Fig. 3

Unique protein expression occurs with EO cell presence in the endosteal niche of tumor-bearing bones. Athymic nude mice were injected via intratibial injection with an admix of MDA-MB-231GFP/Luc2 human breast cancer cells plus either EO-231 cells or MC3T3-E1 osteoblasts, or MDA-MB-231GFP/Luc2 cells alone. Eight weeks later, mice were euthanized and their tibia harvested. Tibia sections from athymic mice were prepared as described in the “Materials and methods” section. Sections were stained for osteopontin, alkaline phosphatase, VEGF, alpha-smooth muscle actin, MMP3, collagen type I, MCP-1, IL-6, and green fluorescent protein via immunofluorescence. The cortical and trabecular bone microenvironment was examined via fluorescent microscopy. As shown on the tibia at the left, the black box represents the positioning of the tumor in the examples shown, whereas the green circle represents the locations in the bone where the images were taken. At least three independent, serial sections were stained per bone and three bones examined per condition. Shown are representative images. Scale bar = 50 μm