Extended Data Figure 2 : Progesterone regulates migration and is linked to branching morphogenesis.

From: Early dissemination seeds metastasis in breast cancer

Extended Data Figure 2

a, qPCR analysis of Pgr, Rank (also known as Tnfrsf11a), Rankl and Wnt4 in normal and transgenic mouse mammary tissue or tumours. Note the increased expression of Pgr, Wnt4 and Rankl in early lesions compared to primary tumours. Only Rank (the Rankl receptor) is strongly expressed in primary tumours. b, Primary cultures of early lesions treated with progesterone, WNT4, and RANKL. WNT4 and RANKL treatment induce the early lesion signature and act synergistically. c, d, PGR (green) staining at 5 and 12 weeks of age (FVB wild-type mice). Scale bars, 100 μm. The percentage of PGR+ cells per duct was quantified (n = 2 mice per age group) in the distal and proximal portions of the gland (relative to the origin). PGR-expressing cells were more abundant in distal ducts (number of analysed ducts per group is displayed above each column). LN, lymph node.e, f, Photomicrograph of migration assay (e) and quantification of migrating cells (f) derived from fresh tissue (e, left) or dissociated spheres derived from primary tumours or early lesions (e, right; f, quantification). Progesterone, WNT4 and RANKL induce migration of early-lesion-derived but not primary-tumour-derived cells (see also Fig. 2a). g, Scheme of combined migration and sphere assay. The lower chamber is filled with serum-free sphere medium and the bottom is covered with poly-HEMA to prevent adhesion and enable sphere formation. After 72 h migration, the insert is removed and the lower chamber is analysed (after 11 days) for mammosphere formation (see Methods). h, Effect of oestrogen and progesterone on migration and sphere formation of mammary cells derived from early lesions. Cells were exposed to 10 nM oestrogen or progesterone or 10 nM oestrogen + 10 nM progesterone inhibitor (RU486). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001 (Student’s t-test); Data are mean ± s.e.m. (d) or mean ± s.d. (other panels).