Dose-response relationship of ultrasound contrast agent in an in vivo murine melanoma model

Abstract

Many factors affect the sensitivity and reliability of tumor vasculature assessment at the small doses of contrast agent necessary for imaging mice. In this study we investigate the dose-response relationship of ultrasound contrast agent for a minimal exposure power Doppler technique (minexPD) in a murine melanoma model. K1735 murine melanomas grown in 25 C3H/HeN mice were imaged by power Doppler ultrasound using different doses of contrast agents, Optison(R) and Definity(R). Six mice were treated with an antivascular agent, combretastatin A4-phosphate (CA4P), and imaged before and after treatment. The color-weighted fractional area (CWFA) of the peak-enhanced image was measured to assess tumor perfusion on a relative scale of 0 to 100. CWFA increased logarithmically with dose (R(2)=0.97). Treatment with CA4P resulted in pronounced reduction in tumor perfusion 2 h after contrast injection, but perfusion recovered in the tumor periphery after 2 days. CWFA was significantly different between pre- and post-treatment for all doses at 2 h and 2 days (p < 0.05, respectively). There was no significant difference detectable between the two contrast agents, Optison(R) and Definity(R) (p = 0.46). In vivo tumor enhancement in mice increases as logarithmic function with dose. Although the extent of enhancement is dose dependent, the difference between pre- and post-therapy enhancement is relatively unchanged and uniform at varying doses. The two contrast agents tested in this study performed equally well. These results suggest that quantitative contrast-enhanced power Doppler imaging is an effective method for monitoring therapy response of tumors in mice.

Figure 1

Dose-dependency of Optison® enhancement of K1735 tumor perfusion. Long axis view of the same K1735 tumor after injection of 10 (top left), 20 (top right), 50 (lower left) and 100 μl (lower right) of Optison®; the transducer is not moved between the injections. Increasing contrast dose results in stronger enhancement with considerable blooming. Note the very uniform vascularity of this tumor model.

Figure 2

Optison® dose relationship to enhancement of tumor perfusion. Dose–response relationship of Optison® in untreated K1735 tumors (n = 25). Color-weighted fractional area (CWFA) parameters (mean ± standard error) plotted over dose show a logarithmic increase of the CWFA with increasing dose as shown by the equation included in the figure.

Figure 3

Effect of treatment with combretastatin A4-phosphate on tumor perfusion. Long axis view of a K1735 tumor before, 2 h after and 2 days after treatment with combretastatin A4-phosphate (CA4P) (from left to right). At 2 h there is profound loss of perfusion, which recovers primarily in the periphery after 2 days.

Figure 4

Dose–response relationship of Optison® enhancement of perfusion in tumors treated and untreated with combretastatin A4-phosphate. Dose–response relationship of Optison® in untreated K1735 tumors (n = 25), and K1735 tumors 2 h and 2 days after treatment with combretastatin A4-phosphate (n = 6). The vascularity is significantly decreased 2 h after treatment and recovers slightly 2 days later. The difference between treated and untreated tumors is evident at all contrast doses.

Figure 5

Contrast enhancement of tumor perfusion by Optison® versus Definity®. K1735 tumor scanned after injection of 20 and 50 μl of Optison® (top row, left to right) and 20 and 50 μl of Definity® (bottom row, left to right). Note the very similar enhancement pattern with subjectively better definition of the vessels using Definity®.

Figure 6

Comparison between contrast-enhanced ultrasound imaging of tumor perfusion and tumor histopathology. K1735 tumor scanned 2 days after treatment with combretastatin A4-phosphate with corresponding hematoxylin and eosin staining of the tumor. Two large areas of necrosis divided by a vascularized zone are seen as perfusion defects on the power Doppler image.