4T1 orthotopic mammary tumor model

Either WT or PDPN-tk female mice, around 3 months old, were used for orthotopic implantation of 4T1 mammary epithelial cancer cells. 4T1 Cells were from American Type Culture Collection (ATCC) and cultured in DMEM with 10% FBS and 100 U/mL penicillin–streptomycin. Cells were examined monthly to ensure a negative result for mycoplasma test. Mice were anesthetized with ketamine/xylazine, the skin near the mammary gland was incised, and 4T1 cancer cells were injected into the mammary glands (in total, 1 × 10⁶ cells per mouse; 5 × 10⁵ cells at each side, for both left and right sides), as previously described [4]. PDPN-tk and WT control mice were treated with daily intraperitoneal injections of 50 mg/kg body weight of GCV (InvivoGen, San Diego, CA, USA), when the sum of the tumor volumes per mouse reached approximately 300 mm³ (approximately 8–9 days post cancer cell inoculation). Tumor volumes were measured every other day using digital calipers and calculated using the equation length × width² × 0.52. Mice were sacrificed when the sum of the tumor volumes reached approximately 1,500 mm³ (approximately 22–25 days post-cancer cell inoculation).