Three-dimensional Dynamic Contrast-enhanced US Imaging for Early Antiangiogenic Treatment Assessment in a Mouse Colon Cancer Model

Abstract

Purpose: To evaluate feasibility and reproducibility of three-dimensional (3D) dynamic contrast material-enhanced (DCE) ultrasonographic (US) imaging by using a clinical matrix array transducer to assess early antiangiogenic treatment effects in human colon cancer xenografts in mice.

Materials and methods: Animal studies were approved by the Institutional Administrative Panel on Laboratory Animal Care at Stanford University. Three-dimensional DCE US imaging with two techniques (bolus and destruction-replenishment) was performed in human colon cancer xenografts (n = 38) by using a clinical US system and transducer. Twenty-one mice were imaged twice to assess reproducibility. Seventeen mice were scanned before and 24 hours after either antiangiogenic (n = 9) or saline-only (n = 8) treatment. Data sets of 3D DCE US examinations were retrospectively segmented into consecutive 1-mm imaging planes to simulate two-dimensional (2D) DCE US imaging. Six perfusion parameters (peak enhancement [PE], area under the time-intensity curve [AUC], time to peak [TTP], relative blood volume [rBV], relative blood flow [rBF], and blood flow velocity) were measured on both 3D and 2D data sets. Percent area of blood vessels was quantified ex vivo with immunofluorescence. Statistical analyses were performed with the Wilcoxon rank test by calculating intraclass correlation coefficients and by using Pearson correlation analysis.

Results: Reproducibility of both 3D DCE US imaging techniques was good to excellent (intraclass correlation coefficient, 0.73-0.86). PE, AUC, rBV, and rBF significantly decreased (P ≤ .04) in antiangiogenic versus saline-treated tumors. rBV (r = 0.74; P = .06) and rBF (r = 0.85; P = .02) correlated with ex vivo percent area of blood vessels, although the statistical significance of rBV was not reached, likely because of small sample size. Overall, 2D DCE-US overestimated and underestimated treatment effects from up to 125-fold to170-fold compared with 3D DCE US imaging. If the central tumor plane was assessed, treatment response was underestimated up to threefold or overestimated up to 57-fold on 2D versus 3D DCE US images.

Conclusion: Three-dimensional DCE US imaging with a clinical matrix array transducer is feasible and reproducible to assess tumor perfusion in human colon cancer xenografts in mice and allows for assessment of early treatment response after antiangiogenic therapy.

Figure 1:

Overall experimental design of 3D DCE US imaging experiments. Subcutaneous human colon cancer xenografts were established in 38 nude mice and then randomized into three experimental groups. The curved lines are the time-intensity curves of 3D bolus (purple curve) or 3D destruction-replenishment (red curve) DCE US techniques.

Figure 2:

Three-dimensional DCE US imaging shows antiangiogenic treatment effect in two representative human colon cancer xenografts by using bolus and destruction-replenishment DCE US imaging. A, After a single dose of bevacizumab, imaging signal (demonstrated on volume rendered displays at peak enhancement [left] and complete replenishment [right] at identical rendering settings) substantially decreased 24 hours after antiangiogenic treatment compared with baseline images by using both DCE US imaging techniques. B, In tumors treated with saline only, the imaging signal did not substantially change before and after saline treatment; yellow scale bar on US images is equivalent to 10 mm. Photomicrographs of CD31-stained tissue sections show decreased percent area of blood vessels in, A, antiangiogenic-treated compared with, B, saline-treated tumor (yellow scale bar on immunofluorescence images is equivalent to 100 µm).

Figure 3:

Box-and-whisker plots show spatial heterogeneity of tumor perfusion for all nine human colon cancer xenografts made visible with DCE US imaging before (black box plots) and 24 hours after (red box plots) antiangiogenic therapy. Three-dimensional data sets were retrospectively segmented into consecutive 1-mm data sets and logarithmically transformed perfusion parameters, including, A, PE, B, AUC, C, T TP, D, rBV, E, rBF, and, F, blood flow velocity, were plotted for each tumor separately. Each box in the plot represents the 25th and 75th quartiles, the line inside each box identifies the median, and the whiskers indicate the 5th and 95th percentile of perfusion parameter measurements. Outliers are represented by *. Note that there is substantial heterogeneity for all six parameters shown by the size of the boxes and the ranges of the values.

Figure 4:

Bar charts plotted for all six perfusion parameters show percent differences in antiangiogenic treatment effects based on data analysis of the central 1-mm plane versus analysis of the volumetric data set for each of the nine tumors treated with antiangiogenic therapy. Ratios of each perfusion parameter after and before antiangiogenic treatment obtained from the central 1-mm plane were compared with the ratios of the perfusion parameter after and before treatment obtained from 3D imaging, and percent differences were plotted. Note that treatment response can be either over-estimated or under-estimated when analyzing the central 2D plane only compared with volumetric data set.