Ex vivo Evans blue assessment of the blood brain barrier in three breast cancer brain metastasis models

Abstract

The limited entry of anticancer drugs into the central nervous system represents a special therapeutic challenge for patients with brain metastases and is primarily due to the blood brain barrier (BBB). Albumin-bound Evans blue (EB) dye is too large to cross the BBB but can grossly stain tissue blue when the BBB is disrupted. The course of tumor development and the integrity of the BBB were studied in three preclinical breast cancer brain metastasis (BCBM) models. A luciferase-transduced braintropic clone of MDA-231 cell line was used. Nude mice were subjected to stereotactic intracerebral inoculation, mammary fat pad-derived tumor fragment implantation, or carotid artery injections. EB was injected 30 min prior to euthanasia at various timepoints for each of the BCBM model animals. Serial bioluminescent imaging demonstrated exponential tumor growth in all models. Carotid BCBM appeared as diffuse multifocal cell clusters. EB aided the localization of metastases ex vivo. Tumor implants stained blue at 7 days whereas gross staining was not evident until day 14 in the stereotactic model and day 28 for the carotid model. EB assessment of the integrity of the BBB provides useful information relevant to drug testing in preclinical BCBM models.

Fig. 1

Brain metastases by stereotactic injection of MDA-231-Br cells. a Using a stereotactic frame, 2 × 10⁵ cells are injected via a burr hole placed within 2 mm of the bregma (right). b CT scan with BLI overlay shows deep intracerebral signal on coronal and sagittal views. c In vivo average flux of 18 mice shows exponential (R² = 0.998) increase in bioluminescence over background (dashed line) days 4 through 25. d Whole brain coronal sections of mice PID 7, 14, 28 corresponding to color box datapoints in c. Evans blue staining of tumors is evident PID 14 and 28, indicating disruption of BBB; respective H&E sections (scale bars 100 µm) and in vivo mouse bioluminescent images. e Micrograph of PID 28 tumor shown in d with inset dark-field microscopy demonstrating fluorescence of cells stained by Evans blue

Fig. 2

Brain metastases generated by implantation of MDA-231-Br tumor fragments. a Depiction of orthotopic MFP tumor (left), the selection of bioluminescent extracted fragment (middle), and implantation of 1 mm³ fragments via burr hole into mouse brain (right). b CT scan imaging with BLI overlay shows intracerebral bioluminescent signal on coronal and sagittal views. c In vivo average flux of six mice BLI over a 35-day period shows (R² = 0.998) exponential increase in bioluminescence over background (dashed line) days 5–35. d Whole brain coronal sections of mice PID 7 and 35, corresponding to color box datapoints in c. Evans blue staining of tumors indicates disruption of the BBB. Parallel frames showing respective H&E sections (scale bars 100 µm) and in vivo mouse bioluminescent images. e Microphotograph of tumor invading brain parenchyma at PID 35 shown in d

Fig. 3

Brain metastases generated by CCA of MDA-231-Br cells. a Depiction of direct injection 5 × 10⁴ cells into the CCA below the bifurcation into external and internal carotid branches (right). b CT scan with BLI overlay shows deep intracerebral bioluminescent signal on coronal and sagittal views. c In vivo average flux of 21 mice over first 42 days, showing exponential (R² = 0.979) increase in bioluminescence over background (dashed line) days 14 and 31. d Whole brain coronal sections of mice PID 17, 28, and 42, corresponding to color box datapoints in c. Evans blue staining was undetectable at 2 weeks but detectable by 4 weeks, indicating disruption of the BBB. Parallel frames show tumor cell reactivity to anti-HLA antibody (scale bars 100 µm) used to identify isolated cell clusters and corresponding mouse in vivo bioluminescence for specified time points. e Microscopic section of brain tumor-interface at PID 42 shown in d

Fig. 4

Evans blue, bioluminescence, and histology findings in three BCBM models. Flow chart of stereotactic (green), implant (red), and carotid (orange) models showing the proportion of mice with visible Evans blue staining at various time points and their corresponding bioluminescence (defined as greater than 1 × 10⁵ p/s). Tumors were identified histologically with H&E (implant and stereotactic injection models) and anti-HLA staining (carotid injection model). *Mouse lost BLI signaling