Additional file 1: Figure S1. of Targeting CXCR4 by a selective peptide antagonist modulates tumor microenvironment and microglia reactivity in a human glioblastoma model

In vivo and ex vivo imaging. In vivo fluorescence images obtained using an IVIS Spectrum bioluminescent and fluorescent imaging system from Xenogen (IVIS Lumina II). Excitation and emission values (640 nm, 700 nm, respectively) were kept constant, and exposure times 7 s were used. A) Example of tumor-bearing mouse at 21th of growth, fluorescence imaging observed at 1 h 30’ after of peptide R (conjugates to VIVOTAG-S 750 fluorochrome) i.v. injected. The animal was placed dorsally under anaesthesia in a light-tight chamber. After final in vivo fluorescence imaging, the animals were euthanized, and the brain of animals were carefully excised, the intensity of fluorescence was increased in brain with tumor (B) respect to the healthy brain (C). Light emitted from the animal/brain appears in pseudocolor scaling. D) Representative multi-slice coronal postcontrast-enhanced (Gd) T1-weighted MR images (4.7 T) of tumor-bearing mouse brain Contrast enhanced-MRI evidences that tumor lesion has almost uniformly disrupted BBB. (TIF 4589 kb

Additional file 2: Figure S2. of Targeting CXCR4 by a selective peptide antagonist modulates tumor microenvironment and microglia reactivity in a human glioblastoma model

A) CD68 expression on U87MG cell line. Representative CLSM analysis of CD68 expression (green) on U87MG cells. Nuclei were stained with DAPI (blue). Scale bars, 47 μM. B) In vivo effect of peptide R on astrocytes reactivity. GFAP (marker of astrocyte reactivity) detection by CLSM (on the left) and immunohistochemistry (on the right) on brain sections of mice treated with peptide R or left untreated (CTRL). Asterisks (*) indicate the tumor-free parenchyma. Scale bars, 30 μM. (TIF 1451 kb

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

Supplementary Figure 1 from Prospective Evaluation of Cetuximab-Mediated Antibody-Dependent Cell Cytotoxicity in Metastatic Colorectal Cancer Patients Predicts Treatment Efficacy

Bland-Altman plot % increase in cetuximab-mediated ADCC: difference SRB assay-Cytotox96 vs average. By plotting the difference between the % ADCC increase determined by each of the methods against the mean of the two determinations it is possible to determine the concordance between both methods. The limits of agreement are also calculated to give a quantitative analysis of the differences between both methods.</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

Additional file 4: of Mutated Von Hippel-Lindau-renal cell carcinoma (RCC) promotes patients specific natural killer (NK) cytotoxicity

Table S3. Detailed characteristics of 28 VHL-WT-RCC patients. (PPTX 71 kb

Ligand-Based NMR Study of C‑X‑C Chemokine Receptor Type 4 (CXCR4)–Ligand Interactions on Living Cancer Cells

Peptide-binding G protein-coupled receptors (GPCRs) are key effectors in numerous pathological and physiological pathways. The assessment of the receptor-bound conformation of a peptidic ligand within a membrane receptor such as a GPCR is of great impact for a rational drug design of more potent analogues. In this work, we applied multiple ligand-based nuclear magnetic resonance (NMR) methods to study the interaction of peptide heptamers, derived from the C-X-C Motif Chemokine 12 (CXCL12), and the C-X-C Chemokine Receptor Type 4 (CXCR4) on membranes of human T-Leukemia cells (CCRF-CEM cells). This study represents the first structural investigation reporting the receptor-bound conformation of a peptide to a GPCR directly on a living cell. The results obtained in the field of CXCL12/CXCR4 are proofs of concept, although important information for researchers dealing with the CXCR4 field arises. General application of the presented NMR methodologies is possible and surely may help to boost the development of new therapeutic agents targeting GPCRs

Ligand-Based NMR Study of C‑X‑C Chemokine Receptor Type 4 (CXCR4)–Ligand Interactions on Living Cancer Cells

Peptide-binding G protein-coupled receptors (GPCRs) are key effectors in numerous pathological and physiological pathways. The assessment of the receptor-bound conformation of a peptidic ligand within a membrane receptor such as a GPCR is of great impact for a rational drug design of more potent analogues. In this work, we applied multiple ligand-based nuclear magnetic resonance (NMR) methods to study the interaction of peptide heptamers, derived from the C-X-C Motif Chemokine 12 (CXCL12), and the C-X-C Chemokine Receptor Type 4 (CXCR4) on membranes of human T-Leukemia cells (CCRF-CEM cells). This study represents the first structural investigation reporting the receptor-bound conformation of a peptide to a GPCR directly on a living cell. The results obtained in the field of CXCL12/CXCR4 are proofs of concept, although important information for researchers dealing with the CXCR4 field arises. General application of the presented NMR methodologies is possible and surely may help to boost the development of new therapeutic agents targeting GPCRs

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