Comparing contrast-enhanced us to markers of angiogenesis in a murine glioma model

Abstract

The purpose of this study was to compare four contrast-enhanced ultrasound (US) techniques to four markers of angiogenesis in glioma tumor xenografts. Twenty-one rats were implanted with a glioma (C6) cell line (ATCC, Manassas, VA). Tumors, approximately 18 mm in diameter, developed 1 to 3 weeks post-inoculation. The US 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) was performed with an Aplio scanner (Toshiba America Medical Systems, Tustin, CA) and a 7.5 MHz linear array. Specimens were stained with immunohistochemical markers corresponding to bFGF, CD31, COX-2, and VEGF. Digital US clips and immunohistochemical stains of each tumor were processed with image-processing software (ImagePro Plus; Media Cybernetics, Silver Spring, MD) to calculate fractional tumor neovascularity as contrast enhanced pixels over total tumor area (for US) and angiogenesis as stained area over total tumor area (for specimens). Linear regression was performed to compare the tumor neovascularity obtained with US to the histologically derived measures of angiogenesis with p-values less than 0.05 considered significant. There was a highly significant correlation between MFI and percent area stained with COX-2 (r=-0.60, p=0.004), as well as for measured fractional areas obtained with MFI and CD31 (r=-0.45, p=0.043). Significant correlation was also shown between FEI and COX-2 (r=-0.48, p=0.026). All other comparisons were not statistically significant (p > 0.17). In conclusion, contrast-enhanced US measures of tumor neovascularity in the glioma xenograft model C6 appear to provide a noninvasive marker for angiogenesis corresponding to the expression of COX-2 and, to a lesser degree, CD31.