Podocalyxin increases the metastatic burden in lungs, liver and bone marrow, but not initial lung seeding. (A) A 50:50 mixture of scrambled shRNA control (shCTRL) and shRNA targeting PODXL (shPODXL) MDA-MB-231 cells were injected into the tail vein of NSG mice in a competitive experimental metastasis model. Mice were killed 3, 7 or 14 days post-injection. Presented is the relative frequency of shCTRL and shPODXL cells detected in the lungs by flow cytometry (as a % of total tumor cells) (n = 6; *P < 0.05). (B) Flow cytometry was performed using an antibody to detect podocalyxin expression on tumor cells in lungs isolated from NSG mice 3, 7 and 14 days post-injection. Histograms displaying the levels of (1) surface podocalyxin expression in shCTRLGFP cells (solid line) compared with shPODXLRFP (dashed line) cells from day 0 to day 14 (upper) and (2) surface podocalyxin expression in shCTRLRFP (solid line) cells compared with shPODXLGFP (dashed line) cells from day 0 to day 14 (lower). The shaded day 0 histograms represent the isotype control. RFI, Relative fluorescence intensity. (C) Representative fluorescence images of lungs showing shCTRLRFP tumor nodules (upper left) and shPODXLGFP tumor nodules (lower left) 6 weeks post-injection. The number of fluorescent tumor nodules on the lung surface was manually counted (right; n = 5; ***P < 0.001). (D) Representative fluorescence images of livers showing shCTRLRFP tumor nodules (upper) and shPODXLGFP tumor nodules (lower) (n = 5) 6 weeks post-injection. (E) Relative frequency of shCTRL and shPODXL tumor cells within the lungs, liver and bone marrow as determined by flow cytometry 6 weeks post-injection (n = 10; ***P < 0.001). The data shown are representative of three independent experiments. All data shown were pooled from mice injected with shCTRLRFP versus shPODXLGFP or shCTRLGFP versus shPODXLRFP run in tandem experiments.