Orthotopic breast tumor models

The murine breast carcinoma 4T1 cells were cultured in Dulbecco’s modified Eagle’s medium (Biological Industries, Israel) containing 10% fetal bovine serum and incubated under the atmosphere with 5% CO₂ at 37℃. Five-week-old female BALB/c mice were purchased from Hunan Sleck Jingda Laboratory Animal Company Limited. Appropriately 2 × 10⁶ 4T1 cells were injected into the right mammary fat pad to establish an orthotopic breast tumor model. In another orthotopic tumor model, primary tumors were generated by orthotopic implantation of 2 × 10⁶ MDA-MB-231 cells in the mammary fat pad of CB-17 SCID female mice. When tumors in these models reached the determined size (approximate 50 mm³), mice were randomly assigned to different treatment groups: (I) control groups, (II) Fe-MOF groups, (III) PAZ groups, and (IV) PAZ@Fe-MOF groups. Tumor volume was measured every 2 days and calculated using the following equation: volume = (length × width × width)/2. After 27 days of careful monitoring, the mice were euthanized, and the tumors were surgically dissected. Finally, tumor samples were photographed, weighed, and used for the following experimental analysis. The animal experiments were approved by the Ethics Committee of Central South University (No. CSU-2024-0085).