Podocalyxin enhances metastasis of 4T1 mammary tumor cells in a syngeneic mouse model. (A) Vector control (VC, black) and shRNA targeting PODXL (shPODXL; dashed line) luciferase-expressing 4T1 (4T1-luc) murine mammary tumor cells were labeled with a conjugated rat anti-mouse podocalyxin antibody and analyzed by flow cytometry to detect surface expression of podocalyxin. Rat igG2B was used as an isotype control (shaded area). Data presented as histograms showing podocalyxin expression on VC cells compared with shPODXL 4T1-luc cells. RFI, Relative fluorescence intensity. (B) VC or shPODXL 4T1-luc cells were injected intravenously into BALB/c mice. Mice were killed after 14 days, and visible tumor nodules on the lung were manually counted using a dissecting microscope (n = 9 and n = 8, respectively; ***P < 0.001). (C) Luciferase expression in the lungs from noninjected, VC and shPODXL 4T1-luc-injected mice was measured using a luminometer (**P < 0.01). The data shown are representative of two independent experiments. RLU, Relative light units.