Journal: Advanced Science

Article Title: Galectin‐1‐Induced Tumor Associated Macrophages Repress Antitumor Immunity in Hepatocellular Carcinoma Through Recruitment of Tregs

doi: 10.1002/advs.202408788

Figure Lengend Snippet: TAMs play a crucial role in the Gal1‐induced tumor progression. A) LGALS1 expression in normal tissues and tumor tissues of HCC patients based on TCGA database. B) Comparison of overall survival between Gal1 high expression and Gal1 low expression groups in TCGA cohort. C) Representative IHC staining of Gal1 in HCC tissue array (left) and comparison of relative Gal1 expression between normal tissues (n = 38) and tumor tissues (n = 62) according to immunohistochemical staining score (right). D) Comparison of Gal1 expression between patients with early stage (TNM stage I/ II, n = 38) and advanced stage (TNM stage III, n = 24). E) Kaplan‐Meier curve analysis of overall survival of HCC patients with high Gal1 expression (IHC staining score ≥ 6, n = 35) compared with patients with low Gal1 expression (IHC staining score < 6, n = 27). F) Western blot analysis of Gal1 protein expression in shNC and shGal1 hepa1‐6 cells. G) Images of HCC tumor (left) and tumor weight (right) in the orthotopic HCC mouse models (n = 6). H) Comparison of overall survival between shNC and shGal1 orthotopic tumor models (n = 6). I) The proliferation of shNC and shGal1 cells measured by CCK8 assay. J) Flow chart of shNC and shGal1 cell implantation and macrophage depletion. K) Representative image of tumor (left) and tumor weight (right) in the orthotopic HCC mouse models with or without macrophage depletion (n = 6). Data were presented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Furthermore, OTX008 (50 μg ml −1 ), anti‐mouse CCL20 antibody (20 μg ml −1 , ab9829, Abcam) or recombinant Gal1 (10 μg ml −1 , 450‐39, PeproTech) was added to these cells.

Techniques: Expressing, Comparison, Immunohistochemistry, Immunohistochemical staining, Staining, Western Blot, CCK-8 Assay

Journal: Advanced Science

Article Title: Galectin‐1‐Induced Tumor Associated Macrophages Repress Antitumor Immunity in Hepatocellular Carcinoma Through Recruitment of Tregs

doi: 10.1002/advs.202408788

Figure Lengend Snippet: TAMs are critical mediators in the establishment of immunosuppressive TME induced by Gal1. A) Analysis of the correlation between Gal1 expression and CD8 + T cell infiltration (left) and Treg infiltration (right) in TCGA database. B) Representative images (upper) of IF staining with Foxp3 (red), CD8 (green), DAPI (blue) in HCC tissues with high Gal1 expression (IHC staining score ≥ 6) and low Gal1 expression (IHC staining score < 6); (lower) quantification of Foxp3 + and CD8 + cells in HCC tissues. C) Flow cytometry analysis of CD4 + CD25 + Foxp3 + Treg infiltration in orthotopic HCC mouse models with or without macrophage depletion (n = 6). D) Flow cytometry analysis of the proportion of CD8 + T cells in orthotopic HCC mouse models (n = 6). E) Representative IHC staining images and quantification of Foxp3 + and CD8 + cells in mouse tumor tissues (n = 6). F) Flow cytometry analysis of the proportion of PD‐1 + LAG‐3 + CD8 + T cells in orthotopic HCC mouse models (n = 6). G) Percentage of IFN‐γ + , TNF‐α + , granzyme B + and perforin + CD8 + T cells assessed by flow cytometry (n = 6). H) Flow cytometry analysis of NK1.1 + cell infiltration in the orthotopic HCC mouse models (n = 6). I) The proportion of CD8 + T cells in the orthotopic HCC mouse models treated with isotype or anti‐CD8 mAb (n = 6). J) Representative image of tumor (left) and tumor weight (right) in the orthotopic HCC mouse models treated with isotype or anti‐CD8 mAb (n = 6). Data were presented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Furthermore, OTX008 (50 μg ml −1 ), anti‐mouse CCL20 antibody (20 μg ml −1 , ab9829, Abcam) or recombinant Gal1 (10 μg ml −1 , 450‐39, PeproTech) was added to these cells.

Techniques: Expressing, Staining, Immunohistochemistry, Flow Cytometry

Journal: Advanced Science

Article Title: Galectin‐1‐Induced Tumor Associated Macrophages Repress Antitumor Immunity in Hepatocellular Carcinoma Through Recruitment of Tregs

doi: 10.1002/advs.202408788

Figure Lengend Snippet: Tumor‐derived Gal1 facilitates Tregs recruitment through upregulating CCL20 expression in TAMs. A) Schematic workflow showing BMDM and tumor cell co‐culture and RNA‐sequencing. B) Volcano plot of differentially expressed genes based on RNA‐sequencing. C) Pathway enrichment analysis of RNA‐sequencing dataset. D) Cluster heatmap of differentially expressed chemokines based on RNA‐sequencing. E) Changes in chemokine levels according to RNA‐sequencing. F) qPCR analysis of CCL20 expression in BMDMs cultured with conditioned medium from shGal1 or shNC hepa1‐6 cells for 24 hours (left, n = 3); ELISA analysis of CCL20 protein expression in supernatants (right, n = 3). G) qPCR analysis of CCL20 expression in shGal1 or shNC hepa1‐6 cells (left, n = 3); ELISA analysis of CCL20 protein expression in the supernatants from shGal1 or shNC hepa1‐6 cells (right, n = 3). H) Flow cytometry analysis of CCL20 expression in TAMs in the orthotopic HCC mouse models (n = 6). I) The proportion of CCR6 + Tregs in the orthotopic HCC mouse models assessed by flow cytometry (n = 6). J) ELISA analysis of CCL20 protein level in tumor tissues from the orthotopic HCC mouse models with or without macrophage depletion (n = 6). K) Schematic diagram of Treg chemotaxis assay. L) Flow cytometry analysis of the number of migrated CD4 + CD25 + Foxp3 + Tregs (n = 3). Data were presented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Furthermore, OTX008 (50 μg ml −1 ), anti‐mouse CCL20 antibody (20 μg ml −1 , ab9829, Abcam) or recombinant Gal1 (10 μg ml −1 , 450‐39, PeproTech) was added to these cells.

Techniques: Derivative Assay, Expressing, Co-Culture Assay, RNA Sequencing, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Chemotaxis Assay

Journal: Advanced Science

Article Title: Galectin‐1‐Induced Tumor Associated Macrophages Repress Antitumor Immunity in Hepatocellular Carcinoma Through Recruitment of Tregs

doi: 10.1002/advs.202408788

Figure Lengend Snippet: Tregs recruited by Gal1‐induced TAMs contribute to impaired functionality of CD8 + T cells. A) Flow chart of Treg depletion in Foxp3DTR mice. B) Representative image of tumor (left) and tumor weight (right) in the orthotopic HCC mouse models with or without Treg depletion (n = 6). C) Infiltration of CD8 + T cells in the orthotopic HCC mouse models was determined by flow cytometry (n = 6). D) Flow cytometry analysis of IFN‐γ + , TNF‐α + , granzyme B + and perforin + CD8 + T cells in the orthotopic HCC models (n = 6). E) Schematic flow diagram of shNC and shGal1 cell implantation and CCL20 neutralization. F) Representative image of tumor (left) and tumor weight (right) in the orthotopic HCC models treated with isotype or anti‐CCL20 antibody (n = 6). G) Flow cytometry analysis of CD4 + CD25 + Foxp3 + Tregs in the orthotopic HCC models (n = 6). H) The proportion of CCR6 + Tregs in the orthotopic HCC models was assessed by flow cytometry (n = 6). I) Representative IHC staining images (upper) and quantification (lower) of Foxp3 + and CD8 + cells in mouse tumor tissues (n = 6). J) Flow cytometry analysis of the infiltration of CD8 + T cells in the orthotopic HCC mouse models (n = 6). K) Flow cytometry analysis of the expression of anti‐tumor cytokines (IFN‐γ, TNF‐α, granzyme B and perforin) in CD8 + T cells in the orthotopic HCC mouse models (n = 6). Data were presented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Furthermore, OTX008 (50 μg ml −1 ), anti‐mouse CCL20 antibody (20 μg ml −1 , ab9829, Abcam) or recombinant Gal1 (10 μg ml −1 , 450‐39, PeproTech) was added to these cells.

Techniques: Flow Cytometry, Neutralization, Immunohistochemistry, Expressing

Journal: Advanced Science

Article Title: Galectin‐1‐Induced Tumor Associated Macrophages Repress Antitumor Immunity in Hepatocellular Carcinoma Through Recruitment of Tregs

doi: 10.1002/advs.202408788

Figure Lengend Snippet: Gal1 upregulates CCL20 through activation of the PI3K/AKT/NF‐κB signaling pathway in TAMs. A) Pathway enrichment analysis of differentially expressed genes based on RNA‐sequencing. B) Protein levels of PI3K/AKT/NF‐κB pathway in BMDMs incubated in the conditioned medium for 24 hours were determined by western blot. C) Representative IF images (left) and quantification (right) of P65 nuclear translocation in BMDMs incubated in the conditioned medium (n = 3). D) ELISA analysis of CCL20 protein expression in supernatants of BMDMs cultured with conditioned medium from shGal1 or shNC hepa1‐6 cells for 24 hours (n = 3). E) Representative image of tumor (left) and tumor weight (right) in the orthotopic HCC models treated with vehicle or BAY 11–7082 (n = 6). F) CCL20 expression in TAMs in the orthotopic HCC models was measured by flow cytometry (n = 6). G) Flow cytometry analysis of the proportion of CCR6 + Tregs in the orthotopic HCC models (n = 6). H) Flow cytometry analysis of CD4 + CD25 + Foxp3 + Tregs in the orthotopic HCC models (n = 6). I) Representative image of tumor (left) and tumor weight (right) in the orthotopic HCC models treated with vehicle or LY294002 (n = 6). J) Flow cytometry analysis of CCL20 expression in TAMs in the orthotopic HCC models (n = 6). K) Flow cytometry analysis of the proportion of CCR6 + Tregs in the orthotopic HCC models (n = 6). L) Flow cytometry analysis of CD4 + CD25 + Foxp3 + Tregs in the orthotopic HCC models (n = 6). Data were presented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Furthermore, OTX008 (50 μg ml −1 ), anti‐mouse CCL20 antibody (20 μg ml −1 , ab9829, Abcam) or recombinant Gal1 (10 μg ml −1 , 450‐39, PeproTech) was added to these cells.

Techniques: Activation Assay, RNA Sequencing, Incubation, Western Blot, Translocation Assay, Enzyme-linked Immunosorbent Assay, Expressing, Cell Culture, Flow Cytometry

Journal: Advanced Science

Article Title: Galectin‐1‐Induced Tumor Associated Macrophages Repress Antitumor Immunity in Hepatocellular Carcinoma Through Recruitment of Tregs

doi: 10.1002/advs.202408788

Figure Lengend Snippet: Blocking Gal1 reverses the anti‐PD1 resistance in HCC. A) Comparison of TIDE scores between Gal1 high expression and Gal1 low expression groups in TCGA cohort. B) Image of HCC tumors (left) and quantification of tumor weight (right) in orthotopic HCC models treated with OTX008, anti‐PD‐1 mAb or combination therapy (n = 6). C) Overall survival of tumor‐bearing mice treated with monotherapy or combination therapy (n = 6). D) Flow cytometry analysis of CCL20 expression in TAMs in the orthotopic HCC models with different treatment (n = 6). E) Flow cytometry analysis of CD4 + CD25 + Foxp3 + Tregs in the orthotopic HCC models (n = 6). F) The percentage of CCR6 + Tregs in the orthotopic HCC models was determined by flow cytometry (n = 6). G) Representative IHC staining images (upper) and quantification (lower) of Foxp3 + and CD8 + cells in the orthotopic HCC models with different treatment (n = 6). H) The number of live CD8 + T cells in the orthotopic HCC models was assessed by flow cytometry (n = 6). I) Intracellular staining of granzyme B, perforin, IFN‐γ and TNF‐α in CD8 + T cells in the orthotopic HCC models was determined by flow cytometry (n = 6). Data were presented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Furthermore, OTX008 (50 μg ml −1 ), anti‐mouse CCL20 antibody (20 μg ml −1 , ab9829, Abcam) or recombinant Gal1 (10 μg ml −1 , 450‐39, PeproTech) was added to these cells.

Techniques: Blocking Assay, Comparison, Expressing, Flow Cytometry, Immunohistochemistry, Staining

Journal: Advanced Science

Article Title: Galectin‐1‐Induced Tumor Associated Macrophages Repress Antitumor Immunity in Hepatocellular Carcinoma Through Recruitment of Tregs

doi: 10.1002/advs.202408788

Figure Lengend Snippet: Schematic representations of Gal1‐induced TAMs in maintaining the immunosuppressive tumor microenvironment in HCC.

Article Snippet: Furthermore, OTX008 (50 μg ml −1 ), anti‐mouse CCL20 antibody (20 μg ml −1 , ab9829, Abcam) or recombinant Gal1 (10 μg ml −1 , 450‐39, PeproTech) was added to these cells.

Techniques: