Longitudinal MRI evaluation of intracranial development and vascular characteristics of breast cancer brain metastases in a mouse model

Abstract

Longitudinal MRI was applied to monitor intracranial initiation and development of brain metastases and assess tumor vascular volume and permeability in a mouse model of breast cancer brain metastases. Using a 9.4T system, high resolution anatomic MRI and dynamic susceptibility contrast (DSC) perfusion MRI were acquired at different time points after an intracardiac injection of brain-tropic breast cancer MDA-MB231BR-EGFP cells. Three weeks post injection, multifocal brain metastases were first observed with hyperintensity on T2-weighted images, but isointensity on T1-weighted post contrast images, indicating that blood-tumor-barrier (BTB) at early stage of brain metastases was impermeable. Follow-up MRI revealed intracranial tumor growth and increased number of metastases that distributed throughout the whole brain. At the last scan on week 5, T1-weighted post contrast images detected BTB disruption in 160 (34%) of a total of 464 brain metastases. Enhancement in some of the metastases was only seen in partial regions of the tumor, suggesting intratumoral heterogeneity of BTB disruption. DSC MRI measurements of relative cerebral blood volume (rCBV) showed that rCBV of brain metastases was significantly lower (mean= 0.89±0.03) than that of contralateral normal brain (mean= 1.00±0.03; p<0.005). Intriguingly, longitudinal measurements revealed that rCBV of individual metastases at early stage was similar to, but became significantly lower than that of contralateral normal brain with tumor growth (p<0.05). The rCBV data were concordant with histological analysis of microvascular density (MVD). Moreover, comprehensive analysis suggested no significant correlation among tumor size, rCBV and BTB permeability. In conclusion, longitudinal MRI provides non-invasive in vivo assessments of spatial and temporal development of brain metastases and their vascular volume and permeability. The characteristic rCBV of brain metastases may have a diagnostic value.

Figure 1. Dynamic susceptibility contrast (DSC) MRI of rCBV.

A. A series of T₂ ^*-weighted images of a mouse brain was acquired before and after a bolus injection of the contrast agent, Gd-DTPA via a tail vein (arrow). Immediately after the injection, significant loss in signal intensity was observed, which gradually recovered to the baseline level 5 mins later. B. The flowchart illustrates the data process of DSC MRI to generate rCBV map. Raw data of DSC MRI signal versus time curve was first plotted, depicting the first pass of the contrast agent via the brain as the dip on the curve. The ΔR₂ ^* was then calculated from the signal time course. FPPM was applied to determine the general trend of ΔR₂ ^* and a three-segment baseline was generated. Finally, Gamma-variate fitting was used to correct ΔR₂ ^*, and the area under the bolus was calculated, which is proportional to CBV. C. An anatomic T₂-weighted image was obtained from a normal mouse brain. A color-coded rCBV map generated from DSC MRI was overlaid on the T₂-weighted image showing symmetric distribution of rCBV between the two hemispheres (D).

Figure 2. Longitudinal MRI monitoring of the initiation and development of intracranial brain metastases.

A. MRI scans of the whole mouse brain were initiated 3 weeks after intracardiac injection of MDA-MB231Br cells and repeated once a week for 2 weeks. Four consecutive coronal MRI sections of a representative mouse brain showed no apparent intracranial lesions on T₂-weighted images. However, follow-up images at week 4 identified multiple lesions with hyper-intensity on T₂-weighted images (arrowhead), but none of them was enhanced on T₁-weighted post contrast images. An increased number of lesions (arrowheads) appeared on the images at week 5, only a few of which (arrowheads) were enhanced post Gd-DTPA. B. Corresponding histological sections of H&E staining showed a good correlation with MRI. C. MRI evaluation of a total of 464 metastases in 9 mice brains indicated that these metastases distributed through the whole mouse brain with a higher incidence in the brain cortex (49%). Note: OB: Olfactory bulb; BG: Basal ganglia; Hippo: Hippocampus.

Figure 3. Significantly lower rCBV in brain metastases than contralateral normal brain.

A. Four weeks after intracardiac injection of 231Br cells, T₂-weighted MRI revealed multiple high signal intensity lesions (arrowheads) on four consecutive coronal sections of a representative mouse brain. Only a few of the lesions (arrowheads) were enhanced on T₁-weighted post contrast images, one (blue arrowhead in the MRI section 3) of which showed partial enhancement, indicating intratumoral heterogeneity of BTB disruption. rCBV maps of the four sections were generated and overlaid on the T₂-weighted images. B. The rCBV values of the metastatic lesions and their contralateral normal brain were obtained and summarized in the table. Note the color presented in the table coincides with the color of arrowhead on each of the MR images. Most of metastatic lesions had lower rCBV values than their contralateral counterparts of normal brain. C. Statistical analysis of rCBV in a total of 212 lesions of 9 animals obtained from the last follow-up MRI showed significantly lower rCBV of the metastatic tumors with a mean value of 0.89±0.03 (s.e.), compared to the contralateral normal brain (mean  = 1.00±0.03; p<0.005).

Figure 4. Lack of correlation between rCBV, tumor size and permeability of brain metastases.

Based on T₁-weighted contrast enhanced MRI, the 212 metastases studied by DSC MRI were separated into the permeable (enhanced, n  = 70) and non-permeable (not enhanced, n  = 142) group. A. A plot of rCBV versus individual tumor size showed no correlation in either the permeable (filled; R² = 0.01) or non-permeable group (empty; R²<0.02). B. The rCBV values of the permeable lesions (median  = 0.84, ranging from 0.34 to 2.10) were not significantly different from those of the non-permeable ones (median  = 0.82, ranging from 0.16 to 2.84; p>0.1). C. Further comparison found no significant difference in tumor size between the permeable (mean  = 0.49±0.11 mm²) and the non-permeable (mean  = 0.47±0.14 mm²; p  = 0.1) metastases.

Figure 5. Longitudinal MRI study of changes in BTB permeability and rCBV of brain metastases.

A. Longitudinal MRI of a representative mouse brain was initiated 2 weeks after intracardiac injection of 231Br cells. At week 3, five small metastases (arrowhead) were identified on four consecutive T₂-weighted coronal images. At week 4, many more lesions appeared on T₂-weighted coronal images, while all the 5 lesions seen on week 3 were found to increase in size (arrowhead). Changes in BTB permeability and rCBV were then evaluated for these five lesions. There was initially no contrast enhancement seen in the five tumors at week 3, indicating an intact BTB. All the tumors except one (yellow arrowhead) still kept BTB intact at week 4. rCBV maps were created and rCBV values of the tumors were presented in the table (B). C. A total of 32 lesions in 5 animals were seen on both scans of weeks 3 and 4. rCBV of brain metastases (solid) was initially similar to that of contralateral normal brain (open; mean  = 1.05±0.05 (se) vs. 0.96±0.04), but decreased significantly (p<0.05) and became significantly lower as compared to their contralateral normal brain in the late scan (mean  = 0.88±0.06 vs. 1.00±0.06; p<0.05).

Figure 6. Immunohistochemical study of microvascular density (MVD) in brain metastases.

A. Anti-CD31 staining was performed on a brain section bearing metastases. A cortical lesion (∼ 600 µm in diameter) was depicted with green fluorescence (GFP). Microvessels (red) within the lesion appeared less dense, as compared to abundant fine vessels in the contralateral normal brain tissues (B). Some of the tumor vessels were irregular in shape and larger in diameter (arrow). C. Quantitative data of MVD showed a significantly lower MVD in brain metastases versus contralateral normal brain (mean  = 669±201/mm² vs. 965±177/mm²; p<0.05).