Amplifying antibody delivery to the cerebellum and orthotopic medulloblastomas using magnetic resonance imaging-guided focused ultrasound

Introduction: Medulloblastoma is the most prevalent form of pediatric brain cancer, with group 3 (G3MB) being one of the most challenging subgroups, harboring a 5-year survival rate of less than 50%. Effective treatment of G3MB remains elusive due to its complex nature, its location within the immunologically and pharmacologically guarded central nervous system, and the substantial risk of long-term developmental impairments stemming from treatment toxicities. Focused ultrasound (FUS) offers a promising strategy for transforming care in the highly vulnerable population of pediatric brain tumor patients that is in critical need of more effective, less toxic therapeutic strategies. FUS offers a non-invasive, non-ionizing, highly controllable method for spatially precise transcranial acoustic energy deposition within the brain. Despite burgeoning applications of this technology in adult brain tumors and even pediatric high-grade gliomas, efforts to develop FUS for cerebellar targeting or MB treatment have thus far been limited. In this proposal, we deploy FUS in combination with microbubbles to achieve temporary blood brain/tumor barrier disruption (BBB/BTB-D) in the cerebellum and a high-fidelity preclinical model of orthotopic G3MB - with the goal of demonstrating safety, feasibility, and potential for improving localized immunotherapy delivery using a model payload. We expect that this study will offer novel and timely insights for clinical translation.

Materials and

Methods: Serial MR imaging and FUS protocols were established in immunocompetent wild-type (C57/Bl6) or orthotopic tumor-bearing mice (stereotactic implantation of Gfi1/c-MYC driven G3MB cells into the cerebellum). Specifically, mice received i.v. albumin-shelled microbubbles (1 × 10 /g B.W.) and underwent multi-target sonication protocols (1.15 MHz, 0.3-0.45 MPa, 0.5% duty cycle, 2 min period) under 9.4T MRI guidance (Fig 1A). FUS treatments were bookended by contrast-enhanced T1-, T2-, and susceptibility-weighted MR imaging. During FUS BBB/BTB-D, heart and respiratory rates were monitored to confirm safety. Additionally, real-time acoustic emissions monitoring was performed via passive cavitation detection (PCD) to contextualize linear (“stable”) and non-linear (“inertial”) oscillation activity of microbubbles (i.e. cavitation) over the full sonication period, thereby providing a signature of mechanical impact associated with each peak negative pressure tested. PCD analysis was retrospectively performed via custom Matlab code calculating harmonic cavitation dose (HCD), ultraharmonic cavitation dose (HCDU), and inertial cavitation dose (ICD) from acoustic emissions readings. A 20 kHz bandwidth centered around the first 4 harmonics and ultra-harmonics of each FUS-treated mouse was used to define HCD and HCDU, respectively. ICD was defined as broadband emissions, i.e. signal excluding 20 kHz bandwidth centered around the first 4 harmonics and ultra-harmonics. For model payload (antibody) delivery studies, fluorescently-labeled IgG was systemically delivered concomitant with microbubbles. Antibody penetrance was evaluated via ex vivo epifluorescence imaging. Terminal histology was performed to assess for acute evidence of microhemorrhage or cellular response following BBB and/or BTB disruption.

Results, Conclusions, and Discussions: Clear evidence of localized BBB/BTB-D was observed with post contrast T1w-MRI by gadolinium enhancement in both naïve cerebella (Fig 1B) and orthtopic G3MB tumors (Fig 1C), confirming feasibility of targeting this anatomical location. PCD analysis suggested a significant increase in HCD, HCDU, and ICD from 0.3 to 0.45 MPa (Fig 1D). However, no notable changes were observed in cardiac and respiratory rates during or after treatments, and FUS did not confer discernible changes in T2 (Fig 1B), susceptibility-weighted, or histopathologic signatures - indicating safety across the pressures deployed. FUS BBB/BTB-D significantly elevated antibody penetrance into naïve cerebellum (Fig 1E) and G3MB (Fig 1F) relative to the controls, indicating improved antibody access. A trend of increased antibody penetrance was observed from 0.3 to 0.45 MPa, suggesting a correspondence of delivery with FUS peak negative pressure and the associated cavitation dose. Our data confirm the safety and feasibility of utilizing FUS BBB/BTB-D in both the naïve and G3MB-bearing cerebellum. We demonstrate that FUS significantly improves the penetration of a large molecular-weight payload (antibody) into naïve cerebellum and G3MB tumors in a FUS exposure-dependent manner. Ongoing investigations are utilizing these insights to deploy delivery of novel immunotherapy candidates with FUS BBB/BTB-D to elaborate immunologic control of G3MBs.

Acknowledgements (Optional): This work was supported by the NIH Director’s Early Independence Award (DP5OD031846) and the Ben & Catherine Ivy Foundation.