ER-positive human breast cancer ZR-75-1/GFP/puro cells, which we know are non-tumorigenic and non-metastatic without exogenous estrogen supplementation in nude mice, were then injected intracardiacally into mice bearing growing tumors formed by MDA-MB-231 or MDA-MB-435-F-L cells two and half weeks after the orthotopic inoculation. This evidence concerns the gene ESR1 and breast cancer.