(A) Radiographs of NCr/SCID mice during progression of tumor growth. Mice (n=3) received intratibial injection of 100,000 MDA-MB-231 BrCa cells and were treated with Bzb or PBS (0.3 mg/kg i.p.; 3 times/week) until sacrifice at 49 days following BrCa injection. Interval radiographs are shown indicating a delay in the onset of osteolysis in Bzb treated mice. There is an absence of clearly defined lesions in Bzb treated mice until day 35, in contrast the control group has clear osteolysis by day 21 (box). Microlytic lesions are seen in the Bzb group on day 35 (box). (B) Quantitation of radiographs. Average score (see methods) from each time point and all reviewers for control and Bzb treated mice is presented. This shows presence of osteolysis from d21 in control treated mice, but delay of osteolysis until day 35 in the Bzb treated group. Additionally, the increase in osteolysis during the final week of the experiment (d42-49) is shown (days 21 and 28 p<0.05, days 35, 42 and 49 p<0.005). (C) Micro-CT images. Transverse section distal to the physis (left panels) and 3D reconstruction of the proximal tibia (right panels) of Bzb and control treated mice (n=3) 49 days following intratibial injection of MDA-MB-231 BrCa cells. The reduction in osteolysis is evident by an unresorbed periosteal surface in Bzb treated as compared to control group. (D) Quantitation of micro-CT images. Relative bone volume at time was assessed by micro-CT analysis of the proximal tibia in two separate experiments at 28 days (n=5) and 49 days (n=3). The relative bone volume was significantly lower in control treated as compared to Bzb treated mice (p<0.05, 28 days, p=0.001, 49 days).

Bortezomib treatment reduces the size of BrCa tumors implanted in the mouse tibia. (A) In vivo positron emission tomography (PET) imaging. 39 days following intratibial injection, mice were injected intravenously with 50 μCi 18F-fluorodeoxyglucose. Representative PET image reconstructions of the tumor containing tibial region of Bzb treated and control mice overlaid on CT image reconstructions of the same region. The color corresponds to the intensity of the 18F signal and is scaled from bottom to top (black to white) as shown in the color bars where black indicates 0% radioactivity uptake and white indicates 100% uptake. Higher intensity in the control (white area) reflects the 3-dimensional size of the tumor, while red areas are those tumor cells with less metabolic activity. Blue is normal tissue cellular activity. Controls exhibited a 50–100% (yellow) range of intensity, while the Bzb group showed isotope labeling within the 25–50% (orange) intensity range. Standard uptake value for the control mouse is 2.94 g/ml and for the Bzb treated mouse is 2.31 g/ml. (B) The effect of Bzb on tumor growth and progression (H & E stained sections). Upper Panels (5×): Representative histologic sections of proximal tibias from n=6 control (left panels) and Bzb treated mice (right panels) showing the tumor size and tibial bone loss. Lower Panels (10×): Representative sections of Bzb and control treated tibias. 1) Muscle of Bzb treated tibia showing tumor growth. 2) Marrow cavity of Bzb treated tibia with necrotic cells. 3–4) Area of similar tumor growth in Bzb (3) and control (4) tibias. (C) Quantification of tumor size and growth. Left panel: Areas of tumor were selected from multiple histologic sections (n=5–7) of Bzb treated and control tibias (n=3) and tumor size (mm²) was calculated. Bzb treated mice had significantly (60%) smaller tumors as compared to control mice (p<0.0001). Right panel: Ki-67 immunohistochemistry was performed on Bzb and control treated tumor sections. The Ki-67 growth fraction was calculated as the % of total cells that were positive for Ki-67. The fraction of growing tumor cells was also significantly less in Bzb tumors than controls (n=4, p<0.05). (D) The effect of Bzb on bone osteolysis as evaluated by TRAP staining. In controls, the cells of the bone surface were already resorbed (Panel 1), only small pieces of fractured bone remained with TRAP positive cells (Panels 2 and 3). In the Bzb treated group, bone architecture was preserved (Panel 1) and osteoclast activity was present on cortical surfaces (Panels 2 and 3). Sections are TRAP stained and presented at 5× (left), 10× (center) and 40× (right).

Micro-CT analysis was performed on the distal femurs of NCr/SCID mice 49 days following injection of MDA-MB-231 BrCa cells into the right proximal tibia. Parameters of trabecular bone formation showed significant (p<0.05) increases in Bzb treated mice as noted by increases bone volume/total volume, trabecular thickness and connectivity density. Structure model index showed a significant (p<0.05) decrease, indicative of a more plate-like architecture in the Bzb treated bone.

(A) Experimental design. The experiment consisted of 3 groups: a control group treated with saline from 21 days prior to intratibial inoculation with BrCa until termination of the experiment at 25 days following inoculation; a Bzb pretreatment group that received 0.3 mg/kg Bzb IP three times a week for the 21 days prior to BrCa cell inoculation with saline injection for the 25 days following inoculation; and a continuous Bzb treatment group that received 0.3 mg/kg Bzb IP three times a week in both the pre-inoculation and post-inoculation periods. (B) Radiographic analysis of NCr/SCID mice taken at 25 days following injection of MDA-MB-231 BrCa cells into the right proximal tibia and the treatment conditions described above. Presented are views of the entire tibia (n=3) and representative higher magnification views of the area in which the majority of the osteolysis occurred. Note the fibular head overlapping the tibia in the area of the osteolytic lesion. The tibias of Bzb pretreated and Bzb continuous treatment show smaller osteolytic lesions than those in the control group at both time points. (C) Radiographs were independently scored based on a grading scale developed for osteolytic lesions (n=7 reviewers). Bzb pretreatment and Bzb continuous groups had a lower grade than the control group. Significantly decreased grade was found in Bzb pretreatment and Bzb continuous groups as compared to control (**p<0.0001) and Bzb pretreatment as compared to Bzb continuous (*p<0.05).

Micro-CT analysis was performed on the bilateral distal femurs of the mice from the 3 treatment groups described in Figure 4 (n=3). In all measured parameters, there is a significant anabolic effect evident in both Bzb pre-treatment, and continuous treatment groups as compared to controls. Note that the right femur, proximal to the tumor bearing tibia, has less bone than the contralateral femur. (* p<0.05, + p< 0.001, # p<0.0005)

(A) The relative number viable cells, necrotic cells, and cells undergoing apoptosis following treatment with Bzb in the range of 0 − 50 nM for 14 h were measured by binding of FITC conjugated annexin V and staining with propidium iodide (PI) followed by FACS analysis. The percentage of gated cells that were annexin V and PI negative (live cell, solid line, diamond); annexin V positive, PI negative (apoptotic cells, dashed line, open square); and annexin V and PI positive (necrotic cells, solid line, circle) are shown as a function of the increasing concentration of Bzb treatment. (B) Bivariate plots of the primary FACS data for ungated cells treated with 0 and 50 nM Bzb. The quadrants, which are defined using FACSCalibur software and represent cells that are annexin V and PI negative (bottom left), annexin V positive, PI negative (bottom right); annexin V negative, PI positive (top left); and annexin V and PI positive (top right), show a far greater proportion of cells in the quadrant representing necrosis in response to Bzb treatment. (C) The amount of transcript of various genes in MDA-MB-231 cells treated with Bzb in the range of 0 –50 nM Bzb for 24 h was measured using qPCR and standardized to the amount of GAPDH transcript in the same cells. The amount of transcript relative to no Bzb treatment is shown for each concentration of Bzb treatment used. The CT values for monitored genes were between 18 and 25, except for MMP9 (CT 35). VEGF= vascular endothelial growth factor, DKK1=Dickkopf 1, MMP9 = matrix metallopeptidase 9, LEF1=lymphoid enhanced binding factor 1. (D) Gene transcript levels were measured in MC3T3 osteoblast-like cells following 24 h treatment with 0–50 nM Bzb using qPCR, as described above. The CT values were all less than 21. Col1a=collagen 1A, DKK3=Dickkopf 3, AP=alkaline phosphatase, TCF4= transcription factor 4 (E). Tumor tissue from mice treated with Bzb or control for 28 days was analyzed for relevant gene expression by qPCR. The amount of transcript of Bzb treated tumors relative to control is shown. The CT values were between 16 and 35 for all the monitored genes (note – all less than 30 except RANKL). Significant differences were seen in RANKL, MMP-9 and LEF1 (p<0.05, n=5) RANKL=Receptor activator for nuclear factor κ β ligand.