Breast Cancer Cell Metastasis Model

The University of Michigan Institutional Care and Use of Animals Committee approved all animal procedures. We used 8- to 10-week-old female NOD-scid IL2Rgamma^null mice originally purchased from The Jackson Laboratory and bred in the colony maintained by the University of Michigan Lab Animal Medicine Program. Before mouse experiments, we cultured MDA-MB-231 WT and MDA-MB-231-GIV KO/WT cells in serum-free DMEM with 25 mM glucose overnight. We verified that these cells did not lose viability after overnight culture in a serum-free medium relative to a medium with 10% serum as determined by cell-based measurements of CBG bioluminescence in an IVIS Lumina (Perkin Elmer). We injected 1 × 10⁵ breast cancer cells per mouse (n = 5 per each cell type), verifying the positioning of the 30 g needle in the left ventricle by the return of pulsatile bright red blood as described (83 (link)). We imaged bioluminescence with an IVIS spectrum (Perkin Elmer) in mice at time points shown in the figure legend and quantified data with Living Image software. For each mouse, we calculated the fold-change in bioluminescence relative to the value obtained one day after injection to normalize for variations in injected amounts of cells. We calculated area-under-the-curve (AUC) ± SEM for total bioluminescence in each group.