Fig 1: MDP-induced metastasis through M2 macrophages is dependent on IL-6 pathway. (A) A schematic representation of MDP adoptive transfer experimental design is shown. GFP-expressing mice implanted with met-high melanoma cells were treated with either IgG control or anti-IL-6 antibodies twice weekly. At end point, FACS-sorted MDPs were intravenously injected into naïve recipient mice (n=5 mice/group). The next day, the recipient mice were implanted with met-low melanoma cells. At end point, mice were sacrificed, and tumors and lungs were extracted. (B) Tumor weights are shown. (C) Representative images of lung sections are shown, bar=100 µm. Arrows indicate metastatic foci. (D) Metastatic foci per lung section were quantified (n=5–7 sections per group). (E–H) GFP+ cells in single-cell suspensions of lung samples were evaluated by flow cytometry to quantify the percentage of dendritic cells (E), total macrophages (F), M1 macrophages (G), and M2 macrophages (H). (I) A schematic representation of MDP versus GMP adoptive transfer is shown. GFP-expressing mice were implanted with met-high melanoma cells. At end point, MDPs or GMPs were sorted by FACS and were subsequently intravenously injected into naïve recipient mice (n=4 mice/group). The next day, the recipient mice were implanted with met-low melanoma cells. At end point, mice were sacrificed, and lungs were removed for the evaluation of metastasis. (J) Representative images of lung sections are shown, bar=100 µm. Arrows indicate metastatic foci. (K) Metastatic foci per lung section were quantified (n=3–4 sections/mouse). Statistical significance was assessed by unpaired two-tailed t-test. Significant p values are shown as *p<0.05; **p<0.01; ***p<0.001. GMP, granulocyte-monocyte progenitor; IL-6, interleukin 6; MDP, monocyte-dendritic progenitor; met-high, high metastatic potential; met-low, low metastatic potential.
Fig 2: Mice lacking ERV1/ChemR23 display increased IL-6 expression. (A) ERV1/ChemR23 mRNA expression in visceral adipose tissue, peritoneal macrophages and liver from wild-type (WT) and KO mice. (B) IL-6 mRNA expression in these tissues from WT and KO mice. The data represent the mean ± SEM of WT (n = 12) and KO (n = 12) mice. *p < 0.05 and **p < 0.01 versus WT.
Fig 3: Increased immunosuppressive macrophages and MDPs correlate with IL-6 expression and aggressive tumors. (A–D) C57BL/6 mice aged 8–10 weeks were implanted with B16 IL-6 overexpressing cells or with corresponding control EV cells. At end point (day 18), mice were sacrificed, lungs were removed, and the bone marrow was harvested (n=4–6 mice/group). (A) Representative images of lung sections are shown, bar=100 µm. Arrows indicate metastatic foci. Metastatic foci per lung section were quantified (n=4–6 sections/mouse). (C) MDP and (D) GMP levels in BM were assessed by flow cytometry. (E) Heat-map of linear correlations in R values between IL-6 expression and M1 or M2 gene signatures as well as single genes CD64 or CD163 representing M1 and M2 subsets, respectively. The data were obtained from human breast cancer samples using the METABRIC data set. (F) Scatter graphs of IL-6 expression in correlation with CD64 or CD163 in human breast cancer samples (METABRIC data set). (G–J) MDP (G, H) and GMP (I, J) levels were analyzed in peripheral blood of patients with breast (G, I) and lung (H, J) cancer segregated based on early and advanced stage disease. Statistical significance was assessed by unpaired two-tailed t-test. Significant p values are shown as *p<0.05. BM, bone marrow; EV, empty vector; GMP, granulocyte-monocyte progenitor; IL-6, interleukin 6; MDP, monocyte-dendritic progenitor; PB, peripheral blood.
Fig 4: Overexpression of ERV1/ChemR23 in omental adipose tissue from obese individuals carrying the C allele. (A) Immunohistochemical staining of ERV1/ChemR23 protein in omental adipose tissue of obese patients carrying the TT (n = 16), TC (n = 16) and CC (n = 17) genotypes. Representative images with a 200X magnification are shown (left). Quantification of the percentage of the positive staining area is shown on the right. (B) Real-time PCR analysis of ERV1/ChemR23 mRNA expression. (C) IL-6 mRNA expression (D) CD68 mRNA expression. The data represent the mean ± SEM.
Fig 5: Circulating levels of cytokines in obese individuals carrying the different ERV1/ChemR23 genotypes. Plasma levels of IL-6 (A), IFN-α2, IL-15 (B), IL-1ra, IL-10 (C), GM-CSF and G-CSF (D), and VEGF (E) determined by the Luminex assay according to the ERV1/ChemR23 SNP genotype. (F) IL-6 expression in polymorphonuclear leukocytes isolated from non-C carrier and C-carrier individuals incubated with LPS (100 ng/ml) in the absence or presence of RvE1 (10 nM) for 2 h. The data represent the mean ± SEM of the individuals with TT (n = 16), TC (n = 16) and CC (n = 17) genotypes. *p < 0.05 and **p < 0.001 versus TT.
Supplier Page from Abcam for Mouse IL-6 ELISA Kit