Blood-Brain Barrier Opening with Ultrasound Augments Doxorubicin and Anti-PD-1 Therapy Efficacy, Intracranial Immune Activation, and Prolongs Survival in Preclinical Glioma Models

INTRODUCTION: The limited drug penetration across the blood-brain barrier (BBB) represents a challenge for the treatment of glioblastoma patients. A novel drug delivery technology based on low-intensity pulsed ultrasound combined with intravenous microbubbles (LIPU/MB) overcomes this challenge by opening the BBB. In this study, we hypothesize that LIPU/MB enhances the therapeutic effect of both liposomal doxorubicin and anti-PD-1 therapy in gliomas by eliciting local immune responses. METHODS: We utilized LIPU/MB to deliver doxorubicin and anti-PD-1 blocking antibodies to murine models and evaluated drug concentrations in the brain. Flow cytometry evaluated IFN-g production by glioma-associated microglia and macrophages in murine gliomas treated with doxorubicin and anti-PD-1 therapy delivered by LIPU/MB. Furthermore, we assessed the efficacy of this drug combination delivered by LIPU/MB in glioma-bearing mice. RESULTS: LIPU/MB induced a 4-fold increase in doxorubicin concentrations in sonicated brains compared to those not sonicated (p = 0.003). In addition, doxorubicin delivered with LIPU/MB elicited an IFN-g phenotype (p < 0.0001) as well as MHC I and PD-L1 expression in glioma-associated microglia and macrophages (p < 0.05). Increased brain concentrations of doxorubicin led to long-term survivors in the majority of glioma-bearing mice. On the other hand, LIPU/MB-mediated BBB opening increased brain concentrations of anti-PD-1 therapy in mice (6.3-fold increase, p < 0.01). The combination of doxorubicin and anti-PD-1 therapy delivered with LIPU/MB resulted in long-term survival (p < 0.0001). This effect was abrogated in CD8-/- mice suggesting that T cells are important for the efficacy of doxorubicin and anti-PD-1 therapy delivered with LIPU/MB. CONCLUSIONS: This preclinical study demonstrates the immense potential of LIPU/MB technology in enhancing intracranial immune responses and drug delivery for glioblastoma treatment using doxorubicin and anti-PD-1 therapy, paving the way for more effective treatment strategies for this brain cancer.