The antivascular action of physiotherapy ultrasound on a murine tumor: role of a microbubble contrast agent

Abstract

This study investigated whether a microbubble-containing ultrasound contrast agent had a role in the antivascular action of physiotherapy ultrasound on tumor neovasculature. Ultrasound images (B-mode and contrast-enhanced power Doppler [0.02 mL Definity]) were made of 22 murine melanomas (K1735(22)). The tumor was insonated (I(SATA) = 1.7 W cm(-2), 1 MHz, continuous output) for 3 min and the power Doppler observations of the pre- and postinsonation tumor vascularities were analyzed. Significant reductions (p = 0.005 for analyses of color-weighted fractional area) in vascularity occurred when a contrast-enhanced power Doppler study occurred before insonation. Vascularity was unchanged in tumors without a pretherapy Doppler study. Histologic studies revealed tissue structural changes that correlated with the ultrasound findings. The underlying etiology of the interaction between the physiotherapy ultrasound beam, the microbubble-containing contrast agent and the tumor neovasculature is unknown. It was concluded that contrast agents play an important role in the antivascular effects induced by physiotherapy ultrasound.

Fig. 1

Power Doppler ultrasound images (group A, mouse A2R) of a melanoma, following the intravenous injection of an ultrasound contrast agent, prior to (a) and following (b) sham insonation. The double ended arrow represents a 1 cm scale. The green box is the region from which Doppler information was acquired, and the boundaries of the neoplasm, traced in white from the corresponding B-mode image, have been copied to these images. In both images, the tumor and the contiguous normal tissues are almost fully perfused. A similar almost full perfusion of the melanoma and adjacent normal tissues is also present in a tumor (c) insonated with physiotherapy ultrasound without the prior injection of a contrast agent (Group B, mouse E2R).

Fig. 2

B-mode and power Doppler ultrasound images following the intravenous injection of an ultrasound contrast agent (Group C, mouse ILIR) prior to (a and b) and following treatment (c and d) with physiotherapy ultrasound. In the initial B-mode image (a) the melanoma is hypoechoic to the surrounding tissues and its distinct borders are outlined with white dots. Following the injection of the contrast agent, the initial power Doppler image shows that the tumor and the contiguous tissues are almost fully perfused (b). 15 min after the contrast microbubbles were no longer detectable in the initial power Doppler image, the tumor was insonated for 3 min with physiotherapy ultrasound. In the post-insonation B-mode image (c), the tumor has a patchy increase in echogenicity and hypoechoic peri-tumor edema is present (arrow). Reduced tumor perfusion is demonstrated in the post-insonation power Doppler image (d) and the peri-tumor edema is again seen (arrow); the perfusion of the normal tissues is unchanged. The observations suggest that the contrast agent played a role in the antivascular effect of physiotherapy ultrasound.

Fig. 3

Effect of insonation on vascular perfusion assessed by power Doppler imaging: (a) represents visual estimation of tumor vascularity, (b) represents measurement of percentage area of flow (PAF) and (c) represents measurement of color-weighted flow area (CWFA). In each figure, the hatched blocks represent the mean size of the vascular area before insonation and the open blocks that after treatment; the bars represent the standard deviation of the mean. Regardless of the method of analysis, there was no significant change in tumor vascularity in either the sham-treated control group (A) or in tumors that were insonated without an initial contrast-enhanced power Doppler study (Group B). There were, however, significant reductions in tumor vascularity in tumors insonated 15 (Group C) or 60 min (Group D) after the contrast microbubbles were no longer detectable in the initial power Doppler images. It appears that the ultrasound contrast agent had a role in the observed antivascular effect of physiotherapy ultrasound on the tumor neovasculature.

Fig. 3

Effect of insonation on vascular perfusion assessed by power Doppler imaging: (a) represents visual estimation of tumor vascularity, (b) represents measurement of percentage area of flow (PAF) and (c) represents measurement of color-weighted flow area (CWFA). In each figure, the hatched blocks represent the mean size of the vascular area before insonation and the open blocks that after treatment; the bars represent the standard deviation of the mean. Regardless of the method of analysis, there was no significant change in tumor vascularity in either the sham-treated control group (A) or in tumors that were insonated without an initial contrast-enhanced power Doppler study (Group B). There were, however, significant reductions in tumor vascularity in tumors insonated 15 (Group C) or 60 min (Group D) after the contrast microbubbles were no longer detectable in the initial power Doppler images. It appears that the ultrasound contrast agent had a role in the observed antivascular effect of physiotherapy ultrasound on the tumor neovasculature.

Fig. 4

Comparison of visual assessment of tumor vascularity with quantitative measures of vascularity: (a) percentage area of flow (PAF) and (b)color-weighted flow area (CWFA). A correlation (R²) of 0.64–0.73 indicated a good agrrement between qualitative and quantitative assessment of tumor vascularity.

Fig. 5

Histologic appearance of untreated melanoma (group A, mouse E1L). The melanoma is highly cellular and compact. It is characterized by spindle shaped cells arranged in a pattern of short, often interlacing, streams or bundles.

Fig. 6

Histologic appearance of a melanoma insonated without a prior contrast-enhanced power Doppler study (group B, mouse A1L). The melanoma is identical to those found in the control group (Fig. 6). Note the pre-existing normal fibers of skeletal muscle (unaffected by the insonation) enveloped by the growing neoplasm and separated by infiltrating neoplastic melanocytes (two arrows).

Fig. 7

Histologic appearance of a melanoma insonated 15 min after contrast microbubbles were no longer detectable in the power Doppler image (group C, mouse C1L). The melanoma shows marked dilation of the capillary bed (three thin arrows), often with surrounding hemorrhage into the neoplastic tissue leading to separation of the neoplastic cells (two circled areas). The decreased compactness of the cells in other regions is related to intercellular edema (three thick arrows).

Fig. 8

Histologic appearance of a melanoma insonated 60 min after contrast microbubbles were no longer detectable in the power Doppler image (group D, mouse B1L). The dilation of the capillary bed (three small arrows), with associated hemorrhage (circled area), is identical to that seen in Fig. 8. Note also the pre-existing nerve fiber that is unaffected by the insonation but is infiltrated with neoplastic cells (open arrow).

Fig. 9

Linear regression analyses (across all tumors) comparing the total % area in which tissue structure had altered and the change in tumor vascularity determined by contrast enhanced power Doppler images. The four black dots represent data from each of the four groups of mice (groups A, B, C and D from left to right). There is high correlation between total % histologic change and the ultrasound measurements of tumor vascularity (PAF and CWFA).

Fig. 10

Schema of cumulative histogram area analysis of contrast-enhanced power Doppler images.

Fig. 11

Linear regression analysis (across all tumors) comparing the color-weighted flow area (CWFA) and cumulative histogram area methods of analyzing contrast-enhanced power Doppler observations of tumor vascularity. There is a full correlation (R² = 1.0) between the two measurements of tumor vascularity.