Intra-Animal Comparison between Three-dimensional Molecularly Targeted US and Three-dimensional Dynamic Contrast-enhanced US for Early Antiangiogenic Treatment Assessment in Colon Cancer

Abstract

Purpose To perform an intra-animal comparison between (a) three-dimensional (3D) molecularly targeted ultrasonography (US) by using clinical-grade vascular endothelial growth factor receptor 2 (VEGFR2)-targeted microbubbles and (b) 3D dynamic contrast material-enhanced (DCE) US by using nontargeted microbubbles for assessment of antiangiogenic treatment effects in a murine model of human colon cancer. Materials and Methods Twenty-three mice with human colon cancer xenografts were randomized to receive either single-dose antiangiogenic treatment (bevacizumab, n = 14) or control treatment (saline, n = 9). At baseline and 24 hours after treatment, animals were imaged with a clinical US system equipped with a clinical matrix array transducer by using the following techniques: (a) molecularly targeted US with VEGFR2-targeted microbubbles, (b) bolus DCE US with nontargeted microbubbles, and (c) destruction-replenishment DCE US with nontargeted microbubbles. VEGFR2-targeted US signal, peak enhancement, area under the time-intensity curve, time to peak, relative blood volume (rBV), relative blood flow, and blood flow velocity were quantified. VEGFR2 expression and percentage area of blood vessels were assessed ex vivo with quantitative immunofluorescence and correlated with corresponding in vivo US parameters. Statistical analysis was performed with Wilcoxon signed rank tests and rank sum tests, as well as Pearson correlation analysis. Results Molecularly targeted US signal with VEGFR2-targeted microbubbles, peak enhancement, and rBV significantly decreased (P ≤ .03) after a single antiangiogenic treatment compared with those in the control group; similarly, ex vivo VEGFR2 expression (P = .03) and percentage area of blood vessels (P = .03) significantly decreased after antiangiogenic treatment. Three-dimensional molecularly targeted US signal correlated well with VEGFR2 expression (r = 0.86, P = .001), and rBV (r = 0.71, P = .01) and relative blood flow (r = 0.78, P = .005) correlated well with percentage area of blood vessels, while other US perfusion parameters did not. Conclusion Three-dimensional molecularly targeted US and destruction-replenishment 3D DCE US provide complementary molecular and functional in vivo imaging information on antiangiogenic treatment effects in human colon cancer xenografts compared with ex vivo reference standards. ^© RSNA, 2016 Online supplemental material is available for this article.

Figure 1:

A, Summary of the time-intensity curve diagrams of 3D VEGFR2-targeted US molecular imaging (USMI) and the two DCE US techniques, along with the imaging parameters measured by using the three different techniques. B, Overall experimental design of 3D molecularly targeted US, bolus DCE US, and destruction-replenishment DCE US imaging experiments. Subcutaneous human colon cancer xenografts were established in 23 nude mice and then randomized into two treatment groups. MB_Nontargeted = nontargeted microbubbles, MB_VEGFR2 = VEGFR2-targeted microbubbles.

Figure 2:

A, US images show the antiangiogenic treatment effect at 3D molecularly targeted US (USMI), bolus DCE US, and destruction-replenishment DCE US in two representative subcutaneous human colon cancer xenografts. After administration of a single dose of bevacizumab, imaging signal (demonstrated at identical volume rendering settings of molecularly targeted US signal [left column], bolus DCE US at peak enhancement [middle column], and destruction-replenishment DCE US at complete replenishment [right column]) substantially decreased 24 hours after antiangiogenic treatment compared with baseline images by using the three imaging techniques. B, In saline-treated tumors, the imaging signal did not change substantially before and after treatment. Scale bar = 10 mm.

Figure 3:

Box and whisker plots of the antiangiogenic treatment effects on A, tumor volume, B, VEGFR2-targeted 3D molecularly targeted US (USMI) signal, and perfusion values obtained with, C, bolus DCE US and, D, destruction-replenishment DCE US. The percentage change from baseline to 24 hours after treatment is plotted in treatment and control groups. Each box in the plot represents the 25th and 75th quartiles; the line inside each box indicates the median, and the whiskers indicate the 5th and 95th percentile of measurements, excluding the outliers (indicated by the X at the end of each line). PE = peak enhancement. * = P < .05, ** = P < .001.

Figure 4:

A, Representative photomicrographs of VEGFR2 (red), CD31 (green), and merged (yellow) staining of human colon cancer tissue slices obtained from the tumor center show decreased VEGFR2 expression levels and percentage area of blood vessels in a tumor treated with antiangiogenic therapy (lower row) compared with tumor treated with saline only (upper row) (scale bar = 100 µm). B, Box plots summarize quantitative ex vivo results of immunofluorescence and percentage area of blood vessels in treated and nontreated tumors. Each box in the plot represents the 25th and 75th quartiles; the line inside each box indicates the median, and the whiskers indicate the 5th and 95th percentile of measurements, excluding the outliers (indicated by the X at the end of each line). * = P < .05.