Spontaneously metastatic breast cancer cells dynamically regulate epithelial cadherin during in vivo primary and metastatic tumor growth. (A) Bioluminescence imaging (BLI) of metastatic 4T1 cells expressing cytomegalovirus (CMV)-driven Renilla luciferase and firefly luciferase driven by the epithelial cadherin (E-cad) promoter. Cells were engrafted onto the mammary fat pads of BALB/c mice, and qualitative E-cad promoter activity (for example, luciferin-derived bioluminescence) was monitored in vivo and ex vivo. E-cad bioluminescence was spatially correlated with E-cad protein expression as determined by IHC. Bars indicate 40x, 100x and 400x magnifications. (B) E-cad promoter activity (for example, luciferin-derived bioluminescence) was quantified by normalization to CMV promoter activity (for example, coelenterazine-derived bioluminescence) in in vitro cultured cells and ex vivo tissues derived from primary tumors and their late-stage metastases (Mets). Data represent ten samples (n = 10, ±SE) derived from five individual mice bearing primary and metastatic tumors, resulting in the indicated P values. (C) Circulating tumor cells (CTCs) were isolated from the blood of 4T1 tumor-bearing mice and selected for resistance to Zeocin. Photomicrographs show the antibiotic-resistant CTCs (CTC ex vivo). The morphologically epithelial cells (black arrow) were physically isolated from the mesenchymal cells (white arrow) and further subcultured as separate populations of mesenchymal-like and epithelial-like cells as indicated. Bars indicate a 100x magnification.