Progesterone increases basal ductal progenitor activity.

<p>(<b>A</b>) Mammospheres formed from mammary epithelial cells (MECs) expressed estrogen receptor alpha (ERα) and progesterone (PR) receptor. (<b>B</b>) Mammospheres treated with E2+P4 significantly upregulated PR expression. PR expression was detected by qPCR (n = 3 patient samples; mean±s.d.). (<b>C</b>) 17β-estradiol (E2) + progesterone (P4) treatment significantly enhanced MEC mammosphere numbers (n = 9 patient samples; mean±s.e.m.). (<b>D</b>) P4 and E2+P4 treatment significantly enhanced secondary mammosphere numbers (n = 6 patient samples; mean±s.e.m.). (<b>E</b>) E2+P4 treatment of MECs significantly increased floating colony growth (n = 9 patient samples; mean±s.e.m.). (<b>F</b>) E2+P4 treatment of MECs significantly enhanced the growth of bi-potent (CK8/14⁺) colonies in adherent culture (n = 3 patient samples; mean±s.e.m.). (<b>G</b>) On collagen, E2, P4, and E2+P4 significantly increased acinar colonies. P4 significantly enhanced ductal colonies (n = 9 patient samples; mean±s.e.m.). (<b>H</b>) E2, P4, and E2+P4 significantly increased acinar colonies. MECs were grown as floating colonies, then plated with hormones on collagen (n = 3 patient samples; mean±s.e.m.). (<b>I</b>) P4 significantly increased ductal colonies. MECs were grown as mammospheres, then plated on collagen with hormones (n = 7 patient samples; mean±s.e.m.). (<b>J</b>) P4 and E+P significantly increased acinar colonies in mature luminal (ML; EpCAM⁺CD49f⁻) cells, and P4 significantly increased ductal colonies in luminal progenitor cells (LPC; EpCAM⁺CD49f⁺), mature basal (MB; EpCAM⁺CD10⁺), and basal progenitor cells (BPC; EpCAM⁻CD10⁺). MECs were sorted and grown on collagen in the presence of hormones (n = 3 patient samples; mean±s.e.m.). Data are represented as a fold change compared to the vehicle control for each cellular population multiplied by the percentage of total acinar or ductal colonies formed. (<b>K</b>) ERα expression was enriched in ML cells; PR expression was enriched in ML and BPCs. MECs were sorted and qPCR performed (n = 6 patient samples; mean±s.d.). (<b>L</b>) PR expression was present in the basal epithelial layer adjacent to p63 expressing epithelial cells (inset). Basally located PR expressing cells also expressed CK8 (inset). (<b>M</b>) Primary epithelial cells (MEC) were isolated from reduction mammoplasty tissues, transduced with GFP lentivirus, and grown in the humanized fat pads of ovariectomized NOD/SCID mice treated with E2, P4, E2+P4, or placebo pellets. Glands from P4 and E2+P4 treated mice demonstrated increased growth of ductal structures (inset; n = 3 experiments). Scale bars = 100 µm.