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Metastatic cell arrest in distant organ microvessels constitutes an essential rate-limiting step in hematogenous cancer metastasis. Previously, in a series of works we demonstrated the importance of interactions between cancer-associated Thomsen-Friedenreich antigen (TF-Ag) and endothelial galectin-3 (Gal-3) in facilitating the initial carbohydrate-mediated tumor cell adhesion to distant organ microvasculature. However, it was unclear which molecules stabilize TF-Ag/Gal-3 mediated adhesion and how these interactions effect subsequent events in a metastatic cascade. Here, the results of a pull down assay using TF-Ag coated gold nanoparticles followed by Western analysis identified endothelial integrin α3β1 being physically associated with TF-Ag/Gal-3 complexes. Blocking α3β1 did not affect the initial tumor cell adhesion to the endothelium, but reduced >10-fold shear force necessary to displace cancer cells stably attached to endothelial monolayers, indicating that α3β1 stabilizes tumor-endothelial cell adhesion initiated by TF-Ag/Gal-3 interactions. Subsequent Quadrupole Time-of-Flight mass spectrometry analysis revealed additional 7 proteins forming a macromolecular complexes with TF-Ag, Gal-3 and α3β1 such as filamin B, talin, IQ motif containing GTPase activating protein 1, vinculin, α actinin 4, zyxin, and T-plastin, all of which map onto focal adhesion pathway. Our data demonstrate an induction of complex signaling pathways downstream of this macromolecular complex facilitating endothelial cell retraction and metastatic cell transendothelial migration. Thus, endothelial α3β1 mobilization downstream of TF-Ag/Gal-3 interactions stabilizes tumor-endothelial cell adhesion, initiates focal adhesion formation, and induces signaling pathways in endothelial cells promoting cancer metastasis. This work was supported in part by Award Number 1I01BX000609 from the Biomedical Laboratory Research & Development Service of the VA Office of Research and Development to VVG and AHA National SDG 0830287N to OVG. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2436. doi:10.1158/1538-7445.AM2011-2436