Podocalyxin promotes primary tumor formation, local invasion and metastasis. (A) Growth curve of subcutaneous (s.c.) flank tumors initiated in NSG mice by scrambled shRNA control (shCTRLRFP; closed squares) or shRNA targeting PODXL (shPODXLRFP; open circles) MDA-MB-231 cells (left; n = 5 for shCTRLRFP group and n = 6 for shPODXLRFP group; growth curves are significantly different with P < 0.001). Similarly, we compared s.c. tumor growth of shCTRLGFP with shPODXLGFP MDA-MB-231 cells in NSG mice (right; n = 6; growth curves are significantly different with P < 0.05). GFP, Green fluorescent protein; RFP, Red fluorescent protein. The final volume of the excised tumors was measured on the dates mice were killed (left; day 21, right; day 20). (B) Wet weight (g) of pooled shCTRL (solid bar) and shPODXL (open bar) tumors weighed immediately after excision (n = 11 for shCTRL group and n = 5 for shPODXL group; *P < 0.05). Representative photograph of excised shCTRL and shPODXL tumors (inset). (C) Representative H&E–stained images of shCTRL (upper left) and shPODXL (upper right) primary tumor sections (scale bar = 1 mm). Representative immunofluorescence images of shCTRL (lower left) and shPODXL (lower right) primary tumor sections showing Ki-67-positive cells (green). DAPI stain was used as a nuclear marker (blue). (D) The number of tumor cells detected in the lungs 20 or 21 days after subcutaneous injection. Data shown are the number of fluorescent tumor cells (×103) per 106 events detected by flow cytometry (n = 11 for shCTRL group and n = 5 for shPODXL group; *P < 0.05). The data shown are representative of three independent experiments.