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. 2023 Jan 29;15(3):819. doi: 10.3390/cancers15030819

Figure 3.

Figure 3

Figure 3

Conditioned media from TNBC cells treated with DHEA modulate macrophage recruitment and Tumor-Associated Macrophage phenotype. (A) Schematic representation of the in vitro co-culture experiments to obtain tumor-associated macrophages (TAM). Conditioned media (CM) were collected from both TNBC (MDA-MB-231 and MDA-MB-436) cells treated with vehicle (231-CM and 436-CM) or with DHEA 10 µM for 24 h (DHEA-treated 231-CM and DHEA-treated 436-CM). Human THP-1 monocytes were stimulated with phorbol 12-myristate 13-acetate (PMA, 100 nM) for 14 h followed by 24 h rest to generate macrophage-like cells and then incubated for 5 days with CM as indicated. (B) Trans-well recruitment of THP-1 cells in response to control media (−), 231-CM, 436-CM, DHEA-treated 231-CM, or DHEA-treated 436-CM was assessed after 12 h incubation. The migrated monocytes were stained with DAPI, and five random fields were captured per well with an Olympus microscope at 10× magnification. (C) MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay was performed in THP-1-derived macrophages incubated for 5 days with the control media (−), 231-CM, 436-CM, DHEA-treated 231-CM or DHEA-treated 436-CM. (D) Real-time qRT-PCR assay for matrix metalloproteinase-9 (MMP-9), interleukin (IL)-6, IL-10, monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF) in macrophages incubated as in C. The values represent the mean ± SEM of three different experiments. * p < 0.05; ** p < 0.005; *** p < 0.0005; **** p < 0.0001.