Wnt3a stabilizes β-catenin in MM cell lines and primary MM plasma cells. The myeloma cells were treated with Wnt3a-CM or Cont-CM for the indicated time (A). Primary CD138-selected plasma cells from 6 patients with MM confirmed by flow cytometry analysis were treated with Wnt3a-CM or Cont-CM for 3 hours (B). Proteins isolated from the above-treated cells were subjected to Western blotting analysis as described in “Wnt3a stabilizes β-catenin in MM cells.” Total β-catenin was determined by using an antibody that recognized the protein. The vertical lines have been inserted to indicate different gels used.

Wnt3a prevents bone loss and reduces tumor burden in myelomatous bones. SCID-hu mice were engrafted with H929/EV or H929/W3A cells. (A) X-ray radiographs of the implanted myelomatous bones taken from 3 representative mice in each group, before cell engraftment (Pre-MM) and at the experiment's end (MM). Note that, although EV-bearing bones were severely resorbed and tumors grew on the outer surface of the implanted bone, implanted bone mass from the H929/W3A-bearing hosts was preserved. (B) Changes in the level of BMD of implanted bones engrafted with H929/EV or H929/W3A cells. (C) Circulating human Ig level of SCID-hu mice engrafted with H929/EV or H929/W3A cells. Error bars represent SEM.

Wnt3a has no effect on subcutaneous MM growth in SCID mice. SCID mice were subcutaneously injected with H929/W3A or H929/EV cells. (A) Circulating human Ig levels were similarly increased in hosts engrafted with EV and W3a cells. (B) Subcutaneous tumor volume. Error bars represent SEM. (C) X-ray radiographs showing similar tumor size (white arrows) in hosts with H929/EV and H929/W3A cells.

Increased canonical Wnt signaling in osteogenic cells in myelomatous bones treated with Wnt3a. Myelomatous human bone sections were immunohistochemically stained with 8E7 antibody that reacts with the active form of β-catenin. (A,B) Bones were engrafted with H929/EV (A) and H929/W3A (B) MM cells. Note expression of active form of β-catenin in certain vascular endothelial cells (VECs) and in cellular components along the bone surface. Bones engrafted with H929/W3A had increased numbers of cells expressing the active form of β-catenin along the bone surface. (C,D) Primary myelomatous bones treated with Wnt3a had an increased number of osteoblastic (OB) cells expressing the active form of β-catenin. Osteoclasts (OCs) as well as MM cells expressed very low levels of β-catenin.

Wnt3a has no direct effect on growth of MM cell lines and primary MM cells. Cells from indicated MM cell lines were cultured in serum-free medium in the absence and presence of rWnt3a for the indicated times. Cell proliferation was measured by MTT assay as described in “Wnt3a does not promote MM growth.” (A) Protein isolated from H929/W3A or H929/EV was subjected to Western blotting analysis using anti-HA antibody to confirm Wnt3a expression (top) or using anti–β-catenin antibody (second panel from top). The same fractions were also blotted with antitubulin antibody as a control for protein loading (bottom). The proteins isolated from H929/W3a or H929/EV was subject to E-cadherin pull-down assay to measure the uncomplexed β-catenin (second panel from bottom). (B) H929/W3A and H929/EV cells were seeded in RPMI supplemented with 10% FBS for the indicated time, and proliferation was measured by MTT assay. (C) MM cell lines H929, INA6, and OPM-2 were incubated for 48 hours in the absence and presence of rWnts (100 ng/mL) and then subjected to MTT assay. (D) Primary CD138-selected plasma cells from 2 patients with MM (Pt no. 1 and Pt no. 2) were cultured in serum-free medium in the presence of Wnt3a-CM or Cont-CM or with rWnt3a protein for the indicated time. Cell proliferation was measured by MTT assay. Results are expressed as means plus or minus SEM.

Wnt3a increases osteoblastogenesis and reduces osteoclastogenesis in myelomatous bones. (A) Decalcified histologic bone sections of bones engrafted with H929/W3a or H929/EV cells stained for TRAP (red) and osteocalcin (brown). OB indicates osteoblast; OC, osteoclast. (B) Numbers of osteocalcin-expressing osteoblasts and TRAP-expressing multinucleated osteoclasts in implanted bones engrafted with H929/EV and H929/W3a cells. Error bars represent SEM.

Wnt3A stimulates bone formation and attenuates primary MM growth in SCID-hu mice. SCID-hu mice engrafted with MM cells from 5 patients were daily injected with Cont-CM and Wnt3a-CM into the surrounding area of the implanted bones (patients 1 and 2) or treated with rWnt3a using Alzet pumps. (A,B) Changes in circulating human kappa Ig level (A) and in the BMD of the implanted bones (B) from pretreatment level. (C,D) X-ray radiographs showing changes in myelomatous bone mass of hosts treated with Cont-CM and Wnt3a-CM (C) and rWnt3a (D). Note that osteolytic lesions were evident before initiation of treatment (pre-Rx) and that, although in control hosts bone loss continued to increase (white arrows), Wnt3a treatment resulted in increased bone mass (white arrows).