Primary macrophage conditioned media stimulates primary breast fibroblast matrix metalloproteinase (MMP)-9 secretion. Primary human macrophages were treated +/- estrogen (10-10 M) in 2% charcoal-stripped serum-containing media and 24 hr-conditioned media were collected. Primary human breast fibroblasts were placed in 2% charcoal-stripped serum-containing media 24 hr prior to treatment with 2% charcoal-stripped serum, control media + estrogen (10-10 M), macrophage-conditioned media, or conditioned media from macrophages treated with estrogen. (A) Gelatin zymography. (B) Western blot analysis. Results are representative of a minimum of three independent experiments with different fibroblasts and macrophage isolations. (C) Murine mammary glands were humanized and +/- the addition of primary human macrophages and exogenous estrogen supplementation. Immunohistochemical analysis using a human-specific MMP9 antibody of paraffin-embedded histosections glands six days post-humanization. Scale bar = 200 μM. FB, fibroblast conditioned media; ϕ, macrophage conditioned media; E2, estrogen treatment.