells ( q ). r Binding of W-GTF01 (MolPort-004-851-686) to MGAT1, highlighting π-stacking interaction between F324 (yellow) and W-GTF01 (magenta). Data (mean ± SEM), images, western blot, and flow cytometry are representative of at least three independent experiments. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test ( i , l – o ) or two-way ANOVAs followed by Tukey’s multiple comparison tests ( p , q ). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies targeting MGAT1 (Sigma-Aldrich, SAB1400165) and CD73 (Cell Signaling Technology, #13160) were applied and incubated overnight at 4 °C on a shaker.

Techniques: Drug discovery, Activity Assay, Inhibition, High Throughput Screening Assay, Binding Assay, Screening Assay, Flow Cytometry, Membrane, Immunostaining, Western Blot, Functional Assay, Comparison

Journal: Nature Communications

Article Title: MGAT1-Guided complex N-Glycans on CD73 regulate immune evasion in triple-negative breast cancer

doi: 10.1038/s41467-025-58524-9

Figure Lengend Snippet: a Schematic of mouse experiments: 4T1 or E0771 breast cancer cells were injected orthotopically into WT BALB/c or C57BL/6 mice. Tumor weight was measured at the endpoint, and tumor growth was monitored. Created in BioRender. Zhu, Y. (2025) https://BioRender.com/y73s493 . b – d Tumor growth ( n = 6) ( b ), endpoint tumor weight ( n = 6) ( c ), and membrane CD73 levels ( d) in 4T1-MGAT1 OE ( n = 5) vs. control tumors ( n = 5). e tSNE visualization of immune cell composition, comparing TEX, Tregs, MDSCs, and TAMs among live CD45 + cells in 4T1-MGAT1 OE ( n = 5) vs. control tumors ( n = 5) at day 25. f Percentages of PD-1 + TOX + and PD-1 + CD101 + CD8 + T cells among CD45 + cells between 4T1 control tumors ( n = 8) and 4T1 MGAT1 OE tumors ( n = 8). g CD163 expression levels were compared in tumor-associated macrophages (TAMs) between 4T1-MGAT1 OE ( n = 5) vs. control tumors ( n = 5). h Comparison of the percentage of IFNγ + /TNFα + among tumor-infiltrated CD4 + or CD8 + T cells between 4T1 control ( n = 5) and MGAT1 OE tumors ( n = 5). i , j Tumor growth ( i ) and endpoint tumor weight ( j ) in 4T1-MGAT1 KD ( n = 8) vs. control tumors ( n = 6). k Percentage of Tim3 + /PD1 + CD8 + T cells in 4T1-MGAT1 KD ( n = 6) vs. control tumors ( n = 5). l Ki-67 + tumor-infiltrating CD8 + T cells in 4T1-MGAT1 KD (n = 6) vs. control tumors( n = 5). m – o Tumor growth ( m ), endpoint tumor weight ( n ), and membrane CD73 levels ( o ) in E0771-MGAT1 OE + CD73WT ( n = 6) vs. CD73-4NQ tumors ( n = 6) in C57BL/6 mice. p , q 4T1-hPD-L1 tumors (the endogenous mouse PD-L1 was replaced with its human counterpart) were treated with W-GTF01 (10 mg/kg, i.p.) twice/week and durvalumab (10 mg/kg, i.p.) three times/week ( n = 8). PBS and IgG were used in the control groups. Tumor growth ( p ) and survival ( q ) were monitored. Data (means ± SEM), images, and flow cytometry are representative of at least three independent experiments with 5–10 independently analyzed mice per group. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test ( n , o ) or two-tailed unpaired t test ( c – h , j – l ) or two-way ANOVAs followed by Tukey’s multiple comparison tests ( b , i , m , p ). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies targeting MGAT1 (Sigma-Aldrich, SAB1400165) and CD73 (Cell Signaling Technology, #13160) were applied and incubated overnight at 4 °C on a shaker.

Techniques: Injection, Membrane, Control, Expressing, Comparison, Flow Cytometry, Two Tailed Test

Journal: Nature Communications

Article Title: MGAT1-Guided complex N-Glycans on CD73 regulate immune evasion in triple-negative breast cancer

doi: 10.1038/s41467-025-58524-9

Figure Lengend Snippet: a Representative staining of MGAT1, CD73, and PanCK, followed by the selection of regions of interest (ROI) ( n = 32) and subsequent auto-segmentation in TNBC tissue sections using GeoMX DSP. b , c CIBERSORTx analysis of the relative abundance of individual cell populations between MGAT1lo and MGAT1hi areas ( b ) or between MGAT1loCD73lo (DL) and MGAT1hiCD73hi areas (DH) ( c ) among nontumor compartments using transcriptomics data. d Representative composite image (left panel) with inset (right) of a breast cancer specimen stained by multicolor IHC comprising CD8, Ki67, CD3, CD73, MGAT1, PanCK, and DAPI. Scale bars: 100 μm. e The analysis of MGAT1 and membrane CD73 protein expression on TNBC ( n = 146) using mIHC shows the positive correlation between MGAT1 with CD73. f Intensity of membrane CD73 was compared between MGAT1hi and MGAT1lo tumor regions. g Touching events between CD3 + CD8 + T cells and PanCK + tumor cells among DH relative to DL tumor regions. h , i UP-KW functional annotation (UniProt KeyWord Functional Annotations) analysis of post-translational modification (PTM) on the DEGs (different expressional genes) in PanCK + epithelial tumor compartment between DL cases and DH cases ( i ), revealing selected Glycoprotein at increased levels in DH tumors ( h ). * p < 0.05. j , k GO Pathway analysis of DEGs in non-tumor areas between DL cases and DH cases, highlighting selected genes involving B cell receptor signaling and T cell activation at increased levels in DL cases ( k ). * p < 0.05. l The proposed working model. MGAT1, a glycosyltransferase, catalyzes CD73 for glycosylation at the Golgi body resulting in CD73 dimerization that enables CD73 plasma membrane translocation mediated by transport vesicles. Overactivation of MGAT1 due to aberrant THBS1 signaling leads to increased membrane-bound abundance of CD73 that enhances adenosine production and subsequently suppresses CD8 + T cell function. Pharmacological blockade of MGAT1 by an MGAT1 inhibitor (W-GTF01) reduces tumor growth by reinstating the capacity to elicit CD8 + effector T-cell responses through inhibition of CD73 glycosylation and membrane translocation. Data (means ± SEM), images, and flow cytometry are representative of at least three independent experiments. Statistical significance was determined using a two-tailed unpaired t test ( b , c ), two-tailed paired t test ( f , g ), or simple linear regression ( e ). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies targeting MGAT1 (Sigma-Aldrich, SAB1400165) and CD73 (Cell Signaling Technology, #13160) were applied and incubated overnight at 4 °C on a shaker.

Techniques: Staining, Selection, Membrane, Expressing, Functional Assay, Modification, Activation Assay, Glycoproteomics, Clinical Proteomics, Translocation Assay, Cell Function Assay, Inhibition, Flow Cytometry, Two Tailed Test

Journal: Scientific reports

Article Title: Increased primary breast tumor expression of CD73 is associated with development of bone metastases and is a potential biomarker for adjuvant bisphosphonate use.

doi: 10.1038/s41598-025-92841-9

Figure Lengend Snippet: Fig. 1. Representative images of immunostaining for CD73 in TMA cores from patients in the AZURE study. (A) Images of protein expression obtained using the anti-CD73 antibody and visualized at a magnification of 20x. Score 0–3 categories were determined by the intensity of staining in the cytoplasmic compartment of tumor cells only. Scale bar = 200 μm. (B) 40x magnification view of from the boxed regions depicted in panel A. Scale bar of core view = 100 μm. Scale bar of 40x = 50 μm.

Article Snippet: After cooling and washing, slides were blocked with normal goat serum for 1 h at room temperature, after which primary antibodies against CD73 (CST, 13160, 1:1000) were applied (overnight, 4 °C).

Techniques: Immunostaining, Expressing, Staining

Journal: Scientific reports

Article Title: Increased primary breast tumor expression of CD73 is associated with development of bone metastases and is a potential biomarker for adjuvant bisphosphonate use.

doi: 10.1038/s41598-025-92841-9

Figure Lengend Snippet: Fig. 2. Prognostic value of CD73 levels in clinical events. Kaplan-Meier association of CD73 expression level in Overall Survival (OS) in (A) control and (B) zoledronic acid arms. Relationship between level of expression of CD73 in Disease-Free-Survival (DFS) in (C) control and (D) zoledronic acid arms. P-value is obtained from the log-rank test for testing quality of survival Q15 functions.

Article Snippet: After cooling and washing, slides were blocked with normal goat serum for 1 h at room temperature, after which primary antibodies against CD73 (CST, 13160, 1:1000) were applied (overnight, 4 °C).

Techniques: Expressing, Control

Journal: Scientific reports

Article Title: Increased primary breast tumor expression of CD73 is associated with development of bone metastases and is a potential biomarker for adjuvant bisphosphonate use.

doi: 10.1038/s41598-025-92841-9

Figure Lengend Snippet: Fig. 3. Kaplan-Meier association of CD73 levels with clinical metastatic events. Relationship between CD73 expression to time to first bone recurrence, – where cancer spread to bone is the first recorded event in (A) control and (B) zoledronic acid arm. Relationship between level of CD73 expression to spread to bone at any time (for which patients may have had spread to non-bone sites first) in (C) control and (D) zoledronic acid arm. Relationship between CD73 expression to time to spread to non-bone sites in (E) control and (F) zoledronic acid arm. P-value is obtained from the log-rank test for testing quality of survival Q15 function.

Article Snippet: After cooling and washing, slides were blocked with normal goat serum for 1 h at room temperature, after which primary antibodies against CD73 (CST, 13160, 1:1000) were applied (overnight, 4 °C).

Techniques: Expressing, Control