ABSTRACT
Given the marginal penetration of most drugs across the blood-brain barrier, the efficacy of various agents remains limited for glioblastoma (GBM). Here we employ low-intensity pulsed ultrasound (LIPU) and intravenously administered microbubbles (MB) to open the blood-brain barrier and increase the concentration of liposomal doxorubicin and PD-1 blocking antibodies (aPD-1). We report results on a cohort of 4 GBM patients and preclinical models treated with this approach. LIPU/MB increases the concentration of doxorubicin by 2-fold and 3.9-fold in the human and murine brains two days after sonication, respectively. Similarly, LIPU/MB-mediated blood-brain barrier disruption leads to a 6-fold and a 2-fold increase in aPD-1 concentrations in murine brains and peritumoral brain regions from GBM patients treated with pembrolizumab, respectively. Doxorubicin and aPD-1 delivered with LIPU/MB upregulate major histocompatibility complex (MHC) class I and II in tumor cells. Increased brain concentrations of doxorubicin achieved by LIPU/MB elicit IFN-γ and MHC class I expression in microglia and macrophages. Doxorubicin and aPD-1 delivered with LIPU/MB results in the long-term survival of most glioma-bearing mice, which rely on myeloid cells and lymphocytes for their efficacy. Overall, this translational study supports the utility of LIPU/MB to potentiate the antitumoral activities of doxorubicin and aPD-1 for GBM.
Subject(s)
Blood-Brain Barrier , Brain Neoplasms , Doxorubicin , Microbubbles , Programmed Cell Death 1 Receptor , Doxorubicin/pharmacology , Doxorubicin/administration & dosage , Doxorubicin/therapeutic use , Doxorubicin/analogs & derivatives , Animals , Humans , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Programmed Cell Death 1 Receptor/metabolism , Mice , Blood-Brain Barrier/metabolism , Blood-Brain Barrier/drug effects , Brain Neoplasms/drug therapy , Brain Neoplasms/immunology , Brain Neoplasms/pathology , Cell Line, Tumor , Glioma/drug therapy , Glioma/immunology , Glioma/pathology , Brain/metabolism , Brain/drug effects , Female , Drug Delivery Systems , Ultrasonic Waves , Glioblastoma/drug therapy , Glioblastoma/immunology , Glioblastoma/pathology , Male , Microglia/drug effects , Microglia/metabolism , Mice, Inbred C57BL , Antibodies, Monoclonal, Humanized/therapeutic use , Antibodies, Monoclonal, Humanized/administration & dosage , Antibodies, Monoclonal, Humanized/pharmacology , Immune Checkpoint Inhibitors/pharmacology , Immune Checkpoint Inhibitors/administration & dosage , Polyethylene GlycolsABSTRACT
BACKGROUND: Cellular functions hinge on the meticulous orchestration of protein transport, both spatially and temporally. Central to this process is retrograde trafficking, responsible for targeting proteins to the nucleus. Despite its link to many diseases, the implications of retrograde trafficking in glioblastoma (GBM) are still unclear. METHODS: To identify genetic drivers of TMZ resistance, we conducted comprehensive CRISPR-knockout screening, revealing ADP-ribosylation factor 4 (ARF4), a regulator of retrograde trafficking, as a major contributor. RESULTS: Suppressing ARF4 significantly enhanced TMZ sensitivity in GBM patient-derived xenograft (PDX) models, leading to improved survival rates (p<0.01) in both primary and recurrent lines. We also observed that TMZ exposure stimulates ARF4-mediated retrograde trafficking. Proteomics analysis of GBM cells with varying levels of ARF4 unveiled the influence of this pathway on EGFR signaling, with increased nuclear trafficking of EGFR observed in cells with ARF4 overexpression and TMZ treatment. Additionally, spatially-resolved RNA-sequencing of GBM patient tissues revealed substantial correlations between ARF4 and crucial nuclear EGFR (nEGFR) downstream targets, such as MYC, STAT1, and DNA-PK. Decreased activity of DNA-PK, a DNA repair protein downstream of nEGFR signaling that contributes to TMZ resistance, was observed in cells with suppressed ARF4 levels. Notably, treatment with DNA-PK inhibitor, KU57788, in mice with a recurrent PDX line resulted in prolonged survival (p<0.01), highlighting the promising therapeutic implications of targeting proteins reliant on ARF4-mediated retrograde trafficking. CONCLUSION: Our findings demonstrate that ARF4-mediated retrograde trafficking contributes to the development of TMZ resistance, cementing this pathway as a viable strategy to overcome chemoresistance in GBM.
