AMD3100 enhances tumor reduction induced by bortezomib in vivo. Mice were treated with AMD3100 (5 mg/kg, daily), bortezomib (0.5 mg/mL, twice weekly), or their combination and were compared with the control untreated group (A). Tumor growth was determined by bioluminescence imaging. Tumor growth in the AMD3100-treated group was similar to the control group, whereas the bortezomib-treated group showed reduction in tumor progression compared with control (P = .041), and the mice treated with the combination of AMD3100 and bortezomib showed significant tumor reduction compared with control (P = .001) and bortezomib alone (P = .021). Each data point represents 3 to 5 mice. Error bars represent SD. (B) Representative bioluminescence images of each treatment group with time. (C) Immunohistochemistry of specimens taken from BM, liver, and spleen. Top panel shows induction of apoptosis of MM cells in the BM detected by TUNEL assay. AMD3100 did not induce apoptosis compared with control, whereas the bortezomib-treated group showed low levels of apoptosis in the BM, and the combination of AMD3100 and bortezomib showed significant induction of apoptosis. The bottom 3 panels represent tumor spread in the BM, liver, and spleen by staining with anti–human CD138, showing that AMD3100 had a minimal effect on reducing the number of plasma cells present in the BM, liver, and spleen. However, bortezomib and more significantly the combination of bortezomib and AMD3100 showed reduction of tumor burden in the BM, liver, and spleen. Images were taken at 40× magnification with Leica DMLB microscope. (D) Quantification of the number of CD138⁺ cells in the BM, liver, and spleen and of TUNEL⁺ cells in the BM. Statistically significant differences were found between numbers of CD138⁺ and TUNEL⁺ cells in the bortezomib-treated group and the bortezomib + AMD3100–treated group. *P < .001; **P = .002; #P = .021; ##P < .016. Error bars represent SD.