Female fvb nj or balb cj mice

Female FVB/NJ or Balb/cJ mice from Jackson Laboratories were injected at 6–8 weeks of age. Two days prior to orthotopic injections, cells were placed in non-selective media. On the day of injection, 1x10⁵ cells were injected orthotopically into the fourth mammary fat pad of age-matched virgin females. After 30 days the mice were euthanized by intraperitoneal injection of 1mL Tribromoethanol with subsequent cervical dislocation. Primary tumors were resected, weighed, and snap frozen in liquid nitrogen. Lungs were resected, surface metastases were counted; lungs were inflated with 10% nitrate-buffered formalin and sent for sectioning and staining. For tail vein injection, 10⁵ were injected into the lateral tail vein, mice were euthanized 22 days post-injection. All procedures were performed under the Animal Safety Proposal (LCBG-004) and approved by the NCI-Bethesda Animal Care and Use Committee.