Abstract Podocalyxin (gene name Podxl) is a CD34-related sialomucin that has been shown to be important in regulating cell adhesion, migration, and polarity of hematopoietic progenitors and vascular endothelia. In breast cancer, we have shown that podocalyxin is overexpressed on a subset of node-negative tumors and its expression is strongly associated with poor overall survival. To determine whether podocalyxin directly regulates breast cancer cell behavior, we ectopically expressed podocalyxin in the human breast cancer cell line, MCF7, which expresses low levels of endogenous podocalyxin, is minimally invasive, and non-metastatic. Upon ectopic expression of podocalyxin, MCF7 “bulge apically”, produce apical and lateral microvilli, are less adhesive in vitro, and are delayed in targeting integrins to the basolateral surface. In contrast to MCF7, MDA-MB-231 is a basal-like breast cancer cell-line, which expresses high levels of endogenous podocalyxin, exhibits poorly polarized monolayer and mammosphere architecture in vitro and forms “metastatic” lung tumors in vivo. Using Podxl-targeted shRNA vectors we show that expression of podocalyxin is required for MDA-MB-231 cells to form distant metastases in a competitive in vivo assay utilizing humanized NOD/SCIDIL-2rγ−/− (NSG) mice. Previously, we observed that Podxl-deficient hematopoietic cells have impaired CXCR4-mediated chemotaxis to CXCL12, a known axis for tumor metastasis. Our current hypothesis is that podocalyxin regulates chemotaxis of tumor-initiating cells (TICs) to the CXCL12-rich tissues of the lungs, liver, and bone marrow. Up-regulation of podocalyxin on breast cancer cells may be a key event in tumor progression and likely enables these cells to sense chemokines and metastasize to peripheral sites. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-05.