EphrinB2 (EFNB2) and EphB4 (EPHB4) are down-regulated in MM MSCs and in osteoblasts and osteoclasts in myelomatous bones. (A-B) Gene expression of the B family of ephrin ligand and Eph receptor genes in MSCs from MM patients (n = 13) and healthy donors (n = 5) analyzed by GEP (A) and quantitative RT-PCR (B). Note down-regulation of EFNB2 and EPHB4 in MM patient MSCs. (C) Protein level of EphB4 as determined by ELISA, demonstrating reduced total amount of this receptor in patient MSCs. (D) Protein level of ephrinB2 as determined by flow cytometry analysis, demonstrating reduced mean florescent intensity of this ligand in patient MSCs. (E) Representative immunohistochemical analysis revealed reduced levels of ephrinB2 and EphB4 in patient MSCs (original magnification ×200). Note cell surface (↑) and cytoplasmic expression (stained brown) of these factors. (F) Bone sections from myelomatous (MM Bone) and nonmyelomatous (Non-MM Bone) human bones in SCID-hu mice were immunohistochemically stained for human EphB4 and ephrinB2. Note strong expression of EphB4 in osteoblasts in nonmyelomatous bones (bottom left panel), whereas myelomatous bones contained fewer osteoblasts that expressed very low levels of EphB4 (bottom right panel). Osteoclasts (OC) did not appear to express EphB4, whereas hematopoietic cells that seem to be of monocytic lineage (red arrows) highly expressed EphB4 and served as an internal positive control. Also note that ephrinB2 was evident in osteoclasts and osteoblasts in nonmyelomatous bones (top left panel), whereas in myelomatous bones low expression of ephrinB2 was detected in osteoclasts and osteoblasts (top right panel). Vascular endothelial cells (EC) expressed high levels of ephrinB2 in myelomatous bones and served as an internal positive control. An Olympus BH2 microscope equipped with a 160×/0.17 numerical aperture objective (Olympus) was used to obtain images with a SPOT 2 digital camera (Diagnostic Instruments).