Fig. 7: ZEB1 promotes bone metastasis in vivo.

a Left panel shows the Bioluminescent images (BLI) of primary tumors of mice injected with one million ZsGreen- or ZEB1-expressing wild-type MCF7 cells into the mammary fat pad. The panel on the right is the corresponding quantitative analysis of the average radiance of multiple primary tumors. An unpaired and two-tailed Student’s t test was applied to determine whether tumor sizes are different (n = 8 independent animals for ZsGreen cells and n = 4 for ZEB1 cells; means ± SEM). b Left panel is the BLI images showing metastatic lesions in hindlimb bones of ZsGreen- or ZEB1-expressing wild-type MCF7 cells, respectively. The corresponding panel on the right is a bar graph of the metastatic indices (the ratio of the average radiance of metastases over that of the corresponding primary tumor; see Methods for details) for bones (n = 8 independent animals for ZsGreen cells and n = 4 for ZEB1 cells; means ± SEM); note that the data are shown after log10 transformation; an unpaired and two-tailed Student’s t test was used for the statistical analysis. c–f IF images of EpCAM, vimentin, ERα, and ZEB1, and DAPI staining of resected primary tumors formed by MCF7-ZEB1 cells (+DOX) following their orthotopic injection into mouse mammary fat pads (Scale bar = 10 μm). g, h IF images of EpCAM and ZEB1, and DAPI staining of metastatic lesions in femurs of mice xenografted with ZEB1-expressing (g) and ZsGreen-expressing (h) cells (scale bar = 10 μm). i Multiphoton confocal microscopy images of an ex vivo bone invasion assay showing the bone explant stained in red with alizarin red and the invading ZsGreen-labeled (green) control (Ctl) or ZEB1-expressing MCF7-V cells. Without ZEB1, the cells remain on top of the bone tissue, whereas ZEB1-expressing cells invade it. The upper left of each panel shows a projection of a Z-stack with 20 images, and in the upper right of each panel a slice of the area delimited by a yellow rectangle. Images were processed and generated with the software Imaris 9.7 to illustrate representative slides and 3D views of bone tissue. Scale bar = 50 μm. j Transwell assay to evaluate the migration of ZsGreen-labeled control (Ctl) or ZEB1-expressing MCF7-V cells stimulated by bone and muscle tissues. Crystal violet dye was used to stain the migrated cells. Decellularized bone served as a negative control. Images are representative of three independent experiments. Source data are provided as a Source Data file. Scale bar = 50 μm.