Background:
The blood-brain barrier (BBB) is a highly exclusive, protective barrier that hinders the delivery of large therapeutic molecules into brain tissue1,2. Promisingly, MR-guided focused ultrasound (MRgFUS) with microbubbles has emerged as a promising modality for non-invasive, non-ionizing, reversible, and repeatable blood-brain barrier opening (BBBO; Figure 1A)1. Indeed, several ongoing and completed clinical trials have evaluated MRgFUS for BBBO across neurological indications (e.g. NCT01698437, NCT03626896, NCT06329570, NCT05615623, NCT05630209). Toward precision BBBO-enabled drug delivery exercises, intraprocedural spatiotemporal mapping of small and large molecule delivery remains a limitation. Indeed, gadolinium enhancement – the gold standard readout for BBBO confirmation following MRgFUS – provides a limited proxy and does not necessarily reflect the transport of a larger or more complex therapeutic agents.

This work elevates the use of PET imaging to fill a critical gap in modalities for molecular feedback during FUS BBBO procedures. PET imaging enables the direct tracing of molecular agents with high sensitivity. However, a key limitation is relatively low spatial resolution. We have recently overcome this limitation through a powerful integration of PET and MRI with FUS instrumentation in a first-in-class trimodal system3. We herein deploy this system for the first time in an in vivo brain tumor model to address critical knowledge gaps related to small versus large molecule deposition in murine glioblastomas with FUS.

 

Hypothesis/Objective:
To leverage a first-in-class trimodal PET/MRI/FUS system for integrated multimodal mapping of small versus large molecule delivery to glioblastomas following FUS BBBO.

 

Methods:
First-in-class trimodal PET/MRI/FUS system comprises a 1.15 MHz FUS transducer and custom-built PET insert electronically integrated with a 9.4T small-animal MRI, operating with a custom tunable RF coil. GL261 and CT2A glioblastomas were established orthotopically in the right striatum or implanted bilaterally in C57/Bl6 mice. MR imaging was performed on tumor-bearing mice 24-hours prior to treatment for group randomization based on tumor volumes. Mice received concomitant i.v. delivery of 64Cu-DOTA (small molecule) or 64Cu-DOTA-IgG (large molecule), gadolinium contrast agent (Dotarem; gold standard), and microbubbles followed by PET/MRI-guided FUS-BBBO (0.3 MPa or 0.6 MPa). Acoustic emissions monitoring was performed via real-time passive cavitation detection. PET/MRI was performed at serial time points (0-24hr) and ex vivo brain and peripheral biodistribution (64Cu-DOTA, 64Cu-DOTA-IgG) was determined via automated gamma counter.

 

Results:
GL261 tumors treated with a single sonication at a fixed peak negative pressure (PNP) of 0.6MPa exhibited ~1.7-fold increase in 64Cu-DOTA uptake with FUS (Figure 1B-C). Qualitatively, co-registered PET/MRI images revealed concordance between gadolinium (MRI) and 64Cu-DOTA (PET) contrast (Figure 1D). Spatiotemporal mapping of small (64Cu-DOTA) versus large (64Cu-DOTA-IgG) molecule delivery using a multi-sonication pattern revealed clear FUS-enabled increases in uptake in bilateral CT2A tumors across two distinct PNPs. PET scans of 64Cu-DOTA revealed strong spatial concordance with standard MRI contrast (Figure 1E-F), while this was diminished for the large molecule tracer (64Cu-DOTA-IgG) – underscoring the high fidelity of PET imaging for informing delivery of larger biologics (Figure 1G-H). Harmonic and broadband acoustic emissions confirmed increasing mechanical energy deposition with increased FUS PNP, and this was well-conserved across small and large molecule delivery contexts (Figure 1I-L). Ex vivo biodistribution analysis revealed markedly increased (~1.5-2x) 64Cu-DOTA uptake at 0.6MPa compared to 0.3MPa (p=0.0383) and sham (p=0.0858) by 24-hours post-treatment (Figure 1M). By contrast, 64Cu-DOTA-IgG uptake exhibited a trending increase with PNP (p = 0.0696), and the augmentation of uptake with FUS was only significant at 0.6 MPa (vs. sham; ~2x) (Figure 1N).

 

Conclusion:
Our findings generate the first proof of concept for a preclinical PET/MRI/FUS system deployed in murine brain tumors to disentangle small versus large molecule delivery with FUS BBBO. We demonstrate the utility of quantitative PET for spatiotemporal mapping of molecular delivery in murine glioblastomas, highlighting contrasts between MRI and PET feedback that elevate the transformative potential of intraprocedural PET imaging. Future studies will leverage this trimodal system to interrogate delivery impacts of acoustic parameters, microbubble selection and therapeutic sequencing across disease and therapeutic contexts.

 

Image/Figure:
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Image/Figure Caption:
Figure 1: (A) Illustration of focused ultrasound (FUS)-mediated blood brain barrier opening (BBBO). (B) Schematic illustrating GL261-tumor bearing implantation, FUS sonication pattern, and the applied peak negative pressure (PNP; 0.6MPa) for 64Cu-DOTA delivery. (C) Quantification of average standard uptake values (SUVs; %ID/mL) extracted from PET/MRI images, depicting a ~1.7-fold increase in 64Cu-DOTA uptake post-FUS compared to sham control. (D) Representative axial (top) and coronal (bottom) contrast-enhanced T1-weighted MRI images, decay-corrected PET images, and co-registered PET/MRI scans depicting clear regions of enhanced 64Cu-DOTA penetrance (white arrows) in GL261 tumors compared to sham. (E) Schematic illustrating bilateral CT2A tumors, the FUS sonication pattern, and the applied PNPs (0.3MPa, 0.6MPa) for 64Cu-DOTA delivery. (F) Representative axial (top) and coronal (bottom) contrast-enhanced T1-weighted MRI, decay-corrected PET, and co-registered PET/MRI images showing increased 64Cu-DOTA penetrance (white arrows) in bilateral CT2A tumors with FUS, with greater contrast signal at 0.6MPa than 0.3MPa. (G) Schematic illustrating bilateral CT2A tumors, the FUS sonication pattern, and the applied PNPs (0.3MPa, 0.6MPa) for 64Cu-DOTA-IgG delivery. (H) Representative axial (top) and coronal (bottom) contrast-enhanced T1-weighted MRI, decay-corrected PET, and co-registered PET/MRI images showing increased 64Cu-DOTA-IgG delivery (white arrows) in FUS-treated groups compared to sham, with higher contrast and tracer signal at 0.6MPa than 0.3MPa. (I-L) Quantification of acoustic emissions signal in the 2f frequency band, indicating increasing microbubble activity with increased PNP. (M-N) Ex-vivo gamma counter quantification of sonicated tumor-bearing brain hemispheres one day post-treatment, confirming differential impacts of FUS on 64Cu-DOTA (M) and 64Cu-DOTA-IgG (N) delivery.

 

Author
Zehra Demir
University of Virginia