Correlation of ultrasound contrast agent derived blood flow parameters with immunohistochemical angiogenesis markers in murine xenograft tumor models

•Ultrasound contrast kinetics were compared to angiogenesis markers.•Three contrast imaging modes were compared with four markers.•Strongest correlation was observed between harmonic imaging and VEGF expression.•These correlations were stronger than previous fractional vascularity measurements.•Strength of correlation depended on both tumor type and implant location. In this study we used temporal analysis of ultrasound contrast agent (UCA) estimate blood flow dynamics and demonstrate their improved correlation to angiogenesis markers relative to previously reported, non-temporal fractional vascularity estimates. Breast tumor (NMU) or glioma (C6) cells were implanted in either the abdomen or thigh of 144 rats. After 6, 8 or 10days, rats received a bolus UCA injection of Optison (GE Healthcare, Princeton, NJ; 0.4ml/kg) during power Doppler imaging (PDI), harmonic imaging (HI), and microflow imaging (MFI) using an Aplio ultrasound scanner with 7.5MHz linear array (Toshiba America Medical Systems, Tustin, CA). Time-intensity curves of contrast wash-in were constructed on a pixel-by-pixel basis and averaged to calculate maximum intensity, time to peak, perfusion, and time integrated intensity (TII). Tumors were then stained for four immunohistochemical markers (bFGF, CD31, COX-2, and VEGF). Correlations between temporal parameters and the angiogenesis markers were investigated for each imaging mode. Effects of tumor model and implant location on these correlations were also investigated. Significant correlation over the entire dataset was only observed between TII and VEGF for all three imaging modes (R=−0.35, −0.54, −0.32 for PDI, HI and MFI, respectively; p