Mouse models of breast cancer progression and metastasis

For primary tumor growth and spontaneous metastasis, 8‐ to 12‐week‐old syngeneic wild‐type BALB/c female mice (BALB/cAnNTac; Taconic Biosciences) were anesthetized with isoflurane, and 10⁶ tumor cells resuspended in 200 μl HBSS were injected into the 4^th mammary fat pad. Mice were euthanized 28 days later with CO₂, and the primary tumors were resected, weighed, and embedded in OCT. Lungs were fixed by tracheal perfusion with HBSS, 4% paraformaldehyde for 15 min, and removed en bloc. The lung lobes were then embedded in OCT or fixed in 4% formaldehyde for 24 h. Lung sections were then prepared, stained with hematoxylin and eosin (H&E), and imaged. The number of metastases per animal was determined by counting metastatic foci in 2 H&E‐stained lung sections per mouse. The metastasis count was then divided by the area covered by the lung sections to yield the number of metastases per mm² of lung tissue. All mice were housed and handled in accordance with approved St. Jude Children's Research Hospital Institutional Animal Care and Use Committee protocols.