MOESM5 of Galectin-3: an early predictive biomarker of modulation of airway remodeling in patients with severe asthma treated with omalizumab for 36Â months

Additional file 5. Changes of abundance levels, expressed as natural logarithm of score fold change (ln[T36/T0]), for Gal-3 in OR and NOR patients at baseline (T0) and after 36 months (T36) of anti-IgE treatment. Negative value indicates decrease of Gal-3 at T36; on the contrary, positive value indicates increase of Gal-3 at T36 (see Additional file 1). OR and NOR classification is related to reduction (OR) or not (NOR) of RBM thickness after 12 months of anti-IgE treatment

Preclinical Development of a Novel Class of CXCR4 Antagonist Impairing Solid Tumors Growth and Metastases

<div><p>The CXCR4/CXCL12 axis plays a role in cancer metastases, stem cell mobilization and chemosensitization. Proof of concept for efficient CXCR4 inhibition has been demonstrated in stem cell mobilization prior to autologous transplantation in hematological malignancies. Nevertheless CXCR4 inhibitors suitable for prolonged use as required for anticancer therapy are not available. To develop new CXCR4 antagonists a rational, ligand-based approach was taken, distinct from the more commonly used development strategy. A three amino acid motif (Ar-Ar-X) in CXCL12, also found in the reverse orientation (X-Ar-Ar) in the vMIP-II inhibitory chemokine formed the core of nineteen cyclic peptides evaluated for inhibition of CXCR4-dependent migration, binding, P-ERK1/2-induction and calcium efflux. Peptides R, S and I were chosen for evaluation in <i>in vivo</i> models of lung metastases (B16-CXCR4 and KTM2 murine osteosarcoma cells) and growth of a renal cells xenograft. Peptides R, S, and T significantly reduced the association of the 12G5-CXCR4 antibody to the receptor and inhibited CXCL12-induced calcium efflux. The four peptides efficiently inhibited CXCL12-dependent migration at concentrations as low as 10 nM and delayed CXCL12-mediated wound healing in PES43 human melanoma cells. Intraperitoneal treatment with peptides R, I or S drastically reduced the number of B16-CXCR4-derived lung metastases in C57/BL mice. KTM2 osteosarcoma lung metastases were also reduced in Balb/C mice following CXCR4 inhibition. All three peptides significantly inhibited subcutaneous growth of SN12C-EGFP renal cancer cells. A novel class of CXCR4 inhibitory peptides was discovered. Three peptides, R, I and S inhibited lung metastases and primary tumor growth and will be evaluated as anticancer agents.</p></div

Peptides R, I, and S inhibit the growth of primary human SN12C-pEGFP tumors.

<p>6 to 8-week-old female CD1 nude mice were injected subcutaneously with 2×10⁶ pEGFP-SN12C cells pre-treated with 10 µM AMD3100 or 10 µM of each peptide. Beginning the following day mice were further treated intraperitoneally for 10 days with 1.25 mg/kg AMD3100 or 2 mg/kg of peptides R, I or S. A week later mice were euthanized and tumor volume evaluated directly from the flank of mice using a Leica MacroFluo fluorescence stereomicroscope (Leica microsystem, Houston, TX) (A). The experiments were repeated three times. B. SN12C-PEGFP cells migration toward CXCL12 in the presence of AMD3100 or Peptide R, S and I.</p

Peptides R, I and S inhibit murine melanoma lung metastases.

<p>(A) B16-CXCR4 tumor cells pre-treated for 30 minutes with AMD3100 (10 µM), peptide R (10 µM), peptide I (10 µM), or peptide S (10 µM) were inoculated into the tail vein of C57/B female mice and the mice were further treated intraperitoneally for 10 days with 1.25 mg/kg AMD3100, or 2 mg/kg peptide R, peptide I or peptide S. Gross examination of representative lungs; (B) Graphical representation of the number of lung metastases in treated mice. Double tailed T-Test was used for statistical analyses. The experiments were repeated three times.</p

Peptides R, I, S, T impair 12G5-CXCR4 binding and CXCL12-induced Ca²⁺ influx in CCRF–CEM cells.

<p>(A) The amount of peptides or AMD3100 bound was assessed indirectly by flow cytometry using the PE-labelled anti-CXCR4 antibody (clone 12G5). Results are expressed as the percent antibody bound. (B) The Ca²⁺ influx assay was performed using CCRF-CEM cells and Fluo-3 AM calcium indicator. CCRF-CEM cells were incubated 30 minutes at 37°C with Fluo-3 AM and 15 minutes with AMD3100 (10 µM)/peptides (10 µM) and then treated with CXCL12. Results are expressed as the percent of Fluo-3 AM fluorescence in presence of CXCL12 alone. Each peptide was tested in at least three different experiments. Double tailed T-Test was used for statistical analyses. Differences were considered significant at <i>P</i><0.05 compared to control. Control (CTRL).</p

Peptides R, I and S inhibit murine osteosarcoma lung metastases.

<p>(A) Twenty-five 6-8-week-old female Balb/c mice were injected via tail vein with 2.5×10⁵ K7M2 cells pre-treated for 30 minutes with AMD3100 (10 µM), peptide R (10 µM), or peptide I (10 µM) or peptide S (10 µM). The animals were then further treated intraperitoneally for 15 days with 2.5 mg/kg AMD3100 or 10 mg/kg peptide R, peptide I or peptide S. (B) Graphical representation of the number of lung metastases in treated mice. Double tailed T-Test was used for statistical analyses. The experiments were repeated three times.</p

Peptides R, I, S, T inhibit CXCL12 dependent cell migration, wound healing and p-ERK induction.

<p>Experiments were conducted using PES43 cells. (A) Migration was assayed in 24-well Transwell chambers using inserts with 8-µm pore membrane. PES43 cells were placed in the upper chamber (2.5×10⁵ cells/well) in IMDM containing 1% BSA (migration media) in the presence of AMD3100 (10 M) or peptides at several concentrations (10 nM, 100 nM, 1 µM, 10 µM); 100 ng/mL CXCL12 was added to the lower chamber. Migrated cells on the lower surface were fixed, stained with H&E and counted microscopically. The results are expressed as the migration index relative to migration in presence of BSA alone. (B) Delay in wound healing after 6 hours in presence of peptides R, I, S ant T compared with CXCL12. Images were acquired with OKO Time Lapse. (C) Effect of peptides R, I, S, and T on CXCL12 p-ERK induction at 5 minutes. PES43 cells were serum-starved and incubated with CXCL12 (100 ng/ml) alone or in presence of AMD3100/peptides. Each peptide was tested in at least three different experiments. Double tailed T-Test was used for statistical analyses. Differences were considered significant at <i>P</i><0.05 compared to control.</p

Peptide sequences, one-letter code names and shortened sequence notations.

*<p>Square brackets indicate cyclization via disulfide-bridge.</p>**<p>Ac = Acetyl,Nam = amide NH₂.</p