Fig. 5.

Discovery of RAGE/PR-3 as a ligand-binding targeting human bone marrow containing cancer cells. (A) Anti-CWELGGGPC antibodies recognize human recombinant RAGE. ELISA with post- and preimmune polyclonal antibodies against CWELGGGPC was performed on immobilized CWELGGGPC, a control peptide, recombinant Fc-tagged proteins, and a control protein. (B) Anti-CWELGGGPC antibodies recognize native human RAGE. Protein extracts from human prostate cancer cell lines PC3 and DU145, or from human bone marrow mononuclear control cells, along with recombinant RAGE protein, were immunoblotted with post- and preimmune polyclonal antibodies against CWELGGGPC. Arrow points to RAGE. (C) Validation of RAGE binding to PR-3 in vitro. Either immobilized PR-3 or control protein (BSA) was subjected to RAGE, BMPRIA, BSA, and anti-PR-3 antibody. Bars represent mean ± SEM (D) RAGE binding to active PR-3 is concentration-dependent. (E) Binding of CWELGGGPC-phage is specifically inhibited by the synthetic peptide. Binding of unrelated control phage, insertless phage, binding to BSA and inhibition with an unrelated peptide served as controls. (F) Relative quantification of RAGE expression on prostate cancer patient samples. Expression of RAGE is represented as low, moderate and high expression according to a standardized pathology score. (G–I) Immunohistochemistry with RAGE-specific antibodies performed on human tissue sections derived from a panel of prostate cancer patients. (G) Organ-confined prostate cancer; (H), lymph node metastasis; and (I), bone marrow metastasis. Asterisks represent lymphoid (H) and bone (I) tissues. (Scale bar, 100 μm).