Summary of the quantitative MRI biomarkers acquired from diffusion-weighted and susceptibility contrast MRI of C6 xenografts in mice treated with vehicle or 20 mg/kg MLN0518 for 10 days.

<p>ADC  =  apparent diffusion coefficient. Mean of median parameter values from each tumour ± 1s.e.m. (n≥5 per treatment group).</p

Summary of the quantitative MRI biomarkers acquired from intrinsic susceptibility MRI of C6 xenografts in mice treated with vehicle or 20 mg/kg MLN0518 for 10 days.

<p>Mean of median R₂* and ΔR₂* values from each tumour ± 1s.e.m. (n≥5 per treatment group). The proportion of voxels in which R₂* changed significantly, either negatively (ΔR₂* <0) or positively (ΔR₂* >0), with carbogen breathing are also shown.</p

C6 tumour growth is slowed by MLN0518 treatment.

<p>The growth rate of tumours in mice treated with 20 mg/kg MLN0518 was significantly slower than tumours in vehicle treated mice over 10 days. Tumour doubling times were calculated on an individual tumour basis (n = 15 per treatment group). Mean ±1s.e.m.</p

Summary of the histological biomarkers assessed in C6 xenografts in mice treated with vehicle or 20 mg/kg MLN0518 for 10 days.

<p>Values are mean ± 1s.e.m. Two frozen sections per tumour were assessed for fluorescence microscopy (n = 15 per treatment group) and a single FFPE section per tumour for α-smooth muscle actin and necrosis assessment (n≥12 per treatment group). ^* p<0.05, ^** p<0.01.</p

Histological assessment of tumour response to MLN0518.

<p><b>A.</b> Tumour sections stained for the perfusion marker Hoechst 33342 (blue), endothelial marker CD31 (red) and pimonidazole adduct formation, a marker of hypoxia (green) demonstrate that the hypoxic area was lower in tumours treated with 20 mg/kg MLN0518 for 10 days than vehicle treated controls. The percentage of the total vessels perfused and the overall perfused vessel area was also lower in treated versus control tumours. Representative composite images are shown. <b>B.</b> Alpha smooth muscle actin (α-SMA) immunohistochemistry demonstrates a significant reduction in α-SMA positive blood vessels in MLN0518 treated tumours compared to controls. Magnification ×200.</p

Dynamic contrast-enhanced MRI is sensitive to the response of C6 tumours to MLN0518.

<p>Representative parametric maps and quantification of initial area under the gadolinium concentration curve (IAUGC) demonstrate a reduction in tumour blood vessel permeability/flow in mice treated with 20 mg/kg MLN0518 for 3 days compared to controls. Mean parameter values from each tumour ± 1s.e.m. (n≥6 per treatment group), * p<0.05.</p

Diffusion-weighted and susceptibility contrast MRI of C6 tumours treated with MLN0518.

<p>Parametric maps of apparent diffusion coefficient (ADC, top panel), fractional blood volume (fBV, middle panel) and vessel size index (R_v, bottom panel) from C6 xenografts in mice treated with vehicle or 20 mg/kg MLN0518 for 10 days show no clear differences between vehicle and treated tumours. Representative maps are shown.</p

CD138 expression changes in response to therapy.

<p><b>A.</b> Percentage of CD138⁺ human myeloma cells measured by flow cytometry in bone aspirates of mice (n = 3), showing a significantly lower percentage of positive cells in both tibias and spine of mice in the two treatment groups than in untreated mice (p<0.05, 2-way ANOVA with Bonferroni post-test). No CD138⁺ cells were observed in the organs of any of the mice. <b>B.</b> Histological analysis of sections from the tibias of mice from each group showed distinct differences. (i) Sections from healthy mice displayed classical architecture, with no CD138⁺ cells. (ii) In comparison, sections from untreated myeloma mice showed a high infiltration of CD138⁺ cells with loss of normal architecture. (iii) Treatment of mice with BZB resulted in the return of normal architecture and loss of CD138⁺ cells. (iv) A similar result was observed in mice treated with tosedostat, but with occasional scattered CD138⁺ cells.</p

CD138 expression and histology validate BLI, paraprotein and MRI results. A.

<p>Flow cytometry histograms show the presence of CD138⁺ cells in the tibia, femur and spine of myeloma mice. Cytometric evaluation of the organs confirmed that the cells were confined to the bone marrow. <b>B.</b> Histological analysis of the tibia and femur confirmed the presence of a high number of CD138⁺ plasma cells (green arrows), resulting in the loss of classical bone marrow architecture.</p

MRI changes in response to therapy.

<p>MRI-derived tumour volumes. <b>A.</b> Tumour was identified as a hyperintense signal enclosed within the cortical bone on T₂-weighted images. MRI images showed a reduction in signal intensity in both treatment groups compared to positive control in both the tibia (T) and femur (F). <b>B.</b> Tumour volume was quantified from regions of interest drawn on the periphery of the hyperintense signal. Data are mean ± SEM, n≥6. Both BZB and tosedostat (TDT) treatment resulted in a significantly lower tumour volume compared to control (p<0.05, 1-way ANOVA with Bonferroni post-test). In addition, there was no significant difference in tumour volume between the BZB treated and negative control group.</p