Molecular Imaging of Ultrasound-Mediated Blood-Brain Barrier Disruption in a Mouse Orthotopic Glioblastoma Model

Abstract

Glioblastoma (GBM) is an aggressive and malignant primary brain tumor. The blood-brain barrier (BBB) limits the therapeutic options available to tackle this incurable tumor. Transient disruption of the BBB by focused ultrasound (FUS) is a promising and safe approach to increase the brain and tumor concentration of drugs administered systemically. Non-invasive, sensitive, and reliable imaging approaches are required to better understand the impact of FUS on the BBB and brain microenvironment. In this study, nuclear imaging (SPECT/CT and PET/CT) was used to quantify neuroinflammation 48 h post-FUS and estimate the influence of FUS on BBB opening and tumor growth in vivo. BBB disruptions were performed on healthy and GBM-bearing mice (U-87 MG xenograft orthotopic model). The BBB recovery kinetics were followed and quantified by [99mTc]Tc-DTPA SPECT/CT imaging at 0.5 h, 3 h and 24 h post-FUS. The absence of neuroinflammation was confirmed by [18F]FDG PET/CT imaging 48 h post-FUS. The presence of the tumor and its growth were evaluated by [68Ga]Ga-RGD₂ PET/CT imaging and post-mortem histological analysis, showing that tumor growth was not influenced by FUS. In conclusion, molecular imaging can be used to evaluate the time frame for systemic treatment combined with transient BBB opening and to test its efficacy over time.

Keywords: PET; SPECT; blood-brain barrier; drug delivery; focused ultrasound; glioblastoma.

Figure 1

Schematic representation of the experimental plan used for the FUS-induced hemispheric BBB permeabilization and imaging study on healthy and tumor-bearing animals. Group “GBM + FUS“: tumor-bearing animals undergoing FUS-mediated BBB permeabilization; Group “GBM only”: tumor-bearing animals without BBB permeabilization (tumor growth control); Group “FUS only”: healthy animals undergoing FUS-mediated BBB permeabilization (BBB opening control).

Figure 2

Imaging of the animal’s brain following BBB disruption and [99mTc]Tc-DTPA intravenous injection in healthy mice 0.5 h later. (A) Brain representative SPECT/CT tomographic images of [99mTc]Tc-DTPA distribution in animals that received single-spot (group A, left panel) or raster scan trajectory (group B, right panel) focused ultrasound. The white square represents the approximate region were FUS were applied for both sonication schemes; (B) Quantification of [99mTc]Tc-DTPA activity 0.5 h, 3 h and 24 h following focused ultrasound. Results are expressed as injected dose per tissue volume (ID/mm³; n = 3; mean ± SEM). Statistical differences are reported as # for comparisons between single spot vs. raster scan trajectory and as * for comparisons between 0.5 h vs. 3 h vs. 24 h (^# p < 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001). The black dotted line is a visual representation of the baseline of an animal that did not receive BBB permeabilization, obtained as an average of the 0.5 h, 3 h and 24 h [99mTc]Tc-DTPA quantifications of the sham animal (7.8 × 10⁻⁸ ± 1.6 × 10⁻⁸).

Figure 3

BBB permeabilization and [99mTc]Tc-DTPA SPECT/CT imaging on healthy and tumor-bearing animals. (A) Brain representative images obtained by [99mTc]Tc-DTPA-mediated SPECT/CT imaging in an animal of group “GBM + FUS” (tumor-bearing animals undergoing FUS-mediated BBB permeabilization) 0.5 h after hemispheric trajectory focused ultrasound. The white dotted line represents the separation between ipsilateral (I) and contralateral (C) brain hemispheres that was used for the quantifications; (B) Quantification of [99mTc]Tc-DTPA activity in animals of group “GBM + FUS” (tumor-bearing animals undergoing FUS-mediated BBB permeabilization, n = 5), group “GBM only” (tumor-bearing animals without FUS-mediated BBB permeabilization, n = 5) and group “FUS only” (healthy animals undergoing FUS-mediated BBB permeabilization, n = 4). Results are expressed as ipsilateral/contralateral ratio (mean ± SEM; * p < 0.05).

Figure 4

BBB permeabilization and [18F]FDG PET/CT imaging on healthy and tumor-bearing animals. (A) Brain representative images of [18F]FDG-mediated PET/CT two days after hemispheric trajectory focused ultrasound in an animal of group “GBM + FUS” (tumor-bearing animals undergoing FUS-mediated BBB permeabilization). The white dotted line represents the separation between ipsilateral (I) and contralateral (C) brain hemispheres that was used for the quantifications; (B) Quantification of [18F]FDG activity in animals of group “GBM + FUS” (tumor-bearing animals undergoing FUS-mediated BBB permeabilization, n = 5), group “GBM only” (tumor-bearing animals without FUS-mediated BBB permeabilization, n = 5) and group “FUS only“ (healthy animals undergoing hemispheric FUS-mediated BBB permeabilization, n = 4). Results are expressed as ipsilateral/contralateral ratio (mean ± SEM).

Figure 5

Tumor growth and [68Ga]Ga-RGD₂ PET/CT imaging following BBB permeabilization by focused ultrasound on tumor-bearing animals. (A) Quantification of [68Ga]Ga-RGD₂ activity in animals of group “GBM + FUS” (tumor-bearing animals undergoing FUS-mediated BBB permeabilization) and group “GBM only” (tumor-bearing animals without FUS-mediated BBB permeabilization) before and after BBB permeabilization. Results are expressed as injected dose per tissue volume (ID/mm³; n = 5 per group; mean ± SEM; ns: not significant); (B) Brain representative images obtained by [68Ga]Ga-RGD₂-mediated PET/CT imaging at day 16 after hemispheric trajectory focused ultrasound in an animal of group “GBM + FUS” (tumor-bearing animals undergoing FUS-mediated BBB permeabilization) and an animal of group “GBM only” (tumor-bearing animals without FUS-mediated BBB permeabilization).

Figure 6

Survival and histological analyses following tumor grafting and BBB permeabilization by focused ultrasound. (A) Kaplan-Meier survival curves of the animals of group “GBM + FUS” (tumor-bearing animals undergoing BBB permeabilization) and group “GBM only” (tumor-bearing animals without FUS-mediated BBB permeabilization), n = 5 per group; ns: not significant; (B) Representative coronal section of Hematoxylin and Eosin staining of tumor-bearing animals that had been exposed or not to FUS-mediated BBB permeabilization (group “GBM + FUS” and “GBM only” respectively) at end point. The a represents relatively low-enhancing glioblastoma tumor cells forming a spheric tumor lesion and the b represents normal brain tissue surrounding the tumor (scale bar: 1 mm).