Tumor vessel compression hinders perfusion of ultrasonographic contrast agents

Abstract

Contrast-enhanced ultrasound (CEUS) is an advanced approach to in vivo assessment of tumor vascularity and is being increasingly adopted in clinical oncology. It is based on 1- to 10 microm-sized gas microbubbles, which can cross the capillary beds of the lungs and are effective echo enhancers. It is known that high cell density, high transendothelial fluid exchange, and poorly functioning lymphatic circulation all provoke solid stress, which compresses vessels and drastically reduces tumor blood flow. Given their size, we supposed that the perfusion of microbubbles is affected by anatomic features of tumor vessels more than are contrast agents traditionally used in dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Here, we compared dynamic information obtained from CEUS and DCE-MRI on two experimental tumor models exhibiting notable differences in vessel anatomy. We found that tumors with small, flattened vessels show a much higher resistance to microbubble perfusion than to MRI contrast agents, and appear scarcely vascularized at CEUS examination, despite vessel volume adequate for normal function. Thus, whereas CEUS alone could induce incorrect diagnosis when tumors have small or collapsed vessels, integrated analysis using CEUS and DCE-MRI allows in vivo identification of tumors with a vascular profile frequently associated with malignant phenotypes.

Figure 1

Morphologic differences between BB1 and A17 tumor histotypes. Two different cell lines were established from mammary tumors, which had spontaneously arisen in FVB/neuNT233 transgenic mice. The BB1 cells exhibited a round to polygonal shape, and grew in tightly junctioned clusters (A). The A17 cells were star- or spindle-shaped and grew in monolayers free of growth constraints (B). The subcutaneous injection of BB1 cells provoked lobular carcinomas resembling spontaneous tumors (C), with large hemorrhagic areas (D), whereas A17 cells generated sarcomatoid lesions (E). At T2-weighted MRI, BB1 parenchyma showed a heterogeneous and notably lower signal intensity than A17 tumors (F and G).

Figure 2

Postmortem histopathologic analysis of BB1 and A17 tumor vasculature. Postmortem analysis of CD31-immunostained sections showed that A17 vasculature was composed of extremely numerous but, on average, very small vessels (A and B). The vessels of BB1 tumors proved less numerous and, on average, larger, and were confined within the stromal compartment of viable areas (C). At ultrastructural analysis, the lumina of A17 vessels frequently appeared flattened (D; arrow) and the endothelial cells exhibited blastic traits such as abundance of polyribosomes (E; black filled arrow). The occurrence of pinocytotic vesicles (E; gray filled arrow) suggests that vessel lumina were functional. In contrast, at ultrastructural analysis, the vessels of BB1 tumors frequently appeared enlarged (F), occasionally close to perivascular edemas (F; arrow) and delimited by morphologically differentiated endothelial cells (G; arrow).

Figure 3

In vivo assessment of BB1 and A17 tumor vasculature by DCE-MRI with Gd-DTPA-albumin. A series of quadrangular ROIs matching the whole tumor section was manually selected on the MRI images (A), and was analyzed as previously described [20] to measure the fractional vascular volume (fPV) and transendothelial permeability (kPS) of tumor vessels. The first parameter is dependent on the intercept between the curve and the y-axis; the latter depends on the slope of the curve (B). Both parameters proved significantly higher, on average, in A17 than in BB1 tumors (C; see also Table 1).

Figure 4

In vivo assessment of BB1 and A17 tumor vasculature by DCE-MRI with Gd-DTPA. DCE-MRI with Gd-DTPA as contrast agent confirmed that A17 tumors showed a higher level of vascularization than BB1 tumors. Unlike DCE-MRI with Gd-DTPA-albumin, the maps of enhancement obtained by pixel-by-pixel subtraction between the last postcontrast and the precontrast images make evident the difference in size and the distribution of vessel network between A17 (A) and BB1 (B) tumors. Two representative time -intensity curves show vascular differences observed between the two tumor histotypes (C).

Figure 5

In vivo assessment of BB1 and A17 tumor vasculature by CEUS with SonoVue contrast agent. CEUS showed that A17 tumors had a lower level of vascularization than BB1 tumors. Signal enhancement after contrast agent injection was less evident in A17 (A, I: precontrast; II: postcontrast) than in BB1 (A, III: precontrast; IV: postcontrast) tumors. Two representative time-intensity curves show differences between time course contrast enhancement observed in A17 (B, top panel) and BB1 (B, bottom panel) tumors. BB1 tumors had a reduced maximal intensity peak (Mi), delayed slope (S), and reduced wash-out (Wo) in comparison with BB1 tumors (C; see also Table 1).