BackgroundTreatment of ER⁺ breast cancer with intramuscular formulation of Formestane (4-OHA) shrinks the tumor within weeks. Since the tedious way of intramuscular administration and side effects are not suited for adjuvant treatment, Formestane was withdrawn from the market. A new transdermal formulation of 4-OHA cream may overcome the defects and retain the effect of shrinking the breast cancer tumor. However, the effects of 4-OHA cream on breast cancer need further confirmatory studies.MethodsIn this work, in vivo, the influence of 4-OHA cream on breast cancer was evaluated using the mode of 7,12-dimethylbenz(a)anthracene (DMBA) induced rat mammary cancer. We explored the common molecule mechanisms of action of 4-OHA cream and its injection formulation on breast cancer through RNA- sequencing-based transcriptome analysis and several biochemical experiments.ResultsThe results showed that the cream substantially reduced the entire quantity, size, and volum of tumors in DMBA-treated rats consistent with 4-OHA injection, and indicated that there were comprehensive signals involved in 4-OHA antitumor activity, such as ECM-receptor interaction, focal adhesion, PI3K-Akt signaling pathway, and proteoglycans in cancer. In addition, we observed that both 4-OHA formulations could enhance immune infiltration, especially CD8⁺ T cells, B cells, natural killer cells, and macrophages infiltration, in the DMBA-induced mammary tumor tissues. The antitumor effects of 4-OHA partly depended on these immune cells.Conclusion4-OHA cream could inhibit breast cancer growth as its injection formulation and may provide a new way for neoadjuvant treatment of ER⁺ breast cancer.