Focused ultrasound for safe and effective release of brain tumor biomarkers into the peripheral circulation

Abstract

The development of noninvasive approaches for brain tumor diagnosis and monitoring continues to be a major medical challenge. Although blood-based liquid biopsy has received considerable attention in various cancers, limited progress has been made for brain tumors, at least partly due to the hindrance of tumor biomarker release into the peripheral circulation by the blood-brain barrier. Focused ultrasound (FUS) combined with microbubbles induced BBB disruption has been established as a promising technique for noninvasive and localized brain drug delivery. Building on this established technique, we propose to develop FUS-enabled liquid biopsy technique (FUS-LBx) to enhance the release of brain tumor biomarkers (e.g., DNA, RNA, and proteins) into the circulation. The objective of this study was to demonstrate that FUS-LBx could sufficiently increase plasma levels of brain tumor biomarkers without causing hemorrhage in the brain. Mice with orthotopic implantation of enhanced green fluorescent protein (eGFP)-transfected murine glioma cells were treated using magnetic resonance (MR)-guided FUS system in the presence of systemically injected microbubbles at three peak negative pressure levels (0.59, 1.29, and 1.58 MPa). Plasma eGFP mRNA levels were quantified with the quantitative polymerase chain reaction (qPCR). Contrast-enhanced MR images were acquired before and after the FUS sonication. FUS at 0.59 MPa resulted in an increased plasma eGFP mRNA level, comparable to those at higher acoustic pressures (1.29 MPa and 1.58 MPa). Microhemorrhage density associated with FUS at 0.59 MPa was significantly lower than that at higher acoustic pressures and not significantly different from the control group. MRI analysis revealed that post-sonication intratumoral and peritumoral hyperenhancement had strong correlations with the level of FUS-induced biomarker release and the extent of hemorrhage. This study suggests that FUS-LBx could be a safe and effective brain-tumor biomarker release technique, and MRI could be used to develop image-guided FUS-LBx.

Fig 1. Experimental setup.

(A) Illustration and (B) picture of the MR-guided FUS system used for the FUS-LBx treatment. A clinical MR-guided FUS system that integrated a clinical MRI scanner with a FUS phased array was configured for small animal study by coupling with a small animal adaptor (see Methods for more details). (C) MR image of a fiber-optic hydrophone used for measuring the acoustic pressures. The hydrophone was placed in a water container that was placed on top of the small animal adapter.

Fig 2. Effect of peak negative pressure on FUS-LBx yield.

Comparison of the circulating eGFP mRNA level in the control mice receiving no treatment and FUS-treated mice at 0.59, 1.29, and 1.58 MPa. eGFP mRNA levels were quantified using quantitative polymerase chain reaction (qPCR) with (A) primer A and (B) primer B.

Fig 3. Effect of peak negative pressure on brain hemorrhage.

(A–D) Representative images of H&E-stained sections from mice in the control, 0.59 MPa, 1.29 MPa, and 1.58 MPa groups, respectively. Boxes in the images on the top indicate the location where the higher magnification images (bottom) were acquired. (E) Illustration of microhemorrhage quantification using the positive pixel detection algorithm (see Methods). Yellow lines highlight the detected microhemorrhage. (F) Microhemorrhage density is significantly higher in mice treated with higher pressures (1.29 MPa and 1.58 MPa) compared to the low pressure (0.59 MPa) and control groups.

Fig 4. Relationships between intratumoral and peritumoral MR image contrast enhancements and microhemorrhage density and eGFP mRNA level.

(A) Overview of MRI analysis workflow. A representative pre-sonication MR image is presented to show the tumor. This image was used to define the intratumoral area mask and peritumoral area mask. Pre- and post-sonication contrast-enhanced MR images were processed following the same procedure described in Method for the quantification of MR contrast enhancement in the intratumoral area and the peritumoral area. Correlation analyses between intratumoral and peritumoral enhancements with microhemorrhage density (B, C) and plasma eGFP mRNA level (D, E) in FUS-treated mice (n = 15) using primer A.