For 3D mono-culture of normal mammary epithelial cells, MCF10A cells at the density of 2.5 × 104 were seeded on top of 200 μL polymerized Matrigel (BD Biosciences, Franklin Lakes, NJ, USA, growth factor reduced) per well of a 4-well plate (1.9 cm2/well) and covered with the growth medium containing 4% Matrigel as described [33]. Cultures were maintained for 10–21 days with addition of fresh medium on alternate days [33]. For 3D mono-culture of mammary fibroblasts (MFs), cells were grown in Matrigel: collagen I (1:1) mixture in the same manner as 3D co-culture (see below) [24]. To determine the effects of the inhibition of NO production, 3D cultures were kept under the treatment of 2.5 mM L-NAME (NOS inhibitor, Nω-Nitro-L-arginine methyl ester hydrochloride, Sigma-Aldrich, St. Louis, MO, USA), vehicle control (PBS) or NO synthase agonist L-arginine (2.5 mM) for 2–3 weeks with medium change every other day [34].

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