The alpha estrogen receptor (ERα) contributes to breast cancer progression and recent guidelines define ER positivity as ≥1% stained cells, and a few tumor tissues show no ERα expression at all or are at 100%. Although ER and aromatase inhibitors are widely used to treat hormone receptor-positive (HR+) breast cancer, their effect on tumor activity at different ERα levels remains unclear. Therefore, we investigated the role of ERα+/ERα- ratios in determining the ERα level. We used ERα stably transfected and wild-type MDA-MB-231 cells (MDA-MB-231Trans-ER and MDA-MB-231WT, respectively) as represented ER+ and ER- cells, respectively, and MCF-7 cells were the positive control. MDA-MB-231Trans-ER and MDA-MB-231WT cells were mixed and cocultured at a ratio of 0%, 20%, 40%, 70%, and 100%. Migration and invasion functions at different cell ratios were evaluated in vitro using the Transwell and scratch test. In a xenograft mouse model, the polarization of the tumor-associated (M2) macrophage and the expression of breast cancer gene 1 (BRCA1), human epidermal growth factor receptor 2 (HER2), vascular endothelial growth factor (VEGF), and tumor necrosis factor (TNF)-α were measured. The results showed that the cell invasion and migration were significantly higher at 40% and 70% than they were at other ratios. Additionally, in vivo, the 70% ERα+/ERα-ratio was a critical indicator of cell activity and cytokine expression. The highest M2 level and expression of VEGR, TNF-α, BRCA1, and HER2 were shown at a ratio of 70%. Moreover, the effects of ERα were not linear in breast cancer, indicating that the ERα status requires continuous monitoring during long-term endocrine treatment. These results indicate that during HR+ breast cancer treatment, the ERα+/ERα- ratio may be a useful predictor and should be evaluated further.Copyright © 2022 Yanchu Li et al.