Fig 1: Therapeutic effects of Gal3 blockade in vivo. Kaplan–Meier survival curves for mice implanted with SKOV3c344, A2780c344, or MDA-MB-231 tumors are shown. (a) For mice implanted with SKOV3c344 tumors, the median survival time for the untreated group was 49 days (95% CI, 45.3–52.6), while the median survival for the group treated with 14D11 and for the group implanted with MUC16 negative SKOV3 cells was not reached after 56 days when the experiment was terminated (p = 0.001). (b) Average tumor growth for animals implanted with SKOV3 cells. (c) For the group implanted with A2780c344 cells, the median survival time for the untreated group was 22 days (95% CI, 19.6–24.4), while the median survival time for the group treated with 14D11 was 31 days (95% CI, not estimable), and median survival was not reached for animals implanted with MUC16 negative A2780 cells (p = 0.0164). (d) Average tumor growth for animals implanted with A2780 cells.
Fig 2: Anti-Gal3 monoclonal antibody, 14D11, inhibits MUC16-mediated tumorigenesis in vitro. (a) Direct ELISA for Gal3 binding to selected antibodies in the presence or absence of lactose. The presence of increasing lactose concentrations progressively inhibited the binding of Gal3 to 14D11, an isotype control antibody (18C6) and 1F5, an antibody reactive to the N terminal 15 amino acids of Gal3. Each lactose condition was compared to the zero lactose condition for the same antibody by paired t-test. (b) Effect of 14D11 on MUC16-mediated activation of ERK/AKT signaling in MUC16-expressing A2780c344 and SKOV3c344 cells after 48-h exposure to antibody. MUC16-negative A2780 and SKOV3 cells were used as controls. Densitometry was used to normalize the results against β-actin. (c) Effect of 14D11 on Matrigel invasion in MUC16-overexpressing SKOV3c344 and A2780c344 cells, and endogenous MUC16-expressing OVCAR3 cells. Anti-MUC16 N-glycosylation site antibody, 18C6, was used as a positive control. The values are the mean of triplicate well from a representative study and the invasion assays were repeated independently ≥ 3times. (d) Effect of 14D11 on Matrigel invasion in MDA-MB-231 and shLGALS3-MDA-MB-231 cells, performed as in 3C.
Fig 3: Knockdown shLGALS3-MDA-MB-231 cells show decreased invasiveness both in vitro and in vivo. (a) LGALS3 knockdown cell line, shLGALS3-MDA-MB-231 and a similar LGALS1 knockdown, shLGALS1-MDA-MB-231 were generated. LGALS3 and LGALS1 silencing was confirmed by Western blot in a manner previously described23. β-Actin normalized densitometry quantification values are shown below each Western blot band. (b) In a Matrigel assay using triplicate chambers with 1 × 104 cells/chamber, invasion of the shLGALS3-MDA-MB-231 and shLGALS1-MDA-MB-231 were compared to the parental MDA-MB-231 wild-type cell line after 48 h (p = 0.005, unpaired t test; error bars represent standard error). Three replicates were performed. (c) Representative luminescence images of all mice inoculated intravenously (by tail vein) with 5 × 106 shLGALS3-MDA-MB-231 cells or wild-type MDA-MB-231 control cells as previously described31. (d) Kaplan–Meier survival curves; median survival for mice (n = 10 female athymic nude mice) implanted with MDA-MB-231 wild-type cells was 60 days (95% CI, 53.8–66.2), while median survival for mice (n = 10 female athymic nude mice) implanted with shLGALS3-MDA-MB-231 cells was not reached (p = 0.02, log-rank test).
Fig 4: Generation of anti-Gal3 monoclonal antibody. (A) ELISA screening strategy for selecting candidate antibodies. The desired antibody screened positive for human Gal3, Peptide 1, his tagged Gal3, and mouse Gal3. It screened negative for human Gal 1, 7, 8, 9. A second antibody targeting N-terminus screened positive for human Gal3 and Peptide 5. (B) Galectin sequences. The carbohydrate binding domain is in red, the 23 amino acid sequence of the CBD that is conserved among species is highlighted in blue. Murine homolog is highlighted in magenta. Peptide 5, the N-terminus sequence, is highlighted in green. (C) Antibodies generated from campaigns 1, 2, and 3. (D) Gal3 crystal structure with N-acetyllactosamine (PDB: 4XBN), pink color: the last 6 amino acids (245-ASYTMI -250) which were omitted for 14D11 antibody screening, 5 amino acids (H158, N160, R162, N174, and E184) are participated in the LacNAc binding and green color is LacNAc. The solvent accessible area of sugar-binding pocket is approximately ~ 400 Å. (E) Computationally predicted VH and VL domain structure of 14D11, blue colors: 3 CDR regions of VH, purple colors: 3 CDR regions of VL, each of VH or VL has one disulfide bridge (orange color: Cys-Cys) and the solvent accessible area is approximately ~ 2500 Å. (F–H) The predicted docking model between Gal3 and 14D11 variable regions with 90 degree rotations, suggesting (1) the variable regions of 14D11 is positioned at a sugar binding pocket and (2) CDR3 of VH and CDR1, CDR2 of VL are occupying the carbohydrate-binding. pocket. Olive color of Gal3: peptide 1 regions (aa: 173-CNTKLDNNWGREERQSVFPFESG-195), which are conserved and a part of the sugar binding pocket.
Fig 5: Therapeutic effects of Gal3 blockade on tumor metastases. (A) For the mice implanted with MDA-MB-231 cells, median survival for the untreated group was 99 days (95% CI, 76.2–121.8), while for group treated with 14D11, median survival was 115 days (95% CI, 56.1–173.9) (p = 0.081). (B) Representative luminescence images of mice at 1, 20, and 34 weeks.
Supplier Page from Abcam for Recombinant human Galectin 3 protein (Active) (His tag N-Terminus)