Fig 1: Soluble CSF-1R-Fc is efficient to trap both IL-34 and CSF-1 and avoid CSF-1R downstream signalization pathways. (A–B) Different formats of CSF-1R, D1-D3 versus D1-D5, monomeric or dimeric through an Fc, were tested. Increasing concentration of each form was pre-incubated with IL-34 (A) or CSF-1 (B), then mixed with THP-1 cells to measure the intracellular level of phosphorylated Akt. Results were normalized against no cytokine (0%) and IL-34 or CSF-1 alone (100%) conditions, and expressed as mean±SEM, n=4 experiments. (C–D) sCSF-1RD1-D5-Fc efficacy to trap either recombinant IL-34 (C) or recombinant CSF-1 (D) was confirmed, in comparison with respective blocking antibodies, on two CSF-1R pathways, Akt and ERK. Results were normalized against no cytokine condition (0%) and IL-34 or CSF-1 alone (100%), expressed as mean±SEM; n=8 experiments; Tukey’s multiple comparisons test; ****p<0.0001. CSF-1R, colony stimulating factor-1 receptor; IL, interleukin.
Fig 2: sCSF-1RM149K-Fc was more efficient than sCSF-1RWT-Fc to inhibit CSF-1 and IL-34 present in pleural effusions of patients with mesothelioma. (A) CSF-1 and IL-34 quantification in pleural effusion and classification of pleural effusions based on the production of IL-34: CSF-1+IL-34+ (circle) versus CSF-1+IL-34− (square). (B–C) Monocytes, sorted from fresh human peripheral blood mononuclear cell, were cultured for 3 days with pleural effusions from patients with pleural mesothelioma, then cell viability was analyzed. Pleural effusions were divided into two categories: CSF-1+IL-34– (B) and CSF-1+IL-34+ (C) based on the production or not of IL-34. Results were expressed as mean±SEM, n=8 experiments using three donors; Tukey’s multiple comparisons test; *p<0.05 **p<0.01 ***p<0.001. CSF-1R, colony stimulating factor-1 receptor; IL, interleukin; mAb, monoclonal antibodies; PE, phycoerythrin.
Fig 3: Characterization of Meso13/ and Meso34/monocytes, MCTS and monocytes inhibition by sCSF-1R-Fc. Meso13 and Meso34 cells, as well as monocytes from healthy donors, were seeded in a low adherence 96-round well plate for 5 days. (A–B) Confocal microscopy pictures after labeling macrophages of Meso34/monocytes MCTS (A) or Meso13/monocytes MCTS (B) with anti-CD45 (left panel, magenta) or anti-CD163 (right panel, green) antibodies. Hoechst (blue) was used to label cell nuclei. Scale bar=100 µm. (C–D) Volcano plot of the differentially expressed mRNAs between Meso34 (C) or Meso13 (D) MCTS without versus with monocytes. Blue dots: p<0.05, gray dots: p>0.05. Dotted lines: gene regulated with a log2 fold change ≥1 or ≤−1. (E–F) mRNA were extracted from Meso34/monocytes MCTS (E) or Meso13/monocytes MCTS (F) and the expression of macrophage markers and IL-10 was measured using real-time PCR. Bar graphs represent mean±SEM of 3 independent experiments. T test *p<0.05; **p<0.01 and ***p<0.001. CSF-1R, colony stimulating factor-1 receptor; IL, interleukin; MCTS, multicellular tumor spheroid; mRNA, messenger RNA.
Fig 4: sCSF-1R-Fc efficacy to trap both IL-34 and CSF-1 to inhibit monocyte viability can be improved by a single amino acid mutation. (A–B) Ribbon representations of IL-34/CSF-1R (A, PDB code 4DKD) and CSF-1/CSF-1R (B, PDB code 4WRL) complexes and close views of the interaction sites showing the amino acids in the vicinity of the residue M149 of CSF-1R. (C–D) Monocytes, isolated from human peripheral blood mononuclear cells were cultured 3 days in the presence of 4 nM of IL-34 (C) or 4 nM of CSF-1 (D) with sCSF-1RWT-Fc or sCSF-1RM149K-Fc at different concentrations. Cell viability was analyzed, and results were expressed as % of live cells, mean±SEM, n=6/8 experiments using 3/4 donors. Tukey’s multiple comparisons test; *p<0.05 ****p<0.0001. CSF-1R, colony stimulating factor-1 receptor; IL, interleukin.
Supplier Page from R&D Systems, a Bio-Techne Brand for Recombinant Human M-CSF R/CD115 Fc Chimera Protein