In vivo characterization of changing blood-tumor barrier permeability in a mouse model of breast cancer metastasis: a complementary magnetic resonance imaging approach

Abstract

Objectives: The current lack of efficacy for any chemo- or molecular therapeutic in the treatment of brain metastases is thought to be due, in part, to the heterogeneous permeability of the blood-brain-barrier (BBB). Little is known about how heterogeneous permeability develops, or how it varies among individual metastases. Understanding the BBB's role in metastasis will be crucial to the development of new, more effective therapies. In this article, we developed the first magnetic resonance imaging-based strategy to detect and measure the volumes of BBB permeable and nonpermeable metastases and studied the development of altered BBB permeability in metastases in vivo, over time in a mouse model of breast cancer metastasis to the brain.

Materials and methods: Animals bearing human experimental brain metastases of breast cancer (231-BR cells) were imaged, using 3-dimensional balanced steady-state free precession to visualize total metastases, and contrast-enhanced T1-weighted spin echo with gadopentetic acid (Gd-DTPA) to visualize which of these displayed contrast enhancement, as Gd-DTPA leakage is indicative of altered BBB permeability.

Results: Metastases detected 20 days after injection showed no Gd-DTPA enhancement. At day 25, 6.1% ± 6.3% (mean ± standard deviation) of metastases enhanced, and by day 30, 28.1% ± 14.2% enhanced (P < 0.05). Enhancing metastases (mid: 0.14 ± 0.18 mm, late: 0.24 ± 0.32 mm) had larger volumes than nonenhancing (mid: 0.04 ± 0.04 mm, late: 0.09 ± 0.09 mm, P < 0.05); however, there was no significant difference between the growth rates of the 2.

Conclusions: A significant number of brain metastases were uniformly nonpermeable, which highlights the need for developing treatment strategies that can overcome the permeability of the BBB. The model developed herein can provide the basis for in vivo evaluation of both BBB permeable and nonpermeable metastases response to therapy.

FIGURE 1.

Comparison of metastasis detection pre- and post Gd-DTPA. Axial MR images of the mouse brain, with arrows indicating metastases. A to C show images of each sequence, before injection of Gd-DTPA contrast agent. Metastases appeared as signal hypointensities in noncontrast SPGR (A). Very few metastases were detected in noncontrast T1wSE, and appeared hypointense (C). Metastases appeared hyperintense in noncontrast bSSFP images (C). D to F show the corresponding images, post Gd-DTPA injection. Metastases appeared either hyperintense or isointense in postcontrast SPGR (D). Metastases were hyperintense in postcontrast T1wSE (E) and bSSFP (F) images. In noncontrast images, metastases are easiest to visualize with bSSFP. However, the only significant (P < 0.05) differences in metastasis detection were seen in noncontrast T1wSE and postcontrast SPGR (G).

FIGURE 2.

In vivo visualization of altered BBB permeability in the same animal over time. Axial MR images of the mouse brain. A to C show Gd-enhanced T1wSE, at each of early, mid, and late time points. D to F show the corresponding noncontrast bSSFP image. A metastasis is visible at early time point with bSSFP (D, arrow), but does not exhibit Gd-enhancement (A). At mid time point, this metastasis exhibited Gd-enhancement (B, large arrow). There are also several smaller metastases visible with bSSFP at mid time point (E, arrows) that do not enhance. At late time point, the large metastasis exhibited extensive Gd-enhancement (C, large arrow); however, the small metastases visible with bSSFP (F, arrows) still did not enhance (F). There were a small number of metastases visible with bSSFP at early time point; however, none of these exhibited Gd-enhancement. There was a significant difference between the number of metastases detected with bSSFP and the number detected with Gd-enhanced T1wSE at mid and late time points (P < 0.01) (G). When taken as a fraction of total metastases detected (H), there was a significant difference between the number of enhancing metastases at mid and late time points (P < 0.05). The ventricles can also be visualized (*) in these images.

FIGURE 3.

Volume measurements of enhancing and nonenhancing metastases. A, At both mid and late time points, the average volume of enhancing metastases was significantly larger than nonenhancing metastases (P < 0.05). However, there was a wide range of volumes for both enhancing and nonenhancing metastases (B). There also appeared to be a minimum volume threshold of enhancing metastases, although being larger than this did not guarantee enhancement. C, 3D volume rendering of a mouse brain in the coronal plane, from the same mouse at each time point. Gd-enhancing metastases are rendered in red and nonenhancing metastases are shown in green. Neither volume nor position in the brain appear to have an influence on whether a metastasis enhanced or not.

FIGURE 4.

Examples of volume and permeability heterogeneity in metastases. Axial MR images of the mouse brain. A, B show Gd-enhanced T1wSE images from 2 different mice. C, D show the corresponding noncontrast bSSFP images. A large metastasis (0.42 mm³) near the lateral ventricle was visible with bSSFP (C, arrow), but did not exhibit Gd-enhancement (A), while a nearby metastasis visible with bSSFP (C) did exhibit Gd-enhancement (A, arrow). Two very small metastases (left: 0.04 mm³, right: 0.06 mm³) in the frontal cerebral cortex of another animal were visible with bSSFP (D, arrows) and exhibited Gd-enhancement (B, arrows).

FIGURE 5.

BBB permeability with Gd-enhanced MRI confirmed with in situ fluorescence. Axial MR images of the mouse brain reveal a metastasis visible with bSSFP (A, arrow) that exhibited Gd-enhancement with T1wSE (B, arrow) at mid time point. C, The same metastasis visualized with fluorescent microscopy, appeared as 2 separate clusters of MDA-231-BR cells (green fluorescence), and exhibited extensive fluorescent dextran permeability in the surrounding parenchyma (red fluorescence). D, The same section stained with H&E, to provide anatomic reference, shows a compact tumor connected to a larger, more diffuse tumor cell cluster.

FIGURE 6.

Non-BBB permeable metastasis detected with MRI confirmed with in situ fluorescence. Axial MR images of the mouse brain reveal a small metastasis that was visible with bSSFP (A, arrow) that did not exhibit Gd-enhancement with T1wSE (B) at early time point. C, The same metastasis visualized with fluorescent microscopy appears as a diffuse collection of several MDA-231-BR cells (green fluorescence) with no fluorescent dextran leakage visible in the surrounding tissue. There is, however, dextran leakage surrounding vessels just above the tumor (red fluorescence). D, The same section stained with H&E reveals large lesions of empty space surrounding the cancer cells.