Metastasis assay

To measure the metastatic potential of MCF7 and various MCF7 transfectants, the cells were injected intravenously into 4- to 6-week-old Rag2^−/−γ_c^−/− mice treated with estrogen pellets (17β-estradiol, Innovative Research of America, Florida, USA), which were placed subcutaneously in the intrascapular region as described.⁵⁸ MCF7-Ctrl, MCF7–ROR1, or MCF7–P841A cells (1 × 10⁶) were injected intravenously (i.v.) through the lateral tail vein in 100 µl of PBS. At the indicated times (1 or 3 weeks after the injection of cells), the mice were euthanized, and their lungs were removed, fixed, and stained with H&E. Metastatic foci were counted by using Nikon microscope (Japan), objective 40×.

To examine the effect of antibodies on the capacity to generate metastatic foci, MCF7–ROR1 cells (1 × 10⁶) were injected intravenously (i.v.) through the lateral tail vein in 100 µl of PBS. Mice received mg/kg of either Ctrl-IgG or cirmtuzumab on days-0 and -14 at 10 mg/kg via the tail vein (i.v.). At the indicated times (1 or 3 week after the injection of the MCF7–ROR1 cells), the mice were euthanized, and their lungs were removed and fixed in formalin for pathology review, as described in the preceding paragraph.