Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Galectin-3 induces pathogenic immunosuppressive macrophages through interaction with TREM2 in lung cancer

doi: 10.1186/s13046-024-03124-6

Figure Lengend Snippet: Tumor derived galectin-3 is a ligand for TREM2. ( A ) Illustration of human lung cancer tissue protein precipitated by anti-TREM2 or IgG antibody ( n = 3), and peptides enriched in each complex were identified by mass spectrometry. ( B ) Venn diagram and tables showing secreted proteins concentrated in the TREM2 enriched complex. ( C ) Immunohistochemical staining of galectin-3 in adjacent normal lung and intratumor areas of lung tissues ( n = 5). Scale bars, 20 μm. ( D ) Galactin-3 levels in lung cancer or normal lung lavage were determined by ELISA. ( E ) 293T cells were transfected with pcDNA3.1-vector/pcDNA3.1-hTREM2-HA/pcDNA3.1-hGalectin-3-Flag plasmids as indicated. Anti-HA antibody was employed for exogenous CO-IP experiments. ( F ) F4/80 + macrophages were sorted from human lung cancer tissue. Anti-TREM2 antibody was employed for endogenous CO-IP experiment. ( G ) 293T cells were transfected with pcDNA3.1-TREM2-HA and pcDNA3.1-Galectin-3-Flag plasmids. The co-localization between TREM2 and Galectin-3 was examined using confocal microscopy. Scale bars, 5 μm. ( H ) 293T cells were transfected with pcDNA3.1-TREM2-HA/pcDNA3.1-TREM2-ΔIg-HA/pcDNA3.1-TREM2-ΔTm-HA/pcDNA3.1-TREM2-ΔCyto-HA/pcDNA3.1-Galectin-3-Flag/pcDNA3.1-vector plasmids as indicated. Anti-Flag antibody was employed for exogenous CO-IP experiments. ( I ) 293T cells were transfected with pcDNA3.1-vector/pcDNA3.1-TREM2-Ig-HA/pcDNA3.1-TREM2- HA/pcDNA3.1-Galectin-3-Flag plasmids as shown. Anti-Flag antibody was employed for exogenous CO-IP experiments. ( J ) 293T cells were transfected with pcDNA3.1-vector/pcDNA3.1-TREM2-HA/pcDNA3.1-Galectin-3-ΔNH2-Flag/pcDNA3.1-Galectin-3-ΔCRD-Flag/pcDNA3.1-Galectin-3- ΔRepeats-Flag plasmids as indicated. Anti-HA antibody was employed for exogenous CO-IP experiments. ( K ) Galectin-3 protein bound to TREM2 in the plate was determined using anti-Galectin-3 antibody. Data represent mean ± SD from three experiments. **, P < 0.01; ***, P < 0.001

Article Snippet: Tyrosine kinase inhibitor (Genistein) (absin, Shanghai, China), Syk inhibitor (R406) (Selleck Chemicals, Houston, USA), Src inhibitor (PP2) (Solarbio, Beijing, China), TREM2 Fc (R&D Systems, Minnesota, USA), GB1107 (Selective Galectin-3 inhibitor) (TargetMol, Boston, Massachusetts), Recombinant mouse Galectin-3 protein (rm Galectin-3) (R&D Systems, Minnesota, USA).

Techniques: Derivative Assay, Mass Spectrometry, Immunohistochemical staining, Staining, Enzyme-linked Immunosorbent Assay, Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Confocal Microscopy

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Galectin-3 induces pathogenic immunosuppressive macrophages through interaction with TREM2 in lung cancer

doi: 10.1186/s13046-024-03124-6

Figure Lengend Snippet: Galectin-3 inhibits TREM2/DAP12 receptor complex to suppress Src/Syk signaling pathway and altered macrophage to an M2-like phenotype. (A , B ) 293T cells were transfected with plasmids as shown, and anti-HA ( B ) or anti-Flag ( B ) antibodies were employed for exogenous CO-IP experiments. ( C-E ) 293T cells were transfected with plasmids as shown and anti-HA ( C ), anti-Flag ( D ), or anti-Myc ( E ) antibodies were employed for exogenous CO-IP experiments, respectively. (F , G ) After 24 h of LLC CM treatment with the addition of GB1107 (5 µM), and 2 h of stimulation with LPS (1 ng/mL), the phosphorylation levels of Src and Syk in indicated time point were analyzed by western blot ( F ). The gray values of p-Syk and p-Src protein bands were analyzed by Image J software, and the relative gray values were standardized to the gray values of Syk and Src ( G ). (H , I ) After 24 h of LLC CM treatment with the addition of GB1107 (5 µM), and 2 h of stimulation with LPS (1 ng/mL), the phosphorylation levels of Src and Syk in WT or TREM2 KO BMDMs were analyzed by western blot ( H ). The gray values of p-Syk and p-Src protein bands were analyzed by Image J image analyses software, and the relative gray values were standardized to the gray values of Syk and Src ( I ). (J , K ) After 24 h of LLC CM treatment with the addition of rm galectin-3 (200 ng/ml), and 2 h of stimulation with LPS (1 ng/mL), the phosphorylation levels of Src and Syk in WT or TREM2 KO BMDMs were analyzed by western blot ( J ). The gray values of p-Syk and p-Src protein bands were analyzed by Image J image analyses software, and the relative gray values were standardized to the gray values of Syk and Src ( K ). ( L ) Phagocytosis assay between WT or TREM2 KO BMDMs which were pretreated with LLC CM supplemented with GB1107 (5 µM) for 24 h and LLC were detected by flow cytometry. ( M ) Following a 24 h-treatment with LLC CM supplemented with GB1107 (5 µM), the F-actin polarization of RAW264.7 cells blocked with anti-TREM2 antibody was observed by confocal microscopy. Scale bars, 5 μm. Quantitative statistics of F-actin polarization were analyzed by Image J image analyses software. ( N-P ) After 24 h of LLC CM which was supplemented with GB1107 (5 µM) treatment, the transcription levels of Ccr2 ( N ), M2-like macrophage markers ( CD206 , Arg1 ) ( O ) and M1-like macrophage markers ( Nos2 , TNFα ) ( P ) in WT or TREM2 KO BMDMs were detected by RT-qPCR assay. Data represent mean ± SD from three experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, no significance

Article Snippet: Tyrosine kinase inhibitor (Genistein) (absin, Shanghai, China), Syk inhibitor (R406) (Selleck Chemicals, Houston, USA), Src inhibitor (PP2) (Solarbio, Beijing, China), TREM2 Fc (R&D Systems, Minnesota, USA), GB1107 (Selective Galectin-3 inhibitor) (TargetMol, Boston, Massachusetts), Recombinant mouse Galectin-3 protein (rm Galectin-3) (R&D Systems, Minnesota, USA).

Techniques: Transfection, Co-Immunoprecipitation Assay, Phospho-proteomics, Western Blot, Software, Phagocytosis Assay, Flow Cytometry, Confocal Microscopy, Quantitative RT-PCR

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Galectin-3 induces pathogenic immunosuppressive macrophages through interaction with TREM2 in lung cancer

doi: 10.1186/s13046-024-03124-6

Figure Lengend Snippet: Combination therapy of the galectin-3 inhibitor GB1107 and TREM2 deficiency significantly inhibit lung cancer progression and reduced the immunosuppressive M2-like TAMs infiltration. ( A ) Schematic representation of an immunocompetent subcutaneous lung cancer model using the murine lung cancer cell line LLC ( n = 5). Mice were orally administered GB1107 (10 mg/kg) daily beginning on day 5 until the mice were sacrificed on day 19. ( B ) Tumor size of each group. ( C ) Tumor weight of each group. ( D ) Tumor growth rate of each group. ( E ) Schematic illustration of an immunocompetent orthotopic lung cancer model using LLC-luc ( n = 3). Mice were orally administered GB1107 (10 mg/kg) daily beginning on day 5 until the mice were sacrificed on day 19. ( F ) Representative images of tumorigenesis in each group. ( G ) Representative HE staining of lung tissues from each group and statistical analyses of tumor nodules. ( H ) In vivo IVIS images of orthotopic lung cancer in each group at corresponding time points and results of quantitative fluorescence analyses. ( I ) Survival rate of orthotopic lung cancer in each group ( n = 8). ( J ) In the orthotopic lung cancer model, the percentage of tumor-infiltrating macrophages was analyzed using flow cytometry. ( K-L ) Representative flow plots ( K ) and quantification ( L ) of CD206, Nos2, CD80, CD86, MHC I and MHC II in tumor-infiltrated macrophages in each group. The data is displayed as MFI. Data represent mean ± SD from three experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Article Snippet: Tyrosine kinase inhibitor (Genistein) (absin, Shanghai, China), Syk inhibitor (R406) (Selleck Chemicals, Houston, USA), Src inhibitor (PP2) (Solarbio, Beijing, China), TREM2 Fc (R&D Systems, Minnesota, USA), GB1107 (Selective Galectin-3 inhibitor) (TargetMol, Boston, Massachusetts), Recombinant mouse Galectin-3 protein (rm Galectin-3) (R&D Systems, Minnesota, USA).

Techniques: Staining, In Vivo, Fluorescence, Flow Cytometry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Galectin-3 induces pathogenic immunosuppressive macrophages through interaction with TREM2 in lung cancer

doi: 10.1186/s13046-024-03124-6

Figure Lengend Snippet: Combination of the Galectin-3 inhibitor and TREM2 deficiency enhanced the infiltration and functionality of anti-tumor CD8 + T and NK cells. ( A ) In the orthotopic lung cancer models, the percentage of anti-tumor CD8 + T and NK cells were analyzed using flow cytometry. ( B-C ) Representative flow plots and quantification of granzyme B ( B ) and perforin ( C ) producing CD8 + T cells in TME. ( D-E ) Representative flow plots and quantification of granzyme B ( D ) and perforin ( E ) producing NK cells in TME. ( F ) The concentrations of perforin and granzyme B in the tumor tissue grinding supernatant were detected by ELISA. ( G ) A propose model to illustrate the mechanism of galetin3-TREM2 axis in promoting lung cancer progression. Data represent mean ± SD from three experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Article Snippet: Tyrosine kinase inhibitor (Genistein) (absin, Shanghai, China), Syk inhibitor (R406) (Selleck Chemicals, Houston, USA), Src inhibitor (PP2) (Solarbio, Beijing, China), TREM2 Fc (R&D Systems, Minnesota, USA), GB1107 (Selective Galectin-3 inhibitor) (TargetMol, Boston, Massachusetts), Recombinant mouse Galectin-3 protein (rm Galectin-3) (R&D Systems, Minnesota, USA).

Techniques: Flow Cytometry, Enzyme-linked Immunosorbent Assay