To determine whether metformin inhibits the stemness of breast cancer cells through the suppression of the Shh signaling pathway, the MDA-MB-231 cells were cultured at a very low density (1 cell/μl) in 96-well plates containing serum-free medium with rhShh (1 μg/ml) alone, metformin (3 mM) alone, or a combination of both for 7 days. The gene discussed is SHH; the disease is breast cancer.