Contrast enhanced maximum intensity projection ultrasound imaging for assessing angiogenesis in murine glioma and breast tumor models: A comparative study

The purpose of this study was to prospectively compare noninvasive, quantitative measures of vascularity obtained from four contrast enhanced ultrasound (US) techniques to four invasive immunohistochemical markers of tumor angiogenesis in a large group of murine xenografts. Glioma (C6) or breast cancer (NMU) cells were implanted in 144 rats. The contrast agent Optison (GE Healthcare, Princeton, NJ) was injected in a tail vein (dose: 0.4 ml/kg). Power Doppler imaging (PDI), pulse-subtraction harmonic imaging (PSHI), flash-echo imaging (FEI), and Microflow imaging (MFI; a technique creating maximum intensity projection images over time) was performed with an Aplio scanner (Toshiba America Medical Systems, Tustin, CA) and a 7.5 MHz linear array. Fractional tumor neovascularity was calculated from digital clips of contrast US, while the relative area stained was calculated from specimens. Results were compared using a factorial, repeated measures ANOVA, linear regression and z-tests. The tortuous morphology of tumor neovessels was visualized better with MFI than with the other US modes. Cell line, implantation method and contrast US imaging technique were significant parameters in the ANOVA model (p < 0.05). The strongest correlation determined by linear regression in the C6 model was between PSHI and percent area stained with CD31 (r = 0.37, p < 0.0001). In the NMU model the strongest correlation was between FEI and COX-2 (r = 0.46, p < 0.0001). There were no statistically significant differences between correlations obtained with the various US methods (p > 0.05). In conclusion, the largest study of contrast US of murine xenografts to date has been conducted and quantitative contrast enhanced US measures of tumor neovascularity in glioma and breast cancer xenograft models appear to provide a noninvasive marker for angiogenesis; although the best method for monitoring angiogenesis was not conclusively established.

Research highlights
► The largest contrast US study to date of 144 murine xenografts has been conducted.
► The best visualization of tumor blood flow was achieved with MFI (p < 0.03).
► Contrast US appears to provide noninvasive, quantitative markers of angiogenesis.
► A single best contrast US mode for monitoring angiogenesis could not be determined.