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: Fig. 3 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 concentrat ed 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 ex amined 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: Agonists and inhibitors 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: Fig. 4 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 transcrip tion 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: Agonists and inhibitors 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: Fig. 7 Combination therapy of the galectin-3 inhibitor GB1107 and TREM2 deficiency significantly inhibit lung cancer progression and reduced the im munosuppressive 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 quan titative 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: Agonists and inhibitors 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: Fig. 8 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: Agonists and inhibitors 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