Localized blood-brain barrier opening in infiltrating gliomas with MRI-guided acoustic emissions-controlled focused ultrasound

Abstract

Pharmacological treatment of gliomas and other brain-infiltrating tumors remains challenging due to limited delivery of most therapeutics across the blood-brain barrier (BBB). Transcranial MRI-guided focused ultrasound (FUS), an emerging technology for noninvasive brain treatments, enables transient opening of the BBB through acoustic activation of circulating microbubbles. Here, we evaluate the safety and utility of transcranial microbubble-enhanced FUS (MB-FUS) for spatially targeted BBB opening in patients with infiltrating gliomas. In this Phase 0 clinical trial (NCT03322813), we conducted comparative and quantitative analyses of FUS exposures (sonications) and their effects on gliomas using MRI, histopathology, microbubble acoustic emissions (harmonic dose [HD]), and fluorescence-guided surgery metrics. Contrast-enhanced MRI and histopathology indicated safe and reproducible BBB opening in all patients. These observations occurred using a power cycling closed feedback loop controller, with the power varying by nearly an order of magnitude on average. This range underscores the need for monitoring and titrating the exposure on a patient-by-patient basis. We found a positive correlation between microbubble acoustic emissions (HD) and MR-evident BBB opening (P = 0.07) and associated interstitial changes (P < 0.01), demonstrating the unique capability to titrate the MB-FUS effects in gliomas. Importantly, we identified a 2.2-fold increase of fluorescein accumulation in MB-FUS-treated compared to untreated nonenhancing tumor tissues (P < 0.01) while accounting for vascular density. Collectively, this study demonstrates the capabilities of MB-FUS for safe, localized, controlled BBB opening and highlights the potential of this technology to improve the surgical and pharmacologic treatment of brain tumors.

Keywords: acoustic emissions; blood–brain barrier; focused ultrasound; glioma; microbubbles.

Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.

MRgFUS-mediated BBBO: (A) Schematic illustration of the transcranial MRgFUS system. The helmet-shaped hemispherical phased array is comprised of 1,024 ultrasound transducers allowing for electronic steering and beam refocusing through the skull. Intracerebral endothelial cells are shown as FUS beams converge toward the target location causing circulating MBs within the acoustic field to oscillate. The resulting stable MB oscillation leads to BBBO. (B, Left) Schematic illustration of the focal spot for an ideal hemispherical phased array as ultrasound beams emanating from the phased array converge forming a theoretical ovoid geometry. The length of the focal spot corresponds to one wavelength along the z-axis and half a wavelength in the XY-plane. At 230 kHz—the center frequency used for BBBO—one wavelength corresponds to ∼6.5 mm. (Middle) Depiction of the subspot grid encompassing nine individual subspots. Focal spots (gradient disks) are shown across the 3 × 3 subspot grid. The arrows indicate the direction of the ultrasound beam steering pattern during sonications, which proceeded in a linear loop pattern. (Right) Axial T1w MR image showing the subspot grid target in two dimensions within the tumor region (enclosed dotted circle). The arrows in the XY-plane indicate the beam steering direction during treatment.

Fig. 2.

Targeted BBBO in infiltrating gliomas: (A) preoperative axial MR images prior to FUS showing T1w, T2w, GRE, T1c, and cerebral blood volume (CBV) sequences. The blue dotted circle indicates the tumor region. (B) Preoperative axial MR images after FUS treatment showing T1w, T2w, GRE/T2*, and T1c sequences. The blue square represents the subspot grid; the inset shows the new contrast enhancement within the target region (e.g., subspot grid). (C) Intraoperative white light images showing the earliest stage of tumor surgery during removal of the MB-FUS–targeted region (blue square). Inset shows fluorescence imaging of this region (visualization score = 3) using the Zeiss YELLOW 560 module. (D) Postoperative axial T1c and T2w MR images showing resection of the intrinsic tumor.

Fig. 3.

Target subspot grid, acoustic energy maps, and BBBO: (Left) Axial view of T1c MRI acquisitions during treatment planning (pre-FUS). (Left Middle) Subspots are positioned across the target subspot grid region during treatment planning. (Middle) Following MB-FUS treatment, the accumulated acoustic energy is calculated from the corresponding spectrograms based on AEM data and represented as a colorometric acoustic energy map; note the relative heterogeneity and conformality within the target regions. (Middle Right) The intraprocedural view of the subspot grid with underlying energy map showing the real-time deposition of acoustic energy across a given target. (Right) The corresponding axial T1c MRI after FUS treatment depicts MB-FUS BBBO.

Fig. 4.

MRI, histology, and fluorescein intensity of intrinsically enhancing, nonenhancing, and FUS-treated tumor regions. (A–C) Preoperative contrast-enhanced T1w MRI (Left) in axial view showing intrinsically enhancing, nonenhancing, and FUS-treated glioma regions, respectively (blue dotted circle). Representative histologic hematoxylin and eosin slices (Middle) from resected tissues are shown next to the MRI images. (Scale bar, 50 µm.) Fluorescein accumulation in the tissue (Right) imaged with a 20× objective at excitation wavelengths of 488 nm. (D) Quantification of sodium fluorescein intensity for intrinsically enhancing, nonenhancing, and FUS-treated glioma regions indicated in arbitrary units (A.U.). (E) Quantification of vessel density in tissue samples resected from intrinsically enhancing, nonenhancing, and FUS-treated glioma regions. Immunofluorescence staining of a vascular marker (CD31, red) and cell nucleus (DAPI, blue) was imaged with a 20× objective at excitation wavelengths of 405 nm and 561 nm for CD31 and DAPI, respectively. We assessed vessel density in the same region by using ImageJ’s tubeness function. Plots show means ± SEM (n = 4). P values were determined by unpaired t tests (*P ≤ 0.01; ****P ≤ 0.0001).

Fig. 5.

Post-FUS T2* changes and correlation with surgical pathology. GRE MRIs for the four study participants with varying degrees of new T2* changes: (A) none/minimal, (B) significant, (C) moderate, and (D) none/minimal. The corresponding hematoxylin and eosin–stained histological sections of the (E–H) FUS-treated (FUS) and (I–L) untreated nonenhancing (No FUS) tumor regions for each of the four patients revealed no significant differences and, more specifically, no evidence of microhemorrhages in the FUS-treated regions. (Scale bar, 50 µm.)

Fig. 6.

Acoustic emissions and HD correlations with MRI findings: (A and B) T1c and GRE/T2* axial MR images before (Left) and after (Right) FUS treatment reveal differences in new vascular permeability corresponding to BBBO accompanied by minimal changes in interstitial components, respectively. Insets show magnified T1c (Lower Left) and GRE/T2* (Lower Right) of the target region indicated by the blue square in the respective MR images. The corresponding spectrograms (Center) and HDs calculated from these spectrograms of 0.5 (patient 3, red box) and 0.8 (patient 1, blue box) show levels correlating with the degree of new T1c. The HD per subspot was calculated as the accumulated harmonic score (over all pulses/sonications) divided by the number of subspots. These values are unitless, as they are referenced against a calibration value. (C and D) To account for within-case correlation of MRI signal values, a generalized estimating-equation modeling was conducted. This analysis revealed a statistically significant association between GRE/T2* and HD (P < 0.001) but not between the new T1c signal and HD (P = 0.07). The insets show the quantification of the corresponding T1c and T2* values for patient 4, respectively. Notably, patients 1, 2, and 3 were treated using ExAblate Neuro’s interface version 7.0, while patient 4 was treated using ExAblate Neuro’s interface version 7.4, which allowed coverage of more complex shaped target grids of up to 32 subspots as well as more uniform energy distribution within a given target grid. Patient 4 was treated at four different HDs. The treatment of HD at 1.1 was not included due to moving artifacts in the MRI acquisitions (MRI data are given in arbitrary units of voxel intensity).