Fig. 5. NHWD-870 downregulated CSF1 expression in tumor cells to inhibit TAM proliferation.

a, b NHWD-870 inhibited tumor cell and TAM proliferation in vitro. A2780 tumor cells were pretreated with 100 nM NHWD-870 and then directly co-cultured with TAMs (A2780:TAMs ratio as 4:1) at the density of 1 × 10⁵ total cells in a 12-well. Shown are immunofluorescent staining of Ki67 and CD68 (a) and quantification of Ki67+ and CD68− or CD68+ cells (b). Scale bar is 50 μm. Data are presented as mean ± SEM from three independent experiments. c–e NHWD-870 significantly inhibited tumor cell supported TAM proliferation. A2780 cells (pre-treated with or without 100 nM NHWD-870 for 48 h) were seeded into the top chamber (transwell size: 0.4 μm) and TAMs (Mac, 40,000 cells per 24-well) in medium containing 2% matrigel were seeded into the bottom chamber pre-coated with Matrigel. No NHWD-870 was added to the co-culture. The upper chamber without A2780 cells was used as a control. Shown are schematics of transwell co-culture experiment (c), representative pictures of TAM spheroid (d: black/white), and quantification of spheroid volumes (e). Scale bar is 50 μm. Data are presented as mean ± SEM from 15 independent experiments. f RT-qPCR analyses of relative CSF1 mRNA level in ovarian cancer cells (ID8, A2780, SKOV3, and ES-2) and melanoma cells (B16, YUSOC, YUGASP, YUAME, YUMAC, and A375) treated with 50 nM NHWD-870 for 48 h. Data are presented as mean ± SEM from three independent experiments. g CSF1 protein levels in supernatant of 10⁶ ID8, B16, A2780, SKOV3, and A375 cells treated with DMSO or 25 nM NHWD-870 for 24 h, as measured by ELISA. Data are presented as mean ± SEM from three independent experiments. h Representative immunofluorescent staining of CSF1 in DMSO or 100 nM NHWD-870 treated A2780 cells. Scale bar is 20 μm. i Quantification of CSF1 staining in DMSO or 100 nM NHWD-870 treated A2780 cells. Data are presented as mean ± SEM from three independent experiments (6 fields per sample). j, k NHWD-870 significantly reduced CSF1 expression in tumors from A2780 tumor-bearing mice treated with vehicle or NHWD-870 for 15 days. Shown are representative immunofluorescent staining of CSF1 (green) and CD68 (red) (j) and quantification of CSF1 staining (k). Data are presented as mean ± SEM from five different tumors. p values were calculated using two-tailed, unpaired t tests in this figure. **p < 0.01; ***p < 0.001. Source data are provided as a Source Data file.