Fig 2: CSF2 activates Stat3 to upregulate CXCL3 expression in adipocytes co-cultured with breast cancer cells. (A) The mRNA expression levels of CSF2Rα and CSF2Rβ in adipocytes co-cultured with MDA-MB-231 cells were measured by q-PCR. (B and C) Cytokine expression (B) and secretion (C) in adipocytes treated with rhCSF2 (20 ng/ml) were detected by q-PCR and ELISA, respectively. (D) The mRNA expression levels of IL-6, CSF2, and CXCLs in normal and paracancerous adipose tissues. (E) Adipocytes were stimulated with rhCSF2 for 12 h and then treated with stattic (10 μM). The mRNA expression levels of CXCLs, IL-6, and IL-1β in adipocytes were detected. (F) Adipocytes were treated with rhCSF2 and stattic for 15 min, and Stat3 phosphorylation was analyzed by western blotting. (G and H) Adipocytes were co-cultured with MDA-MB-231 cells in the presence or absence of CSF2-neutralizing antibody (2 μg/ml). The expression of CXCLs (G) and Stat3 phosphorylation (H) in adipocytes were analyzed by q-PCR and western blotting, respectively. ###P < 0.001, ##P < 0.01, and #P < 0.05 compared to the control group; ***P < 0.001, **P < 0.01, and *P < 0.05 compared to experimental group; n = 3.

Fig 3: Transwell migration assay of macrophages. Cultured B16F10 cells in the lower chamber were treated with three different titers (MOI 1, 10, or 100) of wild type T7 (WT T7) or T7 displaying the homing peptide and harboring an mammalian expression cassette of GM-CSF (T7-pep42_G). Macrophages (RAW264.7) in the upper chamber were allowed to migrate for three different time periods. (A) Staining and visualization of membrane after migration. White pores are seen from the membrane and macrophages are stained with crystal violet. (B) Three random fields were chosen and the migrated cells were quantitated. For statistical analysis Tukey’s test was performed. *Above each vertical bar indicates statistical significance of each test to the control. *Above each horizontal bar indicates statistical significance of each test between corresponding pairs.

Fig 4: Phage preparation, homing, and expression of granulocyte macrophage-colony stimulating factor (GM-CSF) in vitro. (A) Removal of endotoxin after phage preparation. SM buffer was used as a control. (B) Cell viability assay of B16F10 cells after exposure to bacteriophages. Two different concentrations (multiplicity of infection of either 10 or 100) of native T7 or engineered T7 were added to the culture and incubated for 24 h before MTT assay was performed. Control was treated with SM buffer. (C) Homing and internalization of phage particle, and nuclear localization of phage DNA. First and second row: wild type phage T7 (WT T7) or T7 displaying the homing peptide (T7-pep42) was added to in vitro cultured B16F10 melanoma cells and binding was observed under a fluorescent laser scanning confocal microscope. The nucleus was stained with DAPI (blue) and the phage particle was stained with anti-T7 antibody (green). Third row: B16F10 cells were first treated with anti-Grp78 antibody (red) to mask the receptor for pep42. Then T7-pep42 (green) was added and binding was observed. Fourth row: T7-pep42 was produced in the presence of BrdU (green) to label the genomic DNA and added to cultured B16F10 cells. Internalized phage DNA to DAPI (blue) stained nucleus is shown. (D) Real-time PCR (RT-PCR) analysis of mRNA encoding GM-CSF from T7-pep42 or T7-pep42_G transduced B16F10 cells. Relative amounts of mRNA encoding GM-CSF from cells treated with T7-pep42 or T7-pep42_G are shown in black or white bars, respectively. Control was treated with SM buffer. T7-pep42, phage T7 displaying pep42 and T7-pep42_G, phage T7 displaying pep42 and expressing GM-CSF. (E) Western blot analysis of GM-CSF from T7-pep42_G transduced B16F10 cells (lane 2) and empty cell (lane 1). GAPDH was used as an internal control. For statistical analysis, one way ANOVA was performed and then Tukey’s test was conducted. *Above each vertical bar indicates statistical significance of each test to the control. *Above each horizontal bar indicates statistical significance of each test between corresponding pairs.

Fig 5: Ligand–receptor genes differentially expressed after treatment with BRAF inhibitor (BRAFi) associated with antihuman VEGFA antibody (anti‐hVEGFA) in A375 melanoma xenograft. (A) Log2‐transformed, normalized expression value of granulocyte‐macrophages colony‐stimulating factor (GM‐CSF) quantified by microarray in control, BRAFi, anti‐hVEGFA and BRAFi + anti‐h‐VEGFA A375 tumors. GM‐CSF ligand is detected in human dataset; n = 3. (B) Immunofluorescence staining for GM‐CSF (red) and F4/80 (green) protein in A375 xenograft treated with BRAFi in combination with anti‐hVEGFA. (C) Quantification of GM‐CSF expression determined by immunofluorescence staining in A375 tumors treated as indicated. Bar graphs indicated the GM‐CSF+ area/tumor area; n = 3. Scale bar, 40 μm. Data are presented as means ± SEM. Significance was assessed by and Student's t‐test (A) and one‐way ANOVA test followed post hoc pairwise analysis test (C); *P < 0.05, **P < 0.01.